KR20010006280A - Molecules presenting a multitude of active moieties - Google Patents

Abstract

The present invention relates to pharmaceutical compositions that provide a multifunctional therapeutic agent. The pharmaceutical composition contains a multifunctional donor. In one embodiment, the multifunctional donor has Formula 1:

Formula 1

(Y)-(XA) _n

Where

Y is the basic structure

X is a direct bond or a linker,

A is a provided functional group,

n is an integer greater than 10 selected to allow a given group to interact with multiple target binding sites.

The composition may also include a pharmaceutically acceptable carrier. Alternatively, the donor itself can act as its own pharmaceutically acceptable carrier. Also described are methods of treating any disease or condition. The method includes administering a plurality of Group A to a subject to be treated. It is treated by the interaction of a multifunctional donor with a plurality of target binding agents. Another aspect of the present invention includes a packaged multifunctional provider having instructions for use in the method described above and in a method for devising a multifunctional provider useful for the method of the present invention. The multifunctional donor disclosed herein exhibits specificity in binding, which has a number of advantages. In addition, multifunctional donors allow for both positive and negative interactions.

Description

MOLECULES PRESENTING A MULTITUDE OF ACTIVE MOIETIES}

Government support

The study was funded in part by NIH approved GM30367 and GM39589. The NMR facility in Harvard was funded by NIH Approval 1-S10-RR04870-01 and NSF Approval CHE88-14019. The government may have certain rights in the invention.

Related Applications

This application claims priority to US Provisional Patent Application Nos. 60 / 043,781 and 60 / 043,826, filed April 11, 1997 and April 14, 1997, respectively. This application claims U.S. Provisional Patent Application No. 60,043,288, filed April 11, 1997, and "Polyvalent Presenter Combinatorial Libraries and Their Uses," filed April 15, 1997. US Provisional Patent Application No. 60 / 043,918, entitled Field.

Polyvalency is dominant in the biological field and important for the functioning of biological systems. Multiaction is an interaction that occurs simultaneously, for example, through several pairs of individual ligand-receptor bonds between two distinct species (see FIG. 1). The interaction of the ligand with the receptor occurs throughout the organism. Ligands include molecules that carry information or act on proteins in biological systems. Examples of ligand types include drugs, hormones, signal molecules, cell surface markers, toxins, enzyme substrates, bioregulators, neurotransmitters and lymphokines. Receptors include molecules that interact with and receive information from the ligand. Most receptors are proteins, which include protein receptors, antibodies and enzymes. Some receptors are nucleic acids, which include regulatory regions of DNA and RNA.

Most drugs are ligands that interact with a single receptor (some drugs are receptors that interact with a single ligand). In the biological field, many important results are derived from the simultaneous interaction of multiple receptors with multiple ligands. Multifunctional interactions dominate in the biological field. Multiaction is typically included in many interactions involving interactions that occur on the cell surface and / or receptor groups or receptor communities. Multifunctionality can also be important in the interaction of macromolecules involving multiple attachment points simultaneously.

So far, the concept of multifunction has been investigated. This has been investigated in the context of studies aimed at identifying the mechanism by which influenza viruses adhere to cell surfaces (Whitesides, G. M. et al., J. Med. Chem., 1995, 38, 4179-4190); Sigal et al., Journal of the American Chemical Society 1996, 118, 16, 3789-3800. Kiessling et al., Chemistry and Biology 1996, Vol. 3, No. 2, 71-77, presented a structural template for producing multifunctional carbohydrates for studying and regulating biological cognition. Chiesling et al. Considered them as "chemical tools to investigate multifunctional protein-saccharide interactions." In the paragraph entitled "Where do we go from here?", Kiessling et al. "Multifunctional carbohydrates are not only useful for investigating biological recognition processes, but can also be used as probes for biological function". It is written. Matrosovich [FEBS LETTERS 1989, 252, 1, 1-4] proposed the use of multifunctional inhibitors to inhibit microbial adhesion. Matrosovich is well known in the art for "drug delivery systems, such as by coupling monofunctional inhibitory active molecules into multiple copies to solubilize biocompatible polymers or particulate carriers to produce such multifunctional structures. "You can use some principles of design." In addition, Matrosovich describes that "the accuracy and prospects for the practical use of this approach to design antimicrobials may be evaluated in the future."

Summary of the Invention

So far "the idea that high affinity specific binding is a receptor-ligand interaction has been largely dominant" (see page 71 of the literature cited above). Typically, attempts to optimize receptor-ligand interactions have been focused on the binding capacity or binding specificity of specific ligands with specific receptor-binding sites in individual binding. For example, a ligand is selected for interaction based on its known advantageous binding capacity, or a ligand that is considered to be a weak binder is chemically modified to enhance its binding capacity before it is used or selected for use. It became. Weakly binding ligands need not be excluded as a component of the polyvalent presenter of the present invention.

The present invention, at least in part, is based on the understanding of how multi-functional multi-provider providers interact with a group of target binding sites in an exceptional and holistic manner for the inventors of receptor-ligand interaction and multi-function. Based on consideration. Our approach differs from conventional ways of looking at interactions based on more individual basis as separate receptor-ligand interactions. Due to the above exceptional overall manner of considering receptor-ligand interactions and multifunctions in biological systems, the inventors have found that multifunctions can be used as a basis for rational drug design, such as a key basis, and also to many different diseases and It has been found that it can be applied universally in treating diseases. In addition, because of the exceptional overall manner of contemplating receptor-ligand interactions, the inventors do not have to select specific ligands based on their individual binding capacity in designing multifunctional drugs. I found out.

The multifunctional donor of the present invention may be a plurality of group A (e.g., which may be the same or different on the framework (e.g. polymer backbone) that forms the multifunctional donor for treating any disease or condition). Ligands) to form and arrange. The configuration and arrangement of group A in the framework may be attributed to a therapeutic effect due to the blanketing of a group of target binding sites B or a series of target binding sites B (such as a clustered receptor binding site on the cell surface), or one ligand. It can be made based on different types of binding sites for different types of ligands with different types of binding sites for and multiple types of ligand (s) attached in the subject. The shielding of a group of target binding sites B occurs as a result of the compliant interfacial interaction of the multifunctional donor with a group of target binding sites B. Shielding a series of target binding sites occurs as a result of compliant interfacial interactions with multiple groups of target binding sites B. Shielding is not intended to be limited to continuous blocking films, but is discussed in more detail in the later section entitled "Detailed Invention".

For the purpose of illustration, the shielding effect will be discussed in detail for the case corresponding to a multifunctional provider with a polymeric framework. As shown in FIG. 2, the multi-functional donor (P) having the polymer framework (1) is shaped to conform to a surface containing an interface (3), for example a group of target binding sites (5) upon interaction. Can be taken. The one or more attached and compliant multifunctional donors essentially mask a series of target binding sites B (which consists of one or more groups of target binding sites B) to exhibit or enhance the therapeutic effect of the multifunctional donor (s). . The shield may be a physical shield (eg, the multifunctional donor may sieve cover the target binding site B) and / or a steric shield (eg, the target binding site B may fill multiple groups A of the multifunctional provider A). May be). The shield need not be a continuous or tight fit barrier, and may be a discontinuous or loose fit barrier as shown in FIG. 2, and the shield need not completely cover the target binding site. In some cases, the shield will be a gel layer that conforms to a surface containing a series of target binding sites B.

In addition, the present invention relates to a method for treating any disease or condition. In one embodiment, the method comprises administering to the subject a plurality of group A such that the disease or condition is treated by masking a group or series of target binding sites B in the subject. Shielding a group of target binding sites B in a subject occurs through adaptive interfacial interaction of a group of target binding sites B with a group of target binding sites. The shielding of a series of target binding site B occurs through adaptive interfacial interaction of multiple multifunctional donors with different groups of target binding site B. In other embodiments, the methods of the present invention include the use of multifunctional donors that meet certain criteria or the use of specially selected components such as group A, a framework or a linker.

The invention also relates to pharmaceutical compositions for multifunctionally providing a therapeutic agent. The pharmaceutical composition contains a multifunctional donor. The multifunctional provider may have a structure of Formula 1

(Y)-(XA) _n

Where

Y is the basic structure

X is a direct bond or a linker,

A are provided functional groups which may each be the same or different,

n is an integer greater than 10.

The provider is selected such that Group A provided can interact with a group of target binding sites B. The composition may also include a pharmaceutically acceptable carrier. The provider can act as its own pharmaceutically acceptable carrier. In one embodiment the multifunctional donor is selected such that, for example, the multifunctional donor conforms to an interface containing a group of target binding sites B, and masks a group of target binding sites B when administered to a subject and-(XA ) Residues are attached to Y. In another embodiment, X is a linker that is not part of Y or A as an independent residue, and n is an integer greater than 10. Such donors are selected to conform to a group of target binding site B when the multifunctional donor is administered to a subject. In another embodiment, Y is a polymer framework, A is a provided functional group, X is a linker, and n is an integer greater than 10. This structure is chosen to conform to a group of target binding site B when the multifunctional donor is administered to a subject.

Another aspect of the invention is a method of controlling the adhesion between a plurality of surface-bound biological molecules and a plurality of second molecules. The method includes providing a plurality of surface-bound biological molecules and multifunctional donors together. Multifunctional donor includes a basic structure to which a plurality of third molecules are attached. The third molecule may be formed by simultaneous entropy enhanced binding of the third molecule to the plurality of surface-bound biological molecules, and a portion of the plurality of surface-bound biological molecules to which the third molecule is not bound. The steric blocking by the structure regulates the adhesion between the surface-bound biological molecule and the second molecule.

Another aspect of the invention is the above method, wherein the multifunctional donor is a non-saccharide and has an enhancement factor β greater than about 10.

Another aspect of the invention is a multifunctional donor which is pAA (Gal-β), pAA (Gal-α), pBMA (Gal-β) and pBMA (Gal-α).

Another aspect of the invention is a method of preventing adhesion of erythrocytes with lysine. The method comprises contacting lysine with an effective amount of a multifunctional donor selected from the group consisting of pAA (Gal-β), pAA (Gal-α), pBMA (Gal-β) and pBMA (Gal-α). do.

Another aspect of the invention is a method of preparing a multifunctional donor selected from the group consisting of pAA (Gal-β) and pBMA (Gal-β). This method involves reacting Gal-βO-L ₁ NH ₂ with poly (N-acryloyloxysuccinimide) or poly (butadiene-co-maleic anhydride) and quench the reaction.

Another aspect of the invention is a method of making a multifunctional donor selected from the group consisting of pAA (Gal-α) and pBMA (Gal-α). This method involves reacting Gal-αC-L ₂ NH ₂ with poly (N-acryloyloxysuccinimide) or poly (butadiene-co-maleic anhydride) and quench the reaction.

Another aspect of the invention is a multifunctional provider which is pAA (GlcNAc-β).

Another aspect of the invention is a method for inhibiting fertilization in a subject, comprising administering to the subject an effective amount of a multifunctional donor that is pAA (GlcNAc-β).

Another aspect of the invention is a method of preparing a multifunctional provider which is pAA (GlcNAc-β). This method involves reacting GlcNAc-β-L ₁ NH ₂ with poly (N-acryloyloxysuccinimide) and quenching the reaction.

1 is a schematic showing both monofunctional and multifunctional reactions.

FIG. 2 is a schematic diagram illustrating that the polymeric multifunctional donor (s) "shields" a group of target binding sites and a series of target binding sites B. FIG.

3 is a schematic diagram illustrating lysine-cell interaction.

4 is a schematic diagram illustrating a strategy for induction of acrosome reaction of sperm.

5 is a schematic diagram illustrating the synthesis of polymeric multifunctional galactosides.

FIG. 6A is a plot schematically illustrating the molar fraction of Gal of the polymer versus RCA ₁₂₀ versus the aggregation inhibitory activity of the polymeric multifunctional galactoside.

FIG. 6B is a plot schematically illustrating the molar fraction of Gal's adhesion to RCA ₆₀ versus the adhesion inhibiting activity of polymeric multifunctional galactosides.

7 is a schematic diagram showing the structure of multifunctional polymeric n-acetylglucosamine and galactoside.

8 is a schematic diagram illustrating the induction of acrosome response of sperm in mice by mono- and multi-functional GlcNAc.

9A is a schematic showing induction of acrosome response of mouse sperm by multifunctional GlcNAc.

9B is a plot schematically showing the acrosome-reacted sperm (%) obtained by pAA (GlcNAc).

10 is a plot schematically illustrating the inhibition of sperm-egg binding by polymeric multifunctional GlcNAc.

In FIG. 11, 11a is a schematic diagram showing generation of pMVMA (NeuAc).

In FIG. 11, 11b is a schematic diagram showing the production of pMVMA (NeuAc; R).

In FIG. 11, 11c is a schematic diagram showing generation of pAA (Gal).

In FIG. 11, 11d is a schematic diagram showing generation of pBMA (Gal).

In FIG. 12, 12a is a schematic diagram showing generation of pAa (SLe ^X ).

In FIG. 12, 12b is a schematic diagram showing the production of pAA (Bacitracin; R).

Drug design

The present invention is directed to a method of designing a multifunctional provider for administration to a subject for treating any disease or condition. The method includes constructing and arranging at least ten multiple groups A on a framework that forms a multifunctional donor for treating any disease and condition. The construction and arrangement is based on the ability of the designed donor to produce a therapeutic effect due to the shielding of a series of target binding site B. In a preferred embodiment, the series of target binding sites B in the subject is masked with one or more multifunctional donors. The shielding is caused by the interaction of the compliant interface of one multifunctional donor with a group of target binding sites B or the interaction of the compliant interface between a plurality of multifunctional donors and a plurality of target binding sites B.

The “multifunctional donor” of the present invention is described in detail later, which includes multicomponent molecules having at least 11 provided group A (which can bind to at least 11 target binding sites B) attached to the framework. . In summary, the multifunctional donor may have a structure of Formula 2:

Y- (A) _n

Where

"Y" represents a basic structure,

"A" represents a provided functional group,

"n" represents an integer greater than 10 selected to allow a given functional group to interact with multiple binding sites B.

The term "disease or disease" includes a disease or condition that is treated by a multifunctional provider of the invention via multifunctional interactions, such as the interaction of a group A with a multifunctional receptor comprising a target binding site B. . A disease or condition intended as part of the present invention may typically be characterized by the interaction between binding site B and group C, wherein both B and C are multifunctional. “B” herein refers to a plurality of binding sites, and “C” is a residue or group that naturally associates with or interacts with a disease or condition. In some embodiments, group C may be identical to group A present on the multifunctional donor of the present invention, causing competition between poly (C) and poly (A) for binding site B. In other embodiments, group C can be different from group A. Binding site B occurs naturally, but group C may be synthesized or may be naturally occurring. For example, C can be naturally occurring sugars, peptides, proteins (eg, ligands for cell surface receptors such as viral antigens), or can be synthesized (eg, present in silicone implants or implants). Group). Group C can be present on the surface or in solution, but must be multifunctional. The term "disease or disease" also refers to a disease or condition that includes or has been found to be affected by naturally occurring multifunctional molecules, such as α ₂ macroglobulin molecules that inhibit the interaction between influenza virus and target cells. It includes. Biological reactions are complex, but those skilled in the art will recognize that the specific point at which the multifunctional donor acts (ie, the specific point targeted for the multifunctional donor to mediate) is dependent upon the interaction between poly (C) and poly (B). Examination of whether or not related will recognize whether any disease or disorder comprises a multifunctional interaction. Examples of diseases and disorders are described in detail below under the subheading "Method of treating a disease or condition."

The term "subject" includes mammals that are susceptible to or have a disease or condition to be treated or targeted for treatment. As such, the present invention is useful for treating humans, livestock, livestock, zoo animals, and the like. Examples of subjects include humans, cattle, cats, dogs, goats, and mice. In a preferred embodiment, the present invention is used to devise a medicament for treating a human subject.

The expression “treating a disease or condition” includes the treatment of a subject having a disease or condition or the prevention of such a disease or condition that can be corrected by treatment with the multifunctional donor of the invention. The term "treatment" has a beneficial effect on a subject, including a significant reduction in one or more symptoms or indications of a disease or disorder. Thus, "treatment" means including continually alleviating one or more symptoms of such a disease or condition to treat any disease or condition. Treatment also includes the use of a multifunctional donor alone, or in combination with other multifunctional donor (s) or with other treatments with agents that are not multifunctional donors.

The phrase “composing and arranging a plurality of groups A on a framework” refers to manipulating the various components of a multifunctional donor that produce a provider capable of performing its intended function in the treatment of any disease or condition. Include. Such manipulations can be made based on the consideration of the interaction (s) between group A and a group of target sites B in a holistic manner. Manipulation involves positioning, sizing and selecting various components of the multifunctional donor, such as the framework, linker, group A. For example, the expression may be a position relative to the backbone linker used to link any of the linker molecules or monomers of the basic structure used to attach the group A to the basic structure and to the positioning of the group A and the base structure relative to each other. We want to include the setting. The expression is also intended to include the selection of specific types of group A, frameworks and / or linkers.

The framework may be chosen for its ability to form certain types of "shields" for a group of target binding sites B, when they form part of a multifunctional donor. For example, some frameworks will form gel-like physical barriers for a group of binding sites B. Any framework may also be selected based on the different features desired in the designed multifunctional provider. Any framework can be selected based on its "flexibility" and / or its ability to impart flexibility to the multifunctional provider even after group A is attached.

Group A may also be selected based on its ability to impart desired features to the multifunctional provider. Although the binding capacity of group A is one factor in its selection process, it is important to recognize that at least some of the present invention, group A, which binds weakly, may be useful for its multifunctional form.

The positioning of group A in the framework also makes it part of the "configuration and arrangement" of the multifunctional provider. For example, positioning may be based on the spatial arrangement of a group of target sites B predicted or known using molecular modeling of a multifunctional environment. Positioning can be made along several different direction axes with respect to the basic structure. For example, any target site B may be spaced on average 10 mm apart from neighboring target site B, such that group A is spaced along the framework in a horizontal direction suitable for accessing the neighboring target binding site B, or Can be located. It should be noted that group A need not be positioned to match the distance between neighboring target binding sites, but rather properly spaced to approach. Positioning group A on the framework takes into account factors other than distance, such as flexibility of the linker and the contour of the interface with the target binding site. For example, the flexible linker may be adjusted in vivo to approach the target binding site even if the distance between group A does not exactly match the distance between neighboring target binding sites B, or even if the contour of the interface is curved rather than flat. Can be. The depth of the engagement pocket (e.g., 2 to 20 microns) can be predicted or known to position group A along an axis that is substantially perpendicular to the underlying structure (e.g., a particular length of linker can be used). .

"Configuration and Arrangement" is also intended to include selecting the type and length of linkers that attach Group A to the framework. The length of the linker can be selected based on their ability to provide group A to the target binding site B in a binding pocket having a known or predicted depth and / or diameter. The chemical nature of the linker, such as hydrophobicity or hydrophilicity, may also be selected based on knowledge of the environment surrounding the target binding site, for example the linker may have to pass through a channel or environment known to be hydrophobic or hydrophilic. Linkers may also be selected based on their ability to impart desired properties to the multifunctional donor, such as flexibility.

The expression “therapeutic effect” is intended to include the ability of a multifunctional provider to perform its intended function, such as to treat or prevent any disease or condition. The therapeutic effect of the multifunctional donor can be determined using techniques recognized in the art for the particular disease or condition to be treated by the designed multifunctional provider, or using assays designed using techniques known in the art. , Or by the assays described herein that do not fall into one of the above categories. A therapeutic effect is considered to be “due to shielding” when the effect is derived from the ability of the multifunctional donor to conform and engage multifunctionally with a group of target binding sites B.

The term “shielding” includes physical shielding or physical masking of target binding site B, and steric shielding such as steric smoothing of target site (s) B and / or enhancement of occupancy of binding site B by ligand A. Examples of physical shielding include the formation of a gel-like layer or barrier for a group of binding sites B, which prevents other groups present on the multifunctional donor from accessing the binding sites. The physical barrier can also be the spaghetti-shaped barrier shown in FIG. 2. In either case, the formation of a gel-like layer or barrier or spaghetti-shaped barrier is biospecifically produced by specifically binding at least a portion of the binding on the multifunctional donor with the corresponding binding partner on the target.

A “stereoscopic shield” of target binding site B is that binding site B is surrounded by group A, which is attached to the framework in a velcro-like manner. One group A may be bound to a binding site, while the other group A may stericly block the same binding site B or a neighboring target binding site.

The expression “a group of target binding sites B” includes binding sites B to which one multifunctional donor molecule conforms. For example, if one multi-functional donor has 10 groups A attached to the framework, the group of binding sites can be the full length of the binding site B to which the molecule conforms, such as 10 to 12 binding sites. However, only two binding sites may actually be occupied. Note that not all binding sites B need to interact with group A, and some sites may remain unoccupied.

The term "series of target binding sites B" includes one or more groups of target binding sites B on an interface. The series of binding sites B need not be equally spaced or positioned, but rather may be randomly positioned in various directions along the axis in different directions with respect to the interface, depending on factors such as the contour of the interface and the cluster shape of the binding sites. For example, the contour of the interface can be flat, bent or movable.

The term "compliant interfacial interaction" includes interactions that occur while the multifunctional donor is present in conformed form to a group of target binding sites B in the molecular region accessible to the interface, such as group A. The interface may be any surface, but not limited to, and is intended to include boundaries of interaction. The term “compliant” is intended to encompass situations where the interface between the multifunctional donor and the surface is intimately or tightly fitted in whole or in part of the surface. In the latter case, the multifunctional donor may be close to the surface at some parts and less closely at other parts of the surface. The term "interaction" is intended to include both positive and negative interactions, as discussed below. The multifunctional donor of the present invention is flexible enough to be adapted to the contour of the interface and therefore conforms to interfaces such as surfaces such as cell surfaces. A specific number of target binding sites B can be acclimated along the entire length. Preferably, the full length is at least about 50, at least about 100, at least about 1000 to about 10 ⁶ or less target binding sites B. Intermediate ranges of the above recited values, such as about 50 to about 1000, or about 1000 to about 10 ^6, are intended to be part of the present invention. For example, the present invention includes the range of full-length values used by combining any of the above quoted values as the upper limit and / or the lower limit. The range is intended to include both the sites in the group of target binding sites B and the sites in the series of target binding sites B.

The term "interaction" is intended to include negative interactions, for example, inhibitory and influenza interactions of influenza, such as interactions that lead to the transformation of a signal or interactions that pull two surfaces together. For example, the multifunctional donor may bind to a group of target binding sites B on the cell surface and may convert signals within the cell or induce the cell to consume energy. Examples of the effects of such interactions include apotosis (programmed cell death), clonal expansion, such as B-cell or T-cell replication, cell migration, such as movement of cells in a direction, hormones such as insulin, Release of soluble molecules such as cytokines such as Il2; Prostaglandins, endocytosis or cell uptake or exocytosis, active delivery of some substances (internal or external), induction of electrical activity, or differentiation or dedifferentiation of certain cells. Modulation or treatment of all such effects of multifunctional donors is intended to be part of the present invention. In some embodiments, the interaction is not antistick.

The term “stereoscopic stabilization” refers to the mechanism by which one multifunctional donor biospecifically binds to one of two surfaces, thereby stericly inhibiting close access to both surfaces. This is distinguished from the stabilization resulting from the addition of a water swelled polymer to the colloidal mixture, which is used to keep the colloidal particles in solution. In this case, the molecule binds nonspecifically to the surface to be stabilized. See, eg, A. Sung and I Piirma ("Electrosteric stabilization of polymer colloids", Langmuir 1994, 10, 1393-8) and U Genz, Daguano (B D ' See Aguanno, J Mewis and R Klein, "Structure of sterically stabilized colloids" Langmuir, 1994, 10, 2206-12. The concept is also distinguished from grafting polymers to the surface of colloids or liposomes. In the latter case steric stabilization is achieved by covalent bonding of molecules to the surface to be stabilized. Polyethylene glycol is a circular example. See, eg, K Zhulina, O Borisov and V Priamitsyn, "Theory of steric stabilization of colloid dispersions by grafted polymers", Colloid and Interfacial Science 137: 495-511. , 1990.

The expression “multifunctional donor” includes multi-component molecules having at least 11 provided groups A, which can bind to the corresponding target binding site B and are attached to the framework. The multifunctional provider of the present invention can be illustrated by the following formula (2):

Formula 2

Y- (A) _n

In certain embodiments, multifunctional donors of the invention can be represented by Formula 1:

Formula 1

(Y)-(XA) _n

In the above formula, "Y" represents a basic structure, "A" represents a provided functional group, "X" represents any linker that can be used to attach group A to the basic structure Y, and "n" represents a provided group An integer greater than 10 selected to interact with this plurality of binding sites B.

The integer "n" is chosen such that a sufficient number of group A is provided for the treatment of the disease or disorder. The integer "n" is further selected to enable multifunctional provision of a plurality of groups A. The integer "n" is greater than 10, preferably greater than 10 to about 10 ⁶ , more preferably about 50 to about 10 ⁶ , about 100 to about 10 ⁶ , or about 1000 to about 10 ⁶ . The range of "n" which falls in the middle of the listed ranges is also intended to be part of the present invention, such as greater than 10 to about 100, greater than 10 to about 1000, about 100 to 1000, and about 1000 to 100,000. For example, the range of "n" values used in combination of any of the above values quoted as upper and / or lower limits is intended to be included in the present invention.

The term "providing a multifunctional" or "multifunctional mode" is well known in the art and refers to a multifunctional indication of A, B or C such that poly A, B or C acts differently from its monofunctional counterpart. It includes. For example, poly A may cooperate with mono A positively or negatively, or exhibit a non-cooperative biological effect. In addition, the multifunctional donor may act independently of cooperativeness, as demonstrated by the β factor, as described in more detail below. Multifunctional provision or multifunctional modes include the use of multifunctional donors other than sustained release compounds or drug delivery systems known in the art. Multifunctional donors are designed to be multifunctional but not release monofunctional groups that modulate the pharmacological activity of the agent. In addition, due to the release and diffusion of monofunctional groups, sustained release compounds typically function at a site different from the site of administration, which is not necessarily the case for multifunctional donors.

Basic structure

The term "basic structure (Y)" includes a support structure or backbone composed of a substance to which a plurality of groups A may be attached and which may be provided multifunctionally and having a molecular weight greater than about 10,000. It can be attached by means to enable the group to appear multifunctional on the framework. In certain embodiments, Group A is attached to the framework prior to administration to the subject. In another embodiment, group A is administered to a subject and assembled on a framework in vivo to form the multifunctional provider of the invention, such as by self assembly. The framework component should have a sufficient average hydrodynamic radius to span the entire length of the distance between adjacent receptors, and will be greater than 100 ms. These dimensions allow multiple functional groups A attached to the framework to simultaneously bind to target receptors, such as cell surface receptors. Any ordinary skilled person will recognize that the average hydrodynamic radius of a polymer can be measured approximately using standard statistical dynamics methods.

In some embodiments, the “backbone” of the base structure may further comprise a linker (hereinafter referred to herein as the backbone linker) for linking the monomer units of the base structure. Special backbone linkers will be discussed in more detail later with respect to polymeric frameworks. In some embodiments, the backbone linker can be cleaved, such as the backbone linker can be readily hydrolyzed.

A useful type of structure in a multifunctional provider is that group A can be attached and can provide group A multifunctionally. In addition, the basic structures used for in vivo purposes may be suitable for in vivo administration or used for in vivo assembly. Examples of basic types of structures include, but are not limited to, polymers, liposomes, micelles, colloids, dendrimers, and biological particles. These types are discussed briefly below and will be discussed in more detail in the detailed description of covalent and non-covalent base structures. Each of these base structures will be described in detail only under the subheadings Covalent and Non-Covalent Base Structures for each, and is not considered to limit the scope of the base structures. The present invention is intended to include all types of frameworks that can multifunctionally provide Group A described herein.

The term "polymer" or "polymeric" is known in the art and includes structural frameworks composed of repeating monomer units. In order to limit, the polymeric framework must also provide the "A" group multifunctionally so that the disease or disorder is treated. The term also encompasses copolymers and homopolymers, such as copolymers and homopolymers present synthetically or in nature. Linear polymers, branched polymers and crosslinked polymers are also considered to be included. In a preferred embodiment, the polymer is not dextran.

The terms "liposomes", "micels" and "colloids" are recognized in the art. These terms include derived variants such as liposome derivatives, crosslinked liposomes and the like.

The term "dendrimer" is recognized in the art. Dendrimers include a special subclass of branched polymers that include a number of naturally occurring generations. In dendrimers, each spontaneous generation results in multiple branching points. In a preferred embodiment, the framework is not a dendrimer.

In certain embodiments, the framework of the present invention may include "particles of life". The term "particles in living organisms" refers to covalent molecules (eg, sugars, proteins, lipids, small molecules, protein aggregates, and nucleic acids) and non-covalent particles (eg, cells induced, chemically modified, or modified by an exogenous nucleic acid). Or modified viruses such as viral particles. The use of "particles of living things" as the basic structure is distinguished from particles occurring in the natural state, since the basic structure of the present invention is modified to provide functional group A multifunctionally.

Covalent Structure

In one embodiment, the monomer units of the base structure can be bonded by covalent bonds. Examples of covalent frameworks include crosslinked liposomes, biological particles (eg, sugars, proteins, peptides, lipids or small molecules) and polymers (eg, Siraganian, RP, et al., Immunochem. 1975, 12). , 149-155, Wofsy, C, et al., J. Immunol. 1978, 121, 593-601, Barlocco, D, et al., Parmaco 1993. 48. 387-96. , Castagnino, HE et al., Jpn. Heart J 1990, 31, 845-55, Costa, T. et al., Biochem. Pharmacol 1985, 34, 25-30, Dembo Dembo, M, et al., J. Immunol 1979, 122, 518-28, Holgerer, P, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-8, Piergentili, A et al. (Farmaco 1994, 49, 83-7), Portoghese, PS et al., J. Med Chem 1991, 34, 1292-6, Kizuka , H) and Hanson R, N. (see J. Am. Chem. Soc 1987. 30, 722-6).

In certain embodiments, proteins, such as albumin, can be used as a system for providing multiple groups (see Roy, R, Lafemere, CA Can. J. Chem. 1990, 68, 2045-2054). Thus, it may act similar to natural glycoproteins.

In other embodiments, the polymer may be used as the basis for a multifunctional provider. Polymers are basic structural systems with a variety of uses (Spaltenstein et al., 1991, J. Am. Chem. Soc. 113: 686, Mamen et al., 1995. J. Med. Chem. 38: 4179). In a preferred embodiment, group A of the invention is attached by a linker to an underlying structure comprising a polymeric backbone. In certain embodiments, reactive polymers can be used in the "structure" of the present invention as described in more detail below.

Polymers can be prepared using methods known in the art (Sandler, SR, Karo, W. Polymer Syntheses, Harcourt Brace, Boston) , 1994, Shalaby, W, Ikada, Y., Langer, R, Williams, J, Polymers of Biological and Biomedical Significance; ACS Symposium Series 540; American Chemical Society; Washington, DC, 1994). The polymer can be designed to be flexible and the distance between the bioactive side chains and the linker length between the polymer backbone and the group can be designed and controlled.

Polymers offer many advantages as a basis. They can be designed such that multiple groups A simultaneously bind to multiple B group binding sites with a minimum of undesirable strains. Polymeric multifunctional donors can be synthesized easily and quickly and intensively (Spartenstein et al., 1991, J. Am. Chem. Soc. 113: 686 and Mamen et al., 1995, J. Med. Chem. 38: 4179). The polymers also allow for control of various physical properties of the donor, such as conformational flexibility, solubility and hydrophilicity, and compliance and flexibility in solution through changes in temperature and ionic strength.

Chemistry of high molecular weight polymers is a well-developed discipline, and organic polymers provide a very important class of compounds used for multifunctional provision. These compounds are high molecular weight and can provide a very large group of replicas. They can provide more than one group at the same time, and their movement through the biomembrane is usually limited, so the lifespan in their particular compartment can be regulated in vivo. The polymeric framework provides a variety of macromolecules that are easily synthesized and approximates a range of bioactivity.

In a preferred embodiment, the modified polymeric material for use in the present invention has low antigenicity and low toxicity. In a preferred embodiment, the polymeric framework may be chosen to be compatible with water, have a varying molecular weight, and may have a range of different groups attached to the polymer backbone. The polymer backbone of the present invention can be selected to facilitate synthesis.

Inherently biocompatible polymers containing functional groups suitable for side chain addition are preferred (Sarabi, Icada, Langer, R, Williams, Polymers of Biological and Biomedical Significance (ACS Symposium Series 540); American Chemical Society: Washington, DC, 1994). Examples of polymers include polyethylene oxide or polyethylene glycol (Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications .; Plenum: New York, 1992), Horton, D. Advances in Carbohydrate Chemistry and Biochemistry; see Academic Press: San Diego, 1995) as well as derivatives of acrylamide and N-vinylpyrrolidone, linked oligomers of oligoethylene glycol, linked oligomers of dextran, and the like. .

Other preferred structures for use in the present invention have proven utility as, for example, plasma extenders, pharmaceutical excipients or binders, food additives, or inert or corrosive materials used in vivo. For example, poly (ethylene glycol), poly (lactic acid), poly (glycolic acid) and poly (vinyl pyrrolidine) can be used.

Preferred polymers for use in the synthesis of multifunctional donors contain reactive groups such as activated carboxylic acids. Many synthetic and natural polymers may contain carboxylic acid functional groups or be modified to have carboxylic acid functionalities, which have been used in vivo. Such polymers may form covalent bonds with provided Group A, such as amide bonds. Particular preference is given to polymers containing internally cyclic carboxylic acid functional groups, such as anhydrides or succinimide groups. Other preferred polymers include maleic anhydride or subunits derived from maleic acid. Examples of copolymers include styrene-maleic anhydride and alpha-olefin-maleic acid copolymers (eg, divinylether-maleic acid). In another embodiment, sodium carboxymethylcellulose, chondroitin sulfate, and poly (methacrylate / acrylate) materials can be used. In other embodiments, polymers having no activated carboxylic acid, such as dextran sulfate, can be used.

Examples of other polymeric basic structures include poly (ester), poly (anhydrides), poly (carbohydrates), polyols, poly (acrylates), poly (methacrylates), poly (ethers) and poly (amino acids) do.

Examples of other polymeric basic structures include poly (glutamic acid), poly (aspartic acid), dextran, dextran sulfate, poly (maleic anhydride-co-vinyl ether), poly (succinimide), poly (acrylic anhydride) ), Poly (ethylene glycol), poly (lactic acid), poly (glycolic acid), poly (vinyl pyrrolidone), poly (styrene-maleic anhydride), alpha-maleic acid, hyaluronic acid, sodium carboxymethylcellulose, Chondroitin sulfate, poly (acrylate), poly (acrylamide), poly (glycerol) and starch.

Those skilled in the art recognize that any polymeric material capable of providing multiple groups A may be suitable for use in the present invention. The polymers may, for example, be modified as described above or derived using a bifunctional crosslinker to provide suitable functional groups for attaching and providing group A as described in more detail below. Copolymers may be preferred in certain circumstances.

Examples of polymer Poly (ethylene glycol) poly (amide) poly (peptide), poly (amino acid) poly (aspartic acid), poly (glutamic acid) poly (lysine), other protein (gelatin) poly (ester) poly (lactic acid), polyacted Poly (glycolide) poly (caprolactone) poly (tartrate) polysaccharide cellulose, alginate, starch dextran derivative poly (N-vinylpyrrolidone) Poly (ethylene-vinyl acetate) poly (acrylamide) poly (urethane) poly (methacrylate) poly (acrylate) poly (maleic acid copolymer) poly (anhydride) poly (orthoester) copolymers with degradable bonds Encapsulation for encapsulation target for copolymer encapsulation protection with group for copolymer adhesion

Backbone linker

In certain embodiments, it is preferred to include a "linker" which is the linking moiety between monomer units of the main chain. Examples of "backbone linkers" may contain hydrocarbon, carbamate, amide, ether, thioester, thioether, carbonate and ester linkages. In certain embodiments, the backbone residues may be linked by cleavable linkers. The lifetime of a multifunctional material in vivo may depend in part on molecular dimensions. By placing linkers between medium size oligomers and controlling the in vivo stability of these linkers, the lifetime of the polymer in vivo can be controlled. Thus, nonfunctionalized degradable linkages within the polymer backbone can be used to aid in the removal of the polymer. Removable linkers are generally different from those used to link the polymer backbone to group A. This removable linkage will form a functionalized polymeric fraction that is small enough to be removed through the kidney. Degradable linkers include, for example, ester, carbonate or oxalate groups and may include readily hydrolyzable linkers.

In certain embodiments, the framework of the multifunctional provider of the invention may comprise a dendrimer. Dendrimers are well known in the art and include a highly branched low molecular weight polymer (eg, often monodisperse) species. Dendrimers have the advantage that they have sufficiently defined molecular structures and that their solubility is relatively invisible compared to their molecular weights. They can provide groups in a densely packed form compared to linear polymers, which can be advantageous for targeting receptors that cluster on the cell surface. The number of groups A can be precisely controlled in the dendrimer as well as the distance, compliance and stiffness of the internal groups of the dendrimer molecule. Polysaccharides may belong to the dendrimer class (see Sabesan et al., 1992. J. Am. Chem. Soc. 14: 8636). In one embodiment, the head group of dendrimers can be used as the base structure (see Roythe et al., Synthesis and Antigenic Properties of Sialic Acid-Based Dendrimers; Acs Symp. Ser 1994).

Non-Covalent Base Structure

Multiple group A may also be linked to a non-covalent framework. Examples of non-covalent frameworks include liposomes, micelles, colloids, protein aggregates, modified cells and modified virus particles. For example, group A can be bound to liposomes, membranes or head groups of molecules on the surface (Kingery-Wood, JE, et al., J. Am. Chem. Soc. 1992, 114, 7303 -7305, Spevak, W et al., J. Am. Chem. Soc. 1993, 115, 1146-1147, and Spebak et al., J. Med Chem. 1996, 39, 1018-1020 ].

Liposomes and micelles are known in the art and include visible particles of lipid aggregates such as surfactants. In one embodiment, the multifunctional donor can provide groups on liposomes or micelles (Spevac et al., J. Am. Chem. Soc. 1993, 115, 1146-1147, Charych, D. See Chem. & Biol. 1996, 3, 113-120). This system can be designed to provide a surface that closely matches that of the target cell in terms of group type and group density, almost similar to the shape of the target cell. For example, lipid molecules containing group A (eg, nuramic acid (NeuAc)) can be reconstituted into liposomes as polar head groups. Liposomes have advantageous biocompatibility and are fairly easily synthesized. Liposomes can also be designed to act as sensors. Polymerized liposomes can be used to detect morphological changes of liposomes by indicating a change in UV / visible light absorption of the membrane's internal chromophore (eg, crosslinked polydiacetylene). For example, binding of the virus to liposomes can be detected by color change (blue to red) as known in the art.

In other embodiments, biological particles, such as, for example, modified cells or modified viruses, can be used as the framework for providing multifunctional groups of group A. Thus, proteins, peptides, polysaccharides, cell membrane fragments or modified intact cells (eg erythrocytes), modified bacterial cells or modified viruses can be used as the framework in certain embodiments.

Activated framework components

As used herein, an "activated framework" refers to a tactic containing a functional group that is activated by an "activation group" and then reacts with one or more functional groups, ancillary groups, and / or spacer groups. As described herein, including both covalent and non-covalent base component. Suitable functional groups include, for example, carboxyl (acid form and salt), hydroxyl, sulfhydryl, amide, carbamate, amino, ketone, aldehyde, olefin, aromatic, and the like. The polymer may be activated prior to exposure to the functional group (“preliminary activation”) or may be activated in the presence of the functional group (“activity in the same reaction system”).

The activation step may deactivate the polymer by a group capable of reacting with a nucleophilic or electrophilic material. In addition, bipolar addition reactions (eg, 1,3- and 1,4-dipolar addition reactions), cyclic addition reactions (eg, Diels-Alder type reactions), and mechanisms initiated by cations, anions, and radicals It is within the scope of the present invention to activate the polymers so as to effect the polymerization by

Carboxyl groups are, for example, cyclic or linear anhydrides, activated esters (e.g. N-hydroxysuccinimide, nitrophenols, 4-hydroxy-3-nitrobenzene sulfonic acid, etc.), acid chlorides, imidazolides (e.g. carbo Carboxylic acid and ester) can be activated for reaction with nucleophilic materials. Carboxylic acids containing polymers include carboxyl groups and reagents such as dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, alkyl chloroformates, chlorosilanes, pyridinium salts and Bu _It may also be activated by forming an adduct between ₃ N and the like).

The selection of appropriate activation groups for carboxyl functional groups is clearly recognized by those skilled in the art. It is likewise apparent to those skilled in the art which reaction system can be applied or which reaction system requires activation or preactivation in situ.

The hydroxyl groups can be activated using, for example, carbonates formed by reaction with alkyl or acyl haloformates (eg iso-butylchloroformate, p-nitrophenylchloroformate, etc.), cyanogen bromide or phosgene. In terms of using polymers containing polymers containing adjacent diol groups (eg, dextran and other polysaccharides), oxidation reactions using a periodate compound can be used to provide reactive carbonyl residues on the polymeric backbone. have. Other methods of activating polymers containing hydroxyl groups are clearly known to those skilled in the art.

Polymers containing sulfhydryl groups can be activated using dithiobispyridyl compounds such as 2,2'-dithiobis (5-nitropyridine), 2,2'-dithiobis (pyridine) and the like. Further methods useful in activating a polymer containing sulfhydryl are known to those skilled in the art.

The activation reaction is presented by way of example only, and those skilled in the art recognize that there are many other alternatives to the above.

Once the polymeric framework is activated it can react with one or more functional groups, auxiliary groups, spacer groups or mixtures thereof. Alternatively, the activated polymer can react with a combination of functional groups, auxiliary groups and / or spacer groups.

Functional group

The functional group component of the multifunctional provider prepared according to the method of the present invention may be attached to the framework component, auxiliary group and / or spacer group and provided with the multifunctional group in a mode of action, for example for treating a disease or condition. Include groups that exist. The functional group components may be the same or different. The term "multiple functional group components" or alternatively "multiple R ³ groups" is understood to mean one or more functional group components, wherein each of the plurality of functional groups is independently the same or different. The functional groups may be the same or different within the category of functional group types, for example one functional group is a carbohydrate and another functional group may be an antibiotic. The functional groups may also be different within functional groups of the same type or category, such that two different carbohydrates may be functional groups. Functional groups provided in the case of "homomeric donors" are the same, whereas functional groups provided in the case of "heteromeric donors" differ, for example, from different ranges or within the same category. For ease of explanation in the following and claims, the nomenclature of R ³ ₁ to R ³ _n is used to refer to several members of a plurality of R ³ .

As used herein, the functional groups provided by the multifunctional group providers of the invention are functional. In addition, the functional groups act when attached to the framework components of the multifunctional donor. As mentioned above and elaborated herein, this is different from drug delivery systems known in the art, such as polymeric drug delivery systems that release a therapeutic agent and function in the form in which the therapeutic agent is released. For example, in a preferred embodiment of the present invention, the functional groups act by biospecifically interacting with at least 11 (ie multiple) binding sites B, eg, to directly provide a therapeutic effect. In other embodiments, where the multifunctional donor is, for example, a heteromeric donor, some of the functional groups (eg, R ³ _n ) do not interact with B, but instead function differently. For example, functional groups can affect the interaction of R ³ ₁ with binding site B and thus act as an enhancer group. In other embodiments, the functional group (eg, R ³ _n ) can be a functional subgroup (eg, an auxiliary group) that can affect the physical characteristics of the multifunctional donor, such as solubility or ability to cross the cell membrane. In other embodiments, the functional groups can provide a function by providing a label (eg, fluorescent or radiolabel) that can be detected to enable tracking of the subjects of the invention.

The functional component of the present invention may be synthetic or natural. In addition, functional group components may be classified based on physical characteristics such as molecular size such that the functional group may be of low molecular weight, medium molecular weight or high molecular weight. Examples of natural functional ingredients include, but are not limited to, natural sugars, proteins (eg, IgE or erythropoietin), peptides or other known agents. Examples of synthetic functional group components include, but are not limited to, peptide analogs and functional groups synthesized by combinatorial chemistry or trial drug design. Specific types of functional group components that can be used in accordance with the methods of the invention are described in more detail under the subheading "functional group type" below.

Group A

Group A of multifunctional providers includes, for example, a group that may be provided as a multifunctional group in a manner of action for treating a disease or condition and that may be attached to the framework, for example. Group A may be the same or different. The term "multiple groups A" is used to mean one or more groups A, each of which is independently the same or different. Group A may be the same or different within the category of the Group A type, such as one group A is a carbohydrate and another group A may be an antibiotic. In addition, group A may be different in group A of the same type or category, such as two different carbohydrate groups A. Group A provided in the case of a "homomeric donor" is the same, and group A provided in the case of "heteromeric donor" may be different from, for example, different categories or within the same category. For ease of explanation in the following and claims, the nomenclature of A ₁ to A _n is used to refer to various members of a plurality of groups A.

As used herein, “Group A” provided by the present invention is functional. Group A also acts when attached to or on the base of the multifunctional donor. As mentioned above and elaborated herein, this is different from drug delivery systems known in the art, such as polymeric drug delivery systems that release a therapeutic agent and function in the released form. For example, in a preferred embodiment, group A acts in biospecific interaction with at least 11, ie, "multiple" binding sites B, eg to directly provide a therapeutic effect. In certain embodiments, for example, where the multifunctional donor is a heteromeric donor, some functional groups (eg, A _n ) do not interact with B, but instead may provide other functions. For example, A _n can affect the interaction of A ₁ with binding site B and thus acts as an enhancement group. In other embodiments, A _n may be a functional subgroup that may affect the physical characteristics of the multifunctional donor, such as solubility or ability to cross cell membranes. In another aspect, group A may provide a function by allowing tracking of a subject of the invention, such as providing a label that can be detected (eg, a fluorescent or radioactive label).

Group A of the present invention may be synthetic or natural. In addition, group A is classified based on physical characteristics, such as molecular size, such that group A may be of low molecular weight, medium molecular weight or high molecular weight. Examples of natural group A include natural sugars, proteins (eg, IgE or erythropoietin), peptides or other known agents. Examples of synthetic groups include peptide analogs, groups synthesized by combinatorial chemistry or generated by trial drug design. In a preferred embodiment, group A is alone or from any of the groups from the group consisting of carbohydrates, polymyxins (eg polymyxin B), beta-lactams, furan-derived compounds, pyran-derived compounds and antibodies It is not chosen in combination with other members. In another embodiment, group A is not a derivative or fragment of group A described above. The special type of group A is described in more detail under the heading "Type of group A" below.

Number of group A per provider

The various physical properties of the framework can influence the ability of the multifunctional donor to coordinate the interaction between group A and B binding sites. By changing the group equivalent number, the effectiveness of the polymeric donor can be altered so that it can be increased or decreased. For example, many attachment points can cause the donor backbone to degrade on the surface on which the B binding site lies and make steric stability less effective but more effective in competitive blocking of target sites and / or action / antagonism of biological reactions. to be.

Systematic studies of substituent effects on parent polyacrylamide polymers having 0.2 equivalents of sialic acid (SA) have shown that the net charge, size and hydrophobicity of the backbone substituents affect the success of biological functions by the donor. (See Mamen et al., 1995, J. Med. Chem. 38: 4179). In general, changing the properties of the backbone substituents affects both affinity and steric stability. In addition, the effect of the polymeric inhibitor decreases with increasing charge and size of the substituents. Coulombic and steric interactions make the chains more prolonged and less effective in steric stabilization.

Heteromeric donors providing many different groups

Heteromerger provision can be used to provide both greater strength and specificity than interactions of the same monofunctional group. For example, by providing additional group types (eg, A ₂ ), the total number of interactions may increase, and then the total intensity of the interactions may increase. The specificity of the interaction can be increased by differentially controlling the number of groups A ₁ and A ₂ provided. For example, a donor containing both A ₁ and A ₂ may interact more closely to B ₁ and B ₂ than a donor containing A ₁ alone or A ₂ alone.

In addition to the use of different group A, which confers unique pharmaceutical properties to the subjects of the present invention, the use of a number of different group A may provide protection against a different range of pathogens.

For example, in one embodiment the multifunctional donor can provide Group A ₁ which interacts with receptors on the pathogen surface and Group A ₂ which interacts with the second pathogen. In another exemplary embodiment, B ₁ and B ₂ are on the same pathogen.

Type of group A

Group A of the present invention includes groups useful for treating a disease or condition when provided in a multifunctional manner. These groups may be known groups or agents, or may be new groups or agents selected after studying multifunctional interactions related to a particular disease or condition. The present invention utilizes the aforementioned known group A (see Kiessling, LL, Pohl, NL, see Chem. & Biol, 1996, 3, 71-77) and novel The development of groups and the provision of multifunctional groups is provided for combination methods that increase the effect of known monomer groups. It is recognized that some uses for multifunctional donors may be advantageous over others due to easier access to the site of action. It should be understood that a less easy approach will be provided by more direct administration to the site of action.

Groups known to be involved in cell-pathogen interactions, cell-cell interactions, pathogen-extracellular matrix interactions, and pathogen-pathogen interactions can be provided in a multifunctional manner on a subject of the invention.

For example, as shown in the appended examples and described in the appendix, an example of one group is N-acetyl nuramic acid (sialic acid), which is a natural binding site for influenza hemagglutinin. The interaction between sugar and lectins is the first essential step in influenza virus attachment to target cells. Very large numbers of multifunctional sugars are possible groups such as Neu5Ac (2,6) galactose, Neu5Ac (2,6) lactose, Neu5Ac (a2,3) Gal (b1,3) GalNAc, heparin sulfate and galacto Seal ceramides are included, all of which appear to be important for attaching different viral particles to host cells.

In other embodiments, groups comprising multifunctional groups, which are known to modulate other diseases or disorders, can be used on the present invention. For example, group A may be selected for its ability to mediate multifunctional interactions between platelets when desired to treat thrombosis.

In a preferred embodiment, the subjects of the invention may comprise known agents or compounds which, when administered in monofunctional form, do not appear to be very effective in treating a disease or condition. In such embodiments, the medicament may be quite effective when provided with a multifunctional group according to the present invention. Accordingly, the present invention provides for a number of existing drug and non-pharmaceutical groups that can be incorporated into the present invention as Group A. Those skilled in the art will appreciate that group A may have one or more biological effects and / or may be useful for the treatment of one or more types of diseases or disorders. In addition, some group A are useful for treating a disease or condition involving multifunctional interactions and / or some group A is useful for treating a disease or condition not previously identified as involving multifunctional interaction. . The term "pharmaceutical" is used below to refer to group A as possible for ease of discussion.

For example, agents are provided that have an effect on the central nervous system. Treatment of Alzheimer's disease may use, for example, tacrine or donepezil as Group A. In another embodiment, the treatment of alcoholism can use disulfiram as group A. Treatment of acute and / or chronic pain and / or inflammation is analgesic (eg, acetaminophen, aspirin, ibuprofen, naproxen, pentazosin, indomethacin or diflunisal) as an A group or anesthetic (eg lopiacaine or remy) Fentanyl) can be used. Treatment of pain and / or drug addiction can use, for example, hydrocodone, propoxyphenmeperidine, hydromorphone, morphine, methadone or oxycodone as group A. The use of anesthetics such as epinephrine, xylocaine, mepivacaine, metohexital, bupivacaine and novocaine and the like is also shown. Multifunctional donors of the present invention may also utilize cholinsterase inhibitors such as pyridostigmine or niostigmine as group A. In addition, hypnotics may be incorporated into the subject matter of the present invention. For example, fluzepam, pentobarbital, triazolam, temazepam or secobarbital can be used. Group A can also be antitussives, such as pseudoephedrine or codeine. The present invention also provides for the use of group A migraines, such as ergot derivatives (eg ergotamine or methiserguide) or somethepthene, or serotonin (5-HT) antagonists (eg sumittriptan). . Group A may also be a vertigo treatment such as meclizain or scopalamine.

In another use, Group A also contains muscle relaxants such as pyridostigmine, neostigmine, succinylcholine, mibakurium, doxacurium, rocuronium, bekuronium, dantrolene, cyclobenzaprine, Baclofen, chloroxazone, metocarbamol or papaverine. Motion sickness can be treated, for example, with prochlorperazine, chlorpromazine, trimetobenzamine, perfenazine, hydroxyazine or ondansetron. Parasympathetic inhibitors such as biferdene, phenobarbital, ergotamine, dicyclomine, hyaciamine, glycopyrrolate can also be used as group A. Likewise, parasympathetic stimulants such as tacrine, pyrrocaprine, beta nicol, edroponium, yohimbine can be used. Group A may also include parinsonism agents such as trihexhenidyl, ventropin, procclidine, levodopa, bromocriptine, carbidopa, amantadine. The invention also provides for the use of psychotropic agents in the subject matter of the invention. Likewise, anti-anxiety agents such as lorazepam, buspyrone, chlordiazepoxide, meprobamate, chloraseate, diazepam and alprazolam can be incorporated into the present subject matter. Antidepressants such as phenelzin, tranilcipromin, parosetin, fluoxetine, sertraline, amitriptyline, nortriptyline, imipramine or protriptyline can be used. Antipsychotics (e.g., clozapine, procloperazine, haloperidol, oxypine, thiolidazine, flufenazine, risperidone, mesodazine, trifluoroperazine, olanzapine or chlorpromazine) can be used as group A have. The use of mental stimulants such as femoline or methylphenidate is also provided. Sedatives (eg, mepobarbital, secobarbital or temazepam) can also be used as group A. Seizure disorders include, for example, pelbatol, gabapentin, phenytoin, mephenitoin, etotoin, lamotrigine, metsuccinimide, pensuccinimide ethosuccinimide, carbamozepine, phenacemide or carbamazepine It can be treated by a functional group provider. For example, sympathetic blockers such as phentolamine can be used. Group A also includes, for example, anticonvulsants (eg phosphphenytoin); Antidepressants (such as mirtazapine) that can be used to treat complex sclerosis (such as glatiramer) or epilepsy (such as topyranate); Or groups that can be used to inhibit angiogenesis (eg, angiogenesis inhibitors known in the art).

Also provided is the use of group A, which is effective in the cardiovascular system. For example, there are adrenergic drugs such as doxazosin, terrazosin, prazosin, methyldopate, clonidine, and labalol. In another embodiment, angiotensin can be used which converts enzyme inhibitors such as captopril, ricinopril, tradolapril or enalapril. Multifunctional donors can also be made to provide angiotensin II receptor antagonists such as losartan or valsartan. The use of antiarrhythmic agents is also provided, examples being disopyramide, procainamide, quinidine, propaphenone, plecanide, tocanide, propanolol, sotalol, amiodarone, digoxin. In other embodiments, β-blockers such as thiolol, metoprolol and atenolol can be used as group A. In a further aspect, a calcium channel clocker can be used as a provided group. Examples include nifedipine, nicardipine, diltiazam, pelodipine and verapamil.

Diuretics such as acetazolamide, ethacrynic acid, furosemide, spironolactone, amylolide, chlorothiazide and hydrochlorothiazide can also be used as group A. Vasodilators (such as papaverine, hydralazine and amlinone) can also be used in the present subject matter. Also provided is the use of vasoconstrictors (eg, metaramimin, phenylephrine or isoproterenol). In another embodiment, group A may comprise, for example, mecamylamine. Group A can be hypolipidemic such as clofibrate, gemfibrozil, simvastatin, lovastatin, simvastatin, atorvastatin or niacin. Severe vascular thrombosis can be treated using Group A, such as denafaroid. In further embodiments, an antihypertensive agent such as midodrin may be used.

For the treatment of cancer, group A is for example an anti-androgen substance (e.g., loopolide or flutamide), a cytotoxic agent (e.g., adriamycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, α2-interferon) , Anti-estrogen substances (eg tamoxifen), anti-metabolites (eg fluorouracil, methotrexate, mercaptopurine, thioguanine). Group A may also include hormones (eg, hydroxyprogesterone, estradiol, leuprolide, megestrol, octreotide or somatostatin). In another embodiment, group A may comprise, for example, irinotecan, gemcitabine, toptecan, nilandrone or docetaxel.

In another aspect, the subject matter of the present invention can be used in gastrointestinal use. For example, antispasmodic or anticholinergic agents may also be included as group A (eg, dicyclomin, thiosiamine, glycopyrrolate, trihexhenidyl or atropine). Appetite suppressants (eg dextromeamphetamine, benzpetamine, phentermine and omajindol) can also be used as group A. In other embodiments, group A may comprise a branching agent (eg, loperamide, diphenoxylate, octetide). Providers of the invention may also include proton pump inhibitors (eg lansoprazole or omeprazole) or vasodilators (eg opioids).

Group A, which regulates the endocrine system, may be provided as a multifunctional group. For example, there are contraceptives (eg, ethinodiol, ethynyl estradiol, noethyne drone, mestranol, desogestrel, methoxyprogesterone). Group A (eg, glyburide or chlorpropamide) that modulates diabetes can also be used. Anabolic agents such as testosterone or stanozolol may also be incorporated into the present invention. Androgens (eg, methyltertosterone, testosterone or fluoxymesterone) can also be used as group A. Antidiuretics (eg desmopressin) can also be used as group A. Providers of the invention may also represent calcitonin, for example, in a multifunctional manner. Estrogens (eg diethylstilbestol) may also be used. Group A may also be a glucocorticoid (eg, triamcinolone, betamethasone). The use of progestogen as group A is also provided, for example noethine drone, ethinodiol, noethine drone, levonorgestrel and ethynylestradiol. In another embodiment, thyroid agents (eg lyothyronine or levothyroxine) or antithyroid agents (eg methiazole) can be used. In another embodiment, the hyperprolactinemia disorder can be treated using Group A, such as cabergoline. In another embodiment, diabetes can be treated using, for example, miglitol or insulin lispro. The use of hormone lowering agents (eg danazol or goserelin) is also provided. In other embodiments, group A may comprise oxytosix (eg, methylergonobin or oxytocin). Prostaglandins such as, for example, myoprostol, alprostadil or dinoprostone can also be used as group A.

Multifunctional donors of the present invention may also be used in skin applications. Examples of group A with dermatological effects include, for example, anti-acne agents (eg, isotretinoin, adapalene or tretinoin). Other group As can be used, for example, in skin applications for the treatment of pruritus, for example alclomethasone, benzocaine, hydroxyzine, fluticasone, mometasone, fluorocinolone, clovestasol or desox Include cimethasone.

Group A may also affect coagulation. For example, the poly (A) may comprise heparin, an anticoagulant. In other embodiments, group A may comprise an antithrombin III or platelet inhibitor such as, for example, absximab and / or ticlopidine.

In other embodiments, eg, group A may be selected for its ability to affect immune modulation. For example, release of histamine from mast cells and basophils involves multifunctional interactions between allergens and IgE receptors on the cell surface. In such cases, group A may include, for example, antihistamines such as benadryl, loratadine, bromfenyramine, perimatin, promethazine, terfenadine, fexofenadine, azelastine and / or clemastine. In another embodiment, group A may comprise mast cell stabilizers such as rodoxamide and / or chromoline.

Other group As capable of modulating the immune system include, for example, steroids (triamcinolone, beclomethasone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone or clobetasol), histamine H ₂ antagonists (eg famotidine) , Cimetidine, ranitidine), immunosuppressive agents (azathioprine, cyclosporin). Groups with anti-inflammatory activity, such as sulindac, etodolak, ketoprofen, ketorolac can also be used as group A. Antihistamines (eg fexofenadine or azelastine) can also be used.

Group A to adjust the respiratory system can also be used. For example, mucolytic agents (eg guapefensin), anti-inflammatory agents (eg chromoline, flunisolide, beclomethasone, or verdesonide) may also be used in the present subject matter. The use of bronchodilators, such as ifpratropium, metaproterenol, terbutalin, isotarin, metaproterenol, albuterol or theophylline as Group A is also provided. For the treatment of asthma, for example, drugs such as japerulcaste or zileuton can be used.

Group A is for example quinolone (e.g. ciprofloxacin, norfloxacin, opfloxacin, enoxacin, lomefloxacin), sulfonamide (sulfasalazine or sulfamomethazole) or for example polymyxin B, bacitracin, neomycin, oxy Antibacterial agents such as tetracycline, tobramycin, sulfacetamide, fosfomycin and penicillin; Antiviral agents (eg, trifluidine, zivudine (AZT), zalcitabine (ddV), didanosine (ddI), stavudine (d4T); or other reverse transcriptase inhibitors such as those sold by Merck; protease inhibitors (E.g. saquinavir, indinavir, ritonavir), acylclovir, famcicvir, nibavirine, zidovudine, nevirapine, sipofovir or fencyclovir), repellents (e.g., thiavenazole, Chloroquine, sulfadoxin, pyrimethamine, mefloquine, metronidazole, albendazole or ivermectin) or antifungal agents (e.g. butenapin, buttoconazole, terconazole, thioconazole, clotrimazole or myconazole) can do. In certain instances, it is clearly known to the average person skilled in the art that the use of certain antibacterial agents is preferred for treating infections at certain sites, such as eye infections.

In other embodiments, penicillin excipients, probensides can also be used as group A.

Urinary tract agents for use as group A may include, for example, uric acid attractant (eg, sulfinpyrazone). Antibacterial agents such as indanyl carbenicillin, nitrofuratoin, nalidic acid, neomycin, bacitracin or polymyxin B can also be used as group A. In addition, the subject matter of the present invention may provide antispasmodic (eg oxybutynin or flavoxate). Calcium oxalate stones inhibitors such as allopurinol may also be used as group A. Prostatic hypertrophy modifiers such as terrazocin or finasteride may also be used. Providers of the invention can also be used to treat cystitis using Group A, such as pentosan.

In addition, the subject matter of the present invention is useful for ophthalmic use. For example, β-blockers (such as bromide or betaxolol), anti-inflammatory agents (such as clofatadine), or agents useful for the treatment of glaucoma (such as latanoprost) can be used as Group A. The present invention also provides for the use of carbonic anhydrase inhibitors such as dichlorphenamide, metazolamide or doxolamide as group A.

For dental applications such as aphthous ulcers, group A may include, for example, Amlexoxox.

In another embodiment, Group A comprises erythropoietin, tacrolimus, human growth hormone, tissue plasmin activator, platelet activator, interleukin stimulating factor, granulocyte stimulating factor, macrophage stimulating factor, and growth hormone secretagogue, EPO secretion It may be independently selected from the group consisting of small molecule secretagogues targeting protein product receptors such as promoters and insulin secretagogues.

Some specific embodiments of the non-limiting multifunctional donor for illustrative purposes include multifunctional sialic acid against HA on influenza virus, two derivatives of sialyl Le ^x with platelets containing P-selectin, both E and P selectin. Multifunctional sialyl Le ^x to endothelial containing, rubiDate (benzodiazepine) binding GABA _B complex receptor containing two different receptor sites, RGD and sialyl Le ^x binding leukocytes with both integrin and selectin, C _5A and Gal-X-Gal binding tumors (which bind to gal-x-gal) and proteins at complementary stages.

Secondary group

As used herein, “adjuvant group” is a moiety that modifies the characteristics of the multifunctional donor and / or the components that make up the multifunctional donor (eg, a structural component, a functional component, a spacer group, etc.). Properties that can be modified include solubility (in water, fat, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, infrastructure flexibility, antigenicity, molecular size, molecular weight, in vivo half-cycle, in vivo distribution, biocompatibility, immunity, Stability, binding strength to multifunctional targets, and the like.

Those skilled in the art understand that there is not complete but significant overlap between many of the above features and the subgroups affecting these features. For example, introducing one or more poly (ethylene glycol) (PEG) groups into the framework of a multifunctional donor increases hydrophilicity and water solubility, increases both molecular weight and molecular size, and affects the properties of the un PEGylated framework. It is thus predicted that the residence time in vivo can be increased. In addition, PEG is expected to reduce antigenicity and enhance the overall stiffness of the polymeric donor through hydrogen bonding to solvent molecules (eg, water). Those skilled in the art will clearly see similar overlapping areas between the structural features and subgroups that may affect these features.

Auxiliary groups that increase the water solubility / hydrophilicity of the multifunctional donor are useful in practicing the present invention. Thus, auxiliary groups that increase the water solubility and / or hydrophilicity of the multifunctional donor such as poly (ethylene glycol), alcohols, polyols (e.g. glycerin, glycerol propoxylates, monosaccharides, oligosaccharides and sugars including polysaccharides, etc.), It is within the scope of the present invention to use carboxylates, polycarboxylates (eg polyglutamic acid, polyacrylic acid, etc.), amines, polyamines (eg polyglycine, poly (ethyleneimine, etc.)). In a preferred embodiment, the auxiliary group used to increase the water solubility / hydrophilicity is a polyol. In a particularly preferred embodiment, the auxiliary group is poly (ethylene glycol).

It is within the scope of the present invention to incorporate lipophilic auxiliary groups into the structure of the multifunctional donor to increase the lipophilic and / or hydrophobicity of the donor. Lipophilic groups useful in the practice of the present invention include, but are not limited to, aromatic groups and polycyclic aromatic groups. As used herein, “aromatic group” includes both aromatic hydrocarbons and bicyclic aromatic compounds. Aromatic groups may be unsubstituted or substituted with other groups, but are at least substituted with a group that allows covalent attachment to the multifunctional donor. Another group useful in practicing the present invention includes fatty acid derivatives that do not form a bilayer in an aqueous medium.

In a preferred embodiment, the lipophilic auxiliary group is a cyclic group such as a hydrocarbon or a heterocycle. In another preferred embodiment, the cyclic group is a 6 membered ring or two or more fused 6 membered rings. In a particularly preferred embodiment, the auxiliary group is a phenyl or naphthyl group.

It is also within the scope of the present invention to use auxiliary groups to produce multifunctional donors that are incorporated into vesicles such as liposomes or micelles. The term "lipid" refers to any fatty acid derivative that can form two layers such that the hydrophobic portion of the lipid material is oriented towards the two layers while the hydrophilic portion is oriented toward the aqueous phase. The hydrophilic character comes from the presence of phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro and other similar groups. Hydrophobicity can be given by including saturated and unsaturated long chain aliphatic hydrocarbon groups, and groups comprising, but not limited to, those hydrocarbon groups substituted with one or more aromatic, alicyclic or bicyclic group (s). Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, di Palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine or dirinoleoylphosphatidylcholine can be used. Other compounds that do not contain phosphorus, such as sphingolipids and glycosphingolipids, are also included in the group referred to as lipids. In addition, the aforementioned positive lipids can be mixed with other lipids, including triglycerides and sterols.

The flexibility of the multifunctional provider framework can be manipulated by incorporating bulky and / or rigid auxiliary groups. The presence of bulky groups or rigid groups can prevent free rotation around the bonds in the base structure or between the base and auxiliary group (s) or between the base and the working period. Rigid groups may include groups whose morphological instability is limited, for example, due to rings and / or multiple bonds. Other groups that can provide rigidity include polymeric groups such as oligo- or polyproline chains.

Stiffness can also be imparted by static electricity. Thus, if the auxiliary group is negatively or positively charged, the equally charged auxiliary group results in a provider framework in the form of providing a maximum distance between each identical charge. The energy cost of making the same charged groups more closely to each other tends to fix the framework in such a way that the same charged auxiliary groups are continuously separated. In addition, auxiliary groups with opposite charges are likely to be attracted to oppositely charged counterparts to form both intermolecular and intramolecular ionic bonds. This non-covalent mechanism tends to anchor the framework in such a way as to allow binding between charged groups. The addition of charged groups or alternatively those auxiliary groups with potential charges revealed by deprotection, pH change, oxidation, reduction or other mechanisms known to those skilled in the art after addition to the base structure is within the scope of the present invention. have.

Bulk groups are large atoms; Or ions containing large atoms (eg, iodine, sulfur, metal ions, etc.); Polycyclic groups including, for example, aromatic groups, non-aromatic groups; And structures comprising one or more carbon-carbon multiple bonds (ie, alkene and alkyne). Bulk groups may also include oligomers and polymers that are branched chain and linear chain compounds. Branched compounds are expected to increase structural stiffness per unit molecular weight increase over linear polymers.

In a preferred embodiment, the stiffness is imparted by the presence of cyclic groups (eg cyclic hydrocarbons, heterocycles, etc.). In another preferred embodiment, the ring is a six membered ring. In a more preferred embodiment, the ring is an aromatic group such as for example phenyl or naphthyl.

It is within the scope of the present invention to alter the antigenicity of multifunctional donors by appropriate selection of auxiliary group (s). In certain applications, the antigenicity of the multifunctional donor may need to be reduced. As mentioned above, masking groups such as, for example, poly (ethylene glycol) are known in the art to have the ability to lower the antigenicity of both monofunctional and multifunctional compounds. In other applications, it may be desirable to elicit an immune response by increasing the antigenicity of the multifunctional donor. In these uses, auxiliary groups may include groups known in the art to increase the immunity of hapten. Suitable groups for increasing the immunity of multifunctional donors include, but are not limited to, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other groups that can increase the antigenicity of multifunctional donors are known to those skilled in the art.

In vivo half-life and in vivo distribution are a function of many molecular properties, including molecular size, molecular weight, charge, hydrophobicity / hydrophilicity, antigenicity, biodegradable, and the presence or absence of a target group on a multifunctional donor. As used herein, the term "target group" refers to a group having affinity for cellular receptors. Methods of modifying these properties to change the half-life or distribution of the compound administered in vivo as desired are well known in the pharmaceutical and medical chemistry arts.

New group A confirmation method

The present invention also provides a method for synthesizing oligosaccharide groups for use as group A (see Horton's Advances in Carbohydrate Chemistry and Biochemistry; Academic Press: San Diego, 1995). Protein analogs can be synthesized (Maassen, JA, Terhorst, C, C. Eur. J. Biochem 1981, 115, 153-158, Wang, KS, et al. See J. Virol. 1992, 66, 4992-5001, Mastromarino, P et al., J. Gen. Virol. 1987, 68, 2359-2369. Computer-assisted design tools can also be used to synthesize group A (see Chemtracts: Org. Chem. 1995, 8, 374-376) by Jorgensen, W.L.

The invention also provides a method of discovering a new group A in connection with a multifunctional group. For example, a single phage in a phage-display library, a single pin of a spatially provided combinatorial array, or a single bead in a combinatorial library made by a mixing and splitting method. Each has only a single type of group that is provided in the form of a multifunctional group. Thus, the library may be characterized by simple adhesion or activity that causes special consequences (eg adhesion), such as infection, cell death, cell proliferation, morphology change or the generation of a reporter that can be easily detected (eg green fluorescent protein). Can be screened directly in a combined form for a multifunctionally provided B. Typical resin beads may have a loading of 100 pmol or less, or about 60 trillion copies or less. Not all of these replicas are present on the surface of all solid supports, but the support can be selected for porosity or surface functionalization. In contrast, phage may exhibit replicas of peptide groups 3 to 1000 on the surface, depending on the envelope protein to which the library is fused. Any Group A compound found to be worth further analysis can then be tested in soluble polymeric form.

By way of example, beads in libraries to which bacteria, fungi or nucleophilic cells are specifically attached carry groups that may serve as useful groups for the multifunctional donor of the invention. Libraries of up to 1,000,000 different peptides can be prepared by split synthesis, and various libraries of other types of compounds can be obtained. The structure of the selected group can be determined from synthetic history or obtained by sequencing, mass spectrometry, deconvolution or encoding.

For example, phage-displayed peptide libraries can be used (Doyle, MV, et al., The Utilization of Platelets and Whole Cells for the Selection of Peptide Ligands from Phage Display Libraries, Cortese, R.). Combinatorial Libraries: Synthesis, Screening, and Application Potential, Walter de Gruyter, Berlin, 1996, pp. 159-174, Fong et al., Drug Dev. Res. 1994, 33, 64-70, and Goodson et al., Proc. Natl. Acad. Sci. USA 1994, 91, 7129-7133). In one embodiment, the PIII-modified phage (3-5 replicates per particle) is selected for interaction with activated total platelets that allows identifying five different types of platelet-binding peptide groups (“planning”). Can be. Synthetic peptides derived from phage sequences have been tested for inhibition of phage-platelet interactions and prevention of platelet aggregation, but the observed IC ₅₀ values were low.

Combination synthesis of polysaccharides using highly effective glycosyl donors is also described (see Liang et al., Science, 274: 1520, 1996). This method generally permits very high yields of polysaccharide synthesis, which is an essential feature of combinatorial libraries, and is dependent on anomer sulfoxide as a highly reactive glycosyl donor. Libraries can occur on beads and screened for interaction with multifunctional lectins in solution. Sticky analysis or aggregation analysis can be used in the system.

A trial of drug design including the use of computer design tools (A new method for predicting binding affinity in computer-aided drug design, Chemtracts: Org. Chem. 1995, 8, 374-376) provides a multifunctional group Can be used to identify a new group A for

Attachment of group A

Group A may be attached to or formed as part of the base structure using means to allow multi-group provision of the group. Attachment can be direct, for example without a linker, or indirect to which group A is attached using a linker. In addition, group A may be attached to the monomer unit before or after the polymerization reaction. In addition, the order of the attachment steps is not critical to the invention as long as the formed donors represent group A in a multifunctional manner. For example, group A may be attached to the linker first, followed by linker-group A residues to the basic structure or vice versa. Alternatively, group A may be attached to the framework-linker residue after the linker is attached to the framework. Group A can also be incorporated into multifunctional donors using bifunctional monomers containing group A, which can then be polymerized. Alternatively, a polymer can be prepared that is reactive and thus activated in the coupling, and then reacts group A with the polymer (“pre-activation” method).

Preferred embodiments include attaching the linker to group A. As mentioned above, the linker comprises a moiety capable of positioning or attaching the position of group A on the framework. In even more preferred embodiments, the linker is a residue that is independent of group A and the framework. An example of a preferred linker type is a linker that leaves the primary amine at the untethered end of the linker. The modified group can then be attached to the activated carboxylic acid on the polymer to form an amide bond. Amide bonds are relatively stable, occur extensively in nature, and can be formed cleanly by a range of well-developed methods. Additional benefits can be obtained by using specific linkers.

Cationic polymers are likely to be more antigenic than anionic polymers. Modified cationic ligands can be used in limited amounts to ensure that all of the modified groups are consumed, leaving only excess carboxylic acid groups on the polymer, which impart water solubility. As mentioned above, the linker also provides space for group A from the basic structure. In certain embodiments, the linker also imparts flexibility to the multifunctional donor to allow the group to move flexibly. Examples of linkers for use in the present subject matter include poly (gly), alkylamines (such as ethylamine), hydrocarbon residues, thioethers, poly (ethylimines) and poly (asp) and polyethers (such as PEG Chain).

The "preliminary activation" method allows for the fabrication of frameworks, such as polymers, with defined molar fractions of monomer units providing different groups. For example, the process may utilize esters of poly (N-acryloylsuccinimide) prepared from polymerization of N-acryloylsuccinimide or activated poly (acrylic acid) such as pNAS. NHS esters of pNAS in DMF can subsequently react with different primary amines such as R ₁ NH ₂ and R ₂ NH ₂ to form amide bonds. The remaining unreacted ester groups can be converted to carboxamides or carboxylic acids by treatment with excess NH ₃ or OH ⁻ , respectively. The distribution of these groups along the polymer backbone is thought to be random using this method. In addition, the degree of polymerization (total number of monomer units in one polymer) can be kept constant when the effect of different side chain groups on the effect is studied. The efficacy of the amide-forming reaction between the amine and pNAS can be measured using both 1 H-NMR spectroscopy and combustion analysis. This reaction is consistently completed at over 90%.

Another example of direct "copolymerization" is illustrated by using a mixture of monomeric derivatives of acrylamide at the reflux of THF (Spattenstein et al., Supra). The copolymerization reaction can introduce two main parameters that are not controlled in the polymerization reaction, where the main parameters are (i) unknown differences in the rate constants for the copolymerization between different N-substituted acrylamides along the backbone of the polymer. Non-uniform group distribution, and (ii) polydispersity, stereoregularity and length of the polymer may vary as the side chains are modified. This may occur due to the difference in rates associated with the polymerization process (propagation; termination). A "preliminary activation" method may be desirable where pA (R) with uniform structural features is required.

In a preferred embodiment, the polymer is activated using a process that produces high yields and allows for easy removal of by-products from the process. Also considered is the tendency to cause reactive, racemization (eg, using poly (amino acids)) of by-products with attached groups.

For example, there are certain chemical methods for activating carboxylic acids to react with amines. The relative reactivity of the activated carboxylic acid decreases in the following order: RCOHal> (RCO) ₂ O + RCON ₃ > RCO ₂ R> RCONH ₂ > RCOR and also increases in proportion to the nucleophilicity of the attacking amine. In a preferred embodiment, internal anhydrides are used.

By anhydride or succinimide units in the polymer backbone, amide bonds are readily formed when reacting with ami. In addition, intramolecular anhydrides in the peptide can be formed using α-amino-N-carboxylic acid anhydride or thiocarboxylic acid anhydride.

In certain embodiments, mixed anhydrides such as carbodiimide and the like can be used. Common methods for forming mixed anhydrides used in peptide chemistry are carbodiimides (eg, dicyclohexylcarbodiimide (DCC) or 1-ethyl-3- (3-dimethylamino) propylcarbodiimide (EDC). )). EDC may be useful for water soluble polymers. Examples of other anhydrides may be formed from reagents such as chloroformate or quinoline derivatives (EEDQ, IIDQ).

As mentioned above, in other embodiments carboxylic acid azide may be used. If carboxylic acid azide is used, a prior reaction is necessary to form the azide. In other embodiments, imidazoles such as carbonyldiimidazole can be used.

In another embodiment, compounds such as p-nitrophenol or N-hydroxysuccinimide that form activated esters can also be used.

In another embodiment, the polymer used contains hydroxyl groups, and methods for deriving hydroxyl groups can be used. Such methods are known to the average person skilled in the art.

Alternatively, combination methods can be used to generate multifunctional donors. That is, a method of combining in a similar solid state can be used to form a library of main chains containing A ₁ to A _n groups. A combinatorial library of arrays of synthetic multifunctional donors is formed, in which case the multifunctional donors of the arrays differ from each other in terms of composition, structure, properties, function, and the like. When preparing the arrangement of the multifunctional donor, in particular, the chemical structure of the basic structural component, the chemical structure of the functional group component, the chemical structure of the auxiliary group, the chemical structure of the spacer group, the chemical properties of the basic structural component, the chemical properties of the functional group component, Chemical properties of the auxiliary groups, chemical properties of the spacer groups, amounts of the framework components delivered, amounts of the functional group components delivered, amounts of the auxiliary group components delivered, amounts of the spacer groups delivered, the number of different framework components delivered And / or amount, number and / or amount of different functional group components delivered, number and / or amount of different auxiliary groups delivered, number and / or amount of different spacer groups delivered, characteristics and number of links between the various components ( For example, the binding properties of the spacer group), the reaction parameters (eg reactant solvent, reaction temperature, reaction time, reaction initiator, reaction catalyst, atmosphere in which the reaction is carried out) , The pressure at which the reaction is carried out, the rate at which the reaction is quenched), the stoichiometry of the various components, the order in which the different components are delivered, and the like.

In one aspect the present invention provides a method of making an array of multifunctional donors, the method comprising (a) a first activated framework component of a first multifunctional donor and a first of a second multifunctional donor Delivering the activated framework component to the first and second reaction vessels and (b) transferring the first functional group component of the first multifunctional provider and the first functional group component of the second multifunctional provider Delivering to the reaction vessel to form an array of two or more different multifunctional providers. The preactivated reactant may react. This process is optionally repeated with additional components (eg, structural components, functional components, auxiliary groups, spacer groups, etc.) and / or different reaction parameters (eg, different reaction temperatures, reaction catalysts, reaction solvents, etc.). It forms a large array of functional group donors.

The arrangement of the multifunctional provider may be screened / arranged for the multifunctional provider having useful properties once formed. Screening can be made for properties including, but not limited to, biological activity, binding affinity, biological properties, pharmaceutical properties, oral bioavailability, circulating half-life, agonist activity, antagonist activity, solubility, and the like. The arrangement of multifunctional providers can be screened sequentially or simultaneously for useful properties. In addition, the arrangement may be screened in the same place, or alternatively the multifunctional provider may be screened in a manner that is not the same place (eg, the multifunctional provider may be removed from the substrate and then screened). ). Once identified, multifunctional donors with useful properties can be prepared on a large scale.

Spacer group

In another aspect of the invention, the spacer group, ie the linker, lies between the base structure and the functional group and / or between the base structure and the auxiliary group.

Structures of one aspect of the invention have the formula:

R ¹ (-R ² (-R ³ ) _n ) _m

R ¹ (-R ² (-R ³ ) _n ) _m (-R ⁴ (-R ⁵ ) _s ) _t

In both formulas, the R ¹ symbol represents a multifunctional framework that provides multiple attachment sites to the spacer group. Polymers, including dendrimers, polypeptides, polysaccharides, and the like, are generally useful for such frameworks. Due to the large number of attachment sites, the backbone provides amplifying function to the action and / or auxiliary groups.

In both formulas, the R ² and R ³ symbols represent spacer groups and functional groups, respectively. The m symbol represents the number of functional groups attached to each spacer. The n symbol represents the number of spacers and functional groups to which they are attached (attached to the substructure). As mentioned above, the number of functional groups, the number of auxiliary groups and the number of spacers are generally such that there are ten more than the total functional groups and / or auxiliary groups on the multifunctional provider.

In formula (III), R ⁴ and R ⁵ each represent a spacer group and an auxiliary group. The s symbol represents the number of auxiliary groups attached to each spacer. The t symbol represents the number of spacers and auxiliary groups bonded to them (they are attached to the basic structure). In general, the number of functional groups and the number of spacers will be 10 greater than the total functional groups on the multifunctional provider. For example, “m” may be 6 and “n” may be 3 such that there are a total of 18 functional groups.

In the embodiment represented by Formula (II), only the spacer / functional structure is attached to the base structure. In the embodiment represented by Formula III, both the spacer / functional group and the spacer / auxiliary group structure are attached to the base structure. In formula (III), spacers R ² and R ⁴ may be the same or different groups and may be present in a molar ratio of about 1: 1 or in different molar ratios. For simplicity and convenience, the following description is made in terms of R ² , but it should be understood that both R ² and R ⁴ are included in the above description. Correspondingly, discussions about R ³ (functional groups) also include R ⁵ (adjuvant groups).

Spacer R ² may be of any of a wide variety of molecular structures, and is at least bifunctional to allow attachment to both R ¹ and R ³ , optionally via a linker. The spacer may be one that is stable or can be cleaved in vivo by the biological environment. A spacer having a plurality of binding functional groups at the terminus (ie, R ³ terminus) receives a plurality of functional groups and / or auxiliary groups. This type of spacer, although to a lesser extent, performs amplification in a manner similar to the basic structure. Other spacers useful in the present invention may have only one functional group at one end, in which case the value of "n" is 1 and the value of "m" is greater than 10. In addition to the in vivo cleavability described above, a particularly interesting property is hydrophilicity. Another useful property is the ability to lower antigenicity and increase molecular weight. Spacers with other properties can also be used to advantage as will be readily apparent to those skilled in the art.

The functional groups represented by R ³ are as described above for group A. The spacer R ² may be of linear or branched chain structure. Preferred R ² groups are those which comprise linear chains in the structure as whole spacer groups or as the main chain of branched chain groups. The linear chain may be a carbon atom chain or a carbon atom chain that is interrupted by one or more heteroatoms such as oxygen atoms, sulfur atoms or nitrogen atoms. The chain may also be substituted with aromatic groups. The bond forming the chain may be a single bond, a double bond or a triple bond, but a single bond is preferred. The length of the chain is not critical and can vary widely depending on the desired relationship between the molecular weight of the structure and the number of functional groups and / or auxiliary groups included on the structure. Best results can generally be obtained using chain lengths ranging from 4 to 1000 atoms, with preferred chains having from 6 to 100 atoms, most preferably from 10 to 50 atoms. As described above, the chain is the main chain of the spacer itself and does not include atoms, groups or side chains bound to the continuously bonded atoms that form the main chain. However, if a linker is present, the linker is included at the chain end connecting the chain to R ¹ and R ³ .

In certain embodiments of the invention, the spacer is a hydrophilic feature to impart hydrophilicity to the structure. Thus, the spacer can be any hydrophilic group of any spacer known in the art. Examples are polyalkylene glycols optionally substituted with groups that may or may not be added to the hydrophilic character. Among the polyalkylene glycols, polyethylene glycol is a preferred example. Examples of optional substituents are alkyl groups, alkoxy groups and hydroxy groups. Unsubstituted polyethylene glycol is particularly preferred. The length of the optionally substituted polyalkylene glycol is not critical and can vary. The choice of length depends on considerations such as achieving the desired molecular weight for the structure and imparting the desired degree of hydrophilic character. In most applications, polyalkylenes having molecular weights ranging from about 100 Daltons to about 20,000 Daltons provide the best results, with a range from about 200 Daltons to about 1000 Daltons preferred.

In embodiments of the invention wherein the spacer provides in vivo cleavability to the construct, the spacer may be used to chain any of the various groups that are cleaved in the biological fluid at an increased rate relative to the rate of the construct that does not comprise a group that is cleaved in the biological fluid. It may contain as a part. Increased rates of cleavage increase the rate at which the framework is removed from the functional groups and / or auxiliary groups of the multifunctional donor. Such removal can be used to lower the toxicity of the functional group and increase its activity. Although the increase in cleavage rate is not critical to the present invention, a preferred example of such spacers is that at least about 10%, most preferably at least about 35% of the cleavable groups are cleaved in the biological fluid within 24 hours of administration. Preferred cleavable groups are ester bonds and disulfide bonds.

In another aspect of the invention, the spacer imparts hydrophilic character to the structure and includes cleavable groups as described above.

The formulas for the spacers vary widely. The following discussion merely provides one type of spacer useful in carrying out the present invention, but is not intended to be limiting as to the type of spacer used in carrying out the present invention. One group of formulas for spacers that impart hydrophilic properties to structures is that m in formulas II and III described above is 1, and R ² in formulas II and III is Compound is:

XR ⁶ -YR ⁷ -Z

XR ⁷ -YR ⁶ -Z

XR ⁶ -Z

In each of these formulas, the hydrophilic component is represented by R ⁶ , which is a polyethylene glycol group having a weight of formula of about 100 Daltons to about 20,000 Daltons, preferably about 200 Daltons to about 1,000 Daltons.

In Formulas IV and V, the R ⁷ group is a cleavable group that increases the rate of cleavage of the structure in the blood. The group is a disulfide group, an ester group located in two directions, SS or C (O) -O or OC (O). If the structure in which R ² is formula IV is cut, the polyethylene glycol group will remain with the polymeric base structure R ¹ . In contrast, if the structure in which R ² is of formula V is cleaved, the polyethylene glycol group will remain with the functional groups and / or auxiliary groups.

The symbols X, Y, and Z are inert linkers which act to link the R groups together. The nature of these linkers is not critical, but the choice of them is a matter of convenience, as determined by the method of synthesis of the structures. By "inert" in this phrase is meant essentially non-toxic, non-immune and stable in terms of cleavage or degradation during the typical time required for use of the construct in clinical or diagnostic procedures. Examples of inert linkers useful for this purpose include alkylamino or aminoalkyl groups such as (CH ₂ ) _q -NH and NH- (CH ₂ ) _q , NH-C (O) -O and OC (O) -NH and Same carbamoyl groups, and alkyl carbamoyl or carnoylalkyl groups such as (CH ₂ ) _q -NH-C (O) -O and OC (O) -NH- (CH ₂ ) _q . The symbol q in this group can vary, but in most cases is 1 to 10, preferably 2 to 4, particularly preferably 2. In the present invention, such groups may be attributable to the terminal atoms at X or Z, respectively, R ¹ or R ³ . For example, the NH group in the definition of X or Z may be formed from amino functional groups on R ¹ or R ³ or from other N-containing groups that can react to form the NH of the linker. The same applies to the terminal O atom.

Examples of structures defined by Formula IV are as follows:

Examples of structures defined by Formula V are as follows:

In each of these six constructs, the symbol “PEG” refers to a polyethylene glycol segment in which terminal hydroxyl groups have been removed so that both ends are terminated with ethylene groups. In addition, spacer groups of similar general character that do not contain PEG may be useful in the practice of the present invention.

As a modified formula IV, the formula for the spacer supporting at least seven functional groups is formula VII:

XR ⁶ -Y '(-R ⁷ -Z) _r

Wherein R ⁶ , R ⁷ and X are as described above and Z is defined as (CH ₂ ) _q -NH. The symbol 'Y' represents the following general formula (VIII).

OC (O) -NH- (CH ₂ ) _q -CH ₃

Wherein q is 1 to 3, and two or more H atoms linked to the C atom in the (CH ₂ ) _q -CH ₃ moiety of the formula is a substituent NH- (CH ₂ ) _S -having s of 2 to 4 Substituted with NH, wherein the number of such substituents is determined by "m" and "n" in Formulas II and III. As a result, a branched structure is formed in which the spacer comprises two or more reactive NH groups for attaching functional groups and / or auxiliary groups.

The symbol r in formula (VII) is a number of zero or less than n. If r is zero then the spacer has no cleavable group, while if r is not zero each cleavable group is included in each functional NH group of the Z linker to which the functional or auxiliary group is attached.

In a preferred example of formula (VIII), q is 2 to 6, most preferably q is 2 or 3.

As a further modified form, the terminal CH ₃ of formula VIII may be replaced by OH or SH. This forms reactive OH or SH groups useful for attachment of functional and / or auxiliary groups.

Formulas of additional groups for R ² in Formulas II and III are as defined by Formula IX:

X'-R ⁸ -Z '

In formula (IX), R ⁸ is a group of formula (X):

(CH ₂ ) _o -R ^9- (CH ₂ ) _p

Wherein R ⁹ is a cleavable group comprising the same definitions as R ⁷ of Formulas IV and V above, ie located in both directions, which are disulfide groups, SS or C (O) -O or OC (O) Ester group. The indices o and p are the same or different and are zero or positive, with the sum of o + p being two or more.

The symbols X 'and Z' in formula (IX) are the same or different and are inert linkers of a similar category to the inert linkers of the formulas described above. Preferred examples of X 'and Z' are NH-C (O), C (O) -NH, NH-C (S) and C (S) -NH.

Depending on these various formulas, the number and arrangement of functional groups and / or auxiliary groups on a single structure of Formula II or Formula III may vary significantly. The number of functional groups will be equal to the product of m × n. The number of subgroups will be equal to the product of s × t. In general, the structure will have one or both products greater than ten.

The structures according to the invention can also be synthesized according to conventional linker reactions known to those skilled in the art. Examples are provided below wherein the basic structure, R ¹ , is functionalized with a number of amine groups, for example polylysine. In this example, the main chain is referred to as (AMP) (-NH ₂ ) _x, where "AMP" provides an amplifying effect of attaching a plurality of spacers and functional groups and / or auxiliary groups, where x is formula II and formula III Represents the number corresponding to.

Polyethylene glycol (PEG) spacers may be attached to the amplifier using activated esters of PEG, for example α, ω-bis-p-nitrophenoxy esters of PEG:

In the above formula, "PEG" means polyethylene glycol from which terminal hydroxyl groups have been removed as described above. The excess PEG ester X thus induced is reacted with (AMP) (-NH ₂ ) _x to obtain an intermediate of formula XII:

This intermediate is then reacted with excess H ₂ N- (ligand), which provides a ligand of a functional group derived to contain a reactive amine group. The product has the structure of formula XIII:

In an optional scheme for similar products, the intermediate of formula (XI) is converted to a second intermediate having a terminal amine group by reacting with a diamine, for example NH _2- (CH ₂ ) ₂ -NH ₂ . The second intermediate has the structure of formula XIV:

The intermediate of formula (XIV) is then reacted with a carboxyl-activated ligand, for example anhydride of the ligand, to prepare a structure of formula (XV).

Intermediate X is reacted with a diamine containing disulfide therein, such as cystenamine disulfide, NH _2- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -NH ₂ to introduce a cleavable group such as disulfide. Additional intermediates may be prepared:

It may then be reacted with a carboxyl-activated ligand to yield a compound of formula XVII:

There are many additional alternatives to this scheme. To prepare structures comprising esters cleavable in spacers without PEG, for example, amine- or hydroxyl-containing amplifying polymers can be derived to prepare carboxylic acid groups as functional groups. This is obtained by reacting the polymer with maleic anhydride, succinic anhydride or glutaric anhydride using known methods. Derived ligands in combination with derived polymers may be formed by reacting a ligand containing an amino alcohol, HO (CH ₂ ) _n NH ₂ with an isothiocyanate group to place a terminal hydroxyl group on the ligand. . The carboxylic acid group on the derived polymer can then be activated by conventional methods using reagents such as dicyclohexylcarbodiimide or carbonyldiimidazole, and reacted with the derived ligand to obtain ester linkages. The portion of the structure between the amplifying polymer and the ligand acts as a spacer and the length of the spacer is determined by the number of CH ₂ groups in the amino alcohol used to induce the ligand.

In an alternative scheme for forming the reverse ester, the ligand is derived by aminocarboxylic acid, HO ₂ C (CH ₂ ) ₂ NH ₂ , rather than amino alcohol. The resulting carboxylic acid-derived ligand is then activated with dicyclohexyl carbodiimide or carbonyldiimidazole and directly linked to the hydroxyl-containing amplifying polymer.

In these two schemes, the optional amine or hydroxyl group, due to the amplification polymer, is protected to avoid coupling reactions when necessary. This is readily obtained by conventional methods, ie acetylation with acetic anhydride.

Ligands having functional groups for attachment to the spacer can be prepared by conventional methods. Known ligands are readily derived, for example, by methods known to those skilled in the art. After induction, it is desirable to select ligands that retain all or most of their binding affinity.

Modified reaction

As mentioned above, one attempt to prepare a multifunctional provider of the present invention involves the formation of a combinatorial library, wherein the combinatorial library consists of a series of synthetic multifunctional providers and a series of multifunctional groups The donors differ from each other in terms of their composition, structure, properties, function, and the like. In the preparation of a series of multifunctional donors, the chemical structure of the base component, the chemical structure of the functional group component, the chemical structure of the auxiliary group, the chemical structure of the spacer group; Chemical properties of the basic structural components, chemical properties of the functional group components, chemical properties of the auxiliary groups, chemical properties of the spacer groups; The amount of framework components transported, the amount of functional groups transported, the amount of auxiliary groups transported, the amount of spacer groups transported; Number and / or amount of different basic structural components transported, number and / or amount of different functional groups transported, number and / or amount of different auxiliary groups transported, number and / or amount of different spacer groups transported; The nature and number of linkers between the various components (eg, the nature of the connection of spacer groups); Reaction factors (eg, reaction solvent, reaction temperature, reaction time, reaction initiator, reaction catalyst, atmosphere in which the reaction is carried out, rate at which the reaction is terminated, etc.); Stoichiometric amounts of various components; The order in which the different components are shipped may vary. Various reaction components (eg, structural components, functional components, auxiliary groups, spacer groups, etc.) and the various methodologies that can be used are described in more detail below.

In one approach, the activated polymer is reacted with one functional group to consume substantially all of the activation groups on the polymeric framework. In such embodiments, the stoichiometric ratio of functional group: activation group is at least 1: 1. If the ratio of 1: 1 is insufficient to substantially consume all the activation groups, excess functional groups may be added.

In other attempts, the functional group may be added in an amount insufficient to consume all of the activation groups. After the reaction with the first functional group, the second functional group or auxiliary group can be carried and reacted. Also included in this embodiment is the addition of more than one functional group as a mixture or sequentially followed by addition of one or more auxiliary groups, optionally or more than one auxiliary group.

In another approach, the activation group is taken by adding substantially enough functional groups. After attaching the first functional group to the framework component, the framework component is reactivated and a second functional group or auxiliary group is added. In the first step of the scheme of this embodiment, the functional group may comprise an individual functional group, a mixture of different functional groups, or a mixture of functional groups and an auxiliary group, wherein the auxiliary group may comprise a single auxiliary group or a mixture of auxiliary groups. .

In another approach, the activation group is substantially consumed by adding a mixture of two or more functional groups or a mixture of one or more functional groups and one or more auxiliary groups.

In another approach, substantially less than all of the activation groups react with one or more functional groups, one or more auxiliary groups or a mixture of one or more functional groups and auxiliary groups. After this first step, the remaining activation group is consumed by adding additional functional groups, auxiliary groups or mixtures of functional groups and auxiliary groups.

In certain embodiments, the functional groups, auxiliary groups and / or mixtures of functional groups and auxiliary groups are added to the activated framework solution as a dry powder or pure liquid. In another embodiment, a solution of a functional group, an auxiliary group and / or a mixture of functional groups and an auxiliary group is added to a solution of the activated polymer. In another embodiment, a functional group, auxiliary group, or a solution of a mixture of functional groups and auxiliary groups is added to the polymer, wherein the polymer is present as a pure liquid or dry powder.

As the solvent used in this embodiment of the present invention, the nature of the reaction of the activated polymer with the functional group and / or auxiliary group is compatible with the presence of the activation group (i.e., a significant amount does not react with the activated basic structure). And any solvents that are compatible. Such solvents include, for example, arbitrary parts of water, dimethylsulfoxide (DMSO), dimethylformamide (DMF), alcohols, ethers, ketones, hydrocarbons, aromatic hydrocarbons and mixtures.

In another embodiment, temperature can be adjusted to control the rate of reaction between the activated framework and the functional groups and / or auxiliary groups. In other embodiments, other reaction factors (e.g., reaction solvent, reaction temperature, reaction time, reaction initiator, reaction catalyst, atmosphere in which the reaction is carried out, pressure at which the reaction is carried out, rate at which the reaction is completed, etc.) are varied and optimized. Can be.

In yet another aspect, the "handle" of a functional group or auxiliary group is activated using an activation group as described above and reacts with an activation group on the framework or an inactivation group on the framework. As used herein, “handle” is used to refer to a reactive group such as carboxyl (acid or salt), hydroxyl, sulfhydryl, amide, carbamate, amine, ketone, aldehyde, olefin, diene, aromatic ring, and the like. do. Appropriate combinations of the basic structure in which the handles of the reaction group and the functional or auxiliary group are combined will be apparent to those skilled in the art.

As those skilled in the art relate to the use of activated polymers and functional groups, the foregoing descriptions relate directly to the use of monomers (eg, non-derived monomers, spacer groups and derived monomers, spacer groups). Or sufficiently indirectly applies to methods including the use of indirectly derived monomers into auxiliary groups and the use of functional monomers (eg derived monomers containing functional groups, ie R ³ directly or indirectly via spacer groups). It will be understood.

Substrate and Transport System

As used herein, "substrate" refers to a material that has a hard or nearly hard surface, or, optionally, a material including dents, wells, containers, trenches, and the like. In many embodiments, one or more surfaces of the substrate are substantially flat, but in some embodiments, physical separation of synthetic regions for different materials, for example, with potholes, wells, rises, etched trenches, etc. It may be desirable. In some embodiments, the substrate, itself, includes wells, risers, etched trenches, etc., which form all or part of the synthetic region.

More specifically, the substrate is organic, inorganic, biological, abiotic, or any combination of these, which may be particulate, stranded, precipitated, gel, sheet, tube, sphere, inclusion, capillary, pad, Exist as slices, films, plates, slides, and the like. The substrate can have any convenient shape, for example disks, squares, spheres, circles, and the like. The substrate is preferably flat, but may have various optional surface structures. For example, the substrate may include raised or lowered portions where the synthesis of the various multifunctional donors is performed. The substrate and their surfaces preferably form a rigid support to carry out the reactions described herein. The substrate may be any of a variety of materials, such as polymers, plastic resins, pyrex, quartz, resins, silicon, silica or silica based materials, carbon, metals, inorganic glass, inorganic crystals, films and the like. Other substrate materials will be apparent to those skilled in the art after reference herein. The surface of the solid substrate may be composed of the same material as the substrate, or optionally of different materials (ie, the substrate may be covered with different materials). In addition, the substrate surface may comprise an adsorbent thereon that transports the interesting components. Most suitable substrates and substrate-surface materials will depend on the class of material to be synthesized and the choice in any given case will be apparent to those skilled in the art. In a preferred embodiment, a suitable substrate may include, for example, a test tube holder comprising a microtiter plate (eg having 96 wells) or an amount of test tubes sufficient to hold each of a series of multifunctional donors. have.

In separate reaction zones, the reaction components are often inhibited from moving to adjacent reaction zones. Most of the time, this is simply ensured by having sufficient spacing between the reaction zones on the substrate such that the various components cannot inter-diffuse between reaction zones. In addition, this can be ensured by providing a suitable barrier between the various reaction zones on the substrate. In one approach, a mechanical device or physical structure defines various reaction zones on a substrate. For example, walls or other physical barriers can be used to inhibit the movement of reaction components in individual reaction zones to adjacent reaction zones. This wall or physical barrier can be removed after the synthesis is performed. Those skilled in the art will appreciate that it is advantageous to remove the wall or physical barrier at the appropriate time before screening a series of materials.

In another approach, for example, hydrophobic materials can be used to cover the areas surrounding individual reaction zones. This material prevents the aqueous (and other specific polarity) solutions from moving to adjacent reaction zones on the substrate. Of course, if a non-aqueous or non-polar solvent is used, different surface coatings will be required. In addition, by selecting a suitable material (eg, substrate material, hydrophobic coating, reactive solvent, etc.), the contact angle of the droplet to the substrate surface can be controlled. It is preferable that the contact angle is large because the region surrounding the reaction region remains unwetted by the solution in the reaction region.

In the transport system of the present invention, a small amount of each reaction component accurately measured is transferred to each reaction zone. This may be done using various transportation techniques. For example, various reaction components can be transported from the distributor in the form of droplets or powder to the desired reaction zone. Conventional micropipette instruments may be applied, for example, to dispense droplets from various capillaries into various volumes. Such dispensers may also be in the form used in conventional ink-jet printers. Such ink-jet dispenser systems include, for example, distributor systems in the form of pulse pressures, distributor systems in the form of bubble jets, and distributor systems in the form of slit jets. Such ink-jet dispenser systems can transport volumes of various droplets. In addition, such a dispenser system may be manual or may optionally be automatic or semi-automatic using, for example, a robotic technique.

In another embodiment, the reaction solution can be transported from the storage vessel to the substrate using an electrophoretic pump. In such a device, a thin capillary connects the reservoir of the reactant to the nozzle of the dispenser. At both ends of the capillary, an electrode is present to provide the potential difference. As is known in the art, the rate at which chemical species move in the potential gradient of an electrophoretic medium depends on a variety of physical properties, such as the charge density, size and shape of the species to be migrated, as well as the physical and chemical properties of the transfer medium itself. It depends. Under appropriate conditions of dislocation gradients, capillary size, and rheology of the transfer medium, hydrodynamic flow will form in the capillary. Thus, a large amount of fluid containing the desired reactant can be pumped from the reactor to the substrate. By adjusting the proper position of the substrate relative to the electrophoretic pump nozzle, the reactant solution can be accurately transported to a predetermined reaction zone on the substrate.

The distributor can be arranged for the appropriate reactor zone by various conventional systems. Widely used in the field of manufacture and testing of electrophoretic devices, these systems can transport droplets of reaction components to individual reaction zones at a rate of 5,000 droplets / second. The (X-Y) precision of the movement of this system is within 1 μm. The position of the distributor stage of such a system can be adjusted according to the position of the substrate in a variety of ways known in the art. For example, with one or two reference points on the substrate surface, a "dead reckonong" method can be provided to locate each reaction region on the substrate. Reference marks in any such system can be accurately distinguished using capacitive sensors, resistive sensors or optical sensors. Alternatively, a "visible" system using a camera can be used.

In another aspect, the distributor can be arranged for the desired reaction zone in a system similar to that used in the field of magnetic fields and optical storage media. For example, the reaction zone where the reaction components are deposited is identified by the location of the tracks and sectors on the disc substrate. Then, as the disk substrate rotates, the dispenser is moved to the appropriate track. If a suitable reaction zone is present below the distributor, droplets of the reactor solution are released.

In other embodiments, the reaction zone may be further defined by depressions in the substrate surface. This is particularly desirable when the head or other sensing device is in contact with or past the substrate surface. The recess may also act as an identifying mark that directs the dispenser to the desired reaction zone.

Those skilled in the art will appreciate that the construct, ie, the composition of the multifunctional donor, can be determined from the synthesis process or obtained by sequencing, mass spectrometry, delineation, encoding, and the like. Such evaluation methods are described in Thomson et al., Chem. Rev., 1996, 96, 555-600.

Pharmaceutical composition

The present invention also relates to pharmaceutical compositions for multifunctionally providing reagents for treatment. The pharmaceutical composition contains the aforementioned multifunctional provider and a pharmaceutically acceptable carrier. In one embodiment, the multifunctional donor has Formula 1

Formula 1

(Y)-(XA) _n

Where

Y is the basic structure

X is a linkage or linker,

A is a donor functional group,

n is an integer greater than 10, such that the group providing the multifunctional group is chosen to react with the library of target binding site B. The donor, by itself, can act as a pharmaceutically acceptable carrier. In one embodiment, the multifunctional donor is selected for n such that the multifunctional donor is suitable for an interface containing a library of target binding site B and can overwrite the library of target binding site B when administered to a subject Attach the-(XA) residue to Y. In another embodiment, X is a linker that is not part of Y or A as an independent residue, and n is an integer greater than 10 that is selected so that the multifunctional donor is suitable for the library of target binding site B when administered to a subject. In another embodiment, Y is a polymeric framework, A is a donor functional group, X is a linker, and n is an integer greater than 10 selected so that the multifunctional donor is suitable for a library of target binding sites B when administered to a subject to be.

The expression "pharmaceutically acceptable carrier" means a substance which is administered with a multifunctional donor to carry out their intended action. Examples of such carriers include solutions, solvents, dispersion media, retardants, emulsifiers and the like. The use of such media for pharmaceutically active substances is known in the art. In addition, any other conventional carrier suitable for use with the multifunctional donor is within the scope of the present invention. It will be appreciated that many multifunctional providers of the present invention are water soluble. Thus, when formulating the multifunctional donor of the present invention into a pharmaceutical composition, there are fewer problems and easier than other less water-soluble materials.

The expression "therapeutically effective amount" of a multifunctional donor means an amount necessary or sufficient to exhibit the intended action in the subject. The therapeutically effective amount can vary depending on the site of interest, the type of component used (eg, basic structure such as linker, eg Group A), the size of the subject or the severity of the condition. One skilled in the art can study the aforementioned factors and determine the effective amount of the multifunctional donor without unnecessary experimentation. In addition, in vivo or in vitro assays can be used to determine the “effective amount” of a multifunctional donor. Those skilled in the art can select an appropriate amount of multifunctional donor for use in the assays described above.

The results obtained in cell culture assays and animal studies can be used to determine appropriate dosages for use in a subject. It is desirable for the dose of such reagents to lie in the range of circulation or concentration of tissue with ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any reagent used in the methods of the present invention, a therapeutically effective dose can be assessed initially from cell culture assays. Dosages that provide a concentration comprising an IC50 as measured in cell culture (i.e., the concentration of a test control reagent capable of obtaining ½ effect on maximal inhibition of symptoms) will be prescribed in animal models. This information can be used to more accurately measure useful doses in the human body. For example, the amount in plasma may be measured by high performance liquid chromatography.

In addition, the method of administration may affect what constitutes an effective amount. Multifunctional donors can be administered alone or in combination with other reagents. In addition, several divided doses, and differential doses, may be administered daily or in stages, or the dose may be infused continuously. In addition, the dosage of the multifunctional donor can be increased or decreased proportionally depending on the condition of the treatment condition.

Pharmaceutical compositions for use in accordance with the present invention may also be formulated according to conventional methods using one or more pharmaceutically acceptable carriers or excipients as pharmaceutically acceptable carriers. Thus, multifunctional donors and their pharmacologically acceptable salts and solvents are formulated, for example, for topical administration, injection, inhalation or aeration (through the mouth or nose) or for oral, oral, parenteral or rectal administration. It can also be done.

As above, the reagents of the present invention may be formulated in an optional specific mode of administration, eg, as appropriate for systemic and local or topical administration. Techniques and formulations can generally be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection methods including intramuscular, intravenous, perineal and subcutaneous infusion are preferred. For injection administration, the multifunctional donor reagents of the present invention may be formulated in liquid solutions, preferably in pharmacologically compatible buffers such as Hanks' solution or Ringer's solution. In addition, the reagents may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical composition of the multifunctional donor may comprise a binder (eg, pregelled corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); Fillers (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); Lubricants (eg magnesium stearate, talc or silica); Disintegrants (eg potato starch or sodium starch glycolate); Or in the form of, for example, tablets or capsules prepared by conventional methods, including pharmaceutically acceptable excipients such as humectants (eg sodium lauryl sulfate). Tablets may be coated by methods known in the art. Liquid compositions for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be present as dry products for composition with water or other suitable vehicle before use. Such liquid compositions include suspending agents (eg, sorbitol syrup, cellulose derivatives, or hydrogelled edible fats); Emulsifiers (eg lecithin or gum arabic); Non-aqueous vehicles (eg, almond oil, oil esters, ethyl alcohol, or fractionated vegetable oils); And pharmaceutically acceptable additives such as preservatives (eg, methyl or propyl-p-hydroxybenzoate, or sorbic acid). In addition, the composition may suitably contain a buffer salt, a flavoring agent, a coloring agent, and a sweetening agent. Compositions for oral administration may be suitably formulated to control the release of the active modifier.

For administration by aspiration, compositions for use in accordance with the present invention may be prepared by employing a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Can be conveniently injected in the form of an aerosol spray provided in a pressurized package or nebulizer. In the case of a pressurized aerosol, the unit dosage may be determined by providing a valve to transport a metered amount. For use in an inhaler or gas injector, capsules and cartridges of, for example, gelatin may be formulated containing a multifunctional reagent and a suitable powder base such as lactose or starch.

Reagents may also be formulated for parenteral administration, such as injections, for example, round injection or continuous infusion. Formulations for injection may be presented in unit dosage form (eg, ampoules or large dose containers) with a preservative added. The compositions may take the form of suspensions, solutions or emulsions in oil, or aqueous vehicles and may contain formulation reagents such as suspending, stabilizing and / or dispersing agents. Optionally, the active ingredient may be present in powder form for compounding with a suitable vehicle, eg, bactericidal pyrogen-free water, before use.

The reagents may also be formulated as rectal compositions such as suppositories or fixed enema, for example containing conventional suppository bases such as cocoa butter or other glycelides.

In addition to formulations as described above, it may be formulated as a reservoir composition. Such long acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the modification reagent may be combined with a slightly soluble derivative such as a suitable polymeric or hydrophobic material (e.g. emulsion in acceptable oil) or an ion exchange resin, or a slightly soluble salt. .

It can also be carried out systemically, through mucous membranes or through the skin. Administration via mucous membranes or skin uses a suitable penetrant to the barrier membrane to be permeated in the formulation. Such penetrants are generally known in the art and include, for example, bail salts and fusidic acid derivatives for mucosal administration. In addition, a cleaning agent may be used to facilitate the penetration. Mucosal administration may be by spraying the nose or using suppositories. For topical administration, the oligomers of the invention can be formulated into ointments, salbs, gels or creams as is known in the art.

In embodiments where the multifunctional donor is large and does not pass rapidly through the hydrophobic membrane, injection or inhalation may be more suitable than digestion or transport through the skin. The challenge for this transport is related to protein-based medicaments. In embodiments in which the multifunctional donor passes through the GI track, suitable transport methods known in the art may be used.

If desired, the composition may be provided as a packaging or dispersant device, or as a kit with instructions. The composition may contain one or more unit dosage forms containing the active ingredient. Packaging materials, for example foam packaging, may be composed of, for example, metal or plastic foil. The packaging or dispersant device may, for example, be used in combination with instructions for administration for use in the methods described above.

Collection of multifunctional providers

The present invention also relates to a collection of multifunctional providers. Such a library can be used in the method of the present invention. The collection of multifunctional providers includes one or more multifunctional providers that are mixed with each other. The collection may include a number of different multifunctional providers that serve the intended action.

Further, the present invention relates to combinations, such as therapeutic compositions, containing a collection of multifunctional donors and a pharmaceutical carrier as described above. Formulations containing a collection of multifunctional donors can be designed such that different multifunctional donors are chosen for their ability to impart specific properties to the entire formulation.

The collection of multifunctional donors may be in the form of polydispersions. In the description below, the term "polydispersion" is used for the collection, which will be understood to be broadly included in other forms of collection of the multifunctional provider of the present invention.

Polydispersions of multifunctional donors include more than one different multifunctional donor. Polydispersions of different multifunctional donors may be size-specific collections due to differences in the length of the base structure and the degree of substitution with group A, and / or with differences in the combination of group A / linkers. In addition, the Group A exact attachment position may be different. Each component of the collection imparts different properties to the overall formulation, which can be advantageously used to treat a disease or condition. For example, low molecular weight constructs in a collection may promote their desired action more quickly than high molecular weight constructs. At the same time, high molecular weight constructs have a longer duration of action than low-molecular weight constructs due to longer residence times at the site of action, and / or longer lifetimes at the relevant sites. This difference is, in the treatment of a disease or condition, that the multifunctional donor is as a collection as compared to a corresponding number of identical species (e.g., a combination of rapid onset of action and prolonged duration of action, or other combination). It has the advantage of having excellent properties.

How to treat a disease or condition

The invention also relates to a method for treating a disease or condition. The method includes administering to the subject a plurality of group A such that the subject can be treated for the disease or disorder. In one embodiment, the disease or condition is treated by conformal interfacial interaction of the multifunctional donor with a library of binding site B or conformal interfacial interaction of the multifunctional multifunctional donor with a library of binding site B in a subject. Can be. Conformal surface interactions consequently cover a library of binding site B or a series of target binding site B in a subject. In other embodiments, the multifunctional donor that facilitates treatment meets some sort of criteria. The criteria are as follows:

Group A acts as a medicament alone or in admixture with a base structure, for example in combination with a compound which has insufficient binding capacity alone but may be amplified as a result of the invention;

The provision of group A attached to the framework provides an additional advantage to the interaction over the provision of a single group A to multiple binding sites B;

A further advantage is the corresponding effect, which is greater than the sum of the advantages provided by the library of monomers of the same group A, which are dispersed in a homogeneous solution.

Examples of additional benefits include those selected from the group consisting of providing sufficient biological effects in low concentrations of group A, enhanced selectivity for target versus non-target sites, and enhanced biological capacity. Can be mentioned. In a preferred embodiment, the multifunctional donor provides at least two additional advantages and in more preferred embodiments at least three additional advantages. The benefits can be selected from the list above and also describe the benefits described below in describing the pharmacokinetics and / or mechanisms associated with the action of the multifunctional donor (eg, positive cooperation, entropy improvements for binding and Providing steric inhibition).

For methods of treating a disease or condition, binding site B is positioned so that multifunctional interactions can occur. For example, the binding site B that interacts with group A of the multifunctional donor can be intracellular or extracellular (eg, cell surface or extracellular matrix) or can be located on the cell membrane. The multifunctional donor of the present invention may have a variety of actions, such as ease of treatment, and they may be in a state associated with multifunctionality, such as an undesirable state (eg fertilization state) or a disease state (eg , Infectious diseases) can be used to control (e.g., raise or lower). Provided herein may be used for the purpose of a particular biological course of a disease or disorder (eg, prophylactically inhibiting cell-pathogen adhesion), and also provides for a particular disease state (eg, expansion of infection). Can be used to treat a subject with a prophylactic infection to modulate or inhibit it. As such, the subject matter of the present invention has a variety of disassembly uses in terms of controlling this condition.

Regulation of specific biological processes

In certain embodiments, multifunctional donors of the invention can be used, for example, to modulate cell-cell interactions. Many biological processes require cell-cell interactions, and the subjects of the present invention may promote or inhibit such interactions. One particular example is the binding of cells to other cells, such as neutrophils and endothelial cells of the arteries. The attachment of neutrophils initially suspended in rapidly flowing blood to endothelial cells closest to the site of inflammation is via multifunctional interaction (A Varki, J. Clin. Invest. 1997, 99, 158-162, JB Lowe, PA Ward, J. Clin. Invest. 1997, 99, 822-826, and MAGimbrone, T. Naggel, Tarper (JN Tapper, J. Clin. Invest, 1997, 99,2062-2070). By near inflammation, the initially rapidly migrated neutrophils attach to the surface of endothelial cells and then rotate slowly (10-20 μm / minute); In this case, endothelial cells mean that they are lined inside the blood vessel. This rotation is due to the interaction between a number of E-selectins and P-selectins within the surface of endothelial cells, which are normally not present on the surface of these cells but are caused by cytokines in the event of inflammation and the surface of neutrophils. It is mediated by a number of glycoproteins that express sialyl Lewis X (tetrasaccharide) on (MAGimbrone, T. Nagel and JNTopper, J. Clin. Invest. 1997, 99, 2062-2070]. In addition, L-selectin present on the surface of neutrophils interacts with sialyl Lewis X present on endothelial cells. The functionality of this set of interactions may be significantly influenced by the kinetics, dynamics, and selectivity of neutrophil recruitment (LLKiessling and NL Pohl, Chem & Biol, 1996, 3, 71-77). ] Reference). Table 2 below presents other examples of cell-cell action.

Examples of Multifunctional Cell-Cell Interactions Cell-1 Molecules on Cell-1 Cell-2 Molecules on Cell-2 Neutrophils L-selectin, P-selectin Endothelial cells Sulfated Sialyl Le ^X Neutrophils Sulfated Siallyl Le ^X Endothelial cells E-selectin Neutrophil cell-adherent molecules (NCAMs) such as L1, NCAM-H, CD24 and α ⁶ -intergreen Neutrophils E-selectincard herrin Neutrophils Sulfated Sialyl Le ^X T-cell T-cell receptor Antigen present cell Major Histocompatibility Complex (MHC) CD3 MHC CD28 Platelets Ia / Iia Endothelial cells Collagen Platelets IIb / IIIa Platelets IIb / IIIa Tumor cells GalTase Endothelial cells GlcNAc sperm GalTase egg cell GlcNAc Aged Red Blood Cells Desialylated Glycoprotein Heptatosite C-type lectins

In other embodiments, multifunctional donors of the invention can be used to inhibit other forms of cell-cell interactions, such as adhesion. For example, attachment of leukocytes to endothelial cells is mediated by a class of adhesion molecules known as selectins. Selectins are glycoproteins (p-selectin) through membranes expressed on platelets, glycoproteins (L-selectins) through membranes expressed on leukocytes (including neutrophils and other lymphocytes), and glycoproteins via membranes expressed on endothelial cells (E). -Selectin and P-selectin). Selectin mediates selectin rotational interactions in the walls of endothelial tubes, which interactions ultimately induce leukocyte extravasation (Kretzschmar et al., Tetrahedron, 1995, 51: 13015). Reference). Providers of the invention will be useful for inhibiting the initial attachment and subsequent steps, for example, the migration of leukocytes. The multi-functional donor of the present invention can be used to prevent neutrophil-endothelial interactions to regulate conditions related to neutrophil adhesion (eg inflammation, adult respiratory distress syndrome, rheumatic neuralgia, sepsis shock and reperfusion disease). Can be.

In other embodiments, multifunctional donors of the invention can be used to inhibit platelet-platelet interactions. Platelet aggregation plays a prominent role for thrombosis or thromboembolism. ADP, thrombin, epipinrin or collagen agonists are exposed to the fibrinogen-binding site on platelet proteins, GPIIb-IIIa. Small peptides such as RGD or HHLGGAKQAGDV (H ₁₂ ) include fibrinogen recognition features and block the binding of fibrinogen to these sites. Peptides bound to GPIIb-IIIa induce the expression of bovine and ephetopes in the GPIIb-IIIa complex (see Gawaz et al. Arterioscler Thromb Vasc. Biol. 16: 621). Platelet granules are then released, where one of the major glycoproteins of the granules is thrombospondin, which is believed to mediate the aggregation of platelets (Gawaz et al. Arteroscler Thromb Vasc. Biol. 16: 621 ] Reference). Group A may be selected to inhibit one of the interactions that induce platelet aggregation. In a preferred embodiment, the interaction of GPIIb-IIIa with fibrinogen cognitive stimulatory features is regulated. In certain embodiments, it is desirable to cause lack of aggregation in the subject, while in other embodiments it will be desirable to induce an overaggregation state in the subject.

In other embodiments, multifunctional donors of the invention can be used to reduce the ability of cancer cells to metastasize. Metastasis includes detaching tumor cells from a first site, invading them into surrounding tissue, and anchoring to a second site, wherein the multifunctional donor of the subject can act at any stage of this step. The directivity of cancer cells to various organs can be sold by lectins of invasive organs or by lectins of tumor cells (Matrosovich. 989. FEB 252: 1,2: 1-4; Beuth, et al, 1988, Clin Exp. Metastasis. 6: 115-120). For example, β-lactosyl populations have been described as potential tumor metastasis inhibitors (see Dean et al., 1993. Carbohydrate Research 245: 175). Thus, as group A, a multifunctional provider can be prepared that mixes the mediators of this recognition step.

In other embodiments, the subject herein can be used to control infection. The initial stage of most cell-pathogen interactions includes an attachment phase. This is true for all viruses. One example where viruses attach to the surface of cells is influenza and bronchial epithelial cells. In the first stage of infection, influenza virus attaches to bronchial epithelial cells. Adhesion is associated with a number of trimers of hemagglutinin (HA, lectins found to be concentrated on the surface of the virus, about 2-4 copies / 100 nm ² or 600-1200 / virus particles) and silalic acid (SA , Terminal sugars on a number of glycoproteins, densely arranged on the surface of the target cell, resulting from interactions between a number of residues of about 50 to 200 copies / 100 nm ² ). Table 3 provides other examples of cell-virus interactions, where P is a protein and S is a sugar (glycoprotein or glycolipid). Many viruses have been found to be used in interactions for the attachment of one or more target cells.

RNA protein disease Viral molecules Molecules on Host Cells Ecopropic murine retrovirus cancer P: epidermal glycoprotein P: basic amino acid transfer agent Non-ekotropic murine leukemia virus leukemia P: gp70 (surface) HIV-1 AIDS P: gp120-Ig heavy chain region P: gp120-resembles leukemia P: gp120-resembles intestinal vascular peptide P: CD4P of T cells: CD4 molecule reacting with class II HLA-DR MHC molecules S: Calactosyl seramide (or a highly related molecule on epithelial HT29 cells of human colon) P: CR2 (especially in EBV infected cells) P: Chemokine receptors CXCR-4 (T-cell tropic) and CCR-5 (dash cell tropic) (both G-protein coupled receptors through 7-membrane) HIV-2 AIDS P: CD4 Simian Immunodeficiecy virus Immunodeficiency Syndrome Similar to AICD P: SIV mac239 P: CD4 African swine virus P: p12, p72

Attachment of the virus to such a host is the first step in viral infection and involves simultaneously binding a number of molecules on the surface of their host cells on the surface of the virus (M. Tardieu). , RLEpstein, HL Weiner (Int. Rev. Cytol., 1982, 80, 27-61). Molecules embedded on the surface of viruses and cells that mediate adhesion are most directly when such attachment is inhibited by such high concentrations of free monomers of the molecule or monoclonal antibodies to the molecule. It is confirmed. This method is often used when the ligand is a sugar or small molecule. Less direct identification of molecules involved in adhesion can be obtained from the interrelationship of mutants (human-made or natural) that fail to successfully bind to the target cell: that is, mutants of specific proteins bind the virus. If you do not have the ability to do so, it is either direct or indirectly involved in the attachment process. This second method is often used for receptors or ligands that are proteins. It is often not clear that many molecules on the surface are involved in the process of virus-cell recognition. Because viral particles are non-specifically attached to multiple substrates, it is often difficult to distinguish biological specific binding from biological non specific binding. Some viral particles may also invade host cells by non-specific migration (the same mechanism by which cells are used to accept small molecules). Some proteins on the surface of the virus may recognize more than one type of ligand on the surface of the cell. Finally, certain viral particles may bind to proteins that are freely dispersed in solution, which recognizes molecules on the surface of cells (mediated attachment). Viruses are molecules of the surface of most classes of cells; Sugars (see Glycobiol. 1993, 3, 97-130) (see, eg, polyomas (H. Fried, Kalc (LDCahan), JCPaulson, Viral. 1981). , 109, 188-192) and orthomyxoviruses (JCPaulson, JESadler, RLHill, J. Biol. Chem. 1979, 254, 2120-2124). Reference) recognizes sialoligosaccharides; Phosphatidyl lipids (e.g., vesicular gastroenteritis virus, VSV recognize phosphatidylserine and phosphatidlinositol) (mastromarino, C.Conti, P.Goldoni, B. Hauttecoeur) , N. Orsi, J. Gen. Virol. 1987, 68, 2359-2369); And proteins (e.g., HIV is CD4 (AG Dalgleish, PCL Beverley, PR Clapham, DH Crawford, MG Greaves, RA Weiss) And human rhinoviruses recognize intercellular adhesion molecule-1, ICAM-1 (JM Greve, G. Davis, Meyer). (AM Meyer), CP Forte, SC Yost, CW Marlor, ME Kamarck, A. McClelland, Cell 1989, 56, 839-847. ]).

The most widely studied system for viral attachment and inhibition in relation to multifunctionality is orthomixovirus A (X-31) (treated strain of influenza) that adheres to the surface of red blood cells [WJ Lees, Spartenstein, WJE Kingery, White, J. Med. Chem. 1994, 37, 3419-3433; Mammen, G. Dahmann, White, J. Med. Chem. 1995, 38, 4179-4190. This attachment occurs through a number of simultaneous interactions between the HA of the virus and the SA of the cell. Accurate measurements of the affinity between influenza virus and red blood cells have not yet been performed.

Another widely studied virus-cell interaction is the interaction between protein gp120 for human immunodeficiency virus (HIV) and 60K glycoprotein CD4 on the T-cell surface. Most concentrates of solubilized CD4 (CD4 chemically cleaved from the cell surface) inhibit adhesion. HIV is an example of a virus that can use other molecules on the cell surface. HIV can infect glioma and rhabdosarcoma cells, both of which lack CD4. Solubilized CD4 does not block the attachment and subsequent infection of these cell types (Klamfam, JN Weber, D. Whitby, K. McIntosh, DalGracey, PJ Maddon ), KC Deen, RW Sweet, Weiss, Nature 1989, 305, 60-62.

There are many examples of virus mediated binding of cells to cells. One example includes hepatitis B, which in some cases binds to aggregates of serum albumin, which in turn binds to receptors for albumin on the surface of hepatocytes (P. Pontisso, P. Petit, MA Petit). ), MJ Bankowski, ME Peeples, J. Virol. 1989, 63, 1981-1988]. The second well studied example begins with the bivalent attachment of antiviral antibodies to the surface of the virus (examples including Dengue virus, West Nile virus, and HIV). [RMHV Van , G. Hardie, Immunochem. 1976, 13, 503-507. The Fc-receptor on the surface of the target cell then interacts multifunctionally with multiple Fc tails. The mechanism of this attachment is the same as that used by macrophages in the recognition of external pathogens, interestingly, the former is infected and the latter is eliminated.

Bacterial pathogens can be divided into two classes: intracellular (bacterial pathogens that enter and replicate within cells) and extracellulars (bacterial pathogens that exist between cells but do not enter them). The mechanism of infection by bacterial pathogens present in cells is similar to viruses, and infection is initiated by the attachment of pathogens to host cells (Tables 4-5). Extracellular bacteria often migrate to and collect on specific tissues. The concentration of bacteria in a particular tissue (tissue tropism) is caused by specific interactions between molecules on the surface of the bacteria and molecules on the cell surface within the desired tissue or components of the extracellular matrix characteristic of the tissue. Can be. Many specific interactions between bacteria and host cells or extracellular matrices have been previously reviewed (I. Ofek, N. Sharon, Curr. Topics Microbiol. Immunol. 1990, 151, 90-113; OPEC, Sharon, Infect. Immun 1988, 56, 539-547. For viruses, the specificity of the interaction is usually defined by the molecule, which is the best inhibitor of adhesion or aggregation assays. This assay is most often used when an interaction exists between lectins on the bacterial surface and sugars on the host cell or extracellular matrix (Table 4). Currently, bacterial interactions appear to include proteins as ligands and receptors more suitably than viral interactions (Table 5). Such protein-protein interactions are very difficult to study because mainly "monomers" are not known and are not available for research or lose their "form" when removed from the flesh of the membrane. As a result, most of the detailed studies of bacterial and viral interactions to date include lectin-sugar interactions and no protein-protein interactions.

Intestinal bacteria live in the gastrointestinal tract of mammals. In most cases, enteric bacteria express type 1 fimbriae that binds to mannose on the surface of epithelial cells of the digestive tract (eg, intestinal E. Coli, Klebsiella nunu). Klebsiella pneumonia, and Salmonella]. This interaction can be assayed by the aggregation of red blood cells of guinea pigs, the surface of which is densely covered with mannose residues. A well-studied example is the study of urophatogenic E. coli.

Certain other intestinal this coli contains pimbrian hemagglutinin on its surface that specifically binds to various sialic acid containing glycoproteins on the surface of epithelial cells. A well-studied example of sialic acid recognition contains K99 pimbria lectin, which recognizes N-glycolylnuramic acid specifically located on NeuAc (α2,3) Gal (β1,4) Gal (β1) -ceramide. In this coli [M. Lindahl, R. Brossmer, T. Wadstrom, Glycoconj. J. 1987, 4, 51-58. Intestinal strains of Vibrio cholera produce various hemagglutinin (R. A. Finkelstein, L. F. Hanne, Infect. Immun. 1982, 36, 1199-1208. The most prevalent of these hemagglutinin bind to fucose derivatives on the epithelial surface.

Host organisms often have relatively nonspecific defenses that are also multifunctional against these multifunctional pathogens. One example is the Tamm-Horsfall glycoprotein, the most abundant glycoprotein in human urine. These glycoproteins contain a variable number of N-bridged oligomannose units and bind a wide range of bacteria (F. DellOlio, FJJ de Kanter, DH van den Eijnden), F. Serafini-Cessi, Carbohyd. Res. 1988, 178, 327-332.

Most bacterial infections are also initiated by an adhesion step that includes bacterial adhesion and carbohydrate determinants present on host cells (Matrosovich. 1989. FEBS 252: 1,2: 1-4). One example of adhesion of bacteria to the cell surface includes these coli and urethral endothelial cells. Europathogenic strains of bacterial E. coli are attached directly and indirectly to both the surface of epithelial cells in the urethra and bladder using multifunctional interactions (FIG. 2). Several bacterial proteins have been demonstrated to confer this tissue preference. Two examples are P-pimbria (containing protein G) and type I pimbria (containing FimH adhesin), both located on the surface of this coli. We describe multifunctionality using P-pimbria as an example in such a system. First, these europatogenic bacteria are resistant to and specifically attached to multiple copies of a portion of the Gal (β1,4) Gal (P _K antigen) of glycoproteins present on the surface of epithelial cells in the urethra, particularly in the kidneys. Lectin-like protein G present in the acrosome of the P-pimbria filament can be used. Second, many copies of the F-protein on the surface of this coli are attached to fibronectin, a multifunctional soluble glycoprotein. Fibronectin is then multifunctionally bound to the surface of epithelial cells. The coli can collect in these tissues of the urinary tract and replicate there, causing disease (especially pyelonephritis). In general, bacteria bind directly to the cell surface or to molecules in the extracellular matrix of the desired tissue. Bacteria bind to both sugars and proteins.

Examples of bacteria that bind sugar (S) and protein (P) on the surface of host cells Bacteria name disease Molecules on bacteria Molecules on the host Actinomyces naeslundii 12104 and A. viscosus LY7 Chronic oral disease S: GalNAc β-containing glycosphingolipids (GSLs), for example GalNAc (β1,3) Gal (α) -O-ethyl Candida albicans Diseases of the oral cavity and some of the normal fauna of the reproductive system, disease predisposition conditions (e.g., diabetes mellitus, immunodeficiency, long-term catheters, antibacterial substances that reduce normal bacterial fauna) Induce P: 37-kDa laminin receptor P: 58-kDa fibrinogen-binding mannoprotein P: C3d receptor P: ubiquitin S: Fucose-containing glycoside on epithelial surface Chlamydia trachomatis Chronic keratoconjunctivitis, a disease that can progress to scars and blindness Respiratory infections S: 7-9 mannose residue containing polysaccharide P: mannose binding protein Digestive tract coherent Escherichia coli (EAggEC) food poisoning P: hemagglutinin S: sialic acid Europatogenic Escherichia coli Urinary tract infection P: PapG Advice S: Gal (α1,4) GalP on glycolipids: Europlakins Ia and Ib S-pimbriaized Escherichia coli Cerebrovascular complications of meningitis S: NeuGc (α2,3) Gal and NeuAc (α2,8) NeuAcS: NeuGc (α2,3) Gal and NeuAc (α2,8) NeuAc (found above epithelial cells of brain microvascular structure) Helicobacter pylori Duodenal ulcer, stomach cancer P: hemagglutinin P: laminin binding protein S: Lewis (b) blood group antigen S: Neu5Ac (α2,3) Gal (SA dependent strain)

Examples of bacteria that bind sugar (S) and protein (P) on the surface of host cells Bacteria name disease Molecules on bacteria Molecules on the host Streptococcus sanguis Streptococcus sobrinus (oral) Some of the normal flora of the upper airway: deficiency can cause disease S: sialic acid of saliva glycoprotein Yersinia enterocoltica Severe abdominal pain and diarrhea; In some cases, it can cause fatal sepsis S: Gal, GalNAc, Lac in Jean Mucin

Examples of bacteria that bind to derivatives of glycolipid lactosyl ceramides The name of the bacterium Target organization Bacteroides fragilis Leader Bacteroides ovatus Leader Bacteroides vulgatus Leader Bacteroides distasonis Leader Bacteroides thetaiotamicron Leader Lactobacillus fermentum Leader Lactobacillus acidophilus Various places Fusobacterium necrioohorus Leader Fusobacterium varium Leader Clostridium difficile Leader Clostridium botulinum Leader Propionibacterium granulosum Skin, large intestine Propionibacterium acne skin Propionibacterium freudenreichii dairy product Actinomyces viscosus Oral cavity Actinomyces naeslundii Oral cavity Shigella dysenteriae Leader Shigella flexnerii Leader Shigella sonnei Leader Salmonella typhimurium Leader E coli chapter Vibrio cholerae Small intestine Campylobacter jejunii chapter Hemophilus influenzae By breathing Yersinia pseudotuberculosis chapter Yersinia pestis chapter Neisseria gonorrhoeae Reproductive Pseudomonas aeruginosa By breathing

Example of Bacteria That Bind Sugar (S) and Protein (P) in Extracellular Matrix Bacteria name disease Receptor on bacteria (ligand) Ligand (Receptor) on Host Matrix Aspergillus fumigatus conidia Various local fungal infections P: D domain of the fibrinogen molecule Borelia burgdorferi Lyme disease P: integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) (RGD) Bordetella pertussis Pertussis (Pertussis) P: pertactin and filamentous hemagglutinin (FHA); Cell-binding sequence (RGD) Candida albicans Part of the normal fauna of the oral cavity and (cancer) reproductive system, secondary to disease predisposition conditions (e.g. diabetes mellitus, immunodeficiency, long-term catheters, antibacterial substances that reduce normal bacterial fauna) P: 37-kDa laminin receptor P: 58-kDa fibrinogen-binding mannoprotein P: C3d receptor P: ubiquitin S: Cell-wall polysaccharide of Streptococcus gordonii during concurrent infection S: Various oligosaccharides containing beta-1,2 and alpha-1,2 crosslinks Chlamydia trachomatis Chronic keratoconjunctivitis, a disease that can progress to scars and blindness Respiratory infections P: heparin-binding protein S: Heparan sulfate-like GAG with decasaccharide of minimum chain length Enterococcus faecalis diarrhea S: galactose, fucose and mannosemin, not mannose Escherichia coli with S pimbria Gastrointestinal infection P: S-pimbria protein SfaA S: inhibited by NeuAc (α2,3) lactose S: glycolipids containing terminal Gal (3SO ₄ ) β-1 residues Haemophilus influenza Often the influenza virus, followed by upper respiratory tract infection P: high molecular weight proteins (HMW-1 and HMW-2)

Other examples include binding of cells to multifunctional molecules such as, for example, bacteria, antibodies and macrophages. One of the important groups of proteins that make up all classes of antibody-immune systems has a number of equivalent receptor sites: two (IgD, IgE, IgG, IgA), four (IgA), six (IgA) or 10 Dog (IgM). Such antibodies bind to structures that recognize antigens or other ligands present on bacteria, viruses, parasites, drugs, "non-self" cells, or other structures, including non-covalent complexes that are not normally present in the blood circulation Is a widespread feature of immune cognition. This interaction inhibits processes important for infection (eg, adhesion of foreign organisms to target cells) and reduces clearance (by degradation by macrophages and components of other immune systems, or by filtration by the kidneys). Removal rate of foreign particles). Multifunctionality is used for high binding affinity or binding activity for surfaces with repeated epitopes, which define the surface characteristics of almost all pathogens invading here.

Mannose residues on the antibody tail (Fc portion) interact with mannose receptors (Fc receptors) on the surface of macrophages (a type of white blood cell important for removing infectious particles). The interaction of its single Fc moiety with its receptor is too weak to induce a response by macrophages. That is, free (uncomplexed) antibodies in solution do not activate macrophages, and a single antibody binds to the digested fragment of an external pathogen. However, a number of antibodies bound to the surface of the infecting particles strongly interact with a number of receptors on the surface of the macrophages and provide a three-layered structure that is stabilized at both contact surfaces through multifunctional interactions.

In addition, multifunctionality in this system not only allows stability and specificity in the recognition of bacteria by macrophages, but also subsequent action by macrophages is also characteristically dependent on multifunctional interactions. Many of the interactions between macrophages and antibody-coated bacteria lead to cross-linking of surface receptors on macrophages, stimulating internal signals in macrophages to inhale (phage) bacteria and lead to degradation.

Other examples of multifunctional molecules that attach to the cell surface are shown in Tables 6 and 7.

Receptors for bacterial toxins bacteria toxin Ligand Vibrio Cholera Cholera toxin S: GM1: Gal (β1,3) GalNAc (β1,4) (NeuAc (α2,3)) Gal (β1,4) Glc (β) ceramide S: Isorigande NeuAc (α2,3) Gal (β1,3 GalNAc (β) (NeuAc (α2,3) Gal (β1,4) Glc (β) ceramide, Gal (β) GalNAc (β1,4) (NeuAc (α2,8) NeuAc (α2,3) Gal (β1) 4 Glc (β) ceramide, GalNAc (β1,4) Gal (β1,3) GalNAc (β1,4) ((NeuAc (α2,3) Gal (β1,4) Glc (β) ceramide, Fuc (α1) Gal (β1,3) GalNAc (β1,4) ((NeuAc (α2,3) Gal (β1,4) Glc (β) ceramide, Gal (β1,3) GalNAc (β1,4) (R- NeuAc (α2,3) Gal (β1,4) Glc (β) ceramide: R = lucifer yellow CH, rhodamine, or DNP E coli Heat-labile toxins S: GM1 Clostridium tetani Tetanus toxin S: Gal (β1,3) GalNAc (β1,4) ((NeuAc (α2,8) NeuAc (2,3) Gal (β1,4) Glc (β) ceramide S: Isorigande NeuAc (α2,3) Gal (β1,3) GalNAc (β1,4) ((NeuAc (α2,8)) NeuAc (α2,3) Gal (β1,4) Glc (β) ceramide, NeuAc (α2,8) NeuAc (α2,3) Gal (β1,3) GalNAc (β1,4) (NeuAc (α2,8) NeuAc (α2,3) Gal (β1,4) Glc (β) ceramide Clostridium Bottlelinium Bottlinium Toxins A and E S: NeuAc (α2,8) NeuAc (α2,3) Gal (β1,3) GalNAc (β1,4) (NeuAc (α2,8)) NeuAc (α2,3) Gal (β1,4) Glc (β) Ceramide Clostridium Bottlelinium Bottlinium Toxins B, C and F S: NeuAc (α2,3) Gal (β1,3) GalNAc (β1,4) (NeuAc (α2,8)) NeuAc (α2,3) Gal (β1,4) Glc (β) ceramide

Receptors for bacterial toxins bacteria toxin Ligand Clostridium Bottlelinium Bottlinium Toxin B S: Gal (β) ceramide Clostridium perfringens Delta toxin S: GalNAc (β1,4) (NeuAc (α2,3)) Gal (β1,4) Glc (β) ceramide Clostridium difficile Toxin A Shiga-like toxin (SLT) and SLT-II / lic S: Gal (α1,3) Gal (β1,4) GalNAc (β1,3) Gal (β1,4) Glc (β) ceramide S: Gal (α1,4) Gal (β) (P1 disaccharide), Gal (α1,4) Gal (β1,4) GlcNAc (β) (P1 trisaccharide), or Gal (α1,4) Gal (β1,4) Gal (β) (Pk trisaccharide) Shigella dysenteriae or E. coli Shiga Toxin Vero Toxin S: Gal (α1,4) Gal (β) ceramide S: Gal (α1,4) Gal (β1,4) Glc (β) ceramide Bordella pertussis Pertussis Toxin S: NeuAc (α2,6) Gal Shigella Daicenteria I Daicenteria toxin S: GlcNac (β1)

In addition, multifunctionality can be important, ie, to prevent unwanted interactions with multifunctional inhibitors. Where multifunctionality can strongly enhance the desired interaction, it can be a useful strategy, particularly in preventing certain unwanted biological interactions that are multifunctional (where multifunctionality is used against multifunctionality). . For example, fluids that cover the interior of most mammalian lungs contain various mucins (proteins that provide oligosaccharide ends in sialic acid, SA). Such mucins, especially 2-macroglobulin, can bind to influenza and other SA-binding viruses, thereby inhibiting their attachment to target cells.

In the selection of group A for providing multifunctionality, the use of group A to interact with pathogenic particles is preferred over interacting with a host. In one embodiment, the subjects of the present invention are used to block cell-bacterial interaction. In other embodiments, a subject of the invention is used to block cell-fungal interactions. In another embodiment, the subject matter of the present invention is used to block cell-virus interactions. In a further embodiment, the subjects of the present invention are used to block cell-parasite interactions. For example, the binding of Entamoeba histolytica is nourished with host galactose (Gal) and N-acetylgalactosamine (GalNac) residues (Adler et al., 1995, J. Biol). . Chem. 270: 5164]. In some embodiments (eg for gene therapy), it may be desirable to enhance cell-pathogen (eg cell-virus) interaction.

In other embodiments, multifunctional donors of the invention can be prepared to modulate pathogen-extracellular matrix interactions. For example, multifunctional donors that prevent bacterial or fungal interaction with the extracellular matrix can be inhibited. In other embodiments, the subjects of the invention can be used to modulate extracellular matrix interactions.

In other embodiments, multifunctional donors that mediate pathogen-pathogen interactions can be constructed. Such donors will be useful for the treatment of infected diseases or for rupturing of the biofilm of cells, for example, by the use of indwelling tools such as prostheses and catheters.

In other embodiments, cell-toxin interactions can be mediated. For example, Shiga toxins from Shigella Daicenteria type 1 bind to glycoproteins or glycolipids of cells with Galaviose disaccharide (Galα1-4Galβ) determinants. Multifunctional donors of the invention can be prepared as donor group A, which can block any such cell-toxin interaction. In another example, multifunctional donors can be prepared that react with hemorrhagic toxins such as Croatalus viridis viridis.

In other embodiments, the present subject matter can be used to modulate cell reactants that bind multifunctionality. For example, cytokine production by cells can be modulated by replacing stimulating cells, usually in cell-cell interactions leading to cytokine degradation. For example, L-selectin ligands on tumor cells have been shown to stimulate monocytes and macrophages to produce tumor necrosis factor. Although sialyl Lewis x (sLe ^x ) tetrasaccharides have been suggested to act as HEV ligands for L-selectin, native ligands can be more complex (Puts and Mannel, 1996, Scand. J. Immunol. 44: 556-564. Multifunctional donors, including group A, which act similarly to a group of tumor cells recognized by monocytes, could be used to act similar to this response. Similarly, a provider comprising a group A that blocks such a response can be used to inhibit this response. In other embodiments, other conditions linked to multifunctionality in which the multifunctional donor can be used are T cell cytokine production, degranulation of mast cells and / or basophil cells, lymphocyte selection, and T or all of which are states that are multifunctional. B cell killing [Seldtsove and Seledtsova, 1995, Biomed & Pharmacother. 1996. 50: 170.

Treatment of a disease or condition (eg combined with multifunctionality)

In addition to targeting any particular biological phenomenon associated with a disease or condition, one of ordinary skill in the art will devise a multifunctional provider that targets the identification of a specific disease or condition, or biological phenomenon provided by a subject. Could. The present invention is intended to include the use of any group which is provided multifunctionally or a medicament provided as part of a multifunctional provider of the present invention. Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds T. R. Harrison et al. McGraw-Hill N.Y., NY; Goodman and Gilman's Pharmacological Basis of Therapeutics, 9th ed. Eds. Joel GG Hardman, Alfred Gilman, Lee L. Limbird, New York. MacGraw Hill 1995; And The Physicians Desk Reference 50th Edition 1997. Oradell New Jersey, Medical Economics Co. The complete contents of each of these documents are expressly incorporated herein by reference. Their description of the disease or condition and of the possible agents (eg, group A) are even more specifically incorporated herein by reference. For example, multifunctional donors can be designed to modulate an infectious disease, including, for example, inhibiting host-parasite interactions, increasing immunization or prophylactic prescription, and reducing sepsis or septic shock. . More particularly, the subjects of the present invention include, inter alia, infectious diseases of the upper airway; Infective endocarditis; Intraperitoneal infections and abscesses; Acute infectious diarrhea disease and bacterial food poisoning; Sexually transmitted diseases; Pelvic inflammatory disease; Urinary tract infections and pyelonephritis; Infectious arthritis; Infections of osteomyelitis and prosthetic joints; Infections of the skin, muscles and soft tissues; Infectious diseases of rosacea users; Bites, scratches, burns and infections from surrounding organisms; And in the treatment of infectious diseases in hospitals. Thus, a subject of the invention may be a condition caused by a gram positive organism (eg, pneumococcal infection, staphylococcal infection, streptococcal infection, corynebacterial infection, listeria, tetanus, Botulism addictive Clostridium infection, and anthrax) and conditions caused by Gram-negative bacteria (eg, meningitis, gonococcal infection, Moraxella and Kingella infections, Haemophilus infections) , Legionaella infection, whooping cough, infection by intestinal bacteria, Pseudomonas infection, salmonella, bacterial dysentery, Campylobacter infection, cholera and other vibriosis, brucellosis, yato disease, Yersina Infectious diseases, bartonella and donovana. Providers of the present invention include nocardia, actinomycosis, mixed anaerobic infections and mycobacteria and H. pylori infections (eg, tuberculosis, leprosy and Mycobacterium avium infections) It may also be useful in the treatment of. In addition, multifunctional donors may be useful for the treatment of spirocheta disease (eg, syphilis, treponemia, leptospirosis, recursive fever, and Lyme Borreliasis). Rickettsia, Mycoplasma and Chlamydia infections are similarly effective in treatment with the multifunctional donors of the present invention.

In addition, viral infections will be treated by the present subject matter. For example, DNA viruses (eg, herpes simplex, varcella-zoster, Epstein-Barr, cytomegalovirus infections, poxviruses). poxvirus infections, parvoviruses, and human papillomaviruses and RNA viruses (eg, retroviruses, influenza, gastroenteritis, enteroviruses and leoviruses, rubeo, ru Bella, mumps, lavis, labdovirus and marburg-like agents, arbovirus infections, and arenavirus infections) can also be treated.

In another embodiment, a subject of the present invention is a fungal infection (eg, histoplasmosis, coccidiosis and paracoccidiosis, Blascomyses disease, Cryptocox disease, candidiasis, aspergillus) Lupus, mother's disease). In other embodiments, the multifunctional donor may be protozoan infection (e.g., amoeba disease, malaria and barbeciaosis, leishmaniasis, trapazomaosis, toxoplasmosis, pneumocytis cariniosis, flagellosis, cryptosporosis Lydiasis and Trichomoniasis) can be used to treat. In another embodiment, a subject of the present invention is a worm infection (e.g., trichinosis, nematode, intestinal nematode, filamentous nematode, loa nematode, mammary nematode, medina nematodes, schistosomiasis, and other worms Tapeworms). In another embodiment, the subjects of the present invention can be used to treat invasion of external parasites in the body.

In another embodiment, the subjects of the present invention are also useful for modulating the response by both upregulation and downregulation. Thus, multifunctional donors of the present invention will be useful both in the treatment of immunodeficiency diseases as well as in the treatment of autoimmune diseases and the resultant immune-mediated injury. In certain embodiments, a subject of the invention will be useful for inhibiting tissue transplantation.

In another embodiment, the multifunctional donors of the present invention are also useful for the treatment of abnormalities of clotting and thrombosis, and for anticoagulation, fibrin lysis and antiplatelet therapy.

In another embodiment, the subjects of the present invention are also useful for the treatment of neoplasia. In a preferred embodiment, the subjects of the present invention are used to inhibit the metastatic stage of the first tumor.

The first events in the biological action of certain plant-derived bacterial toxins include their multifunctional adhesion to the cell surface, ie binding by multiple interactions of multiple toxin receptors to multiple cellular ligands [loading (Lord, JM), Roberts, LM, Robertus, JD, FASEB 1994, 8, 201-208; Montetecco (C.), Papini, E., Schiavo, G, Experientia 1996, 52, 1026-1032; Kuziemko, G. M., Stroh, M., Stevens, R. C., Biochem. 1996, 35, 6375-6384; Richardson, J. M., Evans, P. D., Homans, S. W., Donohue-Rolfe, A., Nature Struct. Biol. 1997, 4, 190-193. In one approach, multifunctional polymeric galactosides can be used to block the adhesion of cytotoxic lectins to the surface of mammalian cells from Ricinus communis, commonly called lysine. These polymers work by preventing lysine from interacting with galactose residues at the cell surface. The number of receptor-ligand interactions involved in lysine-cell adhesion is small.

Lysine, Ricinus communis aggregate (RCA ₆₀ , RCA ₁₂₀ ) belongs to the class of cytotoxic lectins called ribosomal-inactive proteins. One lysine subtype, RCA ₆₀ (A ₁ B ₁ covalent complex; up to MW 65 kDA), contains A-chains (N-glycosidases that hydrolyze the specific adenine bases of ribosomal RNA) and B-chains (sugars -Binding lectin), consisting of two subunits (Cuziemco, Straw, Stevens, Biochem. 1996, 35, 6375-6384). See also, Baenziger, JU, Fiete, D. J. Biol. Chem. 1976, 254, 9795-9799; Huston, LL, Dooley, TP J. Biol. Chem. 1982, 257, 4147-4151; Ribera-Sagredo (A.), Solis (D.), Diaz-Maurino, T., Jimenez-Barbero (J.), Martin Martin-Lomas, M. Eur. J. BioChem. 1991, 197, 217-228. The second lysine subtype RCA ₁₂₀ (2 × A ′ ₁ B ′ ₁ ; MW 130kDa or less) is very closely related to RCA ₆₀ in many respects of its structure and function, and two non-covalently bound A ′s. ₁ B ' ₁ unit [Endo, Y., Mitsui, K., Motiguki, M., Tsurugi, K. J. Biol. Chem. 1987, 262, 5908-5912. A-chain-catalyzed inactivation of rRNA is a major cause of lysine toxicity. However, this action occurs without toxic cell internalization and this internalization occurs through receptor (B-chain) -mediated endocytosis.

Lysine strongly inhibits the biosynthesis of proteins in mammalian cells. The molecular action of lysine requires internalization of the cell, which begins with adhesion of lysine to the cell surface. This strong toxicity-cell adhesion is caused by the simultaneous binding of lysine's cell surface glycoproteins and glycolipids to multiple galactoside (Gal) groups.

Lysine is the Gal chain of the B chain for a number of β-D-galactose (β-Gal)-and β-N-acetyl-D-galactosamine (β-GalNAc) -terminal oligosaccharides present on the cell surface. It is attached to the cell surface using multifunctional selective binding of binding sites (up to 3 sites per B chain) (Roberts, LM, Lamb, FI, Papin, DJC, Load). , JM) J. Biol. Chem. 1985, 260, 15682-15686] (The number of Gal-binding sites per B subunit has been debated. A number of studies have found that RCA ₆₀ and RCA ₁₂₀ have a Gal of 3 or less (> 2) and 6 or less (> 4), respectively. -Has the area). Binding studies of ¹²⁵ I-labeled lysine showed that lysine is attached to human erythrocytes with a dissociation constant K _{d of} between 0.1 and 0.01 μM. Simple monofunctional homologues of β-D-galactosides have relatively low affinity for RCA ₁₂₀ , RCA ₆₀ and isolated B-chains when studied by various techniques (equilibrium dialysis, fluorescence, and NMR) (K _d is 10 ³ to 10 ² μΜ). As a result of the present invention, multifunctional donors can be prepared to obtain relatively strong binding of lysine to the cell surface.

Lysine is closely related to bacterial toxins (anthrax; cholera; cigar; verotoxin) and other plant-induced cytotoxic lectins (ribosome-inactivated protein; Avrin) in many aspects of structure and mode of action. These toxins are classified as agents that are the subject of current research and are effective for biological treatment as cytotoxic components of immunotoxins developed as anticancer drugs.

In a further embodiment, in turn, the subjects of the present invention are useful for preventing the adhesion of lysine to red blood cells. In this regard, multifunctional donors can be used to block lysine toxicity based on multifunctionality by blocking lysine-cell adhesion. In this way, the first step, the introduction into the cell, is blocked using a multifunctional polymeric inhibitor that can compete with multiple copies of the ligands provided on the cell surface. Synthesis and bioassays of polymeric multifunctional molecules designed to inhibit adhesion of lysine (RCA ₁₂₀ and RCA ₆₀ ) to chicken red blood cells are discussed herein. Inhibitors are derivatives of poly (acrylic acid) and poly (butadiene-co-maleic acid) and provide a number of copies of the galactoside-containing side chains as pAA (GaL) and pBMA (Gal), which are amide groups. One of the inhibitors, pAA (Gal-βO-L ₁ ; χ ^Gal = 0.4), exhibited inhibitory activity against RCA ₁₂₀ , K _i ^HAI = 0.14 μM (calculated per galactoside reference). Soluble galactosides are up to 1500 times more active than monofunctional Gal-β-OMe and 270 times more active than monofunctional Gal-βO-L ₁ NH ₂ . Polymeric multifunctional inhibitors are significantly more active at blocking hemagglutinin induced by RCA ₁₂₀ (having up to 6 Gal receptor sites) compared to by RCA ₆₀ (having up to 3 Gal receptor sites) Enemy The molecular-based enhanced activity of polymeric multifunctional galactosides in blocking hemagglutinin is described as multifunctional (ie multiple, simultaneous) interaction of the Gal portion with the Gal receptor site of lysine on the polymer. We have demonstrated that synthetic glycopolymers can inhibit lysine-cell adhesion. Thus, such polymers are a new class of synthetic antitoxins. The action of these polymers can mimic the action of antitoxin antibodies by blocking access of the lysine Gal-binding site from the surface of the cell to the Gal moiety, but also the possibility of fundamentally new mechanisms of inhibition, entropy-enhanced lysine Gal. Occupancy of the binding site; Induces volumetric stabilization of the unbonded Gal binding site by the surrounding polymer. The observation that these polymers are more active for RCA ₁₂₀ compared to that for RCA ₆₀ suggests that multifunctional inhibitors are more active for receptors with a greater number of ligand binding sites than providing fewer ligand binding sites. do.

A strategy for neutralization of lysine using a schematic and polymeric multifunctional agent summarizing lysine-cell interactions (RCA ₁₂₀ and RCA ₆₀ ) is disclosed in FIG. 3 and includes (a)-(c) below. : (a) Adsorption of receptor-ligand mediated lysine to mammalian cells: Multiple binding of the lysine receptor site (B-chain) to a number of specific cellular ligands (β-galactoside) results in tight toxin-cells Inducing binding and the attachment of lysine to the cell constitutes an early stage in its toxic action. (b) Toxicity mechanisms of lysine: After introduction of toxins into the cell (via phagocytosis) and reductive cleavage of the A-chain from the B-chain, the monomeric A-chain is a hydrolyzate of a specific adenine base of ribosomal RNA. Pirate N-glycosidation is effectively catalyzed, and this damage interferes with protein biosynthesis. (c) Blocking cellular incorporation of lysine by polymeric multifunctional galactosides.

In turn, the synthetic Gal-providing polymers of the present invention can be used as inhibitors to prevent the attachment of lysine to mammalian cells, and such compounds or derivatives of these compounds can serve as protective agents against therapeutics and other exposures to lysine. It is a strong indicator.

Multiple binding of toxin receptors to specific cell-surface ligands is also important in other situations. For example, abrin contains a number of receptor sites per B-chain of toxin molecules, and these toxins selectively bind to galactose residues [Road, Roberts, Roberts, FASEB 1994, 8, 201-208; Sandvig, K., Olsnes, S., Pihl, A. J. Biol. Chem. 1976, 251, 3977-3984. Cholera toxin uses a pentameric B (sugar-binding) subunit, each of which is a GM ₁ pentasaccharide [Gal (β1,3) GalNAc (β1,4) (NeuAc (α2,3)) Gal recognizes (β1,4) Glc (β1,1) -ceramide] residues [Cozymco, Straw, Stevens, Biochem. 1996, 35, 6375-6384, wherein cigar toxins and vero toxins are Gal-containing glycosphingolipids [globoside, Gal (α1,4) Gal (β1,4) Glc (β1,1) -ceramide] Five B-subunits are used to bind to [Richardson, Evans, Homans, Donohue-Rolf, Nature Struct. Biol. 1997, 4, 190-193.

Thus, the present invention is characterized by a toxin-cell interaction (characterized by a low binder, ie, contains less than 10 Gal receptor sites) from pentagonal (cell) -cell binding (characterized by a high binder: about 100 to 1000 sites Can be used in the design of inhibitors.

Multifunctional donors of the present invention can be used to inhibit egg-sperm interactions such that fertilization is inhibited, for example acrosome reactions can be interfered by using multifunctional donors. Thus, a subject of the present invention may inhibit sperm-egg interaction (eg, inhibition of sperm attachment to an egg), or acrosome reactions (eg, reaction of sperm) before interaction between sperm and egg. Early activity), which is useful for preventing conception.

Fertilization is initiated by species-specific adhesion of sperm to the extracellular coat of the egg. Sperm-egg interactions induce sperm to perform a series of acrosome reactions (acrosome process), and the initial reaction appears as extracellular incorporation of sperm acrosome to the egg coat. This acrosome reaction eventually leads to the fusion of the sperm and egg plasma membranes and the introduction of the sperm nucleus into the egg cytoplasm.

In mouse sperm, the surface protein β-1,4-galactosyltransferase (gal-transferase) on the sperm's plasma membrane is specific for the GlcNAc residue of the zona pellucida (ZP) glycoprotein in the egg coat. It is a receptor recognized by. In FIG. 4, the scheme summarizes the approach for the introduction of sperm acute reactions as well as the inhibition of sperm-oval binding using the polymeric multifunctional agent pAA (GlcNAc). (a, b) The adhesion of mouse sperm to ovule is associated with receptor-ligand interaction [Gal-transferase on sperm head surface and terminal N-acetylglucosamine (GlcNAc) residues of ZP3 glycoprotein on Zona Felucida (ZP) Mediated by: (a) Multiple binding of the Gal-transferase receptor to multiple GlcNAc ligands of ZP3 results in aggregation of Gal-transferases, and (b) subsequent sperm via intracellular signal transduction. Induction of acrosomal extracellular import. (c, d) Proposed mechanism for the induction of acrosomal reactions and inhibition of sperm-oval adhesion by poly (acrylic acid), or pAA (GlcNAc), which provides a number of copies of GlcNAc as amide side chains. 4 is not a constant ratio.

Among the various isomers of Zona Felucida's ZP glycoprotein, ZP3 is known as a glycoprotein ligand for Gal-transferase that mediates sperm-egg adhesion. ZP3 contains various multiple copies of the O-bridged N-acetylglucosamine (GlcNAc) moiety covalently linked to the serine and threonine residues of the glycoprotein. GlcNAc populations from multiple ZP3 can simultaneously bind to multiple surface Gal-transferase receptors of sperm head.

Sperm acrosome reactions occur through intracellular signal transduction that is initiated by the activation of specific G-protein coupled receptors. Activation of G-protein receptors requires spatial aggregation of Gal-transferases (FIG. 4). In the usual course of sperm-egg fertilization, ZP3 glycoproteins in ZP of the egg coat bind and aggregate to Gal-transferase. Aggregation is the result of multifunctional interaction of Gal-transferases on the sperm surface with multiple GlcNAc ligands of ZP3.

In the present invention, the multifunctional form of GlcNAc-an analogue of the multifunctional GlcNAc of ZP3-induces acute reaction of sperm (Figure 4). Polymeric multifunctional GlcNAc induces acrosome exocytosis of mouse sperm and also inhibits binding of sperm to eggs in vitro.

In a preferred embodiment, the multifunctional provider of the present invention can be combined with other methods of contraception or can also be used to prevent disease while being used for the prevention of contraception. For example, group A may be a useful ligand for treating herpes, HIV, chlamydia, human papilloma virus, gonorrhea and syphilis.

In yet another embodiment, the subjects of the present invention may have a cardiac shock, angina pectoris, thrombus formation, heart disease, atherosclerosis, hypertension, seizures, restenosis, renal dysfunction on the secretion of erythropoietic factors, allergic reactions in the circulation. , Secondary disease of the mematopoetic system that develops after psoriasis, sickle cell disease, treatment (chemotherapy, side effects of drugs) or other diseases (metastasis, autoimmune disease), renal cancer, prostate cancer, brain cancer, lung cancer, thyroid cancer , Colon cancer, stomach cancer, liver cancer, pancreatic cancer, tumor cell resistance to chemotherapy, adult onset diabetes, growth deficiency, CNS trauma, neurodegenerative disease, schizophrenia, migraine, anxiety, depression, obsessive compulsive disease, idiopathic (no known cause) Fever from unknown sources, diseases with unavoidable etiologies that involve multiple organ abnormalities), eye symptoms (glaucoma, myopia, retinal degeneration, presbyopia), menopause, Diseases (hypertension, pregnancy addiction, etiology to help carry diabetes and maturity, etiology to help delivery), numbness, fertility, wound healing, incontinence, dermatology (acne, dandruff, urticaria), adverse reaction to medicine, and drugs It is also useful for treating or modulating the toxic effects of Further aspects may also include modulating (eg, promoting) cell death, modulating (eg, promoting, inhibiting or augmenting) metabolic pathways (eg, via enzymes) or specific receptor types (eg, Conditions or diseases involving the targeting of seven clustered permeable membrane receptors) or enzymes (eg, intracellular, extracellular or membrane binding).

Assays to Validate and Test Multifunctional Providers

The present invention also provides a method for assaying multifunctional donors that can be used, for example, to verify useful group A or to test the efficiency of a donor. Such assays can be performed ex vivo or in vivo. In addition, assays can be devised to test the ability of the multifunctional donor to mediate the interaction between A and B, or a biological response resulting from the interaction of A and B (eg, cell adhesion assays, Aggregation assay, platelet aggregation assay, ELISA assay, muscle contraction assay, infectious assay, or lymphocyte stimulation assay, and the like.

In vitro irradiation

In certain embodiments, ex vivo assays can be used to test the effect of the multifunctional donor to assay the ability of the multifunctional donor to interact with or inhibit the interaction of binding site B with group C. . For example, assays that test the ability of Group A of a donor to interact with binding site B at a multifunctional surface can be used to examine the donor (Charych, D. et al., Chem. & Biol. 1996, 3, 113-120.

In another exemplary embodiment, capillary electrophoresis (CE) can be used. CE is a convenient high resolution analytical technique that only requires femtomol of the material. CE allows to separate mixtures of molecules (ions, small molecules, polymers, proteins, micelles) based on charge and hydrodynamic forces. It is possible to accurately quantify the binding constant of the group to the binding site by adding group A to the buffer solution with varying concentrations and observing the effect of concentration on the flowability of injected binding site B. This technique is called affinity capillary electrophoresis (ACE). For example, the affinity of the donor for the whole virus expressing binding site B can be determined using ACE. It has also been shown that ACE forms the basis of a very effective library study. In addition, CE is very useful, such as GPC and light scattering, in the analysis of a provider, especially when the carrier carries a charge.

Surface Plasmon Resonance Spectroscopy can be used (see, eg, Murksich, M. et al., Langmair 1995, 4383); Merkschach et al., J. Am. Chem. Soc. 1995, 117: 12009; See Sigal, G. B. et al. (Anal. Chem. 1996, 68: 490). Surface plasmon resonance (SPR) was used to study the binding results at the surface. In addition, model surfaces based on Self-Assembled Monolayers (SAM's) can be used to assay the donor molecules of the present invention. SAM's of alkanthiolates over gold and silver are another model system for studying adsorption or other molecular consequences occurring at the interface (see, for example, Merckwisch, Whites, Ann. Rev. Biophys. Biomol. Struct. 1996, 25:55), Whites and Gorman, CB (Self-Assembled Monolayers: Models for Organic Surface Chemistry; CRC Press: Boca Raton, 1995); Merschach et al., J. Am. Chem. Soc. 1995, 117: 12009; Sigal et al. (Anal. Chem. 1996, 68: 490); See Lopez et al. (J. Am. Chem. Soc. 1993, 115: 5877).

In other embodiments, for quantitative analysis of multifunctional molecules, aggregation using, for example, synthetic beads mixed with pathogens or cells can be tested (equivalent to hemagglutination dropping assays). Aggregation is a very convenient way to investigate cell-cell and pathogen-cell contact, and microtiter plates with 96 wells are a particularly suitable form for the above assay. Automation of this assay is also possible. For example, the beads can be configured to provide a group that specifically interacts with a pathogen or cell surface. The configuration of the beads may consider a three-dimensional approach to the group compared to the background. In addition, appropriate points of attachment to the group can be considered and based on obtainable crystal structure. In addition, the importance of reducing or reducing non-specific interactions through the use of oligoethylene glycol backgrounds and the importance of the surface density of the group can also be considered. Alternatively, target cells (or surrogate target cells) can be used in certain embodiments. For example, influenza virus binds to red blood cells, and red blood cells other than beads may be used.

In addition, the surface providing the binding site B, which leads to the relative and absolute concentrations of the beads providing group A and the mixture to form the "gel" (ie aggregate), can be determined. Colored beads can be used to easily visualize. The multifunctional material may be configured to contain varying mole fractions of the group on the beads or derivatives of the group, or of completely different groups (in the latter case, inhibition may vary with steric stabilization). In general, the ability of the multifunctional material in the assay will depend on the mole fraction of the monomer units of the polymer linked to the active group. The molar fraction with the maximum capacity is expected to be system-dependent.

These same multifunctional materials can be modified to incorporate into various positions that various other groups may occupy on the carrier. The structure and mole fraction of the various “assistant” (eg, A ₂ to A _n ) groups can increase the specificity and ability of the molecule. One mechanism by which this enhancement is possible is by randomly attracting hydrophobic pockets at the surface of the pathogen (or cell) using a small mole fraction of short hydrophobic side chains. Important properties of the polymers expected to modulate such capacity include the mole fraction, charge, hydrophobicity, length of effect, randomness, physical dimension, and number of bonds with water molecules of the active group.

In addition, inhibition assays can be used that measure the extent to which a molecule prevents a biological surface (eg, a virus) from binding to another biological surface (eg, a cell). By binding the molecule completely to the receptor, it can be done as above, and the binding of the surface binding group at the same binding site can be prevented. One exemplary assay is an hemagglutination inhibition assay, which is described in more detail in the accompanying examples and appendages. HAI assays are based on molecules that inhibit viral aggregation (gel formation) of erythrocyte solutions. The lower limit of efficiency that HAI can conveniently measure is 1 nM.

Another exemplary assay is an optical collision (OPTOCOL) assay, which is based on inhibition of adhesion upon collision of one red blood cell and one virus-coated centrosome in the presence of an inhibitor. This assay is performed under an inverted microscope using parallel optical forceps, each forceps holding one of the two colliders. The OPTOCOL assay allows to quantify the efficiency of an inhibitor active at <1 nM.

Both HAI and OPTOCOL assays yield concentrations at which inhibition is partially effective, ie, at which nearly half of the interactions between group A and binding site B are inhibited. In the HAI assay, the inhibition constant is KHA or KOPTOCOL. When using each assay to determine the reliably measurable inhibition constant, KHA is identical to KOPTOCOL. Both of these inhibition constants can be represented by KINH.

OPTOCOL is based on the manipulation of biological particles using parallel dual optical forceps [Mamen et al., 1996 Chem. Biol. 3: 757]. This technique allows, for example, the study of the interaction between a single red blood cell and a single centrosome that provides influenza virus. This technique is particularly useful for very tight binding systems and can also be used to study the multifunctional inhibition mechanism.

Most preferred assays place importance on the characteristics of the multifunctional group that are important for achieving the desired goal. For example, an effective multifunctional molecule for the purpose of aggluting a multifunctional binding site will consist of "n" larger than 10 groups linked through a long flexible linker. In the end, the assay should reflect the ultimate purpose of the multifunctional species.

There are many ways to investigate, evaluate and quantify multifunctional interactions. Some assays can be a direct measure of affinity, and one of these affinities can infer the free energy of interaction. Other assays can measure complex aggregates of features, the only one being the free energy of interaction. Such other features may include the degree of hydration, the ability to stabilize molecules or surfaces in three dimensions, and / or the ability to crosslink multifunctional receptors.

In order to thermodynamically quantify the binding constants (ie to obtain the binding constants), the relative proportions of the uncomplexed and complexed groups (or binding sites) must be measured (directly or indirectly). Depending on the stability of the complex (relative to its survival), different techniques can be used.

Aggregation assays can be used to determine the ability of a multifunctional group to aggregate multifunctional binding sites (precipitation, gel formation, aggregation). For example, the multifunctional donor can precipitate the multifunctional binding site B on the surface in an immunocrystallization assay. Although the affinity of the multifunctional material is important in determining the ability of the multifunctional provider to precipitate, other features may be important. For example, at low concentrations, the donor cannot bind polyB and precipitation occurs at any suitable concentration band, but at higher concentrations, each binding site B is bound by group A and precipitated Does not occur again. In this example, which is the same as the antibody precipitation reaction, only affinity does not determine the pattern of precipitation.

In vivo assay

For example, in vivo assays may be performed that determine the effect of multifunctional donor treatment on a disease or condition in an animal, eg, to measure protection against infection. Such assays include, but are not limited to, determination of inhibitors that prevent multifunctional interactions. For example, molecules can slow infection by blocking attachment to host receptors, as well as slow removal by blocking clearance mechanisms. One exemplary assay is phage infection of bacteria [Chu, Y.-H. et al., Acc. Chem. Res. 1995, 28, 461-468.

Table 8 discloses various assays that can be used to investigate the multifunctional donors of the present invention for useful properties.

In vitro use

The present invention also provides new diagnostics and characterization of systems and components for purification, materials and systems for passive protection against, for example, biological threats, for multifunctional providers for ex vivo applications. In the use as described above, the multifunctional provider of the present invention is ultimately designed to block infection or poisoning of a subject. In certain embodiments, a subject of the present invention may further comprise one or more anti metabolites. For example, passive protection may be provided by the use of protective wraps, gowns, dressings and wash water for hospitals as well as protective masks containing multifunctional donors effective against infectious substances or poison gases. Likewise, the multifunctional donor of the present invention can be incorporated into a solution and processed for purification systems.

In other ex vivo applications, the present subject matter can be used, for example, in panning assays or optical assays based on adsorption at the surface. For such ex vivo applications, a wider range of instruments would be suitable for use if the need for in vivo complementarity is not taken into account.

Mechanism of action

The mechanisms described below are for discussion and should not be regarded as limiting on the claimed invention in any way. In addition, it should be understood that factors such as "Factor B" are more relevant to the objective criteria than the mechanism of action. In a preferred embodiment, the multifunctional donor of the invention exhibits improved affinity for binding site B compared to what appears to be a single A. In another preferred embodiment, multifunctional donors of the invention have greater specificity compared to A provided monofunctionally. By "specificity" is meant that the nonspecific interaction of polyA with the binding site nonB is reduced compared to that observed for single A. In another preferred embodiment, the multifunctional donor of the present invention produces a biological effect at a lower concentration than that observed for A provided in monofunctional form. The multifunctional provider of the present invention may act by one or more mechanisms described below.

For multifunctional interactions as a class, there is no systematic nomenclature allowed. Since there are many different ways in which binding site B (e.g., N receptor sites) can interact with group A and the binding free energy of the interaction is strongly dependent on the details, there is probably no single general systematic naming. As used herein, the interaction between the N ligand and the N receptor contributing to the two entities will be referred to as the N th multifunctional interaction (describing the tertiary multifunctional interaction). Binding free energy is represented by ΔG _N ^poly .

The mean free energy ΔG _avg ^poly of the interaction between a single ligand residue and a single receptor residue in the multifunctional interaction shown is equal to ΔG _N ^poly / N (Equation 3). Monofunctional receptor-ligand interactions occur with a change in free energy of ΔG ^mono , and N monofunctional independent receptors that interact with N monofunctional independent ligands occur with a change in free energy of NΔG ^mono . Comparison of the most useful binding free energies should be considered as a whole if they include the same number of ligands and receptors, and different numbers of ligands and receptors (and different units for equilibrium constants). Equations 3 to 5 give the values of the corresponding binding constants K:

ΔG _avg ^poly = ΔG _N ^poly / N

ΔG = RTln (K)

K _N ^poly = (K _avg ^poly ) ^N

Cooperation: the size of α

The mean free energy (ΔG _avg ^poly ) of the interaction between the ligand residue and the receptor residue in the multifunctional interaction may be greater than, equal to or less than the free energy (ΔG ^mono ) at similar monofunctional interactions ( Equations 6 to 8). According to the systematic nomenclature allowed in biochemistry, this class of multifunctional interactions will be referred to as positively cooperative (synergistic), non-cooperative (additional) or negatively cooperative (interacting). The protocol defining α as the degree of cooperation is shown below (Connors, KA Binding Constants: The Measurement of Molecular Complex Stability; John Wiley & Sons: New York, 1987). The units of α follow the order of the multifunctional interaction.

ΔG _avg ^poly = αΔG ^mono

NΔG _avg ^poly = ΔG _N ^poly = αNΔG ^mono

K _N ^poly = (K _avg ^poly ) ^N = (K ^mono ) ^αN

α = log [log (K _N ^poly ) / log (K ^mono ) ^N ]

Collaboration in biology has been discussed in the art (Koshland, DE, Neet, K. Annu. Rev. Biochem 1968, 37, 359; Perlmutter-Hayman, B. Acc. Chem. Res. 1986, 19, 90-96) . The best studied positive and cooperative systems in biology do not include multifunctionality. For example, the binding of four oxygen (O ₂ ) molecules to hemoglobin, a tetramer, occurs cooperatively, ie the free energy of the second oxygen bond to hemoglobin is more desirable than the first bond, and -ΔG _avg ^Poly > -ΔG _(first) ^mono . The degree of cooperation α in the non-multifunctional system is greater than 1 and there is no unit.

As a class, multifunctional interactions have not been quantified often enough or carefully so that positive co-operation is clearly inferred in one system. Positive collaboration for multifunctional systems is part of the present invention.

One example of a positively cooperating multifunctional interaction (α> 1) may be the binding of a pentameric cholera toxin with GM ₁ , which is part of an oligosaccharide of GM ₁ ganglioside. Cholera toxin consists of five subunits (AB ₅ ). Binding to (a GM ₁ cut from the sera pyrimidyl moiety at the cell surface) monomeric GM ₁ of the toxin is coupled through the improvement purely enthalpy ever provide a well-studied example of a positive cooperation.

The entropy of the first binding result for the pentameric cholera toxin of the monomeric GM ₁ derivative is the same as the entropy of each subsequent binding result. All differences in binding energy for monomeric GM ₁ to toxins are ultimately due to differences in enthalpy. Schoen et al conducted calorimetric studies of binding of pentameric B ₅ to five independent GM ₁ oligosaccharide units in solution (Schoen, A., Freire, E. Biochemistry 1989, 28, 5019-24). ). The binding constant of the first ligand was less than one of four factors compared to the second binding constant. When calculated purely statistically, the binding constant of the first ligand is expected to be 5/2 greater than the second binding constant [For the N-order multifunctional interaction, the i-th binding constant K _i is Is equal to (N + 1 + i) / i. After all, when N = 5, K ₂ / K ₁ = (5/1) / (4/2) = 5/2]. Thus, binding is enhanced enthalpy.

Binding of multifunctional B ₅ to the surface of cells densely covered with GM ₁ residues has been reported to occur inherently irreversibly to pentameric cholera toxin for the five monomeric units of GM ₁ . This is because the enthalpy of interaction is almost the same. For monomeric GM ₁ and surface-immobilized GM ₁ , this difference in ΔG of binding can contribute to the entropy difference between these two types of binding, and the affinity of the multifunctional receptor is greatly enhanced through multipoint attachment. Can be. Since the affinity of the pentameric to the multifunctional surface has not been quantified, the degree of cooperation even if present is unknown.

The following are two examples of possibly cooperative (interfering; α <1) interactions. In other words, unlike in the case of positive cooperation (heroglobin and O ₂ ), the binding of the second ligand to the second receptor occurs less preferably in terms of free energy compared to the binding of the first ligand to the first receptor. A first example is the binding to the ligand of a bifunctional antibody (Ab) found to be densely packed on a biological surface (mammal cell, virus, or solid support phase during an ELISA assay) or immobilized on a polymeric matrix (Tanaka, T .; Suzuno, R .; Nakamura, K .; Kuwahara, A .; Takeo, K. Electrophoresis 1986. 7, 204-209). In general, the monofunctional binding constant of an antibody to small organic ligands varies considerably, but in the range of 10 ⁵ to 10 ⁸ M ⁻¹ . In a negative or positive cooperative system, we expect K ₂ ^bi <(K ^mono ) ² to (10 ⁵ -10 ⁸ ) ² M ^-1 . Karrush et al. Found that bifunctional Abs to surface antigens on Bacillus sp bind 30 times higher affinity than the corresponding monofunctional Abs (monofunctional antibodies are both present in natural proteins). The above binding sites are separated chemically or enzymatically). That is, K ₂ ^bi = 30 K ^mono <(K ^mono ) ² M ^-1 (Karulin, AY; Dzantiev, BB Molecular Immunology 1990, 27, 965-971). Here the combination is eventually negatively cooperative.

Thus, the degree of cooperation defined in the traditional biochemical sense is neither a useful nor descriptive parameter for a multifunctional system compared to that for a monofunctional system. Finally, experimental metering is needed to explain the enhancement of binding of multifunctional systems. By expanding from the cooperativeness parameter α, we call this the new metering β. Thus, although multifunctional interactions may be quantitatively much larger than any of the monomeric interactions contributing to them, these monomeric interactions may still interfere or not affect each other (during affinity chromatography). "Adhesion" of the multifunctional ligand to the affinity gel indicates this case). To establish the nature of cooperativeness it is only possible by quantitative comparison of multi- and mono-functional interactions.

Enhanced Affinity in Multifunctional Interactions: Size of β

In many multifunctional systems, N, the number of ligand-receptor interactions, is unknown. For example, a multifunctional inhibitor of hemagglutination by influenza virus composed of a polyacrylamide backbone having a specific fraction of side chains terminated with sialic acid (SA) may be a multi-cell hemagglutinin (HA) receptor on the surface of the influenza virus. It specifically interacts with the site. These molecules block the interaction of the influenza with the target cell. If the side chain uses a polymer that provides a SA group and a small number of biotin groups (as a ligand for the attachment of enzyme-conjugated streptavidin in a subsequent step of the assay), such polymeric multifunctionality may be employed using an Elisa-like assay. The binding of the inhibitor to the viral surface can be measured. Surface-bound viruses were cultured by varying the concentration of the polymer containing SA, and the amount of binding polymer was measured indirectly through enzyme-associated streptavidin-biotin interactions. The amount measurable by forming a binding isotherm was the concentration of SA and the amount of SA contained on the polymer bound to the viral surface. The number of polymers bound to the virus or the number of SA groups on each polymer bound to the HA receptor site is not exactly known. The interaction of these polymers with the virus can be analyzed by determining the amount of bound polymer as a function of [SA]. At half the maximum binding, 1 / K ^ELISA ≡ [SA], where K ^ELISA is equal to the binding constant (Equation 9). Since the value of N in this multifunctional interaction is not known here, no mention of co-operation α is possible. In this example the value of K ^mono is 5 × 10 ² M ⁻¹ , and the K ^ELISA value of the best inhibitor of the invention is 10 ⁸ M ^−1.20 . That is, the monomeric SA binds by half of the maximum at [SA] = 2 × 10 ⁻³ M, whereas the polymer containing SA has a maximum of the maximum value on the surface of the virus at [SA] = 2 × 10 ⁻⁸ M. Combine by half. Thus, multifunctional inhibitors may eventually be useful regardless of the α value.

Thus, the enhancement factor β, which is defined as the ratio of two binding constants as shown in Equation 9, is preferred for cooperative α in the discussion of enhanced affinity of a multifunctional system, and whether the interactions that produce β values are cooperative Molecules with high values of β whether or not are useful. Although N is unknown, in any system where the total amount of bound multifunctional molecules is known, β will be a useful parameter. Only when the value of N is known can the known exact relationship between ΔG _N ^poly , K _N ^poly and β be calculated (Equations 10 and 11).

K ^ELISA = βK ^ELISA

ΔG _N ^poly = ΔG ^mono -RTln (Nβ)

β = (K _N ^poly / N) / K ^mono

Multifunctional group donors of the invention wherein group A is a saccharide usually have a β value greater than about 10 ⁸ or preferably equal to about 10 ⁸ . The multifunctional donors of the invention wherein group A is not a saccharide, in particular group A is a non-natural ligand, are usually greater than or equal to about 10, preferably greater than about 10 ² , more preferably about 10 ⁴ Has a larger β value. Nonnatural ligands are generally not polyfunctional, although the receptors that bind to them may have multiple functional groups.

Enthalpy of Multifunctional Interactions

ΔG _N ^poly consists of an enthalpy (ΔH _N ^poly ) component and an entropy (ΔS _N ^poly ) component. As a first approximation, the value of ΔH _N ^poly is NΔH ^{mono, which} is the sum of the enthalpy of the N monofunctional interaction. This value can be made larger or smaller by other interactions around the active site.

ΔG _N ^poly = ΔH _N ^poly -TΔS _N ^poly

Enthalpy enhanced binding

In any case, binding of one ligand to a receptor with a given enthalpy can induce the next ligand to bind to the receptor with a greater enthalpy. That is, the value of ΔH _avg ^poly is in this case a negative value (more preferably) greater than the value of ΔH ^mono . The binding was enhanced enthalpy.

Enthalpy reduced binding

If the binding of one ligand to the receptor interferes with the next binding result, the enthalpy of the multifunctional interaction is less desirable than that N is expected for the same monofunctional interaction. This binding is enthalpy reduced. Enthalpy reduced binding can occur when the formation of multiple ligand-receptor interactions between two multifunctional entities requires an energy undesirable molecular shape. As the most important rule, the more conformally robust the multifunctional entity, the more the spatial misalignment between the ligand and its receptor will result in enthalpy reduced binding (unless the geometric registration between the ligand and the receptor is incorrect). .

Entropy of Interaction

Understanding the entropy of multifunctional interactions is essential to understanding the relationship between monofunctional and multifunctional binding. Failure to fully understand entropy in the design of multifunctional inhibitors has produced only a number of synthetic multifunctional molecules that are less effective than their monofunctional counterparts.

The total entropy ΔS _N ^poly of a multifunctional interaction is defined as potential entropy (ΔS _{potential, N} ^poly ), rotational entropy (ΔS _{rotation, N} ^poly ), and steric entropy (ΔS _{conformational, N} ^poly ), at the binding of the receptor and the ligand, and It is thought to change from the entropy of water (ΔS H _{2 O, N} ^poly ) present in the surroundings, often due to entropy of hydrophobic interactions (Equation 13).

ΔS _N ^poly = ΔS _{trans, N} ^poly + ΔS _{rotation, N} ^poly + ΔS _{conformational, N} ^poly + ΔS _{H2O, N} ^poly

Dislocation and Rotational Entropy

ΔS _trans , the potential entropy of a molecule, results from its degrees of freedom, which are independently displaced through space; The value of ΔS _trans is related to the logarithm of its mass, M (ΔS _trans ∝ 1n (M), and is inversely related to the logarithm of its concentration (ΔS _trans ∝ 1n [L] ⁻¹ ). The ΔS _rotation is produced from the degrees of freedom of the particles rotating around three major axes, which are the logarithms of the three major moments of inertia, I _χ , I _γ and I _κ (ΔS _rotation ∝ 1n (I _χ I _γ I _κ ) For the first approximation, the potential and rotational entropy of all particles — receptors, ligands, receptor-ligand aggregates — are the same: when two particles bind, three potential degrees of freedom and three rotations Disregarding the difference in the mass of the particles (often, the mass of the particles is within a factor of 10 and almost always within a factor of 100), the total cost of the potential and rotational entropy of joining the two particles is Same consistency regardless of mono- or multi-functional In biology, the concentration of molecules can vary in order of more than 9 by size (mM to pM) The potential entropy changes only in logarithmic values depending on the concentration, but due to this wide range Essentially knowing the concentration of interactive particles to predict the importance of dislocation entropy; the cost increases with decreasing concentration.

Conformational entropy

Entropy and enthalpy can have a partial compensatory effect on the affinity of multifunctional interactions; In the case of increasing the entropy cost of binding, the conformational flexibility increases as all ligand-receptor interactions occur without modification of force.

This loss in conformational entropy for the binding of multifunctional ligands to multifunctional receptors has been difficult to quantify. The change in entropy for freezing a single rotatable carbon-carbon bond is about 0.5 kcal / mole (ref). The range of values for other single bonds is from 0.1 to 1.0 kcal / mol. Maximum loss in conformational entropy occurs when all freely rotating bonds initially lose all torsional degrees of freedom for complex formation. The upper limit estimate for this loss (ΔS _conf ) is about 0.5 N kcal / mole, where N is the number of single bonds in the two tether binding ligands or receptors. In flexible long chains, the number can be large, and in triethylene glycol spacers, this may be undesirable, such as 10 kcal / mol.

Entropy of solvation

The final contribution to the total entropy of the bonds in the aqueous system is the degree of change in the entropy of the molecules present around the water (ΔS H _{2 O} ). Major contributors to interactions in water (interactions including hydrophobic molecules, and polar groups) release organicized water from the exposed side of biological molecules, thereby increasing entropy. Quantitative measurements and predictions of hydrophobic interactions have been widely reviewed (Blokzijl, W. and Engberts, BFN, Angew. Chem. Int. Ed Engl. 1993, 32, 1545-). 1579 "]. These terms typically refer to individual ligands in mono- and multifunctional systems (unless the linker changes compliance or binds to the surface of the receptor and consequently does not change the binding reaction with the solvent for complexation). Similarly will apply.

There are many classes of multifunctional interactions with distinctly different entropy properties. The interaction can occur if the two species initially diffuse freely in solution (the extent of the initial six degrees of freedom for these two particles is reduced to three degrees of freedom in the following complex formation). Classify these interactions in three dimensions (3D); When one or two kinds are limited in diffusion into the plane (classified as two-dimensional 2D); The type of interaction is limited to lines (classified as one-dimensional 1D). The dislocation and rotational entropy costs of the bonds depend on the number of degrees of dislocation and the dislocation of the degree of freedom for the complex formation. The cost (the combined values of ΔS _trans and _{ΔS rot} ) has the greatest degree of binding to the particles in 3D, slightly less in 2D, and least in 1D. In other words, binding between freely diffusing N ligands (eg, tethered ligands on the surface of the cell) and between freely diffusing N receptors (eg, trans-membrane proteins on the surface of other cells) in 2D. The entropy cost of is less for these kinds of interactions that spread independently in 3D.

Dynamics and improved affinity

In certain embodiments, multifunctional donors of the present invention function by having an extremely low extinction rate such that they are effectively "permanently" bound for a period of time that exhibits biological activity for treatment. The mechanical differences between polyA and monoA, ie lower extinction rates for multifunctional forms, are another mechanism by which the present invention differs from monoA.

Epidemiological studies of high-affinity interactions suggest that interactions improve because, in most cases, the degradation rate (k _off ) of the two multifunctional complexes decreases rather than because of their increased binding rate. In contrast to binding the same antibody to the DNP-covered surface of ΦX174, binding the anti-DNP antibody to DNP-lys results in a value of k _on that binds to the surface given by only one of the two factors. (k _on (surface) about 3.7 x 10 ⁷ M ^-1 s ^-1 , k _on (DNP-lys) about 8 x 10 ⁷ M ^-1 s ^-1 ), whereas by one of the 10 ⁴ factors The value of k _off that binds to a given surface is (k _off (surface) about 3.3 × 10 ⁻⁴ M ⁻¹ s ⁻¹ , k _off (DNP-lys) about 1.0s ⁻¹ ). Because the speed of the process is qualitatively (and, quite often, quantitatively) related to its thermodynamics [Agmon, N and Levine, RD, "Chem, Phyvs, Lett. 1977, 52 , 197-201 "], these measurements are intuitively consistent with polyfunctionality, and the thermodynamic cost of the first ligand-receptor interaction between the two multifunctionals is about the same as the thermodynamic cost of similar monofunctional interactions; This can cause the speed of bonding to be about the same. Degradation of the multifunctionally interacting class must disrupt the N ligand-receptor interaction, so that degradation can occur more slowly than the corresponding monofunctional interaction in the multifunctional interaction.

Steric inhibition

In certain embodiments, multifunctional donors of the invention act by “stereostatic inhibition”. Steric inhibition is a new strategy in the design of effective drugs. In the case of an infectious agent, for example, a multifunctional inhibitor of adhesion may be designed to include certain molecules that bind firmly to the surface of the infectious agent, ie refer to a polymer that provides a molecule that does not need to be included directly in the attachment. For example, by providing a group that binds neuraminidase (NA) on the surface of an influenza virus, a polymer can be prepared that prevents influenza virus from adhering to red blood cells [Choi, S.-K. ), "Chem & Biol. 1996, 3, 97-104". Since NA sites are generally considered to be mediated by those skilled in the art, the antiadhesive effect found in polymers directly involved in them can eventually attach the polymeric gel layer to the viral surface. The effect is "pure" stericly inhibited, i.e., it can be inhibited without entropy improved occupancy of the active site of hemagglutination, the protein the virus usually uses for adhesion. Thus, multifunctional provision of a medicament can change the original mechanism of activity for the medicament. At present, examples of such agents are unknown.

The mechanism of steric inhibition is known to be more involved in colloidal stabilization than in the case of receptor-mediated, although the tracking of the polymer to the appropriate binding site depends on the receptor-specified specificity. The transport of two residues or groups together when one or both are coated by a gel layer is entropy (because the adaptive mobility of the semiswelled polymer is reduced relative to other surfaces) and enthalpy (preferably). Undesired). Polymeric donors include liposomes (Kinggery-Wood et al., J. Am. Chem. Soc. 1992, 114, 7303-7305) and dendrimers (Roy, R. and Tropper, FD). J. Chem. Soc., Chem. Commun. 1988, 1058-1060; Although there are other related mechanisms that may be shown in conjunction with Roy et al., "American Chemical Society: Washington, D.C., 1994", they only work by this type of mechanism.

Adsorption and precipitation

In certain embodiments, the multifunctional donor of the invention acts by mediating the adsorption of binding site B on the surface. By this mechanism, binding site B (eg viral particles) can be effectively and cleanly removed from solution by the present invention. In other embodiments, the multifunctional donor functions including precipitation or aggregation.

Pharmacological Mechanics

The basic structure is chosen such that its original nature can affect the pharmacodynamics of the multifunctional donor. For example, two properties contemplated for the design of the multifunctional donor of the present invention are solubility and size.

Solubility

In most embodiments, the multifunctional donor of the invention will be more water soluble than conventional agents. Solubility (and size as described above) can affect the pharmacodynamics of the multifunctional donor. For example, the solubility of a donor can affect one or more related features described later.

For example, solubility can affect the removal rate profile of the multifunctional donor of the present invention. The removal rate can increase greatly as the solubility of the molecule increases. Elongation tends to filter water soluble molecules more quickly. The rate of drug removal is also directly proportional to the frequency of drug administration.

The water solubility of the donor of the present invention may also affect the duration of activity of the donor. In a preferred embodiment, the subject matter of the present invention has a longer active period than that performed by A monofunctionally.

In another embodiment, the solubility of the polyA can affect the therapeutic index. As used herein, the term "therapeutic dimension" refers to (LD50 / ED50), which can be measured by methods well known in the art. By therapeutic index as used herein is meant calculated on the basis of group A. The therapeutic index is inversely proportional to the frequency of drug administration. Because of the lower rate of elimination of the subjects of the present invention, polyA may be administered to a subject less frequently than monoA. Moreover, because the removal rate of these agents is very slow, polyA will exhibit a lower concentration variation than monoA over time at the desired site in the subject. They can be maintained at more uniform concentrations in the plasma, which means that the difference between the maximum and minimum concentrations at the site where the multi-functional drug is desired is reduced (e.g., the trough-peak deviation is lower). It means. Because of the large size of multifunctional molecules, they have the obvious advantage that their lifespan can be much longer than that found in small molecules. Because of the lower rate of clearance of the present inventive subjects, PolyA will be administered to a subject at a frequency less than monoA. Their longer half-life is advantageous for a variety of reasons. For example, (i) the convenience and euphoria of the subject increase with decreasing frequency of drug administration; (ii) the subject may be discharged from the hospital earlier than current practice; (iii) The agent may be administered at a therapeutic index lower than current practice.

The solubility of the multifunctional donor of the present invention may also affect the fractionation of the multifunctional donor. In addition to solubility that may affect certain fractionation and all forms of fractionation, such as with or without them, other factors are also intended to be considered part of the invention. For example, the partition coefficient between water and organic solvent (similar to the biological environment) is an important factor. In general, high molecular weight polymeric multifunctional species will not effectively cross biological membranes. In certain embodiments, the feature is preferably administered by delivering the desired fraction directly. Alternatively, the property means that the multifunctional donor can be removed from the unwanted fractions. For example, by intravenous injection into the vascular fraction; Injection into the cerebrospinal fluid and central nervous system via intrathecal injection; Injected orally into the gastrointestinal tract; Via eye drops into the ocular fraction; Put into the skin via creams and ointments; Through catterization into the bladder ureter system and the vaginal-uterine system as well as the bile ducts, factors and bladder; Finally, it is injected into the alveolar bronchiole fraction through breathing.

The molecule can be designed to remain outside a certain fraction. Examples of areas where the concept may be useful include obstetricians (circulating the drug except the fetus), and restricting the drug that is toxic to the kidney (need to prevent the drug from being absorbed into the kidneys in the circulation), or Includes cases where the donor is prevented from invading the central nervous system.

Multifunctional donors may also have a tendency to remain localized at the site of interest, which has the advantage of reducing systemic toxicity and maximizing local concentration.

In addition, the molecule may be designed such that the properties are not limited to one fraction in which it is required. For example, for accurate setting, it may be undesirable to have a long-acting agent, but it is important to have the increased potential that a multifunctional agent can have. In these applications, the polymer may be designed to be of medium size, or may comprise a cutable connector. These molecules have the potential but are small enough to be filtered by the kidneys and will be removed from the kidneys.

Some examples of specific fractions include eye (such as tearing agents and antagonists during surgical procedures), gastrointestinal tract (such as peristaltic and antagonists (cholinergic agents)), muscle (before double-controlled barium studies) Diarrhea) agonists and antagonists (glucagon)). Other examples include the CNS, urinary reproductive system (e.g. kidney, ureter, or bladder, vagina, uterus, fallopian tube (e.g. contraception)) and bronchus (e.g. anti-asthmatic or cystic fiber treatment). do. Still other examples of fractions include surfaces (such as skin and mucous membranes for topical application); Ear canal and middle ear (such as antibiotics and antiviral agents), and blood (such as vehicles for subcutaneous and intramuscular delivery as well as intravenous injections). Other examples of fractions include intravascular fluids, extravascular fluids, cerebrospinal fluid, alveolar bronchiole spaces, pleural spaces, peritoneal spaces, ocular fractions, local fractions, urethral canals, reproductive tracts (e.g. vaginal canals, cervical canals, palio tubules). Include.

size

The size of the multifunctional donor can affect the same duration of activity, therapeutic index, fractionation, and dissolution profiles that can be dissolved.

The term "size" is meant to include the molecular size of the carbohydrates (ie, Stokes radius) and the molecular weight (kD) of the protein. Multifunctional donors with a molecular size of 60 kD or more or an average hydrodynamic diameter of 50 kPa or more have a tendency to fractionate more easily in molecules of smaller molecular size. In a particularly preferred embodiment, the multifunctional donor of the invention has a mean hydrodynamic diameter of at least 2 kD and / or 5 nM 50 kPa.

The size of the multifunctional donor can vary depending on the "disease" being treated. For example, low molecular weight compounds (less than about 10,000 MW) for parenteral administration will generally be removed more quickly. Alternatively, larger compounds can be used that include removable linkages that connect units small enough to be removed when released. Molecules larger than 60 to 70 kDa protein cannot be effectively filtered by the kidneys, which is important if, for example, multifunctional donors are used in the bloodstream. When used for oral, pulmonary or topical administration, the substances need not be removed or degraded in vivo.

For drugs that have a near zero removal rate and are not absorbed or removed by the liver because they are too large to be removed by the kidneys, the removal rate may preferably be induced by a number of mechanisms. For example, low molecular weight moieties (of sizes that can be easily removed by the kidneys) can be joined by connectors that hydrolyze at a significant rate in the serum. Alternatively, such low molecular weight moieties may be bound by connectors that are hydrolyzed by agents (eg, enzymes) originally present in the plasma. In other embodiments, such low molecular weight moieties can be joined by connectors that can be cleaved by an agent (eg, a multifunctional or monofunctional second agent) that is absorbed at the time required for removal of the multifunctional donor. Examples of such second agents may be thiols or chelating agents, and examples of linkages acceptable for these agents may be disulfides and organometallic links.

In general, high molecular weight species cannot be introduced into the blood by oral administration. Nevertheless, high molecular weight systems are useful for other applications. In certain embodiments, they will be acceptable to the subject by penetrating the membrane (administered into the nasal or alveolar membrane as an aerosol). Some multifunctional species can be absorbed or administered as suppositories through cells in the gastrointestinal tract, such as by formulating to remain in a digestive response. Large multi-functional agents may have the advantage of limiting side effects because they lead to systemic circulation through the lungs, stomach, or respiratory system.

Additional aspect

In certain embodiments, it will be desirable to treat the subject using one or more multifunctional donors and further monofunctional inhibitors. Such monofunctional inhibitors may or may not interact with binding site B in the same way that group A interacts on the multifunctional donor. For example, a monofunctional inhibitor of influenza neuraminidase (NA), a hydrolytic enzyme present on the surface of an influenza virus, enhances the ability of polyacrylamide to be represented as an HA inhibitor [Choi; Marmen; Whiteside et al., Chem. & Bio 196, 3, 97-104. The NA site on the surface of the virus acts as the second binding site for SA. The addition of monofunctional inhibitors of NA prevents the second binding of SA to increase the efficacy of these polymeric inhibitors, presumably because of increased steric stabilization. In another embodiment, the subjects of the present invention can be used with any other method of treatment.

The onset time of activity for the multifunctional donor can also be important. Multifunctional hapten-carrier conjugates (in some embodiments not considered part of the invention) typically require a long time to elicit a useful biological response. The multifunctional donor of the present invention can elicit a useful biological response or therapeutic effect in one day or preferably 12 hours, 5 hours, 1 hour, or a substantially immediate response within minutes to 1 second, for example when treating asthma. Or can cause an effect.

The following invention is further illustrated by the following examples, which should not be considered as further limiting. The contents of the manuscripts cited throughout this application (see Attachments A and B) and all references (including the "Background" section) provided in pending patent applications and published patents are incorporated herein by reference. It is cited in detail.

Example 1 Preparation of Multifunctional Providers to Promote Treatment of Influenza

Part A-Preparation and Self-Assessment of Libraries of Poly (acrylic Acid) Derivatives Providing Sialosides as Multifunctional Inhibitors of Influenza-Mediated Hemagglutination

Simple small scale methods have been developed for creating and evaluating libraries of derivatives of poly (acrylic acid) (pAA) that provide mixtures of side chains that affect the biological activity of derivatives of poly (acrylic acid) (pAA). Using this method, derivatives of pAAs with N-acetylnuramic acid (NeuAc-L-NH ₂ ) as side chain pAA (NeuAc-L) were subjected to hemagglutination of erythrocytes in chickens by influenza virus A (X-31). Generated and assayed the ability to inhibit (HAI); The constant representing the inhibition (K _i ^HAI ) is calculated based on the total NeuAc group in solution. Using a mixture of NeuAc-L-NH ₂ and one of 26 different basic amines RNH ₂ , various terpolymers (NeuAc-L; R) (χ ^NeuAc-L about 0.05; χ ^R about 0.06) were prepared Assay. These polymers (pAA (NeuAc-L; R)) include a class of novel polymeric multifunctional sialosides that are potential inhibitors of influenza virus against red blood cells.

The method is based on the conversion of poly (acrylic anhydride) (pAAn) into derivatives of pAA by reacting with various amines RNH ₂ in water. These derived polymers were prepared by sonicating the reactants directly into the wells of a microtiter plate and then assayed in the same plate without further manipulation.

Influenza initiates infection by adsorption on the surface of mammalian cells via a multifunctional interaction of viral protein hemagglutinin (HA) with a population of NeuAc residues expressed on the cell surface. Monomeric sialosides (measured by inhibition of hemagglutination) are generally weak inhibitors for the adsorption of viruses (eg, Sauter, N.K. et al., Biochemistry 1989, 28, 8388).

Suspension of poly (acrylic acid anhydride) (pAAn) (0.12 mg / μL) (eg, Johnes, JF, "J. Polymer Sci. 1958, 33, 15"; Brothton, TK, Smith, Jr. J. and Lyn, JW, "J. Org. Chem. 1961, 26, 1283", are incorporated into 250 μL wells of microtiter plates. By sonication with an aqueous solution of amine RNH ₂ (0.1M), pAA was synthesized with a plurality of R groups as side chains to give pAA (R) (monovalent, see Appendix A). A solution of the copolymer pAA (NeuAc-L) was used to prepare NeuAc-L-NH ₂ (1, 2, 3 or 4) using a different number of equimoles of NeuAc-L-NH ₂ to pAAn (monovalent). Prepared by reacting with acrylic acid anhydride) (pAAn). The equimolar number of polymers sonicated solely pAAn in PBS buffer (137 mM NaCl, 2.7 mM KCl, 7.7 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , 0.05% NaN ₃ ) at 12 pH. Homopolymer pAA obtained by hydrolysis). Copolymeric pAAs (NeuAc-L) with an equimolar number greater than or equal to zero were placed in a microtiter plate with 96 conical bottom wells (i) 6 mg pAAn in the wells; (ii) wet the powder with a variable amount (19-100 μl) of 0.1 M NeuAc-L-NH in 12 pH PBS buffer; (iii) the plate was immediately sealed (by tapping the side of the plate with parafilm (trade name) and its cover tightly closed) and then sonicated for 0.5 h (Fisher ultrasonic bath cleaner) ); This sonication was prepared by gradually increasing the temperature of the water (and reactants) in the bath to about 50 ° C. or less. Each solution of the resulting copolymer (about 3 pH) in the wells was neutralized to about 7 pH by adding 60 μl of 1.0 M NaOH, adjusted to 100 or 200 μl (total volume) with PBS and adjusted to 7 pH before HAI assay. It was. The protocol was easily extended to the preparation of the terpolymer; Here, a three-component mixture [pAAn (6 mg), 50 [mu] L of 0.1 M NeuAc-L-NH ₂ (1 or 3) and 30 [mu] L of RNH ₂ 0.2M] was sonicated.

The resulting pAA (R) was analyzed by ¹ H-NMR (D ₂ O) spectra of the lyophilized reaction mixture and subjected to gel permeation chromatography (M _w = 39.5 kDa, polydispersity = 1.91; polysaccharide reference) Characterized. Incorporation of RNH ₂ was observed by comparing the integrated density of NMR signals from free RNH ₂ before and after sonication. The ¹ H NMR signal of R from pAA (R) is a signal of free unreacted RNH ₂ and its form (a line representing the relatively wide polymer-attached species) and their chemical shifts (δ values of CH ₂ or CH groups). Then shifted to the δ value of the amide group). The% incorporation of RNH ₂ as an amide group is based on RNH ₂ :

% Incorporation = [moles of (CONHR) / moles of RNH ₂ used] × 100%

The average value was tested using five different amines RNH ₂ (4-aminobenzoic acid, 6-aminohexanoic acid, N-methylhydroxylamine, (L) -arginine and 1 (NeuAc-L ₁ -NH ₂ )) It was about 90% (± 5). Since amide formation and hydrolysis reactions in the anhydride group take place competitively, the effectiveness of the former method is influenced by the relative reactivity of each RNH ₂ , and also the pH of the aqueous solution of RNH ₂ (to the respective aromatic and aliphatic amines). Optimum about 7 and 12 pH) and RNH ₂ to pAAn equimolar number (proper equimolar number is less than 0.2).

Copolymeric derivatives of pAA which provide NeuAc-L as side chain (pAA (1) -pAA (4)) can be prepared using the above semisolid phase synthesis methods (1-4; NeuAc-L _n) using derivatives of NeuAc having different linking groups. -NH ₂ ; see FIG. 1 or Scheme 1) for the synthesis of NeuAc-L-NH ₂ 1 and 2, Sparks, MA, Williams, KW, Whites. J. Med. Chem. 1993, 36, 778; Ogura, H. et al., Carbohydr. Res. 1986, 158, 37; Lees, WJ et al., J. Am. Chem. Soc. 1994, 37, 3419. NeuAc-L-NH ₂ (3, 4) was prepared as described below. Since the aromatic moieties present in the center of the linkage can enhance the binding affinity of the monomeric NeuAc-L and HA sites, 3 and 4 were used to incorporate into the side chain of the polymer [Watowich, SJ] Skehel, JJ and Wiley, DC, "Structure 1994, 2, 719".

After sonication, the crude polymer solution was immediately assessed for hemagglutination inhibition (HAI) activity using assays based on chicken's red blood cells and influenza virus A (X-31) [eg, Choi, Mamen, White. Literature of the size "Chem. & Biol. 1996, 3, 97"; Lees et al., J. Med. Chem. 1995, 37, 3419; And literature cited herein.

Table 9 below provides the values of K _i ^HAI in various equimolar NeuAc-L-NH ₂ (lowest concentrations of NeuAc-L group from pAA (NeuAc-L) in solution to prevent hemagglutination). The equimolar number of NeuAc-L-NH ₂ is directly related to χ ^NeuAc-L , the molar fraction of ^NeuAc- containing side chains in the polymer. Table 9 also shows the other three derivatives of pAA (pAA (2)-pAA (4)) with HAI activity in the micromolar range (or less). In comparison, all HAI activities of all monomeric sialic acids (1-4) were low (K _i ^HAI was 5 mM or more).

Hemagglutination inhibitory activity of pAA (NeuAc-L) and library of pAA (NeuAc-L; R). The library of pAA (NeuAc-L; R) is shown in Table 9.

B-part multifunctional donor with two types of "A" groups

By extending the method of part A, a tripolymer mixture of NeuAc-L-NH ₂ , RNH ₂ and pAAn is sonicated to provide a multifunctional polypolymer of NeuAc-L and one R group, pAA (NeuAc- A library of L; R) was prepared.

Table 9 shows the K _i ^HAI of pAA (NeuAc-L; R) obtained from a mixture of NeuAc-L-NH ₂ (equal mole = 0.10) and one of 26 different RNH ₂ (equal mole = 0.12) The values are summarized. Some pAA (1; R) (and pAA (3; R)) are enhanced by 100 to about 7000 or more factors relative to the parent copolymer pAA (1) (equal mole of 1 = 0.10) without the R group present (HAI assays require a limited amount of virus and note that the efficacy of inhibitors whose efficacy K _i ^HAI is less than 1 nM cannot be accurately measured). We have determined that, in some control tests, these terpolymers (and excluding pAA (NeuAc-L)) are responsible for high activity, even if activity is measured directly from crude pAA (NeuAc-L; R). Typically, the incorporation of derivatives of hydrophobic or aromatic amino acids greatly enhanced the activity. We have demonstrated that certain structural forms (and not hydrophobic alone) are particularly effective in reducing the value of K _i ^HAI . We concluded that although certain non-sialoside groups do not exhibit HAI activity on their own, they enhance the activity of pAA (NeuAc-L; R) by up to about 10 ⁴ factors.

These pAAs (NeuAc-L; R) are the best of the new class of hemagglutination inhibitors with conventional high activity at the relatively slowest molar fractions of NeuAc-L (about 5%) and R (about 6%), Each 1% of the mole fractions of NeuAc-L or R is equivalent to about 6 side chains (per polymer molecule). Such findings highlight the importance of the combination of side chains that slow down the activity of these multifunctional donors.

The methods described in sections A & B make both the synthesis and assay in the wells of microtiter plates more efficient in the production of derivatives of pAAs and in the evaluation of the biological activity of these polymers. The methods allow for convenient screening of libraries of polymers that provide multiple combinations of side chains at controlled mole fractions. Because microtiter plate assays are common in the biological and medical arts, these methods can generally provide for screening and obtaining other methods and ranges of cohesive interactions that are affected by multifunctional inhibitors. This process is a rapid and economical way to synthesize and screen multifunctional polymeric inhibitors for biological activity.

Example 2

Method for preparing multifunctional donor for promoting inhibition of platelet aggregation

RGD (L-Arg-L-Gly-L-Asp) is a recognition sequence for binding of fibrinogen to platelets. Homologs of RGD act as platelet aggregation inhibitors by inhibiting the interaction between fibrinogen and platelet membrane glycoprotein IIb / IIIa complex (Phillips, D. R., "and Cell, 65: 356 (1991)").

Multifunctional donors providing RGD as ligands were prepared as follows:

RGD immobilized on a solid support at the C-terminus is prepared, for example, on a Wang resin, according to known methods of solid phase peptide synthesis (SPPS). The solid-supported RGD is reacted with succinic anhydride to provide an immobilized succinylated RGD, which is then coupled with a mono-BOC-protected 1,6-diaminohexane (eg with DCC / HOBt). . The derived RGD is cleaved from the resin by acid treatment, at the same time removing the BOC group and then purified (eg by HPLC) to give the RGD functionalized with a linker moiety having a terminal amino group.

Polysuccinimides [prepared according to known methods; See US Pat. No. 5,484,878 for the preparation of polysuccinimides from aspartic acid in suspension (e.g., diethylformamide (DMF) or dimethylacetamide) and stirring the linker-functionalized RGD To the suspension. After completion of the amino degradation of the polysuccinimide, the reaction mixture was diluted with an aqueous base to hydrolyze the amino-undegraded polysuccinimide, followed by dialysis to remove impurities to functionalize the poly-linked RGD. Aspartic acid) backbone was obtained. The degree of substitution of the main chain with RGD and the size of the poly (aspartic acid) polymer prepared by amino degradation are controlled by adjusting the pH of the reaction mixture, the amount of RGD-linkers present in the reaction mixture, the reaction time and the like.

The resulting RGD-poly (aspartic acid) multifunctional donor is screened to determine the multifunctional donor for fibrinogen that binds to the platelet membrane glycoprotein IIb / IIIa complex according to methods known in the art, such as platelet aggregation assays. The effect was measured.

Example 3

Multifunctional Polymeric Galactosides in the Prevention of Adhesion of Lysine to Red Blood Cells

Abbreviation

PBS-phosphate buffered saline; Et-ethyl; MeOH-methanol; h-hour; i-Pr-isopropyl; pBMA-poly (butadiene-co-maleic acid); RCA-Resinus comunis flocculation; BHA-bromelain-depleted hemagglutinin; LDH-lactate dehydrogenase.

General course

All conventional chemicals were used as received from the supplier without further purification unless otherwise indicated. Unless otherwise specified, all chemicals were purchased from Adrich Chemical Co., St. Louis Molly. Erythrocyte cells (RBC or erythrocytes) from chickens two weeks old were purchased from Spafas Inc. Erythrocytes provided as suspensions in storage buffer (approximately 5% v / v) were phosphate buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 7.75 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO) at 7.2 pH. ₄ ) and then resuspended in PBS (about 0.5% v / v). Fluorescein (fluorescein) isothiocyanate (FITC) - labeled stylized Lysine Lysine containing (FITC- labeled stylized RCA _120, FITC- labeled Chemistry _{RCA 60) (RCA 120, RCA} 60) Sigma Co. you Purchased from

Synthesis of Gal-βo-L ₁ NH ₂ and Gal-αo-L ₂ NH ₂ (Scheme 1); Gal-βo-L ₁ NH ₂ : A solution of methylene chloride (180 mL) containing β-D-galaltose pentaacetate (7.8 g, 19.98 mmol) and allyl alcohol (5.6 mL, 58.82 mmol) cooled in a cold bath To BF ₃ · Et ₂ O (4.0 mL, 32.52 mmol) was added dropwise. After stirring (0 ° C. for 4 hours; then at about 20 ° C. for 30 hours), the mixture was poured into cold saturated NaHCO ₃ (200 mL) in a separate funnel. After shaking, the organic layer was separated and washed again with cold saturated NaHCO ₃ (200 mL). After drying over MgSO ₄ , the methylene chloride solution was evaporated in vacuo to give a pale yellow oil (R _f = 0.57 in 5% MeOH / CH ₂ Cl ₂ ). The crude product was dissolved in methanol (100 mL) and then LiOH (2.88 g, 120 mmol) in water (50 mL) was added. After stirring (at about 20 ° C. for 12 hours), the reaction mixture was neutralized by adding 6.0 M HCl (about 20 mL). The aqueous mixture was concentrated in vacuo to give a thick oily residue which was purified by flash column chromatography (silica gel; 5% to 40% MeOH / CH ₂ Cl ₂ ). The product Gal-βo-CH ₂ CH = CH ₂ was obtained as a pale yellow oil in two yields with a yield of 91% (4.0 g) (R _f = 0.80 in 30% MeOH / CH ₂ Cl ₂ ). ¹ H-NMR (300.1 MHz, CD ₃ OD): δ (ppm) 6.00 to 5.89 (m, 1H), 5.36 to 5.29 (dd, J = 17.2, 3.2, 1H), 5.18 to 5.12 (dd, J = 12.1 , 3.2 Hz, 1H), 4.40-4.34 (dd, J = 13.0, 5.2 Hz, 1H), 4.27-4.25 (d, J = 7.3 Hz, 1H; h _1ax ), _4.17-4.10 (dd, J = 13.0, 6.1 Hz, 1H), 3.90-3.83 (m, 1H), 3.80-3.68 (m, 3H), 3.56-3.50 (m, 2H); FAB-MS (glycerol): m / z 221 [M + H] ⁺ ; HRMS: Calcd for C ₉ H ₁₇ 0 ₆ 221.1024 was 221.1025.

Gal-βo-CH ₂ CH ₂ (4.0 g, 18.17 mmol), HSCH ₂ CH ₂ NH ₂ .HCl (6.19 g, 54.5 mmol) and 4,4′-azo in water (50 mL) -methanol (5 mL) A solution of bis (4-cyanovaleric acid) (4.0 g, 1.43 mmol) was degassed under vacuum for 10 minutes and then saturated with N ₂ (by bubbling N ₂ gas through the solution for 30 minutes). The reaction flask containing the mixture was placed in a photochemical reactor (Rayonet®) and irradiated at 254 nm for 10 hours. The irradiated mixture was neutralized by addition of 2.0 M NaOH (28 mL) and evaporated immediately to remove volatiles. Evaporation gave a pale yellow oil which was purified by flash silica gel chromatography (10% MeOH / CH ₂ Cl ₂ to 5% i-PrNH / 40% MeOH / CH ₂ Cl ₂ ). Adduct (Gal-βo-L ₁ NH ₂ ) was obtained as an oil (R _f = 0.33 in 5% i-PrNH ₂ /30% MeOH / CH ₂ Cl ₂ ). ¹ H-NMR (250.1 MHz, CD ₃ OD): δ (ppm) 4.22 to 4.19 (d, J = 7.4 Hz, 1H; H _1ax ), 4.0 to 3.95 (ddd, J = 9.9, 6.1, 6.1 Hz, 1H ), 3.87 to 3.81 (m, 1H), 3.78 to 3.63 (m, 4H), 3.53 to 3.43 (m, 2H), 2.81 to 2.76 (t, J = 6.5 Hz, 1H), 2.67 to 2.59 (q, J = 7.1 Hz, 4H), 1.92-1.82 (quin, J = 6.6 Hz, 2H); ¹³ C-NMR (100.6 MHz, CD ₃ OD):? (ppm) 76.6, 75.8, 72.5, 70.3, 69.2, 62.5, 41.6, 35.6, 31.2, 29.0; FAB-MS (glycerol): m / z 298 [M + H] ⁺ ; HRMS: calcd for C ₁₁ H ₂₄ NO ₆ S 298.1323 was 298.1324.

Gal-αc-L ₂ NH ₂ : Gal-αc-CH ₂ CH ₂ = CH ₂ after α-C-allylation of β-D-galactose pentaacetate [Giannis, A.) and Sandhof (Sandhoff, K) "Tertahedron Lett. 1985, 26, 1479-1482"], followed by hydrolysis of the acetate. ¹ H-NMR (400.0 MHz, CD ₃ OD): δ (ppm) 5.91 to 5.83 (m, 1H), 5.13 to 4.99 (m, 2H), 4.00 to 3.96 (m, 2H), 3.95 to 3.88 (m, 1H), 3.76-3.67 (m, 4H), 2.49-2.34 (m, 2H); ¹³ C-NMR (100.6 MHz, CD ₃ OD): δ (ppm) 136.7, 116.9, 75.7, 74.0, 71.9, 70.1, 70.0, 62.0, 31.0; FAB-MS (glycerol): m / z 298 [M + Na] ⁺ , Gal-αc-CH ₂ CH ₂ was prepared from Gal-αc-CH ₂ CH ₂ = CH ₂ as described above. ¹ H-NMR (400.0 MHz, CD ₃ OD): δ (ppm) 3.95 to 3.63 (m, 7H), 3.12 (t, J = 6.9, 2H), 2.81 (t, J = 3.4 Hz, 2H), 2.66 To 2.59 (m, 2H), 1.81 to 1.62 (m, 4H); ¹³ C-NMR (100.6 MHz, CD ₃ OD): δ (ppm) 75.7, 74.0, 71.9, 70.4, 70.2, 62.5, 39.9, 32.1, 26.8, 24.8, 20.9; FAB-MS (glycerol): m / z 282 [M + H] ⁺ ; HRMS: calcd for C ₁₁ H ₂₄ NO ₅ S 282.1375 was 282.1374.

Synthesis of pAA (Gal-β), pBMA (Gal-β) and pBMA (Gal-α) (FIG. 5):

pAA (Gal-β): The method described below for the synthesis of pAA (Gal-β; 0.4) is a protocol for general synthesis of pAA (Gal). A solution of N, N-dimethylformamide (DMF, 8 ml) or pNAS (500 mg, equivalent to 3 mmoles of NAS) containing poly (N-acryloyloxysuccinimide) [mamen; but; To white-size document "J. Med. Chem. 1995, 38, 4179-4190", after adding Gal-βo-L ₂ NH ₂ (356 mg, 1.2 mmol) dissolved in DMF (2 mL), i-Pr ₂ NEt (0.2 mL, 1.2 mmol) was added. After stirring (at about 20 ° C. for 2 days), the mixture was basified by addition of 1.0 M NaOH (3 mL) followed by stirring at about 20 ° C. for an additional 2 hours. In the final reaction, the mixture was subjected to dialysis bag [MW cutoff about 12-14 kDa; Spectrum® from Spprturm Medical Industries, Inc.] was transferred and dialyzed at about 20 ° C. over three days. H ₂ O (2 × 4 L), 0.05 M NaOH (4 L), 0.5 M NH ₄ Cl (4 L) and H ₂ O (2 × 4 L). The contents of the bag were lyophilized to give pAA (Gal-β; 0.4) as a light white solid (499 mg). ¹ H-NMR (500.1 MHz, D ₂ OD): δ (ppm) 4.3 (t, J = 7.5 Hz; H _1ax ), 3.89 (br s), 3.85 (s), 3.8 to 3.5 (m), 3.4 ( br m), 3.3 (br s), 2.6 (br s), 2.2 to 1.9 (br d), 1.7 to 1.3 (br m); % S: calculated as pAA (Gal-β; 0.4) 6.97. Another pAA (Gal-?) Was prepared following the same procedure. % S: pAA (Gal-β; 0.2) 5.00 was 5.12. The calculated pAA (Gal-β; 0.6) 8.02 was 8.06. The calculated pAA (Gal-β; 0.8) 8.67 was 8.58. The calculated pAA (Gal-β; 1.0) 9.11 was 9.05.

Synthesis of pBMA (Gal-β) and pBMA (Gal-α):

These polymers were synthesized following a slightly different protocol from above and poly (butadiene-co-maleic anhydride) or pBMAn was used instead of pNAS as precursor polymer. An aliquot of pBMAn provided as a solution in acetone (Polysciences, Inc.) was dried under vacuum and re-dissolved in DMF prior to use. pBMA (Gal-β; 0.09): ¹ H-NMR (500.1 MHz, D ₂ O): δ (ppm) 5.6 (br s), 5.4 (br s), 3.9 to 3.5 (br m), 3.4 to 3.3 ( br m), 3.0 to 2.9 (br m), 2.7 to 2.5 (br m), 2.4 to 2.1 (br s), 1.8 (br s), 3.9 to 3.5 (br m); pBMA (Gal-β; 0.09). pBMA (Gal-α; 0.09): ¹ H-NMR (500.1 MHz, D ₂ O): δ (ppm) 5.6 (br s), 5.5 to 5.3 (br d), 4.3 to 3.7 (br m), 3.6 ( br m), 3.3 (br s), 2.9 (br s), 2.8 to 2.4 (br s), 2.4 to 2.0 (br s), 1.8 to 1.4 (br m), 1.0 (br s). % S: pAA (Gal-α; 0.05) 1.62. The calculated value of pAA (Gal-α; 0.09) 2.62 was 2.69. The calculated pAA (Gal-α; 0.17) 4.11 was 4.12. The calculated pAA (Gal-α; 0.22) 4.81 was 4.85.

Lysine-mediated aggregation of RBC in chickens and its protection by pAA (Gal):

(i) Adhesion of lysine to RBC in chickens: PBS solution of fluorescent lysine contained in 1 ml of Eppendorf vial suspension of suspension of RBC (0.5% v / v; 0.4 ml) in PBS at 7.2 pH. Mix well with FITC-labeled RCA ₁₂₀ (40 nM) or FITC-labeled RCA ₆₀ (1.4 μM) 0.4 ml). After 2 hours of incubation at 4 ° C., the mixture was centrifuged at 2000 rpm for 2 minutes. After pouring off the supernatant, the red pellet was washed with 1.0 ml of PBS and slowly resuspended in PBS (0.2 ml). Optical images of absorption and fluorescence of RCA-absorbed RBCs were obtained by obtaining aliquots of suspended pellets on glass slides and examining the samples using light microscopy and fluorescence microscopy (Leica DMRX).

(ii) protection of RBCs by pAA (Gal-β) from cellular uptake of lysine:

A solution of FITC-labeled RCA ₁₂₀ (80 nM; 0.2 mL) in PBS was equivalent to a PBS solution (0.2 mL) (90 μg mL ⁻¹ or about 200 μM of pAA (Gal-β; 0.4) in an Eppendorf vial. Gal]). After incubation (30 min at 4 ° C.), the lysine-polymer mixture was added to a suspension of RBC (0.5% v / v; 0.4 mL) in PBS, then shaken slowly and incubated at 4 ° C. for 2 hours. The incubated mixture was centrifuged at 2000 rpm for 2 hours. The red pellet obtained after removing the supernatant was washed with 1.0 ml of PBS and resuspended in 0.2 ml of PBS before being examined under an optical microscope.

Hemagglutination (lysine-induced) inhibition (HAI) assay:

Titration of the prepared PBS solution of lysine was measured by serial dilution of 50 μl of lysine solution through 12 wells (mgml ⁻¹ ) (8 × 12-well microtiter plate with conical bottom; ICN flow )). Another 50 μl of PBS was added to each well, followed by a suspension of chicken red blood cells in PBS (100 μl). The solution was mixed and incubated at about 20 ° C. for 1 hour. The terminal point of hemagglutinin (HA) is defined as the final well in which lysine remains in an amount sufficient to aggregate red blood cells. A stock of PBS solution (50 μl) or monofunctional galactoside (5 mM) of polymeric galactoside (1-2 mgml ⁻¹ ; about 2-6 mM [Gal]) contains 12 μl of PBS stock solution. Two successive dilutions were made through the microtiter wells. After serial dilution of the solution of polymeric or monomeric galactoside, each well (50 μl) was mixed with 50 μl of RCA ₁₂₀ (16 nM) or RCA ₆₀ (1.9 μM). After incubation at about 20 ° C. for 30 minutes, 100 μl (0.5% v / v) of chicken red blood cell suspension was added to each well, followed by slow shaking (incubation at about 20 ° C. for 1 hour). The end point of the HAI is the final well in which aggregated pellets are observed. The terminal point (K _i ^HAI ) is defined as the lowest concentration of galactoside in solution that inhibits lysine-induced aggregation of erythrocytes. The values of K _i ^HAI were calculated based on at least 5 independent trials.

Conclusion and discussion

Synthesis of Galactoside-providing Polymer:

Derivatives of two D-galactosides (Gal-βo-L ₂ NH ₂ , Gal-αc-L ₂ NH ₂ ) as monomeric precursors were synthesized with a polymeric multifunctional D-galactoside: Gal-βo- L ₂ NH ₂ comprises a β-O-linkage between galactoside (Gal) and an amine-terminated linker (Scheme 1); Gal-αc-L ₂ NH ₂ comprises α-C-glocoside. As the C ₁ -epimeric homologue of β-O-galactoside, Gal-αc-L ₂ NH ₂ was chosen because it can be easily produced with its high stereoselectivity and its large size. C-glycosidic linkages of Gal-αc-L ₂ NH ₂ provide the additional advantage of resistance to chemical and enzymatic hydrolysis. Both epimers of D-galactoside were used to compare their binding affinity for lysine. Two types of polymer-poly (acrylic acid) (pAA; M _w = about 140 kDa, M _w / M _n = 1.91) as polymer scafford (mamen; provided by Whitesize, J. Med. Chem. 1995, 38, 4179-4190 ") and poly (butadiene-co-maleic acid) (pBMA; M _w = 10-15 kDa)-were used to provide monofunctional galactosides of many homologues as amide side chains. . These polymers were referred to as pAA (Gal-β), pAA (Gal-α), pBMA (Gal-β) and pBMA (Gal-α). pAA-based polymers were selected with the expectation that they would be relatively flexible and at least have high ionic strength, and expected that pBMA-derived polymers were hardly soluble. pAA (Gal-β) reacts poly (N-acryloyloxysuccinimide) (pNAS) with Gal-βo-L ₁ NH _{2 in} DMF (for 2 days at about 20 ° C.) and excess of 1.0 M Synthesis was carried out using the method previously described by quenching with NaOH (mamen; only; Whitesize, J. Med. Chem. 1995, 38, 4179-4190). By reacting pNAS with the χ equivalent of Gal-βo-L ₁ NH ₂ on the polymer, the equivalent sugars of the active ester groups (χ = 0.2, 0.4, 0.6, 0.8 and 1.0), (Gal-β; 0.2 to 1.0) of various pAAs Was prepared. The parameter χ is also equal to the mole fraction of Gal in the polymer, which is defined by dividing the number of side chains comprising Gal by the total number of side chains (Scheme 1). The same strategy was applied to poly (butadiene-co-maleic anhydride) (pBMAn) to prepare pBMA (Gal-β; 0.05 to 0.22) and pBMA (Gal-α; 0.05 to 0.22). All polymers are purified by dialysis (MW cutaf about 3.5 kDa), which is characterized by using ¹ H-NMR spectrometer and combustion analysis (sulfur). The yield of amide-forming reactions for pNAS and pBMAn was at least 95% and at least 65% based on the combustion analysis of the polymers, respectively.

Inhibition of adhesion of lysine to red blood cells by galactoside-providing polymer:

Red blood cells (RBCs) from chickens, two weeks old, were used as a model system for mammalian cells. Red blood cells do not synthesize proteins by removing the nucleus. Nevertheless, they provide a good model of the cells targeted by lysine and provide a system for studying the adhesion of lysine to the cell surface; The surface of erythrocytes provides a variety of β-galactoside-containing glyco-conjugates (approximately 2-3-3 10 ⁶ Gal residues per human RBC) [Sandvig, K., Olsnes, S.) and Phil, A., J. Biol. Chem. 1976, 251, 3977-3984.

Representative absorption and fluorescence images were obtained to explain that lysine adheres to the chicken's RBC causing their aggregation and degradation. Labeling lysine with fluorescent isothiocyanate (FITC) resulted in aggregation and degradation due to the action of lysine. It also demonstrated that the multifunctional galactoside pAA (Gal-β; 0.4) prevented these effects. Polymers that inhibit lysine-mediated aggregation using red blood cells (0.5% by volume) of chicken as a suspension in phosphate buffered saline (PBS) solution at 7.2 pH and lysine (RCA ₁₂₀ about 16 nM; RCA ₆₀ about 1.9 μM) The activity of sexual galactosides was assayed. Table 10 summarizes the hemagglutination inhibition (HAI) activity, K _i ^HAI (limited as the lowest concentration of inhibitor required to inhibit hemagglutination) of polymers purified at various mole fractions of carbohydrate-containing side chains (χ ^{carbohydrates} ). . "Value of K _i ^HAI " refers to the lowest concentration of carbohydrate-containing side chain of a polymer that inhibits lysine-induced aggregation of red blood cells in a chicken. Each value represents the average of five independent measurements, with the experimental nonspecificity at about ± 50% at each value. No inhibitory reaction was observed at the concentration designated as " ^b ". The value of " ^c " refers to the concentration of the carboxylic acid side chain of the polymer in solution that inhibits lysine-induced aggregation of erythrocytes in chickens.

Monofunctional Gal-βo-L ₁ NH ₂ is the value of K _i ^HAI 5 to 3 times less than the value of Gal-β-OMe for RCA ₁₂₀ and RCA ₆₀ respectively. Monofunctional galactosides, including the β-O-anomeric batch form, provide a better but less significant difference from the corresponding β-galactoside. This observation means that the binding of the Gal residue to the lysine Gal-binding site is not very sensitive to the nature of the anomeric configuration (β ≧ α), or the atoms attached to the anomeric carbon (O ≧ C).

pAA (Gal-β; 0.2-1.0) exhibited HAI activity (K _i ^HAI ) for RCA ₁₂₀ -induced aggregation at concentrations below micromoles of Gal residues in solution. The same polymer has a HAI activity that is about 50 to about 300 times lower than the activity for RCA ₁₂₀ and the maximum activity for RCA ₆₀ is 50 times better than that of the monomeric Gal derivative. In contrast, pAA (Gal-β; 0.4) was about 1500-fold higher than monofunctional Gal-β-OMe and about 270-fold higher than that of Gal-βo-L ₁ NH ₂ (activity against RCA ₁₂₀₎ . Had pBMA (Gal-β; 0.05 to 0.22) exhibits micromolar (hereinafter) HIA activity against RCA ₁₂₀ and RCA ₆₀ , and activity against RCA ₁₂₀ is better than activity against RCA ₆₀ . However, pBMA (Gal-α; 0.05 to 0.22) had HAI activity against RCA _{60 which} was about four times higher than that for RCA ₁₂₀ . Multifunctional galactosides formed on pBMA, pBMA (Gal-β; 0.22) and pBMA (Gal-β; 0.05) are the most active inhibitors of activity against RCA ₁₂₀ and RCA _60, respectively. pBMA (Gal) is a relatively small polymer with a molecular weight of about 10 to 15 kDa and has a biocompatible polymer backbone (pBMA) [Conroy, CW, Wynns, GC, and Maren, TH). See, Bioorg. Chem. 1996, 24, 262-272.

Other polymers were tested by providing a number of non-galactoside carbohydrate homologs as controls [pAA (NeuAc-α; 0.2) (Mamen, Mann and Whites, J. Med. Chem. 1995, 38, 4179-4190 "), pBMA (NeuAc-α; 0.2 to 1.0), pAA (GlcNAc-β; 0.2 to 1.0); None of them inhibited the aggregation of erythrocytes by lysine at concentrations comparable to those shown in Table 10 (K _i ^HAI > 300 μM). Thus, it was concluded that lysine-induced aggregation of erythrocytes was selectively inhibited by the polymer providing galactosides. Both α and β anomers were considered almost identical in efficacy. These results suggest that the inhibitory reaction by polymeric galactosides is due to the specific binding of galactoside ligands to the Gal receptor of lysine. We believe that some cause of the high activity of polymeric multifunctional galactosides relative to monofunctional galactosides is due to the multifunctional (entropically enhanced) binding capacity of the galactose ligands to multiple receptor sites.

6 summarizes the HAI activity of polymers for lysine. Figure 6a (including RCA ₁₂₀ ) shows that the value of K _i ^HAI of the polymer that inhibits aggregation by RCA ₆₀ is a non-linear function of the mole fraction of Gal (χ ^Gal ) of the polymer. The relationship between activity and polymer χ ^Gal in the aggregation inhibition reaction by RCA ₆₀ seems to be closely related to the description of inhibition by RCA ₁₂₀ . The K _i ^HAI -χ ^Gal relationship of the polymer also depends on the type of polymer backbone: gentle curve with large flexible pAA (Gal-β); Partially accurate quasi-linear relationship with slightly extended pBMA (Gal). PAA (Gal-β; 0.01) (polymerization = DP about 2000), which provides a low density of Gal side chains (about 20 Gals per polymer chain), is still less than the activity of Gal-β-L ₁ NH ₂ by one of 19 factors. It showed great activity (K _i ^HAI about 2.0 μM). We believe that the cause of increased pAA (Gal-β; 0.01) activity compared to monomeric Gal-β-L ₁ NH ₂ is the flexibility of the polymer backbone and the ability to adjust the distance between the galactoside ligands thereby. Regarded as; This flexibility promotes multifunctional binding to the receptor site of lysine.

Under these conditions we used to assay Gal-providing polymers for their ability to inhibit aggregation, the appropriate amount of RCA _120- the minimum concentration to aggregate RBC (0.25% 200 μL suspended in PBS solution)-is 4 nM and , The appropriate amount of RCA ₆₀ was 480 nM. This difference indicated that RCA ₆₀ (one B-chain; about 3 Gal receptor sites) aggregated weaker than that of RCA ₁₂₀ (2 B-chains; about 6 Gal receptor sites). Monofunctional galactosides were more effective against RCA ₆₀ than against RCA ₁₂₀ in preventing lysine-induced aggregation of red blood cells. Because inhibition of aggregation competitively binds monofunctional galactosides to the Gal receptor site of lysine, these differences in activity have suggested two or more hypotheses regarding the adhesion of lysine to cells. First, the difference in the observed HAI activity of monomeric galactosides for RCA ₁₂₀ and RCA ₆₀ is due to the difference in inherent affinity of monomeric galactosides for Gal receptor sites for RCA ₆₀ and RCA ₁₂₀ . . Studies on the binding of monomeric derivatives of several galactosides with two lysines using equilibrium dialysis and fluorescence techniques have shown that the two lysines have a similar affinity for monofunctional galactosides (10 Factors less than)) (Baenziger, JU, Fiete, D., J. Biol. Chem. 1979, 254, 9795-9799); Houston, LL, Dooley, TP, J. Biol. Chem. 1982, 257, 4147-4151; Rivera-Sagredo (A.), Solis (D.), Diaz-Maurino, T., Jimenez-Barbero (J.), Martin Martin-Lomas, M., J. Biochem. 1991, 197, 217-228. Second, the low value of K _i ^HAI of monofunctional galactoside for RCA ₆₀ compared to RCA ₁₂₀ was determined by the blocked Gal receptor site of lysine, which is required for inhibition of aggregation to the total number of Gal receptor sites of lysine. It may refer to the number n. In other words, if you block the same number of Gal region for RCA ₁₂₀ and RCA _60, resulting in a large fraction of the Gal region than for the blocked RCA _60, and removes the RCA ₆₀ and RCA ₁₂₀ of the aggregation ability more effectively.

Polymeric galactosides gave lower K _i ^HAI values for RCA ₁₂₀ than for RCA ₆₀ . Relative enhancement of the activity of galactoside moieties (values of K _i ^HAI of polymers compared to values in monomers) for multifunctional ^presentation was shown for RCA ₁₂₀ higher than for RCA ₆₀ . These results suggest that multifunctional ligands are more effective as inhibitors against targets with higher costs (RCA ₁₂₀ ) than one target with lower costs (RCA ₆₀ ).

In RCA ₆₀ , several polymers (pAA (Gal-β; 0.6 to 1.0), pBMA (Gal-β; 0.17 to 0.22), pBMA (Gal-α; 0.05) are blocking agents whose corresponding monofunctional galactosides are Little effective in lysine-mediated aggregation (per one basal Gal) These results indicate that for some polymeric multifunctional inhibitors, BHA (3 receptor sites), LDH (4 receptor sites) and RCA ₆₀ (about The binding force to a small number of receptor sites provided by the three receptor sites cannot be so entropy enhanced, and the ligand provided on the polymer backbone does not have an inhibitory polymer (which is an unbounded interaction between the polymer backbone and the protein). We support the conclusion that these ligands (because of their action) may have a lower affinity for the receptor than they do.

The fact that many polymers are more effective at blocking lysine-mediated aggregation than the corresponding monofunctional galactosides suggests that factors other than partial occupancy of the receptor site determine K _i ^HAI . . Since each polymer provides a number of Gal moieties, the binding of the polymer to lysine can, of course, bind relatively firmly as compared to the relatively weak binding of the individual Gal groups. Since the experiments provided herein do not directly measure the occupancy of Gal receptor sites on lysine, the cause of inhibition of hemagglutination is related to other receptors associated with non-receptors such as entropy-enhanced binding or steric inhibition of these Gal-binding receptor sites. It may be due to the effect.

Certain naturally occurring structural complex glycoproteins and oligosaccharides bind firmly to the B-chain of lysine. One example is a glycopeptide derived from fetuin (a class of glycoproteins that provide various carbohydrate populations) and these compounds have a degradation constant for lysine (and for the B-chain), K _d about 10 To 0.1 [mu] M [Wenseeger, Piete, "J. Biol. Chem. 1979, 254, 9795-9799"). These glycopeptides are multi-branched oligosaccharides and oligopeptides that provide copies of a number of galactose and N-acetylgalactosamine residues. Thus, galactoside-containing glycoconjugates are structurally multifunctional with respect to the number and intramolecular distribution of galactosides per molecule, and we believe that these structural features are those glycopeptides for lysine relative to monomer galactotosides. It is believed that the high binding affinity can be explained. The galactoside-providing polymers of the present invention, pAA (Gal) and pBMA (Gal), are well characterized by their hemagglutination inhibitory activity, multifunctional species with a K _i ^HAI of about 0.1 μM or less. These polymers can be conveniently synthesized, in particular polymers that provide C-galactosides are relatively chemically and enzymatically stable compared to fetuin-derived enzyme-sensitive glycopeptides.

In summary, the results indicate that synthetic polymers, which provide replicas of simple derivatives of a large number of β-D-galactose as side chains, can be applied to red blood cells in chickens when examined by optical / fluorescence microscopy and quantitatively determined by hemagglutination (HAI) assay. It is demonstrated that the adhesion of lysine to the drug is effectively inhibited. Following the conversion of monofunctional galactosides, which themselves are weak inhibitors of lysine-cell adhesion, to the polymeric multifunctional galactosides, the activity of the polymer-linked galactoside residues is determined by these sugar units in the assay solution. Calculations based on the total concentration of were increased by about 10 ² to 10 ³ factors for RCA ₁₂₀ , but 50 factors for RCA ₆₀ . The activity of polymeric galactosides is influenced by a number of factors such as the mole fraction of the Gal-containing side chains of the polymer, the C-1 epimeric arrangement of the galactosides (β ≧ α) and the polymer backbone.

Example 4 Multifunctional Polymeric N-acetylglucosamine Induction of Extracellular Incorporation of Acrosomes in Mouse Sperm

Abbreviation:

Gal-transferase, β-1,4-galactosyltransferase; ZP, Jonah Pelusida; GlcNAc, N-acetylglucosamine; Gal, galactose; pA, poly (acrylamide); pAA, poly (acrylic acid); pAA (GlcNAc-β), poly (acrylic acid), which provides a copy of a number of β-O-linked GlcNAc as amide side chains; pAA (Gal-β), a poly (acrylic acid) that provides a copy of a number of β-O-linked Gal as the amide side chain.

Substances and Methods

Polymeric Multifunctional N-Acetylglucosamine (GlcNAc) and Galactoside (Gal): Poly (acrylic acid) —pAA (GlcNAc; χ) and pAA (Gal; χ): χ to give homologs of multiple GlcNAc or Gal = 0.2, 0.4, 0.6, 0.8, 1.0-were prepared following a procedure similar to that described in Example 3 for preparing polymeric multifunctional compounds. 7 shows the structure of the multifunctional polymeric N-acetylglucosamine (GlcNAc) and galactoside (Gal). pAA (GlcNAc) and pAA (Gal) refer to poly (acrylic acid), which provides a number of homologues of each GlcNAc-β-L ₁ -NH ₂ and Gal-β-L ₁ -NH ₂ as amide side chains.

Procedure of acrosome reaction of mouse sperm: Mouse sperm were left to fertilize at 35 ° C. for 30 minutes and incubated at 37 ° C. with or without polymer. Each experiment had the following controls: 1) phosphate buffer (typically subtracted from all other measurements to less than 5% in assays) used only to examine the extent of the "voluntary" acrosome reaction; 2) a polymer backbone—poly (acrylyl amide) or pA—used in the same mass used to produce the desired moles of GlcNAc in pAA (GlcNAc); 3) Zona Felucida glycoprotein (ZP), a "native" inducer; And 4) Calcium Yonophore A23187, used to induce the reaction mixture for 0, 15, 30, and sometimes 60 minutes. The values (%) of acrosome-reacted sperm by ZP and yonopores were 27.1 (± 0.10) and 53.7, respectively. Poly (acrylamide) or pA is tested as a negative control and the values on the x-axis represent the concentration of carboxamide groups of pA. Sperm samples were dried on slides, intact acrosome stained, and the number of stained versus unstained cells measured at 200 sperm for each sample.

Results and discussion

Induction of Acrosomal Extracellular Transduction by Multifunctional GlcNAc: FIG. 8 summarizes the activity (acrosome-reacted sperm,%) of monofunctional GlcNAc and multifunctional GlcNAc in induction of acrosome reactions. pAA (GlcNAc; 0.2) induces a significant amount (9-17%) of the acrosome reaction at a concentration of 100 μM ([GlcNAc]), while the monofunctional GlcNAc or polymer backbone (pA) has the same amount of acrosome reaction under the same experimental conditions. Not much induced (less than 5%). The sample was synthesized where a sperm acrosome reaction required multifunctional binding of Gal-transferase (partial aggregation on the surface) to the GlcNAc residue of the egg coch, providing a copy of a number of single O-linked derivatives of GlcNAc. Can be induced by a polymer.

9A shows the activity of a series of pAA (GlcNAc; χ) (χ = 0.2, 0.3, 0.6 and 1.0) and pAA (GlcNAc; χ) (χ = 0.3, 0.6 and 1.0) in the induction of acrosome reactions in mouse sperm -Sperm responded (%) are summarized. Polymers of pAA (GlcNAc) were shown to be weak to slow the activity (4-10%) at 0.1-100 μM ([GlcNAc]). At the 100 μM they showed increased activity (17-27%). In addition, these results showed that the activity of pAA (GlcNAc) (acrosome-reacted sperm,%) accurately increased at about 100 μM of GlcNAc. FIG. 9A also shows the activity of poly (acrylic acid) giving O-linked galactoside, pAA (Gal). The low activity (inactivity) was probably due to the low-affinity (non-affinity) binding of the Gal residues of pAA (Gal) to Gal-transferase.

9b shows the activity (acrosome-reacted sperm of pAA (GlcNAc; χ) (χ = 0.2, 0.3, 0.6 and 1.0) at 100 μM ([GlcNAc]) against the mole fraction χ of GlcNAc of pAA (GlcNAc). %) Plot. The plot is derived from FIG. 9A and shows a predetermined value of 14% for pAA (NeuAc; 1.0). The plot uses a concentration of 100 μM ([GlcNAc]) because of the threshold concentration causing a large increase in the activity of pAA (GlcNAc). The relationship between activity and χ is nonlinear; (a) an exact increase in activity between χ = 0 and χ = 0.2; (b) After reaching maximum (23%) activity, the activity gradually decreases.

FIG. 10 summarizes the activity of inhibition of sperm-egg binding by multifunctional polymeric GlcNAc. Three polymers—pAA (GlcNAc; χ = 0.3, 0.6 and 1.0) —were tested at 100 μM ([GlcNAc]) for in vitro and in vitro activity in the inhibition of sperm-egg binding, which ranges from 26 (control, and pA) to about The number of 10, 5, and 5 sperm (coupled per egg) was reduced. These results suggest that polymeric multifunctional GlcNAc induces acrosome reactions and inhibits sperm-egg binding in mouse sperm. The polymer backbone, pA, was used as a control at 100 μM ([-CONH ₂ ]).

conclusion

(1) pAA (GlcNAc) induces acrosome exocytosis but not monofunctional GlcNAc; Multifunctionality is involved in the acrosome reaction.

(2) pAA (Gal), which provides non-specific ligands for Gal-transferases, is not effective in inducing acrosomal exocytosis.

(3) There is a non-linear relationship between the activity (acrosome-reacted sperm,%) and the molar fraction (p) of pAA (GlcNAc) at specific concentrations of GlcNAc.

(4) pAA (GlcNAc) inhibits sperm-egg binding, and the mechanism may comprise acute reaction of sperm following multifunctional GlcNAc.

Example 5 Generation of pMVMA (NeuAc) Using a Semi-Solid Phase Reaction (see 11a of FIG. 11)

Copolymer of poly (methyl vinyl ether-co-maleic acid (NeuAc-L ₁ ) or solution of pMVMA (NeuAc-L ₁ ), RNH ₂ (NeuAc-L ₁ NH ₂ ) to poly (methyl vinyl ether-co- Preparation of maleic anhydride) and reaction, or to react with pMVMAn using different such as the number of moles of RNH ₂ anhydride group of pMVMAn (such as molar number = [mole number of RNH _2] / [the number of moles of anhydride groups in pMVMAn]), and Or by adjusting the pH to 2 using an aqueous solution of amine A copolymer pMVMA (NeuAc) with an equimolar number greater than or equal to 0 was prepared in a microtiter plate with wells with 96 conical bottoms as follows: (i ) 3 mg pMVMAn (M _n = 67000, 80000, 311000, 485000 or 1130000 g mol ⁻¹ ) were placed in the well; (ii) the powder was allowed to change in the variable amount of RNH ₂ (NeuAc-L ₁ NH ₂ ) in PBS buffer at 12 pH ( 10 to 38 mL) (iii) The plate was immediately sealed and the mixture sonicated for 0.5 h. Each solution of the prepared copolymer (about 3 pH) was neutralized to about 7 pH by adding 30 mL of 1.0 M NaOH, adjusted to 100 or 200 mL (total volume) with PBS of 7.2 pH, and then influenza of chicken red blood cells. Inhibition assay of virus-induced aggregation was performed.

Example 6 Generation of pMVMA (NeuAc; R) Using Semisolid Phase Reactions (see 11b of FIG. 11)

The protocol used in Example 5 was used to extend the preparation of the terpolymer pMVMA (NeuAc-L ₁ ; R). The tricomponent mixture pMVMAn, NeuAc-L ₁ NH ₂ , R ₂ NH ₂ (aliphatic amine, aromatic amine, amino acid, amino sugar or peptide) was sonicated. Trimolecular polymer pMVMA (NeuAc; R ₂ ) with an equimolar number of NeuAc-L ₁ NH ₂ and RNH versus anhydride groups of pMVMA greater than zero was prepared in a microtiter plate with 96 conical bottomed wells as follows: i) 3 mg pMVMAn (M _n = 67000, 80000, 311000, 485000 or 1130000 g mol ⁻¹ ) are placed in the well; (ii) powder was prepared in 10 ml of NeuAc-L ₁ NH ₂ and 0.1 M RNH ₂ (e.g., naphthylaniline, phenylaniline, cyclohexylamine, phenylethylamine, 4-aminobenzoic acid or mannoseamine) in PBS buffer at pH 12. Wet with a variable amount () of 2-20 ml); (iii) Immediately after sealing the plate, the mixture was sonicated for 0.5 h. Each solution of the terpolymer produced in the well (pH about 3) was neutralized to about 7 pH by adding 30 mL of 1.0 M NaOH, adjusted to 100 or 200 mL (total volume) with PBS of 7.2 pH, and then Inhibition assay of influenza virus-induced aggregation of erythrocyte cells was performed.

Example 7 Generation of pAA (Gal) Using a Semi-Solid Phase Reaction (see 11c of FIG. 11)

A solution of a copolymer of pAA (Gal), using a different, such as the number of moles of RNH ₂ RNH _2; a _{(Gal-bL 2 NH 2 Gal} -aL 2 NH 2) poly (acrylic anhydride) (pAAn; M _n = 20700g Mol- ¹ , M _w = 39500 g mol- ¹ ) to prepare anhydride groups of pAAn (equal moles = [moles of RNH ₂ ] / [moles of anhydride groups of pAAn]), or using an aqueous solution of amine Prepared by adjusting the pH to 12. Copolymeric pAAs (Gal) having an equimolar number greater than or equal to zero were prepared in microtiter plates having wells with 96 conical bottoms as follows: (i) 6 mg pAAn was placed in the wells; (ii) soak the powder in varying amounts (10-100 mL) of 0.1 M RNH ₂ (Gal-bL ₂ NH ₂ or Gal-aL ₃ NH ₂ ) in PBS buffer at pH 12; (iii) Immediately after sealing the plate, the mixture was sonicated for 0.5 h. Each solution of the resulting copolymer (pH about 3) in the wells was neutralized to about 7 pH by addition of 60 mL of 1.0 M NaOH, adjusted to 100 or 200 mL (total volume) with PBS of 7.2 pH, and then Inhibition assay of influenza virus-induced aggregation of erythrocyte cells was performed.

Example 8 Generation of pBMA (Gal) Using a Semi-Solid Phase Reaction (see 11D in FIG. 11)

The protocol used in Example 7 was extended similarly to the above for the preparation of copolymers of poly (butadiene-co-maleic acid) (Gal) or copolymers of pBMA (Gal). A solution of a copolymer of pBMA (Gal) was used as a poly (butadiene-co-maleic anhydride) (pBMAn) using RNH ₂ (Gal-bL ₂ NH ₂ ; Gal-aL ₃ NH ₂ ) using different equimolar numbers of RNH ₂ . M _w = 10000 to 15000 g mol ^-1 ) to prepare anhydride groups of pBMAn (equal moles = [moles of RNH ₂ ] / [moles of anhydride groups of pBMAn]), or pH using an aqueous solution of amine Prepared by adjusting the to 12. Copolymeric pBMA (Gal) with an equimolar number greater than or equal to zero was prepared in a microtiter plate with wells with 96 conical bottoms as follows: (i) 6 mg pBMAn was placed in the well; (ii) the powder was moistened with a variable amount (10-100 mL) of 0.1 M RNH ₂ (Gal-bL ₂ NH ₂ ; Gal-aL ₃ NH ₂ ) in PBS buffer at 12 pH; (iii) Immediately after sealing the plate, the mixture was sonicated for 0.5 h. Each solution of the resulting copolymer (about 3 pH) in the wells was neutralized to about 7 pH by addition of 60 mL of 1.0 M NaOH, adjusted to 100 or 200 mL (total volume) with PBS of 7.2 pH, and then the red blood cells of the chicken. Inhibition assay of influenza virus-induced aggregation of cells was performed.

Example 9 Generation of pAA (SLe ^x ) Using a Semi-Solid Phase Reaction (see 12A in FIG. 12)

The protocol used in Example 7 was extended similarly to the above for the preparation of copolymers of pAA (SLe ^x ). A solution of a copolymer of pAA (SLe ^x), such as a different RNH _2, RNH (SLe ^x -NH ₂₎ by using the number of moles react with the anhydride groups of the pAA pAAn of ₂ (such as molar number = [mole number of RNH _2] / [moles of anhydride groups of pAAn]) were prepared or by adjusting the pH to 12 using an aqueous solution of amine. Copolymeric pAAs (SLe ^x ) having an equimolar number greater than or equal to zero were prepared in microtiter plates having wells with 96 conical bottoms as follows: (i) pAAn (M _n = 20700 gmol ⁻¹ , M _w = 39500 g Molar- ¹ ) 6 mg in the well; (ii) the powder was moistened with a variable amount (10-100 mL) of 0.1 M RNH ₂ (SLe ^x -NH ₂ ) in 12 pH PBS buffer; (iii) Immediately after sealing the plate, the mixture was sonicated for 0.5 h. Each solution of the resulting copolymer (about 3 pH) in the wells was neutralized to about 7 pH by adding 60 mL of 1.0 M NaOH, adjusted to 100 or 200 mL (total volume) with PBS of 7.2 pH, and then to endothelial cells. Assays for inhibition of binding of neutral leukocytes were performed.

Example 10: Generation of pAA (bacitracin; R) using a semisolid phase reaction (see 12B of FIG. 12)

The protocol used in Example 6 was extended similarly to the above for the preparation of the terpolymer of pAA (bacitracin; R). A three-component mixture comprising pAAn, bacitracin and RNH ₂ (aliphatic amines, aromatic amines, amino acids, amino sugars or peptides) is sonicated. Trimeric pAAs (bacitracin; R) having an equimolar number of at least zero for the anhydride groups of pAAn and RNH ₂ were prepared in microtiter plates with 96 conical bottomed wells as follows: (i) 3 mg of pAAn (M _n = 20700 g mol ⁻¹ , M _w = 39500 g mol ⁻¹ ) were placed in the well; (ii) powder in varying amounts (2 to 20) of 0.1 M RNH ₂ (e.g., naphthylaniline, phenylaniline, cyclohexylamine, phenylethylamine, 4-aminobenzoic acid or mannoseamine) in PBS buffer at 12 pH; Ml); (iii) Immediately after sealing the plate, the mixture was sonicated for 0.5 h. Each solution of terpolymer produced in the wells (about 3 pH) was neutralized to about 7 pH by adding 30 mL of 1.0 M NaOH, adjusted to 100 or 200 mL (total volume) with PBS of 7.2 pH, and then Inhibition assays were performed.

Countermeasures and Methods

Those skilled in the art can ascertain many of the same as the specific aspects and methods described herein without using anything other than routine experimentation. Such same aspects and methods are included within the scope of the following claims.

Claims (43)

(a) constructing and arranging a plurality of groups A on a basic structure, wherein the configuration and arrangement is such that the group A and a group of target binding sites B are multifunctionally interacting to allow a group of target binding sites B in any subject Is based on the therapeutic effect caused by blanketing, which masking is optionally caused by adaptive interfacial interaction of a plurality of polyvalent presenters with multiple groups of target binding sites B). Forming a multifunctional provider for treating a disease or condition, optionally (b) The basic structure is based on the ability of the multifunctional donor to form a gel-like barrier that surrounds a series of target binding sites B when the group interacts with the group of target binding sites B adaptively. Organizing and arranging a plurality of groups A to (c) constructing and arranging a plurality of groups A on the basic structure based on the ability of the multifunctional donor to interact with a known or expected spatial arrangement of a group of target binding sites B on an interface within the subject, (d) the type of group A that is a weak binder based on the analysis of the individual interactions between one group A and multiple target binding sites B, provided that multiple groups A are multifunctional donors and a group of target sites B Must satisfy the collective binding threshold when the interface interacts adaptively), (e) constructing and arranging a plurality of groups A on the basic structure based on imparting flexibility to the multifunctional donor such that the multifunctional donor can be adapted to a group of target binding sites B, and (f) selecting one or more of the types of basic structures for the ability to impart desired characteristics to the multifunctional donor, which is optionally selected from the group consisting of increasing flexibility and solubility of the multifunctional donor. Additionally included, A method of making a multifunctional provider administered to said subject to treat said disease or condition. The method of claim 1, Multiple groups A are attached to the framework via linkers, optionally (a) selecting a type of linker based on the linker's ability to impart flexibility to the multifunctional donor, (b) selecting the type of linker based on the linker's ability to provide group A to a group of target binding sites B in the subject, and (c) one factor affecting the ability of a linker to provide group A to a group of target binding sites B in a subject, which is optionally selected from the group consisting of hydrophobicity, hydrophilicity and linker diameter, The selection is based on the hydrophobicity or hydrophilicity of the environment in the subject approaching target binding site B, and the selection of the diameter of the linker is based on the known or expected diameter of the channel in the subject approaching target binding site B. Further comprising one or more of selecting the length or type of linker on the basis of. Any disease by compliant surface interaction of the multifunctional donor with a group of binding sites B in any subject, such a surface being masked by a group of or a series of target binding sites B in the subject, or Administering a plurality of Group A to the subject, optionally in the form of a multifunctional donor, to treat the disease. Any disease by compliant surface interaction of the multifunctional donor with a group of binding sites B in any subject, such a surface being masked by a group of or a series of target binding sites B in the subject, or The use of a plurality of group A, optionally in the form of multifunctional donors, for preparing a medicament for treating said disease or disorder in said subject so that the disease is treated. Any disease by compliant surface interaction of the multifunctional donor with a group of binding sites B in any subject, such a surface being masked by a group of or a series of target binding sites B in the subject, or A plurality of group A used in the treatment of the disease or condition and optionally in the form of multifunctional donors, which are administered to the subject so that the disease is treated. Administering multiple groups A to the subject such that any disease or disorder is treated by interaction of the multifunctional donor comprising multiple groups A attached to the framework with multiple target binding sites B in any subject Including, at this time The multifunctional provider i) group A is functional and acts alone or in combination with the basic structure as a medicament, ii) providing at least one, preferably two, or three additional advantages over providing a single group A to a plurality of target binding sites B by providing a group A attached to the framework (these further advantages over group A Providing sufficient biological effect in low use, enhancing specificity for targeted versus untargeted sites, positive coordination in binding, enhancing entropy of binding, and providing low off-rate Is optionally selected from the group consisting of iii) meeting the criterion of synergistic advantage in that said further advantage is greater than the additional benefit provided by the same group of monomers of the same group A dispersed in a homogeneous solution. A method for treating said disease or condition. For treating any of the diseases or conditions such that any disease or condition is treated by the interaction of a multi-functional donor comprising a plurality of groups A attached to the framework with a plurality of target binding sites B in any subject. Manufacturing drugs The multifunctional provider is i) group A is functional and acts alone or in combination with the basic structure as a medicament, ii) providing at least one, preferably two, or three additional advantages over providing a single group A to a plurality of target binding sites B by providing a group A attached to the framework (these further advantages over group A Providing a sufficient biological effect in low use, enhancing specificity for targeted versus untargeted sites, positive coordination in binding, enhancing entropy of binding, and producing a carrier with low extinction rates Is optionally selected from), iii) the further advantage satisfies the criterion of a synergistic advantage in that it is greater than the further advantage provided by the same group of monomers of the same group A dispersed in a homogeneous solution) Use of multiple groups A. Is administered to the subject such that any disease or condition is treated by interaction of a multifunctional donor comprising a plurality of groups A attached to the framework with a plurality of target binding sites B in any subject, wherein The multifunctional provider i) group A is functional and acts alone or in combination with the basic structure as a medicament, ii) providing at least one, preferably two, or three additional advantages over providing a single group A to a plurality of target binding sites B by providing a group A attached to the framework (these further advantages over group A Providing a sufficient biological effect in low use, enhancing specificity for targeted versus untargeted sites, positive coordination in binding, enhancing entropy of binding, and producing a carrier with low extinction rates Is optionally selected from), iii) meeting the criterion of synergistic advantage in that said further advantage is greater than the additional benefit provided by the same group of monomers of the same group A dispersed in a homogeneous solution. A plurality of group A used to treat the disease or condition in the subject. By interaction of a group of target binding sites B with a multifunctional donor comprising two or more different types of groups (A ₁ and A ₂ ) on which the structure is A ₁ and A ₂ are not carbohydrates Administering groups (A ₁ and A ₂ ) to any subject such that any disease or condition is treated. Any interaction by the interaction of the target binding site B with a multifunctional donor comprising at least two different types of groups (A ₁ and A ₂ ) on the framework, wherein A ₁ and A ₂ are not carbohydrates Use of groups A ₁ and A ₂ for the manufacture of a medicament for treating a disease or condition such that the disease or condition is treated. Any interaction by the interaction of the target binding site B with a multifunctional donor comprising at least two different types of groups (A ₁ and A ₂ ) on the framework, wherein A ₁ and A ₂ are not carbohydrates A group (A ₁ and A ₂ ) used to treat a disease or condition, wherein the disease or condition is administered to any subject for treatment. A group of three or more different types of the base structure group is any disease or disorder treated by (A _1, A ₂ and A ₃₎ are functional groups providing member and the target binding interaction with the arm B comprising a (A _1, A ₂ and A ₃ ) is administered to any subject. The disease or condition so that any disease or condition is treated by the interaction of a target binding site B with a multifunctional donor comprising at least three different types of groups (A ₁ , A ₂ and A ₃ ) on a framework Use of groups (A ₁ , A ₂ and A ₃ ) for the manufacture of a medicament for treating a disease. To any subject such that any disease or condition is treated by interaction of the target binding site B with a multifunctional donor comprising at least three different types of group groups (A ₁ , A ₂ and A ₃ ) on the framework Groups (A ₁ , A ₂ and A ₃ ) used to treat the disease or condition, which are administered. The interaction of a multifunctional donor having a group A with a group of target binding sites B on its basic structure facilitates the treatment of any disease or condition, where A is a carbohydrate, carbohydrate derivative, antibody or fragment thereof Or Y is not dextran and the multifunctional donor is not a vaccine or A is not a carbohydrate or polymixin B). A method of facilitating the treatment of said disease or condition. The interaction of a multifunctional donor having a group A with a group of target binding sites B on its basic structure facilitates the treatment of any disease or condition, where A is a carbohydrate, carbohydrate derivative, antibody or fragment thereof Or Y is not dextran and the multifunctional donor is not a vaccine or A is not a carbohydrate or polymyxin B), the use of a plurality of group A for the manufacture of a medicament for treating said disease or condition. Administration to any subject to facilitate treatment of any disease or condition by interaction of a multifunctional donor having a group A on a framework with a group of target binding sites B, wherein A is a carbohydrate, carbohydrate derivative, Not an antibody or fragment thereof, Y is not dextran, the multifunctional donor is not a vaccine or A is not a carbohydrate or polymyxin B), Multiple groups A used for the treatment of the disease or condition. The method according to any one of claims 3, 6, 9, 12, and 15, A method comprising one or more of any of the following variations: (a) Group A attached to the framework as part of the multifunctional donor is administered, (b) the multifunctional donor is assembled in vivo, (c) multiple target binding sites B are on the same interface, (d) multiple target binding sites B are on different interfaces, (e) the target binding site B comprises one or more types of binding sites, (f) the multifunctional donor exhibits nonspecific binding of group A to a binding site other than B which is reduced compared to the specific binding of group A to binding site B, (g) the multifunctional donor of group A is effective at a lower concentration than group A provided monofunctional at the same molar concentration of A, (h) optionally non-competitive steric inhibition is included in enhancing the therapeutic effect, (i) the multifunctional donor of group A has a higher affinity for the target binding site B than the monofunctionally provided group A, (j) the multifunctional donor modulates the aggregation of the entity in which the target binding site B is present, (k) the basic structure of the multifunctional donor comprises non-covalent bonds, the basic structure optionally comprising a constituent selected from the group consisting of liposomes, liposome derivatives, micelles, colloids, protein aggregates and cells, (l) the basic structure of the multifunctional donor comprises covalent bonds, said basic structure optionally comprising a biological particle selected from the group consisting of sugars, proteins, lipids and small molecules, (m) the basic structure of the multifunctional donor further comprises a linker, optionally an amide linker, (n) linkages, dendrimers, optionally poly (esters), poly (anhydrides), carbohydrates, polyols, poly (acrylates), poly (methacrylates), poly (ethers) , Poly (amino acid), poly (glutamic acid), poly (aspartic acid), dextran, dextran sulfate, poly (maleic anhydride-co-vinyl ether), poly (succinimide), poly (acrylic anhydride), poly (Ethylene glycol), poly (lactic acid), poly (glycolic acid), poly (vinyl pyrrolidone), poly (styrene-maleic anhydride), poly (α-olefin-maleic acid), hyaluronic acid, sodium carboxymethyl And a chemical moiety selected from the group consisting of polymers, copolymers, and homopolymers comprising a member selected from the group consisting of cellulose, chondroitin sulfate, poly (acrylamide), poly (glycerol), starch and derivatives thereof Wherein the polymer is optionally Attached to group A by means of a bond), (o) the multifunctional donor is water soluble, optionally in the milligram / milliliter (mg / mL) range or the gram / milliliter (g / mL) range, (p) the distribution volume of the multifunctional donor in the subject is predictable, (q) the molecular weight of the multifunctional donor is greater than 60 kD, (r) the properties of the multifunctional donor (which are selected from the group consisting of duration of action and therapeutic index) are greater than those of A functionally provided, (s) the multifunctional donor has a longer half-life than A provided monofunctionally in the subject, in certain portions optionally selected from the group consisting of blood, central nervous system, lung, gastrointestinal tract, and kidney, (t) The average hydrodynamic diameter of the multifunctional donor is greater than 50 kPa. The method according to any one of claims 4, 7, 10, 13 and 16, Use of group A comprising one or more of any of the following variations: (a) Group A attached to the framework as part of the multifunctional donor is administered, (b) the multifunctional donor is assembled in vivo, (c) multiple target binding sites B are on the same interface, (d) multiple target binding sites B are on different interfaces, (e) the target binding site B comprises one or more types of binding sites, (f) the multifunctional donor exhibits nonspecific binding of group A to a binding site other than B which is reduced compared to the specific binding of group A to binding site B, (g) the multifunctional donor of group A is effective at a lower concentration than group A provided monofunctional at the same molar concentration of A, (h) optionally non-competitive steric inhibition is included in enhancing the therapeutic effect, (i) the multifunctional donor of group A has a higher affinity for the target binding site B than the monofunctionally provided group A, (j) the multifunctional donor modulates aggregation of the entity in which the target binding site B is present, (k) the basic structure of the multifunctional donor comprises non-covalent bonds, said basic structure optionally comprising a constituent selected from the group consisting of liposomes, liposome derivatives, micelles, colloids, protein aggregates and cells, (l) the basic structure of the multifunctional donor comprises covalent bonds, said basic structure optionally comprising a biological particle selected from the group consisting of sugars, proteins, lipids and small molecules, (m) the basic structure of the multifunctional donor further comprises a linker, optionally an amide linker, (n) linkages, dendrimers, optionally poly (esters), poly (anhydrides), carbohydrates, polyols, poly (acrylates), poly (methacrylates), poly (ethers), poly ( Amino acids), poly (glutamic acid), poly (aspartic acid), dextran, dextran sulfate, poly (maleic anhydride-co-vinyl ether), poly (succinimide), poly (acrylic anhydride), poly (ethylene glycol ), Poly (lactic acid), poly (glycolic acid), poly (vinyl pyrrolidone), poly (styrene-maleic anhydride), poly (α-olefin-maleic acid), hyaluronic acid, sodium carboxymethylcellulose, chond A chemical moiety selected from the group consisting of polymers, copolymers, and homopolymers comprising a member selected from the group consisting of lyotin sulfate, poly (acrylamide), poly (glycerol), starch and derivatives thereof, wherein , The polymer is optionally covalently bonded It is attached to the group A) by, (o) the multifunctional donor is water soluble, optionally in the milligram / milliliter (mg / mL) range or the gram / milliliter (g / mL) range, (p) the distribution volume of the multifunctional donor in the subject is predictable, (q) the molecular weight of the multifunctional donor is greater than 60 kD, (r) the properties of the multifunctional donor (which are selected from the group consisting of duration of action and therapeutic index) are greater than those of A functionally provided, (s) the multifunctional donor has a longer half-life than A provided monofunctionally in the subject, in certain portions optionally selected from the group consisting of blood, central nervous system, lung, gastrointestinal tract, and kidney, (t) The average hydrodynamic diameter of the multifunctional donor is greater than 50 kPa. The method according to any one of claims 5, 8, 11, 14 and 17, Group A wherein the treatment comprises one or more of any of the following variations: (a) Group A attached to the framework as part of the multifunctional donor is administered, (b) the multifunctional donor is assembled in vivo, (c) multiple target binding sites B are on the same interface, (d) multiple target binding sites B are on different interfaces, (e) the target binding site B comprises one or more types of binding sites, (f) the multifunctional donor exhibits nonspecific binding of group A to a binding site other than B which is reduced compared to the specific binding of group A to binding site B, (g) the multifunctional donor of group A is effective at a lower concentration than group A provided monofunctional at the same molar concentration of A, (h) optionally non-competitive steric inhibition is included in enhancing the therapeutic effect, (i) the multifunctional donor of group A has a higher affinity for the target binding site B than the monofunctionally provided group A, (j) the multifunctional donor modulates aggregation of the entity in which the target binding site B is present, (k) the basic structure of the multifunctional donor comprises non-covalent bonds, said basic structure optionally comprising a constituent selected from the group consisting of liposomes, liposome derivatives, micelles, colloids, protein aggregates and cells, (l) the basic structure of the multifunctional donor comprises covalent bonds, said basic structure optionally comprising a biological particle selected from the group consisting of sugars, proteins, lipids and small molecules, (m) the basic structure of the multifunctional donor further comprises a linker, optionally an amide linker, (n) linkages, dendrimers, optionally poly (esters), poly (anhydrides), carbohydrates, polyols, poly (acrylates), poly (methacrylates), poly (ethers), poly ( Amino acids), poly (glutamic acid), poly (aspartic acid), dextran, dextran sulfate, poly (maleic anhydride-co-vinyl ether), poly (succinimide), poly (acrylic anhydride), poly (ethylene glycol ), Poly (lactic acid), poly (glycolic acid), poly (vinyl pyrrolidone), poly (styrene-maleic anhydride), poly (α-olefin-maleic acid), hyaluronic acid, sodium carboxymethylcellulose, chond A chemical moiety selected from the group consisting of polymers, copolymers, and homopolymers comprising a member selected from the group consisting of lyotin sulfate, poly (acrylamide), poly (glycerol), starch and derivatives thereof, wherein , The polymer is optionally covalently bonded It is attached to the group A) by, (o) the multifunctional donor is water soluble, optionally in the milligram / milliliter (mg / mL) range or the gram / milliliter (g / mL) range, (p) the distribution volume of the multifunctional donor in the subject is predictable, (q) the molecular weight of the multifunctional donor is greater than 60 kD, (r) the properties of the multifunctional donor (which are selected from the group consisting of duration of action and therapeutic index) are greater than those of A functionally provided, (s) the multifunctional donor has a longer half-life than A provided monofunctionally in the subject, in certain portions optionally selected from the group consisting of blood, central nervous system, lung, gastrointestinal tract, and kidney, (t) The average hydrodynamic diameter of the multifunctional donor is greater than 50 kPa. The method according to any one of claims 3, 6, 9, 12, and 15, Multifunctional donors may be cell-cell interactions, fertilization, cell-pathogen interactions, cell-extracellular stromal interactions, pathogen-extracellular stromal interactions, neutrophil-endothelial cell interactions, inflammation, cancer metastasis, and platelet-platelets Prevent or inhibit biological interactions selected from the group consisting of the interactions, the multifunctional donor optionally modulate cell migration or block biological residues selected from the group consisting of integrin and platelet receptors, optionally acute thrombosis, low A method for treating a disease selected from the group consisting of agglomerated and hyperaggregated states. The method according to any one of claims 4, 7, 10, 13 and 16, Multifunctional donors may be cell-cell interactions, fertilization, cell-pathogen interactions, cell-extracellular stromal interactions, pathogen-extracellular stromal interactions, neutrophil-endothelial cell interactions, inflammation, cancer metastasis, and platelet-platelets Prevent or inhibit biological interactions selected from the group consisting of the interactions, the multifunctional donor optionally modulate cell migration or block biological residues selected from the group consisting of integrin and platelet receptors, optionally acute thrombosis, low aggregation state and Use of group A for the manufacture of a medicament for the treatment of a disease selected from the group consisting of a high aggregation state. The method according to any one of claims 5, 8, 11, 14 and 17, Multifunctional donors may be cell-cell interactions, fertilization, cell-pathogen interactions, cell-extracellular stromal interactions, pathogen-extracellular stromal interactions, neutrophil-endothelial cell interactions, inflammation, cancer metastasis, and platelet-platelets Prevent or inhibit biological interactions selected from the group consisting of the interactions, the multifunctional donor optionally modulate cell migration or block biological residues selected from the group consisting of integrin and platelet receptors, optionally acute thrombosis, low aggregation state and Group A used for the treatment of a disease selected from the group consisting of a high aggregation state. Administering a plurality of group A to any subject such that the conception is inhibited by the interaction of a multifunctional donor having a group A (which optionally includes GlcNAc) on a framework with a plurality of target binding sites B Inhibition of conception. Of a plurality of group A to prepare a medicament for inhibiting the conception so that the conception is inhibited by the interaction of a multi-functional donor having group A (which optionally includes GlcNAc) with a plurality of target binding sites B on its basis Usage. Multiple used to inhibit conception, administered to any subject such that the conception is inhibited by the interaction of a multi-functional donor having group A (which optionally includes GlcNAc) on the framework with multiple target binding sites B Groups A. The method according to any one of claims 3, 6, 9, 12, and 15, The multifunctional donor prevents biological interaction selected from the group consisting of cell-pathogen attachment and pathogen-extracellular interstitial attachment, wherein the pathogen is optionally selected from the group consisting of bacteria, fungi, viruses and parasites. The method according to any one of claims 4, 7, 10, 13 and 16, The use of group A wherein the multifunctional donor prevents biological interaction selected from the group consisting of cell-pathogen attachment and pathogen-extracellular interstitial attachment, wherein the pathogen is optionally selected from the group consisting of bacteria, fungi, viruses and parasites. The method according to any one of claims 5, 8, 11, 14 and 17, Group A, wherein the multifunctional donor prevents biological interactions selected from the group consisting of cell-pathogen attachment and pathogen-extracellular interstitial attachment, wherein the pathogen is optionally selected from the group consisting of bacteria, fungi, viruses and parasites. Including a multi-functional group having a formula (1), Formula 1 (Y)-(XA) _n [Wherein, Y is optionally a polymer as a basic structure, X is a direct bond or a linker, which linker is optionally an independent moiety and is not part of Y or A, A is a provided functional group, n is an integer greater than 10] Randomly (a) the integer is selected to conform to a group of target binding site B when the multifunctional donor is administered to any subject, and optionally to mask a series of target binding site B when administered to any subject, and (XA) Is attached to Y, (b) said integer is selected to conform to a group of target binding sites B when the multifunctional donor is administered to any subject, (c) further comprising one or more of the modifications, optionally further comprising a pharmaceutically acceptable carrier, Pharmaceutical compositions for multifunctionally providing a therapeutic agent. A compliant multifunctional provider comprising a plurality of groups A on a framework that forms a multifunctional provider that is compliant to mask a group of target binding sites in a subject. A pharmaceutical composition comprising two or more groups of different multifunctional donors and a pharmaceutically acceptable carrier. Providing together a plurality of surface-bound biological molecules and one multifunctional donor, wherein the multifunctional donor optionally has an enhancement factor β greater than about 10 ² as a non-polysaccharide, and a plurality of third molecules (The third molecule has a basic structure by which a third molecule is simultaneously bound to some of the plurality of surface-bound biological molecules, and a portion of the plurality of surface-bound biological molecules to which the third molecule is not bound. Blocking the three-dimensionally to control the adhesion between the surface-bound biological molecule and the second molecule); Optionally adhesion occurs between cells and entities selected from the group consisting of bacteria, viruses, influenza viruses, second cells and multifunctional molecules, A method of controlling adhesion between a plurality of surface-bound biological molecules and a plurality of second molecules. A multifunctional provider selected from the group consisting of pAA (Gal-β), pAA (Gal-α), pBMA (Gal-β), pBMA (Gal-α) and pAA (GlcNAc-β). red blood cells, comprising contacting lysine with an effective amount of a multifunctional donor selected from the group consisting of pAA (Gal-β), pAA (Gal-α), pBMA (Gal-β), and pBMA (Gal-α) Method for preventing the adhesion of lysine. (a) reacting Gal-βO-L ₁ NH ₂ with poly (N-acryloyloxysuccinimide) or poly (butadiene-co-maleic anhydride), and (b) quenching the reactants; A method of making a multifunctional donor selected from the group consisting of pAA (Gal-β) and pBMA (Gal-β). (a) reacting Gal-αC-L ₂ NH ₂ with poly (N-acryloyloxysuccinimide) or poly (butadiene-co-maleic anhydride), and (b) quenching the reactants; A method of making a multifunctional donor selected from the group consisting of pAA (Gal-α) and pBMA (Gal-α). A method of inhibiting fertilization in a subject comprising administering to any subject an effective amount of a multifunctional donor that is pAA (GlcNAc-β). Use of a multifunctional donor that is pAA (GlcNAc-β) to prepare a composition for inhibiting fertilization. Multifunctional donor A used to inhibit fertilization, which is pAA (GlcNAc-β) and is administered in an amount effective to inhibit fertilization in any subject. (a) reacting GlcNAc-β-L ₁ NH ₂ with poly (N-acryloyloxysuccinimide), and (b) quenching the reactants; A method of making a multifunctional donor which is pAA (GlcNAc-β). (a) placing the first activated framework component of the first multifunctional provider and the first activated framework component of the second multifunctional provider into the first reaction vessel and the second reaction vessel, (b) placing the first functional group component of the first multifunctional provider and the first functional group component of the second multifunctional provider into the first and second reaction vessels, and (c) reacting the activated components to form a series of two or more different multifunctional donors, optionally (d) placing a first group of spacers of said first multifunctional provider in said first reaction vessel, (e) placing a first spacer group of the second multifunctional provider in the reaction vessel, (f) placing an auxiliary group of said first multifunctional donor into said first reaction vessel, and (g) further comprising placing an auxiliary group of said second multifunctional donor into said second reaction vessel, (1) repeating the step of injecting "n" functional groups of said first multi-functional donor and said second multi-functional donor into said first reaction vessel and said second reaction vessel, respectively, (2) repeating the step of injecting "n" spacer groups of said first multifunctional donor and said second multifunctional donor into said first reaction vessel and said second reaction vessel, respectively, (3) one of any of the variations of repeating the step of introducing the " n " auxiliary groups of the first multifunctional donor and the second multifunctional donor into the first reaction vessel and the second reaction vessel, respectively. A method of producing a series of multifunctional providers comprising the above modifications. 43. A multifunctional or a series of multifunctional providers which can be prepared by the method according to any of claims 1, 2, 36, 37, 41 or 42.