EP0973551A2 - Molecules presentant une pluralite de groupes fonctionnels actifs - Google Patents

Molecules presentant une pluralite de groupes fonctionnels actifs

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Publication number
EP0973551A2
EP0973551A2 EP98918079A EP98918079A EP0973551A2 EP 0973551 A2 EP0973551 A2 EP 0973551A2 EP 98918079 A EP98918079 A EP 98918079A EP 98918079 A EP98918079 A EP 98918079A EP 0973551 A2 EP0973551 A2 EP 0973551A2
Authority
EP
European Patent Office
Prior art keywords
groups
polyvalent
group
polyvalent presenter
presenter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98918079A
Other languages
German (de)
English (en)
Inventor
George Whitesides
James B. Tananbaum
John Griffin
Mathai Mammen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard College
Innoviva Inc
Original Assignee
Harvard College
Advanced Medicine Inc
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Filing date
Publication date
Application filed by Harvard College, Advanced Medicine Inc filed Critical Harvard College
Publication of EP0973551A2 publication Critical patent/EP0973551A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/00756—Compositions, e.g. coatings, crystals, formulations

Definitions

  • Polyvalency is pervasive in biology and is important to the functioning of biological systems. Polyvalency is the interaction, e.g. occurring simultaneously, between two discrete species through multiple pairs of individual ligand-receptor binding events. (See Figure 1). Interactions of ligands with receptors occur throughout biology.
  • Ligands include molecules that convey information in biological systems or that are acted on by proteins. Examples of types of ligands are drugs, hormones, signaling molecules, cell surface markers, toxins, enzyme substrates, bioregulators, neurotransmittors and lymphokines.
  • Receptors include molecules that interact and receive the information from the ligands. Most receptors are proteins and include protein receptors, antibodies, and enzymes. Some receptors are nucleic acids and include the regulatory regions of DNA and RNA.
  • Matrosovich (FEBS LETTERS 1989,252,1, 1-4) proposed the use of polyvalent inhibitors for inhibiting microbial attachment.
  • Matrosovich sets forth that "[t]o produce such polyvalent structures, one might utilize some principles of the design of the well known 'drug delivery systems', for example, coupling the monovalent inhibitory active molecules in multiple copies to soluble biocompatible polymers or microparticulate carriers”.
  • Matrosovich further sets forth that" [t]he correctness of and the prospects for the practical use of this approach to the design of antimicrobial agents could be evaluated in the future."
  • the present invention is based, at least in part, on our viewing of receptor-ligand interactions and polyvalency in a non-conventional, global manner based on an understanding of how a multicomponent polyvalent presenter interacts with a collection of target binding sites.
  • Our approach differs from the conventional manner of viewing such interactions on a more individualistic basis as separate receptor-ligand interactions.
  • This non-conventional, global manner of viewing receptor-ligand interactions and polyvalency in biological systems led us to the realization that polyvalency can be used as a basis, e.g., a primary basis, for rational drug design and even further has universal applications for treating many different diseases or conditions.
  • This non-conventional, global manner of viewing receptor-ligand interactions has even further led us to the realization that the choice of a particular ligand based on its individual binding capability need not be the most important parameter in designing a polyvalent drug.
  • the polyvalent presenters of the invention are formed by constructing and arranging a plurality of groups A (e.g., ligands), which may be the same or different, on a framework (e.g., a polymeric backbone) forming a polyvalent presenter for treating a disease or condition.
  • groups A e.g., ligands
  • the construction and arrangement of the groups A on the framework can be made based on a therapeutic effect due to the blanketing of a collection of target binding sites B or an array of target binding sites B (e.g., clustered receptor binding sites on a cellular surface) or multiple type of binding sites for a single ligand and multiple types of binding sites for multiple types of ligands with the attached polyvalent presenter(s) within a subject.
  • the blanketing of a collection of target binding sites B occurs as the result of the conformal interface interaction of a polyvalent presenter with a collection of target binding sites B.
  • the blanketing of an array of target binding sites occurs as the result of the conformal interface interactions with a plurality of collections of target binding sites B.
  • the blanketing is not intended to be limited to a continuous barrier and is discussed in further detail in the Detailed Description below. For the purpose of illustration, the blanketing effect will be discussed in detail as it pertains to a polyvalent presenter having a polymeric framework.
  • a polyvalent presenter P having a polymeric framework 1 is capable of assuming a configuration which conforms to an interface 3, e.g., surface, containing a collection of target binding sites 5 as it interacts.
  • One or more attached conformed polyvalent presenters essentially blanket an array of target binding sites B (made up of more than one collection of target binding sites B) enhancing or providing the therapeutic effect of the polyvalent presenter(s).
  • the blanket can be a physical blanket, e.g., a physical covering up of the target binding sites B with the polyvalent presenter and/or a steric blanket, e.g., a crowding of the target binding sites B with multiple groups A of the polyvalent presenter.
  • the blanket does not have to be a continuous or a tightly conforming barrier but can be a non-continuous or loosely conforming barrier as shown in Figure 2, and the blanket does not have to cover the target binding sites completely.
  • the blanket will be a gel layer that conforms to the surface containing the array of target binding sites B.
  • the present invention even further pertains to methods for treating a disease or condition.
  • the method involves administering to a subject a plurality of groups A such that the treatment of the disease or condition occurs by the blanketing of a collection of or an array of target binding sites B within the subject.
  • the blanketing of a collection of target binding sites B within the subject occurs via the conformal interface interaction of a single polyvalent presenter with a collection of target binding sites B.
  • the blanketing of an array of target binding sites B occurs via the conformal interface interactions of a plurality of polyvalent presenters with collections of target binding sites B.
  • the methods involve the use of polyvalent presenters that meet certain criteria or the use of polyvalent presenters having particularly selected components, e.g., groups A, frameworks or linkers.
  • the present invention also pertains to pharmaceutical compositions for polyvalently presenting an agent for therapy.
  • the pharmaceutical compositions contain a polyvalent presenter.
  • the polyvalent presenter can have a formula as follows:
  • Y is a framework
  • X is a direct bond or a linker
  • A is a presented functional group and each A may be the same or different
  • n is greater than ten and is an integer.
  • the presenter is selected such that the presented groups A can interact with a collection of target binding sites B.
  • the composition also includes a pharmaceutically acceptable carrier.
  • the presenter itself can serve as its own pharmaceutically acceptable carrier.
  • the polyvalent presenter is made, e.g., n is selected and the -(X- A) moieties are attached to Y, such that the polyvalent presenter conforms to an interface containing a collection of target binding sites B and blankets the collection of target binding sites B upon administration to a subject.
  • X is a linker group that is an independent moiety and is not part of Y or A and n is greater than ten and is an integer.
  • Y is a polymeric framework, A is a presented functional group and X is a linker group and n is greater than ten and is an integer. This structure is selected such that the polyvalent presenter conforms to a collection of target binding sites B upon administration to a subject.
  • Another aspect of the present invention is a method of modulating adhesion between a plurality of a surface-bound biological molecule and a plurality of a second molecule.
  • the method comprises providing in combination a plurality of a surface-bound biological molecule and a polyvalent presenter.
  • the polyvalent presenter comprises a framework to which is attached a plurality of a third molecule.
  • the third molecule modulates the adhesion between the surface-bound biological molecule and the second molecule by simultaneous entropically enhanced binding of the third molecule to a portion of the plurality of the surface-bound biological molecule and by sterically blocking by the framework of a portion of the plurality of the surface bound biological molecule to which the third molecule is not bound.
  • Another aspect of the present invention is a method as described above wherein the polyvalent presenter is a non-saccharide and has an enhancement factor ⁇ greater than about 10.
  • Another aspect of the present invention is a polyvalent presenter that is pAA(Gal- ⁇ ), pAA(Gal- ), pBMA(Gal- ⁇ ) or pBMA(Gal- ⁇ ) as defined below.
  • Another aspect of the present invention is a method for preventing the adhesion of ricins to erythrocytes.
  • the method comprises contacting the ricins with an effective amount of a polyvalent presenter selected from the group consisting of pAA(Gal- ⁇ ), pAA(Gal- ⁇ ), pBMA(Gal- ⁇ ) and pBMA(Gal- ⁇ ).
  • Another aspect of the present invention is a method for preparing a polyvalent presenter selected from the group consisting of pAA(Gal- ⁇ ) and pBMA(Gal- ⁇ ). The method comprises reacting Gal- ⁇ o-L ⁇ NH 2 with poly(N-acryloyloxysuccinimide) or poly(butadiene- co-maleic anhydride and quenching the reaction.
  • Another aspect of the present invention is a method for preparing a polyvalent presenter selected from the group consisting of pAA(Gal- ⁇ ) and pBMA(Gal- ⁇ ).
  • the method comprises reacting Gal- ⁇ c-L 2 NH 2 with poly(N-acryloyloxysuccinimide) or poly(butadiene-co-maleic anhydride) and quenching the reaction.
  • Another aspect of the present invention is a polyvalent presenter that is P AA(Glc ⁇ Ac- ⁇ ).
  • Another aspect of the present invention is a method for inhibiting fertilization in a subject comprising administering to the subject an effective amount of a polyvalent presenter that is pAA(GlcNAc- ⁇ ).
  • Another aspect of the present invention is a method for preparing a polyvalent presenter that is pAA(GlcNAc- ⁇ ).
  • the method comprises reacting GlcNAc- ⁇ -L ⁇ NH 2 with poly(N-acryloyloxysuccinimide) and quenching the reaction.
  • Figure 1 is a schematic depicting both a monovalent and polyvalent reaction.
  • Figure 2 is a schematic depicting the "blanketing" of a polymeric polyvalent presenter(s) over a collection of binding sites and an array of target binding sites B.
  • Figure 3 is a schematic depicting ricin-cell interactions.
  • Figure 4 is a schematic depicting a strategy for induction of acrosome reaction of sperm.
  • Figure 5 is a schematic depicting the synthesis of polymeric polyvalent galactosides.
  • Figure 6a is a schematic depicting plots of agglutination inhibition activity of polymeric polyvalent galactoside versus mole fraction of Gal of the polymer against RCA ⁇ o.
  • Figure 6b is a schematic depicting plots of agglutination inhibition activity of polymeric polyvalent galactoside versus mole fraction of Gal of the polymer against RCA ⁇ o.
  • Figure 7 is a schematic depicting the structure of polyvalent polymeric ⁇ - acetylglucosamine and galactoside.
  • Figure 8 is a schematic depicting the induction of acrosome reactions of mouse sperm by monovalent and polyvalent Glc ⁇ Ac.
  • Figure 9a is a schematic depicting the induction of acrosome reactions of mouse sperm by polymeric polyvalent Glc ⁇ Ac.
  • Figure 9b is a schematic depicting a plot of acrosome-reacted sperm (%) obtained with pAA(GlcNAc).
  • Figure 10 is a schematic depicting a plot of the inhibition of sperm-egg binding by polymeric polyvalent GlcNAc.
  • Figure 1 la is a schematic depicting the generation of pMVMA(NeuAc).
  • Figure 1 lb is a schematic depicting the generation of pMVMA(NeuAc;R).
  • Figure 1 lc is a schematic depicting the generation of pAA(Gal).
  • Figure l id is a schematic depicting the generation of pBMA(Gal).
  • Figure 12a is a schematic depicting the generation of pAa(SLe x ).
  • Figure 12b is a schematic depicting the generation of pAA(Bacitracin;R).
  • the present invention pertains to a method for designing a polyvalent presenter for administration to a subject for treating a disease or condition.
  • the method involves constructing and arranging a plurality greater than ten of groups A on a framework forming a polyvalent presenter for treating a disease or condition.
  • the construction and arrangement is made based on the designed presenter's ability to provide a therapeutic effect due to the blanketing of a collection of target binding sites B.
  • an array of target binding sites B within the subject is blanketed with one or more of the polyvalent presenters.
  • the blanketing results from the conformal interface interaction of a polyvalent presenter with a collection of target binding sites B or the conformal interface interactions of a plurality of polyvalent presenters with collections of target binding sites B.
  • polyvalent presenter of the present invention is described in detail below and includes multi-component molecules having eleven or more presented groups A (capable of binding to eleven or more target binding sites B) attached to a framework.
  • a polyvalent presenter can have a formula as follows:
  • Y-(A) n wherein "Y” represents a framework; “A” represents a presented, functional group; and “n” represents an integer greater than ten selected such that the presented group can interact with the plurality of binding sites B.
  • the language "disease or condition” includes diseases or conditions whose treatment with the polyvalent presenters of the present invention occurs via a polyvalent interaction, e.g., the interaction of groups A with a polyvalent receptor comprising target binding sites B.
  • Diseases or conditions which are intended to be part of this invention typically can be characterized by an interaction between binding sites B and groups C, wherein both B and C are polyvalent.
  • binding sites B represent a plurality of binding sites and “C” represents a moiety or group associated with the disease or condition in nature and which interacts with B.
  • groups C can be the same as groups A, which are presented on the polyvalent presenter of the invention, leading to competition between poly (C) and poly (A) for binding sites B.
  • groups C can be different from groups A.
  • binding sites B are naturally occurring, groups C can be either synthetic or natural.
  • C can be a naturally occurring sugar, peptide, protein (e.g., a ligand for a cell surface receptor, such as a viral antigen) or can be synthetic (e.g., can be a group presented on a silicon implant or a prosthesis).
  • Groups C can be on a surface, or can be in solution, but must be polyvalently presented.
  • the language "disease or condition” also is intended to include conditions or diseases identified as involving, or being affected by, a naturally occurring polyvalent molecule, e.g., the ⁇ 2 macroglobulin molecule that inhibits interaction between the influenza virus and a target cell. While biological processes are complex, one of ordinary skill in the art would recognize whether or not a disease or condition involves a polyvalent interaction by examining whether or not the specific point at which the polyvalent presenter acts (i.e., the specific event targeted for intervention with a polyvalent presenter) was associated with an interaction between poly(C) and poly(B).
  • the term "subject” includes mammals susceptible to or having the disease or condition being treated or targeted for treatment. As such the invention is useful for the treatment of humans, domesticated animals, livestock, zoo animals, etc. Examples of subjects include humans, cows, cats, dogs, goats, and mice. In preferred embodiments the present invention is used to design drugs for treating human subjects.
  • the language "treating a disease or condition” includes the treatment of a subject having the disease or condition amenable to treatment with a polyvalent presenter of the present invention or prophylaxis of such a disease or condition.
  • treatment includes providing a beneficial effect to the subject, e.g., a significant reduction of at least one symptom or manifestation of the disease or condition. Therefore, “treatment” is meant to include anything along a continuum from amelioration of at least one of the symptoms of the disease or condition to cure of the disease or condition.
  • the treatment also is intended to include the use of a polyvalent presenter, alone, in conjunction with other polyvalent presenter(s) or even in conjunction with other treatments with agents which are not polyvalent presenters.
  • the language "constructing and arranging a plurality of groups A on a framework” includes the manipulation of various components of the polyvalent presenter to produce a presenter capable of performing its intended function in the treatment of a disease or condition.
  • the manipulations can be made based upon a viewing of the interactions) between the groups A and the collection of target sites B in a global manner.
  • the manipulation includes the positioning, sizing and selection of various components, e.g. framework, linkers, groups A, of the polyvalent presenter.
  • such language is intended to cover the positioning of the groups A and the framework relative to each other or relative to an optional linker molecule used to attach the groups A to the framework or a backbone linker used to connect monomers of the framework.
  • the language also is intended to include the selection of particular types of groups A, framework, and/or linker.
  • a framework can be selected for its ability to form a particular type of
  • frameworks over a collection of target binding sites B when it is part of a polyvalent presenter. For example, some frameworks will form gel-like physical barriers over a collection of binding sites B. A framework also can be selected based upon a different characteristic that would be desirable to have in the polyvalent presenter being designed. A framework can be selected based on its "flexibility" and/or its ability to impart flexibility to the polyvalent presenter even after attachment of groups A.
  • the groups A also can be selected based on their ability to impart desirable characteristics into the polyvalent presenter. It is important to emphasize that the binding capability of the group A is a factor in it's selection process but at least part of the present invention is the recognition that a weakly binding group A can be useful in its polyvalent form.
  • the positioning of the groups A on the framework also is intended to be part of the "constructing and arranging" of the polyvalent presenter. The positioning can be made based on the known or predicted, e.g. using molecular modeling of the polyvalent environment, spatial arrangement of a collection of target sites B. The positioning can be along several different directional axes relative to the framework.
  • the target sites B may be spaced an average of 10 angstroms apart from their neighboring target binding sites B and therefore the groups A can be spaced or positioned along the framework in the horizontal direction appropriate for providing access to neighboring target binding sites B.
  • the groups A do not have to be positioned to match the distance between neighboring target binding sites but rather are spaced appropriately to provide access.
  • Other factors besides distance are considered when positioning the groups A on the framework such as the flexibility of the linker and the contour of the interface containing the target binding sites.
  • a flexible linker can make adjustments in vivo for providing access to the target binding sites even if the distance between the groups A does not exactly match the distance between the neighboring target binding sites B or if the contour of the interface is curved rather than flat.
  • the depth of the binding pocket also may be known or predictable allowing for the positioning of the groups A along an axis substantially perpendicular to the framework, e.g., a particular length of linker can be used.
  • the "constructing and arranging" also is intended to include the selection of a type and length of linker that attaches the groups A to the framework.
  • the length of the linker can be selected based on its ability to present the group A to a target binding site B within a binding pocket having a known or predicted depth and/or diameter.
  • the chemical nature of the linker e.g. hydrophobicity or hydrophilicity, also can be selected based on knowledge regarding the environment surrounding the target binding site, e.g. the linker may have to pass through a channel or environment known to be hydrophobic or hydrophilic.
  • the linker also may be selected based on its ability to impart a desired property into the polyvalent presenter, e.g. flexibility.
  • therapeutic effect is intended to include the ability of the polyvalent presenter to perform its intended function, e.g. treat or prevent the disease or condition.
  • the therapeutic effect of the polyvalent presenter can be measured using an art- recognized assay for the particular disease or condition targeted for treatment by the polyvalent presenter being designed or by using an assay designed using art-recognized techniques and/or by an assay described herein which would not fit into one of these categories.
  • the therapeutic effect is considered “due to the blanketing" when the effect is derived from the ability of the polyvalent presenter to conform to and engage in multivalent binding with the collection of target binding sites B.
  • blanketing includes both physical blanketing or the physical covering up of the target binding sites B and steric blanketing, e.g., the steric smothering of target site(s) B and/or by enhancing the occupancy of site B by ligand A.
  • Examples of physical blanketing include the formation of a gel-like layer or barrier over the collection of binding sites B which prevents access to such sites by groups other than those present on the polyvalent presenter.
  • the physical barrier also can be the spaghetti-like barrier depicted in Figure 2. In either case the formation of a gel-like layer or barrier or a spaghetti-like barrier results biospecifically by the specific binding of at least some of the groups on the polyvalent presenter with a corresponding binding partner on the target.
  • the "steric blanketing" of a target binding site B is the surrounding of the binding site B with groups A attached to a framework in a velcro-like manner.
  • a group A can be bound to the binding site but other group A's can be sterically blocking the same binding site B or a neighboring target binding site.
  • selection of target binding sites B includes the binding sites B over which a single polyvalent presenter molecule conforms. For example, if a polyvalent presenter has ten groups A attached to a framework then the collection of binding sites would be that span of binding sites B over which this molecule conforms, e.g., ten to twenty binding sites although only as many as two binding sites may actually be occupied. It should be noted that not all binding sites B have to interact with a group A and some sites may be left unoccupied.
  • an array of target binding sites B includes more than one collection of target binding sites B on an interface.
  • the binding sites B within the array do not have to be equally spaced or positioned but rather can be randomly positioned in various directions along different directional axes relative to the interface, e.g., depending on such factors as the contour of the interface and the clustering configuration of the binding sites.
  • the contour of the interface can be flat or can be curved and also can be mobile.
  • the language “conformal interface interaction” includes an interaction which occurs while a polyvalent presenter is in a conformed configuration over a collection of target binding sites B at an interface, e.g., the molecular region accessible to groups A.
  • the interface can be, but is not limited to a surface, but is intended to include the boundary of interaction.
  • conformal is intended to include situations wherein the interface between the polyvalent presenter and the surface is close or tight with respect to all or a part of the surface. In the latter instance the polyvalent presenter may be close to the surface in some parts and less close in other parts of the surface.
  • interaction is intended to include both positive and negative interactions as discussed in detail below.
  • the polyvalent presenters of the present invention conform to an interface, e.g., a surface such as a cell surface, by being flexible enough to adjust to the contour of the interface. This conformation can be along a span of a particular number of target binding sites B.
  • this span is at least about 50, at least about 100, at least about 1000, up to about at least about 10 ⁇ target binding sites B.
  • Ranges intermediate to the above recited values e.g., at least about 50 to about 1000, or at least about 1000 to about 10 ⁇ , also are intended to be part of this invention.
  • ranges of span values using a combination of any of the above- recited values as upper and/or lower limits are intended to be included. The above ranges are intended to include both sites within a collection of target binding sites B and sites within an array of target binding sites B.
  • interaction is intended to include both negative interactions, e.g., inhibitory, e.g., influenza, and positive interactions, e.g., interactions that result in the transduction of a signal or an interaction that brings two surfaces together.
  • the polyvalent presenter can bind to a collection of target binding sites B, e.g., on a cell surface, and transduce a signal within a cell or induce the cell to expend energy.
  • effects of such an interaction include apotosis (programmed cell death), clonal expansion, e.g., B- cells or T-cells multiplication, cell migration, e.g., movement of a cell in some direction, release of soluble molecules such as hormones, e.g., insulin, cytokines, e.g., 112; prostaglandins, endocytosis or pinocytosis or exocytosis, active transport (inward or outward) of some substance, induction of electrical activity, or differentiation or dedifferentiation of a particular cell. Modulation or treatment of all such effects with a polyvalent presenter is intended to be part of this invention. In some embodiments, the interaction is not anti-adhesive.
  • steric stabilization refers to a mechanism by which a polyvalent presenter sterically inhibits the close approach of two surfaces by binding biospecifically to one of the surfaces. This is distinguished from stabilization resulting from the addition of water-swollen polymers to colloidal mixtures, which is utilized to keep colloidal particles in solution. In such cases, the molecule binds non-specifically to the surface to be stabilized. See, for example, A. Sung and I Piirma "Electrosteric stabilization of polymer colloids" Langmuir 1994, 10, 1393-8; U Genz, B D'Aguanno, J Mewis and R Klein “Structure of sterically stabilized colloids” Langmuir, 1994, 10, 2206-12.
  • the present concept is also distinguished from grafting polymers to the surface of colloids or liposomes.
  • steric stabilization is attained by covalent attachment of a molecule to the surface to be stabilized.
  • Polyetheylene glycol is a prototypical example. See, for example, 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.
  • polyvalent presenter includes multi-component molecules having eleven or more presented groups A, which are capable of binding to corresponding target binding sites B, and which groups A are attached to a framework.
  • a polyvalent presenter of the present invention can be exemplified by the following structure:
  • a polyvalent presenter of the present invention can be represented by the following structure:
  • Y represents a framework
  • A represents a presented, functional group
  • X represents an optional linker group which can be used to attach groups A to the framework Y
  • n represents an integer greater than ten selected such that the presented group can interact with the plurality of binding sites B.
  • the integer “n” is selected such that a sufficient number of groups A are presented for treatment of a disease or condition.
  • the integer “n” is further selected to allow the polyvalent presentation of a plurality of groups A.
  • the integer "n” is greater than ten, preferably greater than ten to about 10 ⁇ , more preferably about 50 to about 10 ⁇ , about 100 to about 10 ⁇ , or about 1000 to about 10 ⁇ .
  • n values intermediate to those listed also are intended to be part of this invention, e.g., greater than ten to about 100, greater than ten to about 1000, about 100 to 1000, and about 1000 to 100,000.
  • ranges of "n” values using a combination of any of the above values recited as upper and/or lower limits are intended to be included.
  • the language "polyvalent presentation” or “polyvalent manner” is art- recognized and includes the polyvalent display of A, B, or C, such that the poly A, B, or C functions differently than its monovalent equivalent.
  • polyA may show a biological effect that is either positively cooperative or negatively-cooperative or non- cooperative from monoA.
  • polyvalent presenters may act independently of cooperativity as demonstrated by the ⁇ factor as explained more fully below.
  • Polyvalent presentation or polyvalent manner includes the use of polyvalent presenters which differ from the slow release compounds or drug delivery systems known in the art. Polyvalent presenters are designed to function polyvalently, not to release monovalent groups that mediate the drugs pharmacological activity. In addition, because of the release and diffusion of monovalent groups, slow release compounds typically act at a site that is different from the site of administration and this does not have to be true in the case of polyvalent presenters.
  • framework includes a support structure or backbone made of a material having a molecular weight greater than about 10,000 to which, a plurality of groups A can be attached and can be presented polyvalently.
  • the attachment can be by a means that allows the polyvalent display of the group on the framework.
  • groups A are attached to a framework prior to administration to a subject.
  • groups A are administered to a subject and assembled on a framework in vivo to form a polyvalent presenter of the invention, e.g., by self assembly.
  • the framework component must be of sufficient mean hydrodynamic radius to span the distance between adjacent receptors, and will be 100 angstroms or greater.
  • the "backbone" of a framework can further comprise linkers (hereinafter backbone linkers) for joining monomer units of the framework together. Specific backbone linkers will be discussed in detail below in the context of polymeric frameworks.
  • the backbone linkers are cleavable, e.g. the backbone linkers are hydrolytically labile.
  • the types of frameworks useful within the polyvalent presenter are those capable of having groups A attached thereto and capable of polyvalently presenting the groups A.
  • the frameworks used for an in vivo purpose also are suitable for in vivo administration or are available in vivo for assembly.
  • types of frameworks include, but are not limited to, polymers, liposomes, micelles, colloids, dendrimers, and biological particles. These types are discussed briefly below and in more detail even further below under the description of covalent and noncovalent frameworks. The detailed description of each of these frameworks are provided under the headings of covalent and noncovalent frameworks only for ease of discussion and should not be construed as limiting the scope of frameworks. It should be understood that the present invention is intended to encompass all types of frameworks capable of polyvalently presenting groups A as described herein.
  • polymer or “polymeric” are art-recognized and include a structural framework comprised of repeating monomer units. To qualify, the polymeric framework must further be capable of polyvalently presenting "A" groups such that the treatment of a disease or condition occurs.
  • copolymers and homopolymers e.g., synthetic or naturally occurring. Linear polymers, branched polymers, and cross-linked polymers are also meant to be included. In a preferred embodiment, the polymer is not dextran.
  • liposome liposome
  • micelles and “colloids” are art-recognized. These terms also include derivatized versions, e.g., liposome derivatives, cross-linked liposomes and the like.
  • dendrimer is art-recognized. Dendrimers include a specific subclass of branched polymers that possess multiple generations. In dendrimers, each generation creates multiple branch points. In a preferred embodiment the framework is not a dendrimer. In certain embodiments, frameworks of the present invention can comprise
  • biological particles includes both covalent molecules, e.g., sugars, proteins, lipids, small molecules, protein aggregates, and nucleic acids, and noncovalent particles, e.g., modified cells (e.g., which have been derivatized, modified chemically or transfected with an exogenous nucleic acid) or modified viruses, e.g. viral particles.
  • modified cells e.g., which have been derivatized, modified chemically or transfected with an exogenous nucleic acid
  • modified viruses e.g. viral particles.
  • the use of “biological particles” as frameworks is distinguished from such particles as they occur in their natural state because the subject frameworks are modified to polyvalently present functional groups A.
  • the monomeric units of a framework can be joined covalently.
  • covalent frameworks include crosslinked liposomes, biological particles (e.g., sugars, proteins, peptides, lipids, or small molecules), and polymers, (see e.g., Siraganian, R. P., et al, Immunochem. 1975,12, 149-155; Wofsy, C, et al, J. Immunol. 1978,727, 593-601; Barlocco, D., et al, Farmaco ⁇ 993.48. 387-96; Castagnino, H. E., et al, Jpn.
  • proteins e.g., albumin can be used as a system for presenting large numbers of groups; (Roy, R; Lafemere, C. A. Can. J. Chem. 1990, 68, 2045-2054) thus mimicking natural glycoproteins.
  • polymers can be used as the framework for a polyvalent presenter.
  • Polymers are a versatile framework system. (Spaltenstein et al. 1991, J. Am. Chem. Soc. 113:686; Mammen et al. 1995. J. Med. Chem. 38:4179).
  • the groups A of the present invention are attached to a framework comprising a polymeric backbone by a linker group.
  • reactive polymers can be used in a "framework" of the present invention as described in more detail below.
  • Polymers can be prepared using methods known in the art (Sandier, S. R; 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). Polymers can be designed to be flexible; the distance between the bioactive side chains and the length of a linker between the polymer backbone and the group can be designed and controlled.
  • Polymers provide a number of advantages as a framework. They can be designed to allow multiple groups A to bind simultaneously to multiple group B binding sites with minimal unfavorable strain. Polymeric, polyvalent presenters are easily, rapidly and convergently synthesized (Spaltenstein et al. 1991, J. Am. Chem. Soc. 113:686; Mammen et al. 1995. J. Med. Chem. 38:4179). Polymers also allow modulation of various physical properties of the presenter, for example, conformal flexibility; solubility; hydrophilicity, and the modulation of conformation and flexibility in solution through variations in temperature and ionic strength.
  • high molecular weight polymers are well-developed science, and organic polymers provide a very important class of compounds to use for polyvalent presentation. These compounds have high molecular weights, and can present very large numbers of copies of the group; they can present more than one group simultaneously; their transport across biological membranes is typically limited, and, thus, their lifetime in particular compartments can be controlled in vivo.
  • Polymeric frameworks offer a variety of easily synthesized macromolecules, and access to a range of biological activities.
  • modified polymeric materials for use in the present invention have low antigenicity and low toxicity.
  • polymeric frameworks can be selected to be compatible with water, will be capable of having varied molecular weight, and will be capable of having a range of different groups attached to the polymer backbone.
  • Polymer backbones of the present invention may also be selected for ease of synthesis.
  • Intrinsically biocompatible polymers containing functional groups appropriate for the addition of side chains are preferred (Shalaby, W.; Ikada, Y.; Langer, R; Williams, J. Polymers of Biological and Biomedical Significance (ACS Symposium Series 540); American Chemical Society: Washington, DC, 1994).
  • Exemplary polymers include polyethylene oxide or polyethylene glycol (Harris, J. M. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications.; Plenum: New York, 1992; Horton, D.
  • poly(ethylene glycol), poly(lactic acid), poly(glycolic acid), and poly(vinyl pyrrolidone) can be used.
  • Preferred polymers for use in the synthesis of polyvalent presenters contain reactive groups, such as activated carboxylic acids.
  • a number of synthetic and naturally occurring polymers contain carboxylic acid functionality or are capable of being modified with such, and these have been used in vivo.
  • Such polymers are capable of forming covalent linkage with a presented group A, e.g., an amide bond.
  • Polymers containing internally cyclized carboxylic acid functionality are especially desirable.
  • Other preferred polymers include subunits derived from maleic anhydride or malic acid.
  • Exemplary copolymers include styrene-maleic anhydride and alpha-olefin-maleic acid copolymers (such as divinylether-maleic acid).
  • sodium carboxymethylcellulose, chondroitin sulfate and poly (methacrylate/acrylate) materials can be used.
  • polymers without activated carboxylic acids can be used, e.g., dextran sulfate.
  • Other exemplary polymeric frameworks include: poly(ester), poly(anhydride), poly(carbohydrate), polyols, poly(acrylate), poly(methacrylate), poly(ether) and poly(amino acid)
  • Still other exemplary polymeric frameworks include: poly(glutamic acid), poly(aspartic acid), dextran, dextran sulfate, poly(maleic anhydride-co-vinyl ether), poly(succir_i_nide), poly(acrylic anhydride),poly(ethylene glycol), poly(lactic acid), poly(glycolic acid), poly(vinyl pyrrolidone), poly(styrene-maleic anhydride), alpha-maleic acid, hyalauronic acid, sodium carboxymethylcellulose, chondroitin sulfate, poly(acrylate), poly(acrylamide), poly(glycerol), and starch.
  • any polymeric material that is capable of presenting a plurality of groups A may be suitable for use in the present invention.
  • Polymers can be modified, e.g., as described above or by derivatization, e.g., with bifunctional cross-linking reagents, to provide functionalities suitable for attaching and presenting A groups, as described in more detail below. In certain situations copolymers may be preferred.
  • Backbone linkers In certain embodiments it will be desirable to include connecting moieties, "linkers" between monomeric units of the backbone.
  • Exemplary “backbone linkers” can include hydrocarbon, carbamate, amide, ether, thioester, thioether, carbonate, and ester connections.
  • the backbone moieties can be linked with a cleavable linker.
  • the lifetime of the polyvalent material in vivo may in part depend on its molecular dimensions. By placing linkers groups between medium-sized oligomers, and by controlling the in vivo stability of these linking groups, the lifetime of the polymers in vivo can be controlled.
  • non-functionalized, degradable connectors in the polymer backbone can, therefore, be used to assist clearance of the polymer.
  • the cleavable linker will, in general, be different from that which is used to link the polymer backbone to the group A. Such a cleavable linkage will cause the formation of smaller, polymeric functionalized fragments that will be small enough to clear through the kidney.
  • Degradable linkers can include, e.g., hydrolytically labile linkers including ester, carbonate or oxalate groups.
  • the framework of a subject polyvalent presenter can comprise a dendrimer.
  • Dendrimers are art-recognized and include a class of low molecular weight, highly branched, polymers, e.g. often monodisperse. Dendrimers have the advantage that they are of well-defined molecular structure, and that their solutions are relatively non- viscous for their molecular weight. They can present groups in a densely packed fashion relative to a linear polymer, which can be advantageous for targeting receptors that cluster on cell surfaces.
  • the number of groups A can be precisely controlled in a dendrimer, as well as the regulation of the inter-group distance, conformation and rigidity of the dendrimer molecule.
  • Polysaccharides may belong to a class of dendrimer Sabesan, et al, 1992 J. Am. Chem. Soc. 14:8636.
  • the head groups of a dendrimer (Roy, R, et al, Synthesis and Antigenic Properties of Sialic Acid-Based Dendrimers; Acs Symp. Ser 1994) can be used as a framework.
  • Noncovalent Frameworks A plurality of groups A also can be joined to a non-covalent framework.
  • Exemplary noncovalent frameworks include liposomes, micelles, colloids, protein aggregates, modified cells, and modified viral particles.
  • groups A can be tethered to the head groups of molecules in liposomes, membranes, or surfaces (Kingery-Wood, J. E., et al, J. Am. Chem. Soc. 1992,114, 7303-7305; Spevak, W., et al, J. Am. Chem. Soc. 1993, 775, 1146-1147; Spevak, W., et al, J. Med Chem. 1996, 39, 1018-1020.).
  • Liposomes and micelles are art-recognized and include macroscopic particles made up of aggregates of lipids, e.g. surfactants.
  • the polyvalent presenter can present groups on a liposome or micelle (Spevak, W., et al, J. Am. Chem. Soc. 1993, 775, 1146-1147; Charych, D., et al, Chem. ⁇ Biol. 1996, 3, 113-120). This system mimics closely the shape of the target cell, and can be designed to present a surface that closely matches that of the target cell in group type and group density.
  • lipid molecules containing groups A can be reconstituted into liposomes.
  • Liposomes have favorable biocompatibility; and are fairly easy to synthesize.
  • liposomes can be designed to act as sensors.
  • Polymerized liposomes can be used to sense the conformational change of the liposome by displaying the change in UV/vis absorption of the internal chromophore (e.g., a cross-linked polydiacetylene) of the membrane.
  • the binding of virus to the liposome could be detected by the shift in color (blue to red) as is art-recognized.
  • biological particles including, for example, modified cells or modified viruses can be used as framework for polyvalent presentation of the groups A.
  • proteins, peptides, polysaccharides, fragments of cell membranes, or modified intact cells (for example, erythrocytes), modified bacterial cells or modified viruses can be used as framework in certain embodiments.
  • the term "activated framework” refers to the framework component as described above, including both covalent and noncovalent framework components, that contain functionality which can be activated, by means of an "activating group,” and subsequently reacted with at least one functional group, ancillary group and/or spacer group.
  • Appropriate functionality includes, for example, carboxyl (acid form and salts), hydroxyl, sulfhydryl, amide, carbamate, amino, ketone, aldehyde, olefin, aromatic, etc.
  • the polymers may be activated prior to exposing them to the functional group (“preactivation"), or may be activated in the presence of the functional group ("in situ”).
  • the activation step can entail derivatizing the polymer with groups capable of undergoing reactions with nucleophiles or electrophiles. Further, it is within the scope of the present invention to activate polymers such that they are able to participate in dipolar additions (e.g., 1,3- and 1,4-dipolar addition), cycloaddition reactions (e.g., Diels-Alder type reactions) and polymerization reactions by cationic, anionic or radical initiated mechanisms.
  • dipolar additions e.g., 1,3- and 1,4-dipolar addition
  • cycloaddition reactions e.g., Diels-Alder type reactions
  • polymerization reactions by cationic, anionic or radical initiated mechanisms.
  • Carboxyl groups may be activated for reaction with nucleophiles by the use of, for example, cyclic or linear anhydrides, activated esters (e.g., N-hydroxysuccinimide, nitrophenol, 4-hydroxy-3 -nitrobenzene sulfonic acid, etc.), acid chlorides, imidazolides (e.g., from carbonyldiimidazole), carboxylic acid and esters.
  • activated esters e.g., N-hydroxysuccinimide, nitrophenol, 4-hydroxy-3 -nitrobenzene sulfonic acid, etc.
  • acid chlorides e.g., imidazolides (e.g., from carbonyldiimidazole), carboxylic acid and esters.
  • Carboxylic acid containing polymers may also be activated by forming adducts between the carboxyl group and agents such as, dicyclohexylcarbodiimide, 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide, alkyl chloroformates, chlorosilanes, pyridinium salts and Bu 3 N, etc.
  • agents such as, dicyclohexylcarbodiimide, 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide, alkyl chloroformates, chlorosilanes, pyridinium salts and Bu 3 N, etc.
  • Hydroxyl groups may be activated by the use of carbonates formed by reaction with, for example, alkyl or acyl haloformates (e.g., /so-butylchloroformate, p- nitrophenylchloroformate, etc.), cyanogen bromide or phosgene.
  • alkyl or acyl haloformates e.g., /so-butylchloroformate, p- nitrophenylchloroformate, etc.
  • cyanogen bromide or phosgene e.g., cyanogen bromide or phosgene.
  • oxidation using periodate compounds can be used to provide reactive carbonyl moieties on the polymeric backbone. Additional methods of activating polymers bearing hydroxyl groups will be apparent to those of skill in the art.
  • Polymers bearing sulfhydryl groups can be activated using dithiobispyridyl compounds such as, for example, 2,2'-dithiobis(5-nitropyridine), 2,2'-dithiobis(pyridine), etc. Additional methods of use in activating sulfhydryl-bearing polymers will be apparent to those of skill in the art.
  • the polymeric framework Once the polymeric framework is activated, it can be reacted with at least one functional group, ancillary group or spacer group or a mixture thereof. Alternatively, the activated polymer can be reacted with a combination of functional groups, ancillary group and/or spacer groups.
  • the functional group component of the polyvalent presenter prepared in accordance with the methods of the present invention includes those groups capable of being attached to a framework component, an ancillary group and/or a spacer group, and of being polyvalently presented in a functional manner, e.g., for treating a disease or condition.
  • the functional group components can be the same or different.
  • the language "a plurality of functional group components" or, alternatively, "a plurality of group R 3 " is intended to cover more than one functional group component, wherein each functional group within the plurality is independently the same or different.
  • the functional group can be the same or different within categories of types of functional groups, e.g., one functional group can be a carbohydrate and another functional group can be an antibiotic.
  • the functional groups can also be different within the same type or category of functional groups, e.g., two different carbohydrates can be functional groups. In the case of a "homomeric presenter,” the functional groups presented are the same, whereas in the case of a “heteromeric presenter,” the functional groups presented are different, e.g., from different categories or different within the same category.
  • R 3 ⁇ to R 3 n will be used to denote various members of a plurality of R 3 .
  • the functional groups presented by the subject polyvalent presenters are functional.
  • the functional groups also provide their function when attached to the framework components of the polyvalent presenters.
  • the functional groups provide function by biospecifically interacting with eleven or more (i.e., a plurality of) binding sites B to, for example, directly provide a therapeutic effect.
  • the polyvalent presenter is, for example, a heteromeric presenter
  • certain of the functional groups may not interact with B, but instead provide another function.
  • the functional groups may influence the interaction of R 3 ⁇ to binding site B and, thus, function as an enhancer group.
  • the functional groups can be a functional auxiliary group (e.g., an ancillary group) that is capable of influencing the physical characteristics of the polyvalent presenter, e.g., the solubility or ability to cross cell membranes.
  • the functional groups can provide function by allowing for tracking of the subject presenters, for example, by providing a label that can be detected (e.g., a fluorescent or radioactive tag).
  • the functional group components of the present invention can be either synthetic or natural.
  • the functional group components can be categorized based on physical characteristics, e.g., molecular size, i.e., the functional group can be of low, medium or high molecular weight.
  • natural functional group components include, but are not limited to, naturally occurring sugars, proteins (e.g., IgE or erythropoeitin), peptides and other known drugs.
  • Examples of synthetic functional group components include, but are not limited to, peptide mimetics, functional groups which are synthesized by combinatorial chemistry techniques or rational drug design techniques. Specific types of functional group components which can be used in accordance with the methods of the present invention are discussed in detail below under the heading "Types of Functional Groups".
  • the groups A of the polyvalent presenter include groups capable of being polyvalently presented in a functional manner, e.g., for treating a disease or condition, and being on, e.g., attached to, a framework.
  • the groups A can be the same or different.
  • the language "a plurality of groups A" is intended to cover more than one group A wherein each group A within the plurality is independently the same or different.
  • the groups A can be the same or different within categories of types of groups A, e.g., one group A can be a carbohydrate and another group A can be an antibiotic.
  • the groups A also can be different within the same type or category of group A, e.g. two different carbohydrate group A's.
  • the A groups presented are the same and in the case of a
  • heteromeric presenter the A groups presented are different, e.g., from different categories or different within the same category.
  • nomenclature Ai to A n will be used to denote various members of a plurality of groups A.
  • groups A presented by the subject presenters are functional.
  • the groups A also provide their function when attached to or on a framework of a polyvalent presenter. As discussed above and reiterated here, this differs from the art- recognized drug delivery systems, e.g. polymeric drug delivery systems, which release their therapeutic agents and wherein the therapeutic agents provide a function in released form.
  • groups A provide function by biospecifically interacting with eleven or more, i.e., a "plurality of binding sites B, e.g., to directly provide a therapeutic effect.
  • certain of the groups may not interact with B, but instead provide another function.
  • a n may influence the interaction of A ⁇ to binding site B, and thus function as an enhancer group.
  • a n can be a functional auxiliary group which is capable of influencing the physical characteristics of the polyvalent presenter, e.g., solubility or ability to cross cell membranes.
  • groups A can provide function by allowing for tracking of the subject presenters, for example, provide a label which can be detected, e.g., a fluorescent or radioactive tag.
  • the groups A of the present invention can be either synthetic or natural.
  • groups A can be categorized based on physical characteristics, e.g., molecular size, e.g., groups A can be of low, medium, or high molecular weight.
  • natural groups A include naturally occurring sugars, proteins (e.g., IgE, or erythropoeitin), peptides, or other known drugs.
  • synthetic groups include, e.g., peptide mimetics, groups that are synthesized by combinatorial chemistry, or generated through rational drug design.
  • the groups A are not selected from the group consisting of carbohydrate, a polymyxin, e.g., polymyxin B, beta-lactam, a furan-derived compound, a pyran-derived compound, and antibody, alone or in combination with any other member of the group.
  • the groups A are not derivatives or fragments of the aforementioned groups A. Specific types of groups A are discussed in detail below under the heading "Types of Groups A". Number of groups A per presenter
  • Various physical properties of the framework can influence the ability of polyvalent presenters to modulate interactions between groups A and binding sites B.
  • the effectiveness of polymeric presenters can be altered, i.e., can be made to either increase or decrease.
  • large number of attachment points can cause the presenter backbone to collapse onto the surface on which binding sites B are displayed and become less effective in steric stabilization but more effective in competitive Blocking of target sites and/or agonism/antagonism of biological responses.
  • Heteromeric presenters which present multiple, different groups. Heteromeric presentation can be used to provide both greater strength and specificity than equivalent monovalent interactions. For example, by presenting an additional type of group (e.g., A2), the total number of interactions is increased, then the total strength of the interaction may increase.
  • the specificity of the interaction can increase by differentially regulating the number of groups of A ⁇ and A2 that are presented. For example, a presenter bearing both A ⁇ and A2 may interact more tightly with to B ⁇ and B2 than a presenter bearing A ⁇ alone or A2 alone.
  • a polyvalent presenter may present groups
  • Bi and B 2 are on the same pathogen.
  • the groups A of the present invention include groups useful for treating a disease or condition when presented in a polyvalent manner. These groups can be known groups or drugs or can be novel groups or drugs selected after studying a polyvalent interaction involved in a particular disease or condition.
  • the present invention provides for the use of known groups A which have been described, (Kiessling, L. L.; Pohl, N. L. Chem. &Biol 1996, 3, 71-77) and combinatorial methods of developing new groups, and of enhancing the effectiveness of known monomeric groups by presentation in polyvalent form. It is recognized that some applications for the polyvalent presenters may be more favored than others due to more facile access to the site of action. It should be understood that less facile access may be addressed by more direct administration to the site of action.
  • groups known to be involved in cell-pathogen interaction, cell-cell interaction, pathogen-extracellular matrix interaction, and pathogen- pathogen interaction can be presented in a polyvalent manner on the subject presenters. For example, as shown in the appended example and described in the
  • N- Acetyl Neuraminic acid (a sialic acid), which is the natural binding site for influenza hemagglutinin.
  • the interaction between this sugar and its lectin is the first essential step in influenza viral attachment to its target cell.
  • a very large number of polyvalent sugars are possible groups, including Neu5Ac(2,6)Galactose, Neu5Ac(2,6)Lactose, NeuAc(a2,3)Gal(bl,3)GaINAc, heparin sulfate, and galatosyl ceramide, which have all been shown to be important in the attachment of different viral particles to host cells.
  • groups known to modulate other diseases or conditions involving polyvalency can be employed on the subject presenters.
  • groups A can be selected for their ability to mediate the polyvalent interactions between platelets in situations where it is desirable to treat thrombosis.
  • the subject presenters can comprise known drugs or compounds which have been shown not to be significantly effective in treating a disease or condition when administered in their monovalent form.
  • the drug becomes significantly effective when presented polyvalently in accordance with the present invention. Therefore, the present invention provides for numerous, existing drugs and non- drug groups that can be incorporated into the subject presenters as Groups A. It will be understood by one of ordinary skill in the art that groups A may have more than one biological effect, and/or may be useful in the treatment of more than one type of disease or condition. It also should be understood that some groups A will be useful for treating diseases or states involving a polyvalent interaction and/or some groups A will be useful for treating diseases or states not previously identified as involving polyvalent interactions. The term "drug" is used below to refer to possible groups A for ease of discussion.
  • drugs that have effects in the central nervous system are provided for.
  • the treatment of Alzheimer's disease can employ, e.g., tacrine or donepezil as groups A.
  • treatment of alcohol dependence can employ disulfiram as groups A.
  • Treatment of acute and/or chronic pain and/or inflammation can employ analgesics as groups A (e.g., acetaminophen, aspirin, ibuprofen, naproxen, pentazocine, indomethacin, or diflunisal) or anesthetics (e.g., ropiacaine or remifentanil).
  • analgesics e.g., acetaminophen, aspirin, ibuprofen, naproxen, pentazocine, indomethacin, or diflunisal
  • anesthetics e.g., ropiacaine or remifentanil
  • Treatment of pain and/or narcotic dependence can employ, e.g., hydrocodone, propoxyphenemeperidine, hydromorphone, morphine, methadone, or oxycodone as groups A.
  • anesthetics is also provided for, such as: epinephrine, xylocaine, mepivacaine, methohexital, bupivacaine, and novocaine.
  • the subject polyvalent presenters can also use cholinesterase inhibitors as groups A, such as, for example, pyridostigmine or neostigmine.
  • Hypnotics can also be incorporated into the subject presenters.
  • Groups A can also be antitussives, for example, pseudoephedrine or codeine.
  • the present invention also provides for the use of anti- migraines as groups A, such as ergot derivatives (e.g., ergotamine or methysergide) or sometheptene, or serotonin (5-HT) antagonists e.g., sumitriptan.
  • Groups A can also be motion sickness remedies, such as meclizine or scopalamine.
  • groups A can also be muscle relaxants, such as: pyridostigmine, neostigmine, succinylcholine, mivacurium, doxacurium, rocuronium, vecuronium, dantrolene, cyclobenzaprine, baclofen, chlorzoxazone, methocarbamol, or papaverine.
  • Nausea can be treated with the use of, e.g., prochlorperazine, chlorpromazine, trimethobenzaminde, perphenazine, hydroxyzine, or ondansetron.
  • Parasympatholytics can also be used as groups A, such as biperiden, phenobarbital, ergotamine, dicyclomine, hyoscyamine, glycopyrrolate.
  • groups A such as biperiden, phenobarbital, ergotamine, dicyclomine, hyoscyamine, glycopyrrolate.
  • the parasympathomimetics e.g., tacrine, pilocarpine, bethaneechol, edrophonium, yohimbine can be used.
  • Groups A can also comprise parinsonism drugs, e.g., trihexyphenidyl, bentropine, procyclidine, levodopa, bromocriptine, carbidopa, amantadine.
  • the invention also provides for the use of psychotropics in the subject presenters.
  • antianxiety agents e.g., lorazepam, buspirone, chlordiazepoxide, meprobamate, clorazepate, diazepam, and alprazolam
  • Antidepressants e.g., phenelzine, tranylcypromine, parozetine, fluoxetine, sertraline, amitriptyline, nortriptyline, imipramine, or protriptyline can be used.
  • Antipsychotics e.g., clozapine, prochlorperazine, haloperidol, loxipine, thioridazine, flupenazine, risperidone, mesoridazine, trifluoperazine, olanzapine, or chlorpromazine
  • the use of psychostimulants e.g., pemoline or methylphenidate
  • Sedatives e.g., mephobarbital, secobarbital, or temazepam
  • groups A can also be used as groups A.
  • Seizure disorders can be treated by polyvalent presenters bearing, e.g., felbatol, gabapentin, phenytoin, mephenytoin, ethotoin, lamotrigine, methsuximide, phensuximideethosuximide, cabamozepine, phenacemide, or cabamazepine.
  • Sympatholytics such as, e.g., phentolamine can also be used.
  • Groups A can also include, e.g., anticonvulsants (e.g., fosphenytoin), antidepressants (e.g., mirtazapine) groups which can be used to treat multiple sclerosis (e.g., glatiramer) or epilepsy (e.g., topiranate), or groups which can be used to inhibit angiogenesis (e.g., art-recognized angiogenesis inhibitors).
  • anticonvulsants e.g., fosphenytoin
  • antidepressants e.g., mirtazapine
  • angiogenesis e.g., art-recognized angiogenesis inhibitors
  • adrenergic agents such as doxazosin, terazosin, prazosin, methyldopate, clonidine, labetalol.
  • angiotensin converting enzyme inhibitors such as captopril, lisinopril, tradolapril, or enalapril can be used.
  • Polyvalent presenters can also be made to present angiotensin II receptor antagonists, such as losartan or valsartan.
  • antiarrhythmics is also provided for; examples include: disopyramide, procainamide, quinidine, propafenone, flecainide, tocainide, propanolol, sotalol, amiodarone, digoxin.
  • ⁇ -Blockers such as timolol, metoprolol, and atenolol can be used as groups A.
  • calcium channel dockers can be employed as presented groups. Examples include nifedipine, nicardipine, diltiazam, felodipine, and verapamil.
  • Diuretics e.g., acetazolamide, ethacrynic acid, furosemide, spironolactone, amiloride, chlorothiazide, and hydrochlorothiazide
  • Diuretics can also be used as groups A.
  • Vasodilators e.g., papaverine, hydralazine, and amrinone
  • vasopressors e.g., metaraminol, phenylephrine, or isoproterenol
  • groups A can comprise e.g., mecamylamine.
  • Groups A can be a hypolipidemic, e.g., clofibrate, gemfibrozil, simvastatin, lovastatin, simvastatin, atorvastatine, or niacin.
  • Deep vein thrombosis can be treated by the use of e.g., danaparoid as groups A.
  • antihypertensives such as midodrine can be used.
  • groups A can include, for example, anti- androgens (e.g., leuprolide or flutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, ⁇ 2-interferon) anti-estrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate, mercaptopurine, thioguanine).
  • anti- androgens e.g., leuprolide or flutamide
  • cytocidal agents e.g., adriamycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, ⁇ 2-interferon
  • anti-estrogens e.g., tamoxifen
  • antimetabolites e.g., fluorouracil
  • Groups A can also comprise hormones (e.g., medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide, or somatostatin).
  • groups A can include, e.g., irinotecan, gemcitabine, toptecan, nilandrone, or docetaxel.
  • the subject presenters can be used in gastrointestinal applications.
  • antispasmodics or anticholinergics can also be included as groups A (e.g., dicyclomine, hyoscyamine, glycopyrraolate, trihexyphenidyl or atropine).
  • Appetite suppressants can also be used as groups A (e.g., dextroamphetamine, benzphetamine, phente ⁇ nine, and ormazindole.
  • groups A can also include anti- diarrhea agents (e.g., loperamide, diphenoxylate, octreotide.
  • the subject presenters can also include, e.g., proton pump inhibitors (e.g., lansoprazole or omeprazole) or vasodilator, e.g., opioids.
  • proton pump inhibitors e.g., lansoprazole or omeprazole
  • vasodilator e.g., opioids.
  • Groups A that modulate the endocrine system can also be polyvalently presented.
  • contraceptives e.g., ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone.
  • Groups A that modulate diabetes can also be used (e.g., glyburide or chlorpropamide).
  • testolactone or stanozolol can also be incorporated into the subject presenters.
  • Androgens e.g., methyltestosterone, testosterone or fluoxymesterone
  • Antidiuretics e.g., desmopressin
  • the subject presenters can also display e.g., calcitonins in a polyvalent manner.
  • Estrogens e.g., diethylstilbesterol
  • Groups A can also be glucocorticoids (e.g., triamcinolone, betamethasone).
  • progestogens as groups A is also provided for, such as, norethindrone, ethynodiol, norethindrone, levonorgestrel, and ethinylestradiol.
  • thyroid agents e.g., liothyronine or levothyroxine
  • anti-thyroid agents e.g., methimazole
  • hyperprolactinemic disorders can be treated using e.g., cabergoline as groups A.
  • diabetes can be treated, using e.g., miglitol or insulin lispro).
  • groups A can include oxytocics (e.g., methylergonovine or oxytocin).
  • Prostaglandins can also be employed as groups A, e.g., mioprostol, alprostadil, or dinoprostone.
  • the subject polyvalent presenters can also be used in dermatological applications.
  • Exemplary groups A with dermatological effects can include, e.g., anti-acne agents (such as, isotretinoin, adapalene or tretinoin).
  • Other groups A can be employed for dermatological applications, e.g., for the treatment of pruritis, include, e.g., alclometasone, benzocaine, hydroxyzine, fluticazone, mometasone, fluocinolone, clobestasol, or desoximetasone.
  • Groups A can also affect clotting.
  • poly (A) can comprise the anticoagulant, heparin.
  • groups A can comprise e.g., antithrombin III, or a platelet inhibitor, such as abciximab and/or ticlopidine.
  • the groups A may be chosen for their ability to affect immunomodulation.
  • the release of histamine from mast cells and basophils involves the polyvalent interaction between an allergen and IgE receptors on the cell surface.
  • groups A may comprise, e.g., antihistamines, such as benadryl, loratadine, brompheniramine, periactin, promethazine, terfenadine, fexofenadine, azelastine and/or clemastine.
  • groups A can comprise mast cell stabilizers, e.g., lodoxamide and/or cromolyn.
  • groups A which modulate the immune system can include: e.g., steroids (triamcinolone, beclomethazone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclomethasone, or clobetasol), histamine H2 antagonists (e.g., famotidine, cimetidine, ranitidine), immunosuppressants (azathioprine, cyclosporin). Groups with anti-inflammatory activity, such as, for example, sulindac, etodolac, ketoprofen, ketorolac can also be used as groups A.
  • Antihistamines e.g., fexofenadine or azelastine
  • Groups A that modulate the respiratory system can also be used.
  • mucolytics e.g., guaifenesin
  • Anti-inflammatory agents e.g., cromolyn, flunisolide, beclomethasone, or budesonide
  • bronchodilators e.g., ipratropium, metaproterenol, terbutaline, isoetharine, metaproterenol, albuterol, or theophylline as groups A is also provided for.
  • zafirlukast or zileuton can be employed.
  • Groups A can comprise antimicrobial agents such as, e.g., quinolones (e.g., ciprofloxacin, norfloxacin, ofloxacin, enoxacin, lomefloxacin), sulfonamides (sulfasalazine or sulfamethoxazole) or, for example, polymyxin B, bacitracin, neomycin, oxytetracycline, tobramycin, sulfacetamide, fosfomycin, and penicillin, antivirals (e.g., trifluridine, zidovudine (AZT), zalcitabine (ddV), didanosine (ddl), stavudine (d4T) or other reverse transcriptase inhibitors, e.g., sold by Merck, protease inhibitors (e.g., saquinavir, indinavir, ritonavir), acyclovir
  • the penicillin adjuvant, probenecid can also be employed as groups A.
  • Urinary tract agents for use as groups A can include, e.g., uricosuric agents, e.g., sulfinpyrazone.
  • Antimicrobials e.g., indanyl carbenicillin, nitrofurantoin, nalidixic acid, neomycin, bacitracin, or polymyxin B can also be used as groups A.
  • the subject presenters can also present antispasmodics (e.g., oxybutynin or flavoxate). Calcium oxalate stone preventatives can also be used as groups A, e.g., allopurinol).
  • Prostatic hypertrophy modifiers e.g., terazosin or finasteride
  • terazosin or finasteride can also be employed.
  • the subject presenters can also be used in the treatment of cystitis, using e.g., pentosan as groups A.
  • the subject presenters are also useful in ophthalmic applications.
  • ⁇ -Blockers e.g., brominide or betaxolol
  • anti-inflammatories e.g., clopatadine
  • drugs useful in the treatment of glaucoma e.g., latanoprost
  • the invention also provides for the use of carbonic anhydrase inhibitors, e.g., dichlorphenamide, methazolamide, or dorxolamide as groups A.
  • groups A can include, for example, amlexanox.
  • the groups A can be independently selected from erythropoeitin, tacrolimus, human growth hormone, tissue plasmin activating factor, platelet activating factor, interleukin stimulating factors, granulocyte stimulating factors, macrophage stimulating factors, small molecules secretagogues which target the receptors of protein products such as growth hormone secretagogues, EPO secretagogues, and insulin secretagogues.
  • polyvalent presenters include polyvalent sialic acid against HA on flu virus, two derivatives of Sialyl Le x with platelet containing P-selectin, polyvalent Sialyl Le x against endothelium containing both E and P selectins, barbiturate (benzodiazepine) binding the GABA B complex receptor containing two different receptor sites, RGD and Sialyl Le x binding leukocyte having both integrins and selectins, and C 5A and Gal-X-Gal binding tumor (which binds gal-x-gal) and also proteins of complement cascade.
  • an "ancillary group” is a moiety which alters a characteristic of the polyvalent presenter and/or the components making up the polyvalent presenter (e.g., the framework component, the functional group component, the spacer group, etc.). Properties which can be modified include, for example, solubility (in water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, framework flexibility, antigenicity, molecular size, molecular weight, in vivo half-life, in vivo distribution, biocompatibility, immunogenicity, stability, strength of binding to the polyvalent target, etc.
  • ancillary groups which enhance the water solubility/hydrophilicity of the polyvalent presenter are useful in practicing the present invention.
  • ancillary groups such as, for example, poly(ethylene glycol), alcohols, polyols (e.g., glycerin, glycerol propoxylate, saccharides, including mono-, oligo- and polysaccharides, etc.), carboxylate, polycarboxylates (e.g., polyglutamic acid, polyacrylic acid, etc.), amines, polyamines (e.g., polyglycine, poly(ethyleneimine, etc.) to enhance the water solubility and/or hydrophilicity of the polyvalent presenter.
  • the ancillary group used to improve water solubility/hydrophilicity will be a polyol.
  • the ancillary group will be a poly(ethylene glycol).
  • Lipophilic groups of use in practicing the instant invention include, but are not limited to, aromatic groups and polycyclic aromatic groups.
  • aromatic groups incorporates both aromatic hydrocarbons and heterocyclic aromatics.
  • the aromatic groups may be either unsubstituted or substituted with other groups, but are at least substituted with a group that allows their covalent attachment to the polyvalent presenter.
  • Other groups of use in practicing the instant invention include fatty acid derivatives that do not form bilayers in aqueous medium.
  • the lipophilic ancillary group will be a cyclic group such as a hydrocarbon or heterocycle.
  • the cyclic group will be a six-membered ring or two or more fused six-membered rings.
  • the ancillary group will be a phenyl or a naphthyl group.
  • lipid refers to any fatty acid derivative that is capable of forming a bilayer such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, and other like groups.
  • Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).
  • Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine or dilinoleoylphosphatidylcholine could be used.
  • lipid Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.
  • the flexibility of the polyvalent presenter framework can be manipulated by the inclusion of ancillary groups that are bulky and/or rigid.
  • ancillary groups that are bulky and/or rigid.
  • the presence of bulky or rigid groups can hinder free rotation about bonds in the framework or bonds between the framework and the ancillary group(s) or bonds between the framework and the functional groups.
  • Rigid groups can include, for example, those groups whose conformational lability is restrained by the presence of rings and/or multiple bonds.
  • Other groups that can impart rigidity include polymeric groups such as oligo- or polyproline chains.
  • Rigidity can also be imparted electrostatically.
  • the ancillary groups are either negatively or positively charged, the similarly charged ancillary groups will force the presenter framework into a configuration affording the maximum distance between each of the like charges.
  • the energetic cost of bringing the like-charged groups closer to each other will tend to hold the framework in a configuration that maintains the separation between the like-charged ancillary groups.
  • ancillary groups bearing opposite charges will tend to be attracted to their oppositely charged counterparts and will enter into both inter- and intramolecular ionic bonds. This non-covalent bonding mechanism will tend to hold the framework into a conformation that allows bonding between the oppositely charged groups.
  • Bulky groups can include, for example, large atoms or ions (e.g., iodine, sulfur, metal ions, etc.) groups containing large atoms, polycyclic groups, including aromatic groups, non-aromatic groups and structures incorporating one or more carbon-carbon multiple bonds (i.e., alkenes and alkynes).
  • Bulky groups can also include oligomers and polymers that are branched- or straight-chain species. Species that are branched are expected to increase the rigidity of the structure more per unit molecular weight gain than are straight-chain polymers.
  • rigidity is imparted by the presence of cyclic groups (e.g., cyclic hydrocarbons, heterocycles, etc.).
  • cyclic groups e.g., cyclic hydrocarbons, heterocycles, etc.
  • the ring is a six-membered ring.
  • the ring is an aromatic group such as, for example, phenyl or naphthyl.
  • the antigenicity of the polyvalent presenter may need to be decreased.
  • masking groups such as, for example, poly(ethylene glycol) are known in the art to have the capacity to lower the antigenicity of both monovalent and polyvalent compounds.
  • the ancillary group may comprise a group known in the art to enhance the immunogenicity of a hapten.
  • Groups appropriate for enhancing the immunogenicity of a polyvalent presenter include, but are not limited to, proteins such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other groups capable of enhancing the antigenicity of a polyvalent presenter will be known to those of skill in the art.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the in vivo half-life and in vivo distribution are functions of numerous molecular properties including molecular size, molecular weight, charge, hydrophobicity/ hydrophilicity, antigenicity, biodegradability and the presence or absence of targeting groups on the polyvalent presenter.
  • targeting groups refers to groups that have an affinity for a cellular receptor. Methods of altering these properties to achieve desired changes in the half-life or distribution of compounds administered in vivo are well known in the pharmaceutical and medicinal chemistry arts. Approaches for Novel group A Identification.
  • the present invention provides for the synthesis of oligosaccharide groups for use as group A (Horton, D. Advances in Carbohydrate Chemistry and Biochemistry; Academic Press: San Diego, 1995).
  • Protein analogs can be synthesized (Maassen, J. A.; Terhorst, C. Eur. J. Biochem 1981,115, 153-158; Wang, K. S., et al, J. Virol. 1992, 66, 4992-5001; Mastromarino, P., et al, J. Gen. Virol. 1987, 68, 2359-2369).
  • Computer based design tools can be also used to synthesize groups A (Jorgensen, W. L. Chemtracts: Org. Chem. 1995, 8, 374-376).
  • the invention also provides methods for discovery of new groups A within the context of polyvalency.
  • a single phage in a phage-display library, single pins in a spatially addressed combinatorial array, or single beads in a combinatorial library produced by the mix-and-split method each bear only one type of group, which is presented in polyvalent form.
  • libraries can be screened directly in bound form against polyvalently presented B for activities such as simple adhesion, or adhesion which leads to a specific result, such as infection, cell death, cell proliferation, morphological change, or production of an easily detectable reporter such as green fluorescent protein.
  • a typical resin bead can have a loading of -100 pmol, or about 60 trillion copies.
  • phage can display 3-1000 copies of peptide groups on the surface, depending on the coat protein to which the library is fused. Any species of group A identified as a candidate worthy of further analysis could be subsequently tested in soluble, polymeric form.
  • beads in a library to which bacterial, fungal, or neutrophil cells specifically adhere carry groups that may serve as useful groups for a polyvalent presenter of the present invention.
  • Libraries of ⁇ 1,000,000 different peptides can be prepared by split synthesis, and diverse libraries of other types of compounds may be obtained.
  • the structures of selected groups could be determined from the synthesis history or could be obtained by sequencing, mass spectrometry, deconvolution, or encoding.
  • phage-displayed peptide libraries can be used (see e.g., Doyle, M.V., et al, "The Utilization of Platelets and Whole Cells for the Selection of Peptide
  • Pill-modified phage (3-5 copies per particle) can be selected (“panned") for interaction with whole, activated platelets, leading to the identification of five different classes of platelet-binding peptide groups.
  • Synthetic peptides derived from phage sequences have been tested in inhibition of phage-platelet interactions and to prevent platelet aggregation, although the observed IC50 values were low.
  • Combinatorial synthesis of polysaccharides using highly efficient glycosyl donors has also been described. (Liang, et al, Science, 274:1520, 1996). Such a strategy depends on anomeric sulfoxides as glycosyl donors, which are highly reactive and allow synthesis of polysaccharides in very high yield, which is an essential characteristic of combinatorial libraries in general. Libraries can be created on beads and screened for interaction to polyvalent lectins in solution. Adhesion assays or agglutination assays can be used in this system.
  • the groups A can be attached to or formed as part of a framework using a means which allows for polyvalent presentation of the groups.
  • the attachment can be direct, e.g., without a linker, or can be indirect in that the groups A are attached using linkers.
  • the groups A also can be attached to monomer units before polymerization or after polymerization.
  • the order of the attachment steps also is not critical to the invention as long as the formed presenter displays the groups A in a polyvalent manner.
  • the group A can be attached first to a linker and then the linker-group A moiety can subsequently be attached to a framework or vice versa.
  • the linker can be attached to the framework and then the groups A can be attached to the framework-linker moiety.
  • Groups A also can be incorporated into the polyvalent presenters using bifunctionalized monomers bearing groups A, which can then be polymerized.
  • a polymer can be prepared that is reactive, i.e., activated for coupling and then groups A can be caused to react with the polymer (a "preactivation" strategy).
  • a preferred embodiment involves attaching a linker to the groups A.
  • the linker includes moieties capable of positioning or attaching a group A on a framework.
  • the linker is a moiety independent of the group A and the framework.
  • An example of a preferred type of linker is a linker that leaves a primary amine at the untethered end of the linker.
  • the modified group can then be attached to an activated carboxylic acid on the polymer, forming an amide linkage.
  • Amide bonds are relatively stable, occur widely in natural systems, and can be formed cleanly by a range of well-developed methods. Additional advantages can be obtained using a particular linker.
  • Cationic polymers tend to be more antigenic than anionic polymers.
  • the cationic modified ligand can be used in limiting quantities to ensure the consumption of all of the modified groups, leaving only excess carboxylic acid groups on the polymer, which imparts water solubility.
  • the linkers also provide spacing of the group A from the framework.
  • the linkers also impart flexibility to the polyvalent presenter, e.g., flexible movement of the group.
  • Exemplary linkers for use in the subject presenters include poly(gly), alkyamines, e.g., ethyla ine, hydrocarbon moieties, tbioether, poly (ethylimine) and poly(asp) and polyethers such as PEG chains.
  • the "preactivation" method allows one to construct frameworks, e.g., polymers, with a defined mole fraction of its monomer units presenting different groups.
  • the method can utilize an activated ester of poly(acrylic acid), such as poly(N-acryloylsuccinimide) or pNAS, made from polymerization of N-acryloylsuccinimide.
  • the NHS ester of pNAS in DMF can be reacted sequentially with different primary amines, (e.g., RjNH2 and R2NH2) to form amide bonds.
  • the remaining, unreacted ester groups can be converted to carboxamides or carboxylic acids by treating with excess NH3 or OH", respectively.
  • the distribution of these groups along the polymer backbone is thought to be random using this method. Furthermore, the degree of polymerization (the total number of monomer units in one polymer) can be held constant as the influence of different side groups on effectiveness is examined. The efficiency of the amide-forming reaction between amines and pNAS can be estimated by use of both 1H-NMR spectroscopy and combustion analysis.
  • Copolymerization may introduce two major uncontrolled variables in polymerization: (i) unknown differences in rate constants for copolymerization among differently N-substituted acrylamides might result in non-uniform distribution of group along the backbone of the polymer; (ii) the polydispersity, tacticity and length of the polymer might vary as the side chain is altered. This can occur owing to the differences in rates involved in the polymerization process (propagation; termination).
  • the "preactivation" method may be preferable where the pA(R) with uniform structural features are needed.
  • polymers will be activated using a process that has a high yield and allows for easy removal of by-products from the method. Further considerations include reactivity of by-products with the attached groups, tendency to cause racemization (e.g., with poly(amino acids)).
  • Intramolecular anhydrides in peptides also can be formed using ⁇ -amino-N-carboxylic anhydrides or thiocarboxylic anhydrides.
  • mixed anhydrides can be used, e.g., carbodiimides and others.
  • Common approaches to forming mixed anhydrides used in peptide chemistry include forming carbodiimides (using, for example, dicyclohexylcarbodiimide (DCC) or l-ethyl-3-(3- dimethylamino) propylcarbodiimide (EDC)).
  • EDC may be useful for water-soluble polymers.
  • Other exemplary anhydrides can be formed from reagents such as chloroformates or quinoline derivatives (EEDQ, HDQ).
  • carboxylic acid azides can be used.
  • prior reaction is required to form the azide.
  • imidazoles such as carbonyldimidazole can be employed.
  • compounds such as p-nitrophenol or N- hydroxysuccinimide, that form activated esters, may also be used.
  • the polymers used contain hydroxyl groups, and so methods for derivatization of hydroxyl groups can be employed. Such methods are known to the ordinarily skilled artisan.
  • combinatorial methods can be used to create polyvalent presenters. Briefly, a quasi-solid-state, combinatorial approach can be used to generate libraries of backbone bearing groups Aj to A n
  • Combinatorial libraries are formed that consist of an array of synthetic polyvalent presenters, wherein the polyvalent presenters in the array differ from one another in terms of their compositions, structures, properties, functions, etc.
  • the chemical structure of the framework component the chemical structure of the functional group component, the chemical structure of the ancillary group, the chemical structure of the spacer group, the chemical nature of the framework component, the chemical nature of the functional group component, the chemical nature of the ancillary group, the chemical nature of the spacer group, the amount of framework component delivered, the amount of functional group component delivered, the amount of ancillary group component delivered, the amount of spacer group delivered, the number and/or amount of different framework components delivered, the number and/or amount of different functional group components delivered, the number and/or amount of different ancillary groups delivered, the number and/or amount of different spacer groups delivered, the nature and number of the linkages between the various components (e.g., the nature of the linkages of the spacer group), the reaction parameters (e.g., reactant solvents, reaction temperatures, reaction times, reaction initiators, reaction catalysts, the atmosphere in which the reactions are carried out, the pressure at which the reactions are
  • the reaction parameters e.g., reactant solvents, reaction temperatures,
  • the invention provides a method of making an array of polyvalent presenters, the method comprising: (a) delivering a first activated framework component of a first polyvalent presenter and a first activated framework component of a second polyvalent presenter to first and second reaction vessels; and (b) delivering a first functional group component of the first polyvalent presenter and a first functional group component of the second polyvalent presenter to the first and second reaction vessels, thereby forming the array of at least two different polyvalent presenters.
  • the preactivated reactants are allowed to react.
  • the array of polyvalent presenters can be screened/assayed for polyvalent presenters having useful properties. Properties which can be screened for include, but are not limited to, the following: biological activities, binding affinities, biological properties, pharmacological properties, oral bioavailabilities, circulatory half-lives, agonist activities, antagonist activities, solubilities, etc.
  • the array of polyvalent presenter can be screened for useful properties sequentially or in parallel.
  • the array can be screened in situ or, alternatively, the polyvalent presenters can be screened in other than an in situ manner (e.g., the polyvalent presenters can be removed from the substrate and then screened). Once identified, the polyvalent presenters having useful properties can be prepared on a large-scale.
  • spacer groups or, interchangeably, linker groups are interposed between the framework and the functional group and/or the framework and the ancillary group.
  • R 1 in both of these formulae represents the multifunctional framework providing a multitude of attachment sites for spacer groups.
  • Polymers including dendrimers, polypeptides, polysaccharides and others, are generally useful for this framework. With its multitude of attachment sites, the backbone serves an amplifying function for the functional and/or ancillary groups.
  • the symbols R 2 and R 3 represent a spacer group and a functional group, respectively.
  • the symbol m represents the number of functional groups attached to each spacer.
  • the symbol n represents the number of spacers, and their associated functional groups, which are attached to the framework.
  • the number of functional groups and the number of ancillary groups and the number of spacers are such as to achieve greater than ten total functional groups and/or ancillary groups on the polyvalent presenter.
  • the symbols R 4 and R 5 represent a spacer group and an ancillary group, respectively.
  • the symbol s represents the number of ancillary groups attached to each spacer.
  • the symbol t represents the number of spacers, and their associated ancillary groups, which are attached to the framework.
  • the number of functional groups and the number of spacers are such as to achieve greater than ten total functional groups on the polyvalent presenter. For example, "m" may be six and "n" may be 3 so that a total of eighteen functional groups are present.
  • both the spacer/functional group construct is attached to the framework.
  • both the spacer/functional group and spacer/ancillary group constructs are attached to the framework.
  • the spacers R 2 and R 4 can be either the same group or different and may be present in an approximately 1 : 1 molar ratio, or a different molar ratio.
  • R 2 the discussion is phrased in terms of R 2 , however, it should be understood that both R 2 and R 4 are encompassed within this discussion.
  • R 3 functional groups
  • R 5 ancillary groups
  • the spacer R 2 can be any of a wide variety of molecular structures, and will be at least bifunctional to permit attachment to both R 1 and R 3 , optionally through linkage groups.
  • Spacers can be those that are stable or can be cleaved in vivo by the biological environment. Spacers with multiple binding functionalities at the terminus (i.e., the R 3 end) accommodate a multitude of functional groups and/or ancillary groups. Spacers of this type serve an amplifying function in a manner similar to that of the framework, although to a lesser extent.
  • Other spacers useful in the invention may have only a single functionality at either end, in which case the value of "n" will be 1 and the value of "m" will be greater than 10 .
  • hydrophilicity In addition to the property of in vivo cleavability mentioned above, a property of prominent interest is hydrophilicity. Still other useful properties are the ability to lower antigenicity and to increase molecular weight. Spacers with still further properties can be utilized to advantage as well, 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 either a straight-chain or a branched-chain structure.
  • Preferred R 2 groups are those which include a straight chain within their structures, either as the entire spacer group or as the backbone of a branched-chain group.
  • the straight chain may be a chain of carbon atoms or of carbon atoms interrupted with one or more heteroatoms such as oxygen atoms, sulfur atoms or nitrogen atoms.
  • the chain can also be substituted with aromatic groups.
  • the bonds forming the chain may be single bonds, double bonds or triple bonds, although single bonds are preferred.
  • the length of the chain is not critical and may vary widely, depending on the desired relationship between the molecular weight of the construct and the number of functional and/or ancillary groups included on the construct. Best results will generally be obtained with chain lengths ranging from 4 atoms to 1,000 atoms, with preferred chains being those of 6 atoms to 100 atoms, and the most preferred being those of from 10 atoms to 50 atoms.
  • the chain as thus described is the backbone of the spacer itself, and does not include atoms, groups or side chains bonded to the serially bonded atoms forming the backbone. It does, however, include linking groups at the chain termini joining the chain to R 1 and R 3 , when such linking groups are present.
  • the spacer will be hydrophilic in character to impart hydrophilicity to the construct.
  • the spacer may thus be any hydrophilic group among those known in the art.
  • Examples are polyalkylene glycols, optionally substituted with groups that may or may not add to their hydrophilic character.
  • polyalkylene glycols polyethylene glycol is a preferred example.
  • Examples of the optional substitutions 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 may vary. Selection of the length will be governed by such considerations as achieving the desired molecular weight for the construct 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 will provide the best results, with a range of from about 200 daltons to about 1,000 daltons preferred.
  • the spacer may contain any of a variety of groups as part of its chain which will cleave in a biological fluid at a rate which is enhanced relative to that of constructs which lack such groups. Accelerated rates of cleavage enhance the rates of removal of the framework from the functional and/or ancillary groups of the polyvalent presenter. Such removal can be used to reduce toxicity or enhance activity of the functional groups. While the degree of cleavage rate enhancement is not critical to the invention, preferred examples of these spacers are those in which at least about 10% of the cleavable groups are cleaved in the biological fluid within 24 hours of administration, most preferably at least about 35%. Preferred cleavable groups are ester linkages and disulfide linkages.
  • the spacer both imparts a hydrophilic character to the construct and includes a cleavable group as referred to above.
  • Structural formulas for the spacer vary widely. The discussion below is offered only as an example of one type of spacer useful in practicing the instant invention and is not intended to serve as a limitation on the types of spacers of use in practicing the instant invention.
  • One group of structural formulas for spacers which impart a hydrophilic character o the construct are those in which m of Formulae II and III above is 1, and R 2 of Formulae II and III is represented by either of Formulas IV, V or VI below:
  • the hydrophilic component is represented by R 6 , which is a polyethylene glycol group having a formula weight of about 100 daltons to about 20,000 daltons, preferably from about 200 daltons to about 1,000 daltons.
  • the group R 7 represents a cleavable group that increases the rate of cleavage of the construct in blood.
  • the group is either a disulfide group S — S, or an ester group oriented in either direction, i.e., C(O) — O or O — C(O).
  • the polyethylene glycol group will remain with the polymeric framework R 1 .
  • the polyethylene glycol group will remain with the functional and/or ancillary group.
  • the symbols X, Y and Z represent inert linking groups which serve to join the
  • linking groups are not critical, and their selection will be largely a matter of convenience as determined by the means of synthesis of the construct.
  • inert in this context means essentially non-toxic, non-immunogenic, and stable with respect to cleavage or dissociation over the typical period of time required for use of the construct in a clinical or diagnostic procedure.
  • inert linking groups useful for this purpose are alkylamino or aminoalkyl groups such as (CH 2 ) q — NH and NH — (CH 2 ) q , carbamoyl groups such as NH — C(O) — O and O — C(O) — NH, and alkylcarbamoyl or carbanoylalkyl groups such as (CH 2 ) q — NH— C(O) — O and O— C(O) — NH— (CH 2 ) q .
  • the symbol q in these groups may vary, but in most cases will generally range from 1 to 10, with 2 to 4 preferred, and 2 particularly preferred.
  • terminal atoms in X or Z may be native to R 1 or R 3 , respectively.
  • a terminal NH group in the definition of X or Z may be formed from an amino functional group on R 1 or R 3 or other N-bearing group which can react to form the NH of the linking group.
  • a terminal O atom may be defined such that terminal atoms in X or Z may be native to R 1 or R 3 , respectively.
  • a terminal NH group in the definition of X or Z may be formed from an amino functional group on R 1 or R 3 or other N-bearing group which can react to form the NH of the linking group.
  • a terminal O atom may be defined such that terminal atoms in X or Z may be native to R 1 or R 3 , respectively.
  • PEG refers to a polyethylene glycol segment with terminal hydroxyl groups removed, thereby terminating in ethylene groups at both ends. Spacer groups of a similar general motif that do not contain PEG will also be useful in practicing the instant invention.
  • R 6 , R 7 , and X are as defined above, with Z restricted to (CH 2 ) q — NH.
  • Y' represents a group of the formula
  • r of Formula VII is either zero or a number equal to n.
  • the spacer lacks a cleavable group, whereas when r is other than zero, a cleavable group is included for each functional NH group on the Z linker to which a functional group or ancillary group is attached.
  • q is 2 to 6, and in most preferred examples q is 2 or 3.
  • terminal CH 3 of Formula VIII may be replaced by OH or SH. This results in a reactive OH or SH group available for the attachment of functional and/or ancillary groups.
  • R 8 is a group having the formula
  • the indexes o and p are the same or different and are either zero or a positive integer, such that the sum of o + p is at least 2.
  • the symbols X and Z' in Formula IX are the same or different and are inert linking groups of scope similar to the inert linking groups of the previous formulas.
  • Preferred examples of X and Z' are NH— C(O), C(O) — NH, NH— C(S) and C(S) — NH.
  • the number and arrangement of functional and or ancillary groups on a single construct of either Formula II or Formula III may vary considerably.
  • the number of functional groups will equal the product of m x n.
  • the number of ancillary groups will equal the product of s x t.
  • constructs will be those in which either one or both products are greater than 10.
  • Constructs in accordance with the present invention may be synthesized in accordance with conventional linkage reactions that are well known among those skilled in the art.
  • the backbone is referred to as (AMP)( — NH 2 ) X , "AMP” denoting the amplifying effect of permitting the attachment of a multitude of spacers and functional groups and/or ancillary groups, and x representing a number corresponding to n in Formulae II and III.
  • PEG polyethylene glycol
  • the above intermediate XI is converted to a second intermediate with a terminal amine group, by reaction with a diamine such as NH 2 — (CH 2 ) 2 — NH 2 .
  • the second intermediate has the structure:
  • a cleavable group such as disulfide can be introduced by reacting the intermediate X with a diamine containing an internal disulfide, such as cystenamine disulfide, NH_— (CH 2 ) 2 — S— S— (CH 2 ) 2 — NH 2 , to yield the further intermediate:
  • an amine- or hydroxyl- containing amplifying polymer can be derivatized to produce carboxylic acid groups as the functional groups. This is readily achieved by reacting the polymer with maleic, succinic or glutaric anhydride using established procedures.
  • a derivatized ligand to combine with the derivatized polymer can be formed by reacting a ligand bearing an isothiocyanate group with an amino alcohol, HO(CH 2 ) n NH 2 , to place a terminal hydroxyl group on the ligand.
  • the carboxylic acid group on the derivatized polymer can then be activated by conventional methods using such agents as dicyclohexylcarbodiimide or carbonyldiimidazole, and reacted with the derivatized ligand to achieve the ester linkage.
  • the section of the construct between the amplifying polymer and the ligand serves as the spacer, and the length of the spacer is determined by the number of CH 2 groups in the amino alcohol used to derivatize the ligand.
  • the ligand is derivatized with an aminocarboxylic acid, HO 2 C(CH 2 ) 2 NH 2 , rather than an amino alcohol.
  • the resulting carboxylic acid-derivatized ligand is then activated with dicyclohexylcarbodiimide or carbonyldiimidazole and coupled directly to a hydroxyl-containing amplifying polymer.
  • Ligands with functional groups for attachment to the spacer can be prepared by conventional methods.
  • Well-known ligands for example are readily derivatized by methods known to those skilled in the art. It is preferable to select a ligand that retains all or most of its binding affinity even after derivatization.
  • one approach to preparing the polyvalent presenters of the present invention involves the formation of combinatorial libraries, which consist of an array of synthetic polyvalent presenters, wherein the polyvalent presenters in the array differ from one another in terms of their compositions, structures, properties, functions, etc.
  • the chemical structure of the framework component the chemical structure of the functional group component, the chemical structure of the ancillary group, the chemical structure of the spacer group; the chemical nature of the framework component, the chemical nature of the functional group component, the chemical nature of the ancillary group, the chemical nature of the spacer group; the amount of framework component delivered, the amount of functional group component delivered, the amount of ancillary groups delivered, the amount of spacer group delivered; the number and amount of different framework components delivered, the number and/or amount of different functional group components delivered, the number and/or amount of different ancillary groups delivered, the number and/or amount of different spacer groups delivered; the nature and number of the linkages between the various components (e.g., the nature of the linkages of the spacer group); the reaction parameters (e.g., reactant solvents, reaction temperatures, reaction times, reaction initiators, reaction catalysts, the atmospheres in which the reactions are carried out, the rates at which the reactions are quen
  • the reaction parameters e.g., reactant solvents, reaction temperatures,
  • the activated polymer is reacted with one functional group in a manner that consumes substantially all of the activating groups on the polymeric framework.
  • the functional group to activating group stoichiometry is at least 1 :1.
  • An excess of functional group may be added if a 1 : 1 ratio is insufficient to substantially consume all of the activating groups.
  • the functional group can be added in an amount insufficient to consume all of the activating groups.
  • a second functional group or an ancillary group can be delivered and reacted.
  • a second functional group or an ancillary group can be delivered and reacted.
  • more than one functional group either as a mixture or sequentially, followed by the addition of an ancillary group or, alternatively, more than one ancillary group as a mixture or sequentially.
  • the activating groups are substantially consumed by the addition of a sufficient amount of a functional group.
  • the framework component is reactivated and a second functional group or an ancillary group is added.
  • the functional group(s) may comprise an individual functional group, a mixture of different functional groups, or a mixture of functional groups and ancillary groups, wherein the ancillary groups may comprise either a single ancillary group or a mixture of ancillary groups.
  • the activating groups are substantially consumed by the addition of a mixture of at least two functional groups or a mixture of at least one functional group and at least one ancillary group.
  • activating groups are reacted with a mixture of at least one functional group, at least one ancillary group or at least one functional group and at least one ancillary group.
  • the remaining activating groups are substantially consumed by the addition of a further functional group, ancillary group or mixture of functional and ancillary groups.
  • the functional group(s), ancillary group(s) and/or mixtures of the functional and ancillary group(s) are added as dry powders or neat liquids to a solution of the activated framework.
  • solutions of the functional groups, ancillary groups and/or mixtures of functional and ancillary group(s) are added to a solution of the activated polymer.
  • solutions of the functional group(s), ancillary group(s) or mixtures of the functional and ancillary group(s) are added to the polymers, wherein the polymer is present as a neat liquid or a dry powder.
  • Solvents of used in this aspect of the invention include any solvent is compatible with the presence of the activating groups (i.e., does not react with the activated framework in a significant amount) and the nature of the reaction between the activated polymer and the functional and or ancillary groups.
  • solvents include, for example, water, dimethylsulfoxide (DMSO), dimethylformamide (DMF), alcohols, ethers, ketones, hydrocarbons, aromatic hydrocarbons and mixtures, in any proportions, of these solvents.
  • the temperature can be controlled to control the rate of reaction between the activated framework and the functional group(s) and/or ancillary group(s).
  • reaction parameters e.g., reactant solvents, reaction temperatures, reaction times, reaction initiators, reaction catalysts, the atmosphere in which the reactions are carried out, the pressure at which the reactions are carried out, the rates at which the reactions are quenched, etc.
  • a "handle" on the functional group or the ancillary groups is activated by means of an activating group as described above and reacted with either an activated group on the framework or an unactivated moiety on the framework.
  • handle is used to refer to a reactive group such as, for example, carboxyl (acid or salt), hydroxyl, sulfhydryl, amide, carbamate, amine, ketone, aldehyde, olefin, diene, aromatic ring, etc.
  • a reactive group such as, for example, carboxyl (acid or salt), hydroxyl, sulfhydryl, amide, carbamate, amine, ketone, aldehyde, olefin, diene, aromatic ring, etc.
  • Appropriate combinations of framework-bound reactive moieties and functional group or ancillary group handles will be apparent to those of skill in the art.
  • Substrate refers a material having a rigid or semi-rigid surface or, alternatively, a material having dimples, wells, containers, trenches, etc. In many embodiments, at least one surface of the substrate will be substantially flat, although in some embodiments, it may be desirable to physically separate synthesis regions for different materials with, for example, dimples, wells, raised regions, etched trenches, or the like. In some embodiments, the substrate itself contains wells, raised regions, etched trenches, etc., which form all or part of the synthesis regions.
  • the substrate can be organic, inorganic, biological, nonbiological or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc.
  • the substrate can have any convenient shape, such a disc, square, sphere, circle, etc.
  • the substrate is preferably flat, but may take on a variety of alternative surface configurations.
  • the substrate may contain raised or depressed regions on which the synthesis of diverse polyvalent presenters takes place.
  • the substrate and its surface preferably form a rigid support on which to carry out the reactions described herein.
  • the substrate may be any of a wide variety of materials including, for example, polymers, plastics, pyrex, quartz, resins, silicon, silica or silica-based materials, carbon, metals, inorganic glasses, inorganic crystals, membranes, etc.
  • Other substrate materials will be readily apparent to those of skill in the art upon review of this disclosure.
  • Surfaces on the solid substrate can be composed of the same materials as the substrate or, alternatively, they can be different, i.e., the substrates can be coated with a different material.
  • the substrate surface can contain thereon an adsorbent (for example, cellulose) to which the components of interest are delivered.
  • adsorbent for example, cellulose
  • suitable substrates include, for example, microtiter plates (e.g., having 96 wells) or a test tube holder containing therein test tubes in an amount sufficient to hold each of the polyvalent presenters of the array.
  • the reactant components in the individual reaction regions must often be prevented from moving to adjacent reaction regions. Most simply, this can be ensured by leaving a sufficient amount of space between the reaction regions on the substrate so that the various components cannot interdifuse between reaction regions. Moreover, this can be ensured by providing an appropriate barrier between the various reaction regions on the substrate.
  • a mechanical device or physical structure defines the various reaction regions on the substrate.
  • a wall or other physical barrier can be used to prevent the reactant components in the individual reaction regions from moving to adjacent reaction regions. This wall or physical barrier can be removed after the synthesis is carried out.
  • a hydrophobic material for example, can be used to coat the region surrounding the individual reaction regions.
  • Such materials prevent aqueous (and certain other polar) solutions from moving to adjacent reaction regions on the substrate.
  • aqueous or nonpolar solvents when non-aqueous or nonpolar solvents are employed, different surface coatings will be required.
  • appropriate materials e.g., substrate material, hydrophobic coatings, reactant solvents, etc.
  • a small, precisely metered amount of each reactant component is delivered into each reaction region.
  • the various reactant components can be delivered to the reaction regions of interest from a dispenser in the form of droplets or powder.
  • Conventional micropipetting apparatuses can, for example, be adapted to dispense various droplet volumes from a capillary.
  • the dispenser can also be of the type employed in conventional ink-jet printers.
  • Such ink-jet dispenser systems include, for example, the pulse pressure type dispenser system, the bubble jet type dispenser system and the slit jet type dispenser system. These ink-jet dispenser systems are able to deliver various droplet volumes.
  • such dispenser systems can be manual or, alternatively, they can be automated or semi-automated using, for example, robotics techniques.
  • the reactant solutions can be delivered from a reservoir to the substrate by an electrophoretic pump.
  • a thin capillary connects a reservoir of the reactant with the nozzle of the dispenser.
  • electrodes are present to provide a potential difference.
  • the speed at which a chemical species travels in a potential gradient of an electrophoretic medium is governed by a variety of physical properties, including the charge density, size, and shape of the species being transported, as well as the physical and chemical properties of the transport medium itself. Under the proper conditions of potential gradient, capillary dimensions, and transport medium rheology, a hydrodynamic flow will be set up within the capillary.
  • bulk fluid containing the reactant of interest can be pumped from a reservoir to the substrate.
  • the reactant solution can be precisely delivered to predefined reaction regions on the substrate.
  • the dispenser can be aligned with respect to the appropriate reaction regions by a variety of conventional systems.
  • Such systems which are widely used in the microelectronic device fabrication and testing arts, can deliver droplets of reactant components to individual reaction regions at rates of up to 5,000 drops per second.
  • the translational (X-Y) accuracy of such systems is well within 1 ⁇ m.
  • the position of the dispenser stage of such systems can be calibrated with respect to the position of the substrate by a variety of methods known in the art. For example, with only one or two reference points on the substrate surface, a "dead reckoning" method can be provided to locate each reaction region on the substrate.
  • the reference marks in any such systems can be accurately identified by using capacitive, resistive or optical sensors.
  • the dispenser can be aligned with respect to the reaction region of interest by a system analogous to that employed in magnetic and optical storage media fields.
  • the reaction region in which the reactant component is to be deposited is identified by its track and sector location on the disk substrate. The dispenser is then moved to the appropriate track while the disk substrate rotates. When the appropriate reaction region is positioned below the dispenser, a droplet of reactant solution is released.
  • the reaction regions may be further defined by dimples in the substrate surface. This will be especially advantageous when a head or other sensing device must contact or glide along the substrate surface.
  • the dimples can also act as identification marks directing the dispenser to the reaction region of interest.
  • composition of polyvalent presenters
  • structure i.e., composition
  • synthesis history can be obtained by sequencing, mass spectrometry, deconvolution, encoding, etc.
  • evaluation strategies are described, for example, by Thompson et al, Chem. Rev., 1996, 96, 555-600, the teachings of which are incorporated herein by reference.
  • the present invention also pertains to pharmaceutical compositions for polyvalently presenting an agent for therapy.
  • the pharmaceutical compositions contain a polyvalent presenter, as described in detail above, and a pharmaceutically acceptable carrier.
  • the polyvalent presenter can have a formula as follows:
  • Y is a framework
  • X is a direct bond or a linker
  • A is a presented functional group
  • n is greater than ten and is an integer selected such that the presented groups can interact with a collection of target binding sites B.
  • the presenter itself can serve as its own pharmaceutically acceptable carrier.
  • the polyvalent presenter is made, e.g., n is selected and the - (X-A) moieties are attached to Y, such that the polyvalent presenter conforms to an interface containing a collection of target binding sites B and blankets the collection of target binding sites B upon administration to a subject.
  • X is a linker group which is an independent moiety and is not part of Y or A and n is greater than ten and is an integer selected such that the polyvalent presenter conforms to a collection of target binding sites B upon administration to a subject.
  • Y is a polymeric framework, A is a presented functional group and X is a linker group and n is greater than ten and is an integer selected such that the polyvalent presenter conforms to a collection of target binding sites B upon administration to a subject.
  • pharmaceutically acceptable carrier is intended to include substances capable of being coadministered with the polyvalent presenter(s) and allows it to perform their intended function.
  • examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like.
  • Any other conventional carrier suitable for use with the polyvalent presenter(s) also falls within the scope of the present invention. It should be understood that many of the polyvalent presenters of the present invention are water soluble. Accordingly, formulation of the polyvalent presenters of the invention into pharmaceutical compositions is rendered more facile with fewer problems than with other materials that are less water soluble.
  • the language "therapeutically effective amount" of the polyvalent presenter is that amount necessary or sufficient to perform its intended function within the subject.
  • the therapeutic effective amount can vary depending on such factors as the type of site being targeted, the type of components (e.g., frameworks, e.g., linkers, e.g., groups A) employed, the size of the subject, or the severity of the symptom(s).
  • One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the polyvalent presenter without undue experimentation.
  • An in vitro or in vivo assay also can be used to determine an "effective amount" of the polyvalent presenter. The ordinarily skilled artisan would select an appropriate amount of the polyvalent presenter for use in the aforementioned assay.
  • the data obtained from cell culture assays and animal studies can be used in formulating an appropriate range of dosages for use in subjects.
  • the dosage of such agents lies preferably within a range of circulating or tissue concentrations that include the 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.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a concentration range that includes the IC50 (i.e., the concentration of the test modulating agent which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC50 concentration of the test modulating agent which achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the regimen of administration also can affect what constitutes an effective amount.
  • the polyvalent presenter can be administered alone or in conjunction with other agent(s). Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused. Further, the dosages of the polyvalent presenter(s) can be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation.
  • compositions for use in accordance with the present invention may also be formulated in conventional manner using one or more physiologically acceptable carriers or excipients as the pharmaceutically acceptable carrier.
  • the polyvalent presenters and their physiologically acceptable salts and solvates may be formulated for administration by, for example, topical application, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal admimstration.
  • the agents of the invention can be formulated in a manner appropriate for a specific mode of administration chosen, including, e.g., systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA.
  • the polyvalent agents of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the agents may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophihzed forms are also included.
  • the pharmaceutical compositions of polyvalent presenters may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g
  • Liquid compositions for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid compositions may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the compositions may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Compositions for oral administration may be suitably formulated to give controlled release of the active modulating agent.
  • compositions for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
  • the agents can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the agents may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the agents may also be formulated as a depot composition. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the modulating agents may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • compositions may, if desired, be provided in a pack or dispenser device, or as a kit with instructions.
  • the composition may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration, e.g., for use in the methods described herein.
  • a collection of polyvalent presenters includes more than one polyvalent presenter combined together.
  • the collections can have as many different polyvalent presenters that allow it to perform its intended function.
  • the present invention even further pertains to formulations, e.g., therapeutic compositions containing a collection of polyvalent presenters and a pharmaceutical carrier as defined above.
  • the formulation containing a collection of polyvalent presenters can be designed such that different polyvalent presenters are selected for their ability to impart a particular characteristic into the overall formulation.
  • the collection of polyvalent presenters can be in the form of a polydispersion.
  • a polydispersion of polyvalent presenters contains more than one different polyvalent presenter.
  • the polydispersion of different polyvalent presenters can be a collection of sizes because of such factors as differences in the length of the frameworks and degree of substitution with groups A, and/or difference in group A/linker combinations. There also may be differences in the precise positions of group A attachments.
  • Each member of the collection can impart different properties to an overall formulation, which can be used as an advantage in treating a disease or condition. For example, lower-molecular-weight members of the collection may access their targets of action more rapidly than do higher molecular weight members.
  • high-molecular-weight members may have a longer duration of action than lower-molecular-weight members due to longer residence time at the site of action and/or lifetime in the relevant compartment.
  • the present invention also pertains to a method(s) for treating a disease or condition.
  • the method involves administering to a subject a plurality of Groups A such that the treatment of the disease or condition occurs.
  • the treatment of the disease or condition can occur by the conformal interface interaction of a polyvalent presenter with a collection of binding sites B or the conformal interface interactions of a plurality of polyvalent presenters with collections of binding sites B within the subject.
  • the conformational surface interaction(s) result in the blanketing of either a collection of binding sites B or an array of target binding sites B within the subject.
  • the polyvalent presenter that facilitates treatment meets a set of criteria.
  • the groups A are functional and act as a drug, alone, or in combination with the framework such as, e.g., a compound that may be of insufficient affinity alone but amplified as a result of the present invention; the presentation of the groups A attached to the framework provide an additional benefit to the interaction relative to the presentation of a single group A to a plurality of binding sites B; and the additional benefit is a synergistic benefit in that the benefit is greater than the additive benefit that would have been provided by a collection of monomers of the same group A dispersed in a homogenous solution.
  • additional benefits include a benefit selected from the group consisting of the provision of a sufficient biological effect at a lower concentration of groups A, the enhancement of specificity for a targeted versus non-targeted site, and the enhancement of biological potency.
  • the polyvalent presenter provides at least two additional benefits and in an even more preferred embodiment at least three additional benefits.
  • the benefits can be selected from the above list and also from those benefits described below in the sections describing the pharmacodynamics and/or the mechanisms related to the functioning of the polyvalent presenters, e.g. positive cooperativity, entropic enhancement of binding and provision of steric inhibition.
  • the binding sites B are located such that a polyvalent interaction can occur.
  • the binding sites B with which the groups A of the polyvalent presenter interact can be intracellular, or extracellular (e.g., on the cell surface or on extracellular matrix) or localized within cellular membranes.
  • the polyvalent presenters of the present invention can have a variety of functions in terms of facilitation of treatment, they can be used to modulate (e.g., up or downregulate) a state or condition associated with polyvalency, for example, an unwanted state (e.g., fertilization) or a disease state (e.g. infection).
  • the subject presenters can be used to target specific biological events of the disease or condition (e.g., prophylactic prevention of cell-pathogen attachment) or can be used to treat specific disease states (e.g., a patient presenting with a pathogenic infection to control or reverse the spread of infection). Likewise, the subject presenters have a variety of ex vivo applications in the modulation of such states. Modulation of specific biological events
  • the polyvalent presenters of the present invention can be used, for example, to modulate cell-cell interactions. Numerous biological processes require cell-cell interactions and the subject presenters can either promote or inhibit such interactions. One particular instance involves the binding of cells to other cells such as, for example, neutrophils and arterial endothelial cells.
  • This rolling is mediated by interactions between multiple E- and P-selectins on the surface of the endothelial cell (these selectins are not normally present on the surface of these cells; they are induced by cytokines during inflammation) and multiple glycoproteins displaying sialyl Lewis X (a tetrasaccharide) on the surface of the neutrophil.
  • sialyl Lewis X a tetrasaccharide
  • NCAM Neutrophil Cell- Adhesion Molecule
  • Neutrophil E-selectin Neutrophil Sulfated sialyl Le x Cadherins
  • T-cell T-cell Receptor Antigen Presenting Major Cells Histocompatibility Complex MHC
  • the subject polyvalent presenters can be used to inhibit another type of cell-cell interaction, e.g., attachment.
  • attachment e.g., attachment
  • the selectins are transmembrane glycoproteins expressed on platelets (P-selectin), leukocytes (including neutrophils and other lymphocytes, L-selectin) and on endothelial cells (E- and P-selectins).
  • P-selectin transmembrane glycoproteins expressed on platelets
  • leukocytes including neutrophils and other lymphocytes, L-selectin
  • E- and P-selectins endothelial cells
  • the selectins mediate rolling interactions on endothelial vessel walls, an interaction that ultimately leads to leukocyte extravasation (Kretzschmar, et al, 1995. Tetrahedron 51:13015).
  • the subject presenters will be useful in inhibiting both initial attachment events and events which follow thereafter, e.g., leukocyte migration.
  • the polyvalent presenters of the present invention can be used to interfere with neutrophil- endothelial cell interaction and, thus, modulate states associated with neutrophil adhesion, such as, e.g., inflammation, adult respiratory distress syndrome, rheumatoid arthritis, septic shock, and reperfusion injury.
  • the subject polyvalent presenters can be used to inhibit platelet-platelet interaction.
  • Platelet aggregation plays a salient role in thrombotic or thromboembolic events.
  • ADP, thrombin, epinephrine, or collagen agonists expose fibrinogen-binding sites on the plate protein, GPIIb-IIIa.
  • Small peptides, such as RGD or HHLGGAKQAGDV (Hi 2) contain fibrinogen recognition motifs and can block fibrinogen binding to these sites.
  • Peptide binding to GPIIb-IIIa induces the expression of cryptic epitopes on the GPIIb-IIIa complex (Gawaz, et al, 1996. Arterioscler Thromb Vase. Biol. 16:621).
  • Platelet granules are then released; one of the major glycoproteins of granules is thrombospondin, which has been shown to mediate platelet aggregation. (Gawaz, supra).
  • Groups A can be chosen to inhibit any of the interactions leading to platelet aggregation.
  • the interaction of GPIIb-IIIa and a fibrinogen recognition motif is modulated. In certain embodiments it will be desirable to induce hypocoagulation in a subject, while in other embodiments it will be desirable to induce a hypercoagulative state in a subject.
  • the polyvalent presenters of the instant invention can be used to reduce the ability of cancer cells to metastasize. Metastasis involves the detachment of tumor cells from primary sites, their invasion into neighboring tissues, and their settlement at secondary sites, and the subject polyvalent presenters can act at any of these stages.
  • the tropism of cancer cells for various organs can be mediated by lectins of the invaded organs or of the tumor cells (Matrosovich. 989. FEB 252:1,2: 1-4; Beuth, et al, 1988. Clin. Exp. Metastasis. 6:115-120).
  • ⁇ -lactosyl clusters have been described as potential tumor metastasis inhibitors (Dean, et al, 1993. Carbohydrate Research 245: 175). Accordingly, polyvalent presenters can be made which incorporate modulators of these recognition events as groups A.
  • the subject presenters can be used to modulate infection.
  • the initial step in most cell-pathogen interactions involves attachment. This is true for all viruses.
  • One example of adhesion of a virus to the surface of a cell is influenza and bronchial epithelial cells.
  • influenza virus attaches to the surface of a bronchial epithelial cell.
  • the attachment occurs by interaction between multiple trimers of the hemagglutinin (HA, a lectin found densely packed on the surface of the virus, about 2-4 copies/100 nm ⁇ or 600-1200 per virus particle) and multiple moieties of sialic acid (SA, the terminal sugar on many glycoproteins and one that is also arranged densely on the surface of the target cell, ⁇ 50-200 copies/ 100 nm ⁇ ).
  • HA hemagglutinin
  • SA sialic acid
  • P protein
  • S - sugar glycoprotein or glycolipid
  • P PreS portion of Env protein
  • P IL-6 receptor
  • P Residues 21-47 of preS
  • P PreS1 sequence of large S
  • P Hepatocyte receptor for polymeric IgA protein
  • Picornaviridae P Residues 95-105 of VP1 P: Member of immunoglobulin Enterovirus capsid protein superfamily, not CD44 as previously Poliovirus Polio reported.
  • Rhinovirus Common Cold P Residues of VP1 and VP3 P: ICAM-1 Human rhinovirus major capsid proteins lining a
  • HIV-1 AIDS P gp120-resembling Ig heavy
  • P CD4 of T cell chain regions
  • P CD4 molecule interacting with class II
  • Neuroleukin S Galactosyl ceramide (or closely related
  • P gp120-resembling vasoactive molecule on human colon epithelial intestinal peptide HT29 cells
  • P CR2
  • Chemokine receptors CXCR-4 (T-cell trophic) and CCR-5 (Macrophage trophic) both are 7-transmembrane G-protein coupled receptors
  • HIV-2 AIDS P CD4
  • African swine virus P p12, p72
  • Viral attachment to its host is the first step to viral infection, and involves the simultaneous association of multiple molecules on the surface of the virus with multiple molecules on the surface of its host cell (M. Tardieu, R L. Epstein, H. L. Weiner, Int. Rev. Cytol. 1982, 80, 27-61).
  • the molecules involved on the surfaces of the virus and cell that mediate attachment are most directly identified when this attachment is inhibited by either a high concentration of corresponding free monomer of that molecule or by monoclonal antibodies against that molecule.
  • Such a strategy is used often when the ligand is a sugar or small molecule.
  • a less direct characterization of the molecules involved in attachment comes from correlations of mutations (either man-made or natural) with an inability to bind successfully to the target cell; that is, if a mutation of a particular protein eliminates the ability of the virus to attach, it is involved in the attachment either directly or indirectly.
  • This second strategy is often used for receptors or ligands that are proteins.
  • a growing number of molecules on surfaces have been implicated in virus-cell recognition, there are often ambiguities. Because viral particles adhere to many substances non- specifically, it is often difficult to distinguish non-biospecific from biospecific binding. Some viral particles may enter a host cell by non-specific transport (the same mechanism that a cell uses to internalize small molecules).
  • Viruses bind to almost all classes of molecules on cellular surfaces: sugars (A. Varki, Glycobiol. 1993, 3, 97-130) (for example, polyoma (H. Fried, L. D. Cahan, J. C. Paulson, Virol 1981, 109, 188-192) and orthomyxoviruses (J. C. Paulson, J. E. Sadler, R. L. Hill, J. Biol. Chem.
  • phosphatidyl lipids for example, vesicular stomatitis virus, VSV, recognizes phosphatidylserine and phosphatidylinositol (P. Mastromarino, C. Conti, P.
  • the system for viral attachment and inhibition that has been most extensively studied in the context of polyvalency is the orthomyxovirus A (X-31) (an engineered strain of influenza) attaching to the surface of erythrocytes (W. J. Lees, A. Spaltenstein, W. J. E. Kingery, G. M. Whitesides, J. Med. Chem. 1994, 37, 3419-3433; M. Mammen, G. Dahmann, G. M. Whitesides, J. Med. Chem. 1995, 38, 4179-4190).
  • This attachment occurs through multiple simultaneous interactions between viral HA and cellular S A. Accurate measurements of the affinity between influenza virus and erythrocyte have so far not been performed.
  • HIV human immunodeficiency virus
  • CD4 solubilized CD4
  • HIV is an example of a virus that can use alternate molecules on the surface of cells: HIV can infect glioma and rhabdosarcoma cells, both of which lack CD4.
  • Solubilized CD4 does not block attachment and subsequent infection of these cell types (P. R. Clapham, J. N. Weber, D. Whitby, K. Mclntosh, A. G. Dalgleish, P. I Maddon, K. C. Deen, R. W. Sweet, R. A. Weiss, Nature 1989, 305, 60-62).
  • mediated binding of virus to cells There are many examples of mediated binding of virus to cells.
  • One example involves Hepatitis B virus, that in some cases bind to aggregates of serum albumin; these aggregates in turn bind to receptors for albumin on the surface of hepatocytes (P. Pontisso, M. A. Petit, M. J. Bankowski, M. E. Peeples, J. Virol. 1989, 63, 1981-1988).
  • a second well-studied example begins with the bivalent attachment of anti-virus antibodies to the surface of the virus (examples include Dengue virus, West Nile virus, and HIV) (R. M. H. V. Van, G. Hardie, Immunochem. 1976, 13, 503-507).
  • Fc-receptors on the surface of the target cell then interact polyvalently with multiple Fc tails. This mechanism of attachment is the same as that used by macrophages in recognition of foreign pathogens: interestingly, where the former leads to infection, the latter leads to clearance.
  • Bacterial pathogens can be divided into two classes: intracellular (those that enter a cell and multiply there) and extracellular (those that live among cells, but not inside them).
  • intracellular tissue that enter a cell and multiply there
  • extracellular tissue that live among cells, but not inside them.
  • the mechanism of infection by those that are intracellular is similar to that of viruses: infection is initiated by attachment of the pathogen to the host cell (Tables 4-5).
  • Bacteria that are extracellular often migrate to and collect in particular tissues. Concentration of bacteria in specific tissues (tissue tropism) can occur by specific interactions between molecules on the surface of the bacteria and either molecules on the surfaces of cells in the preferred tissue, or components of the extracellular matrix characteristic of that tissue. Many specific interactions between bacteria and either host cell or extracellular matrix have been reviewed previously (I. Ofek, N. Sharon, Curr. Topics Microbiol. Immunol.
  • Enteric bacteria are those that take up residence in the gastrointestinal tract of mammals. In most cases, the enteric bacteria express type 1 fimbriae, which bind to mannose on the surface of epithelial cells in the gut (for example, enteric E. Coli, Klebsiella pneumonia, and Salmonella). This interaction can be assayed by agglutination of guinea pig erythrocytes (the surface of which is densely coated with mannose residues). A well-studied example is that of the uropathogenic E. Coli.
  • enteric E. Coli contain fimbrial hemagglutinins on their surface that bind specifically to various sialic acid-containing glycoproteins on the surface of the epithelial cell.
  • a well-studied example of sialic acid recognition occurs on anE. Coli containing the K99 fimbrial lectin (M. Lindahl, R. Brossmer, T. Wadstrom, and Glycoconj. J. 1987, 4, 51-58), which recognizes N-glycolylneuraminic acid located specifically on ⁇ euAc( ⁇ 2,3)Gal( ⁇ l,4)Glc( ⁇ l)-ceramide.
  • Enteric strains of Vibrio cholera produce a variety of hemagglutinins (R. A. Finkelstein, L. F. Hanne, Infect. Immun. 1982, 36, 1199- 1208). The most prevalent of these hemagglutinins binds to fucose derivatives on the epithelial surface.
  • the host organism often mounts a relatively nonspecific defense that is also polyvalent against these polyvalent pathogens.
  • One example is the Tamm-Horsfall glycoprotein, the most abundant glycoprotein in human urine. This glycoprotein contains a variable number of N-linked oligomannose units, and binds a wide range of bacteria (F. DellOlio, F. J. J. de Kanter, D. H. van den Eijnden, F. Serafini-Cessi, Carbohyd. Res. 1988, 178, 327-332).
  • P-fimbrae contaimng protein G
  • type I fimbrae containing the FimH adhesin
  • these uropathogenic bacteria can use the lectin-like protein G located on the tips of their P-fimbrial filaments to adhere strongly and specifically to multiple copies of the Gal( ⁇ l,4)Gal (Pj antigen) portion of a glycolipid present on the surface of the epithelial cells in the urinary tract, especially the kidney.
  • the fibronectin in turn, binds polyvalently to the surface of the epithelial cell.
  • the E. coli collect in these tissues of the urinary tract, multiply there and may cause disease (especially pyelonephritis).
  • bacteria bind either directly to a cellular surface, or to molecules in the extracellular matrix of preferred tissues. Bacteria bind both to sugars and to proteins.
  • GalNAc beta-containing naeslundii 12104 and glycosphingolipids e.g. A. viscosus LY7 GalNAc( ⁇ 1 ,3)Gal( ⁇ )-0-ethyl
  • a predisposing mannoprotein condition e.g. diabetes mellitus, P: C3d receptor immunodeficiency, long-dwelling
  • P Ubiquitin catheters, antimicrobials that eliminate normal bacterial fauna
  • Uropathogenic Urinary Tract Infection P PapG adhesin S: Gal( ⁇ 1 ,4)Gal on glycolipid Esche ⁇ chia coli P: FimH adhesin P?: Uroplakins la and lb
  • Hemagglutinin S Lewis(b) blood group antigen cancers
  • P Laminin binding protein S: Neu5Ac( ⁇ 2,3)Gal (SA dependent strains)
  • Bordetella pertussis Whooping cough (pertussis) P Pertactin and filamentous hemagglutinin (FHA); cell-binding sequence (RGD)
  • Candida albicans Part of normal flora of the oral P: 37-kDa laminin receptor S: Cell-wall polysaccharide of and (female) genital tract, and P: 58-kDa fibrinogen-binding mannoprotein Streptococcus gordonii during causes disease secondary to a P: C3d receptor coinfection predisposing condition (e.g. P: Ubiquitin S: various oligosaccharides diabetes mellitus, containing both beta-1 ,2 and immunodeficiency, long-dwelling alpha-1,2 linkages catheters, antimicrobials that eliminate normal bacterial fauna)
  • Heparin-binding protein S Heparan sulfate-like GAG with can proceed to scarring and minimum chain length blindness decasaccharide
  • Enterococcus faecalis Diarrhea S Galactose, fucose, and mannosamine, but not mannose
  • HMW-1 High-molecular-weight proteins
  • P Lactoferrin-binding protein
  • P Fibronectin, collagen type 1 and (lactoferrin inhibits the binding) type IV and laminin
  • Salmonella enteritidis Severe diarrhea (common food P: Fibronectin poison from chicken and chicken eggs)
  • Staphylococcus aureus Major human pathogen P: Three repeats of a 38-residue D motif .
  • P N-terminal 29 kDa fragment of responsible for wide range of P: Fibronectin-binding proteins (galel A and fibronectin (Fn29K) infections.
  • P Fibronectin meningitis, endocarditis and P: 60 kDa protein
  • P Bone sialoprotein (BSP) (small osteomyelitis
  • BSP Bone sialoprotein
  • CBD Collagen binding domain
  • P Collagen receptor containing either two glycoprotein or three copies of a 187-amino-acid
  • P Collagen of cartilage repeat motif
  • Collagen P N-terminal region (heparin binding domain) of fibronectin
  • Staphylococcus Urinary tract infections S Adherence is partially inhibited saprophyticus by mannose
  • Streptococcus Wide range of gastrointestinal and P Collagen type I respiratory infections
  • P PAM (related to the M proteins) fibronectin
  • Nephritis(+) and Glomerulonephritis P Nephritis plasmin binding protein (NPBP)
  • NPBP Nephritis plasmin binding protein
  • P Human plasmin (the binding is nephritis(-) group A blocked by ⁇ -amino caproic acid) streptococci
  • Streptococcus Not a human pathogen P: Surface protein (ca. 200 kDa)
  • S Cell-secreted extracellular matrix defectivus (ECM)
  • Streptococcus Pneumonia P Laminin, collagen types 1, II and pneumoniae IV, fibronectin, and vitronectin
  • Streptococcus Major pathogen responsible for P Protein F (X-ray structure known)
  • P Fibronectin pyogenes wide range of systemic and local
  • P Streptococcal fibronectin-binding protein
  • P Basal laminae of human cardiac infections, and associated with (Sfb protein, 37-amino-acid sequence) muscle post-streptococcal immunological
  • P 9 kDa glycosaminoglycan-binding protein disorders (GAG-BP)
  • Veillonella atypica Oral infections S Co-aggregations with certain
  • Another example involves the binding of cells to polyvalent molecules such as, for example, bacteria, antibodies and macrophages.
  • All classes of antibodies one of the key groups of proteins making up the immune system — have multiple equivalent receptor sites: two (IgD, IgE, IgG, IgA), four (IgA), six (IgA) or ten (IgM).
  • Polyvalent binding to the structures that these antibodies recognize antigens or other ligands present on the surfaces of bacteria, viruses, parasites, drugs, "non-self cells, or other structures including non-covalent complexes not usually present in the blood circulation — seems to be an ubiquitous characteristic of immune recognition.
  • Galf ⁇ l .3 ⁇ GalNAc( ⁇ 1.4HR-NeuAcfa2.3)Gal( ⁇ 1.4)Glcr ⁇ )Ceramide: R Lucifer yellow CH, Rhodamine, or PNP
  • Clost ⁇ dium tetani Tetanus toxin S Gal( ⁇ 1 ,3)GalNAc( ⁇ 1 ,4)((NeuAc(a2,8))NeuAc(2,3)Gal( ⁇ 1 ,4)Glc( ⁇ )Ceramide S: Isoligands
  • Shigella dysente ⁇ ae Shiga toxin S Gal( 1,4)Gal( ⁇ )Ceramide or E. coli Vero toxin S: Gal( ⁇ 1 ,4)Gal( ⁇ 1 ,4)Glc( ⁇ )Ceramide
  • FGF Fibroblast Growth Factor
  • EGF Epidermal growth factor dimerizes EGF receptor.
  • NP Epidermal growth factor
  • SCF Cytokine Stem Cell Factor
  • TGF Transforming Growth Factor dimerizes TGF receptor.
  • NP Transforming Growth Factor
  • Platelet-derived growth factor dimerizes PDGF receptor. (P, 1:2)
  • hGH Human Growth Hormone
  • Hepatocyte Growth Factor dimerizes c-Met receptor. Heparin may provide a polyvalent template for HGF (see FGF above). (P, N:N)
  • VEGF Vascular Endothelial Growth Factor
  • M-CSF Human Macrophage Colony-Stimulating Factor
  • Lactogenic hormone can dimerize p" ⁇ lactin receptors (PRLR). (P, 1 :2)
  • G-CSF monovalent granulocyte-colony stimulating factors
  • NP Automatic Nervous System
  • Interferon actions Interferon-alpha dimerizes its receptor. (P, N:N)
  • T lymphocyte receptors CD28 and CTLA-4 bind and are oligomerized by co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen presenting cells. (P, N:N)
  • P, 1:2 Heterobivalent ligand dimerizes two surface receptors
  • P, N:N N-valent ligand oligomerizes N surface receptors
  • P probably polyvalent, b with unknown ligand-receptor stoichiometry
  • NP Possibly a non-polyvalent mechanism.
  • Polyvalency also may be important in preventing undesired interaction, i.e., with polyvalent inhibitors. Where polyvalency can strongly promote desired interactions, it can also be a useful strategy in preventing certain undesired biological interactions, especially those that are themselves polyvalent (here, polyvalency is used against polyvalency).
  • polyvalency is used against polyvalency.
  • the fluid coating the interior of the lungs of most mammals contains several mucins (proteins presenting oligosaccharides terminated in sialic acid, SA). These mucins, especially ?2-macroglobulin, can bind to influenza and other S A-binding viruses, and thereby inhibit their attachment to target cells.
  • the subject presenters are used to block cell-bacteria interactions.
  • the subject presenters are used to block cell-fungus interactions.
  • the subject presenters are use to block cell-virus interactions.
  • the subject presenters are used to block cell-parasite interactions. For example, the binding of Entamoeba histolytica trophozoites to host galactose (Gal) and N- acetylgalactosamine (GalNac) residues. (Adler et /. 1995. J. Biol. Chem. 270:5164).
  • polyvalent presenters of the present invention can be made which modulate pathogen-extracellular matrix interactions.
  • polyvalent presenters that prevent the interaction of bacteria or fungi with extracellular matrix can be inhibited.
  • the subject presenters can be used to modulate cell- extracellular matrix interactions.
  • polyvalent presenters that modulate pathogen- pathogen interactions can be constructed. Such presenters will be useful in the treatment of, e.g., infectious states which result from the use of indwelling devices, such as prostheses and catheters, or in the disruption of biofilms ex vivo.
  • cell-toxin interactions may be modulated.
  • shiga toxin from Shigella dysenteriae type 1 binds to cellular glycoproteins or glycolipids having the galabiose disaccharide (Gal ⁇ l-4Gal ⁇ ) determinant.
  • Polyvalent presenters of the present invention can be made to present groups A which can block any such cell-toxin interaction.
  • a polyvalent presenter that reacts with a hemorrhagic toxin, such as that produced by Croatalus viridis viridis can be made.
  • the subject presenters can be used to modulate cellular responses which are associated with polyvalency.
  • cytokine production by cells can be modulated by replacing a stimulator cell in a cell-cell interaction that normally leads to cytokine secretion.
  • the L-selectin ligands on tumor cells have been shown to stimulate monocytes and macrophages to produce tumor necrosis factor.
  • Lewis x (sLe x ) tetrasaccharide has been proposed to function as an HEV ligand for L- selectin, but the natural ligand may be more complex (Putz and Mannel. 1996. Scand. J. Immunol. 44:556-564).
  • Polyvalent presenters bearing A groups which mimic the tumor cell groups recognized by mononuclear cells could be used to mimic this response.
  • presenters bearing A groups which block this reaction could be used to inhibit this response.
  • other states associated with polyvalency in which a polyvalent presenter could be used include T cell cytokine production, mast cell and/or basophil degranulation, lymphocyte selection, and T or B cell apoptosis (Seledtsov and Seledtsova. 1995. Biomed & Pharmacother. 1996. 50:170), all of which are states associated with polyvalency.
  • polyvalent presenters can be designed to modulate infectious disease, including e.g., inhibiting host-parasite interactions, augmenting immunization or vaccination strategies, and reducing sepsis or septic shock.
  • the subject presenters will be useful in the treatment of, inter alia: infectious diseases of the upper respiratory tract; infective endocarditis; intraabdominal infections and abscesses; acute infectious diarrhea diseases and bacterial food poisoning; sexually transmitted diseases; pelvic inflammatory disease; urinary tract infections and pyelonephritis; infectious arthritis; osteomyelitis and infections of prosthetic joints; infections of the skin, muscle, and soft tissues; infections in injection drug users; infections from bites, scratches, burns, and environmental organisms; and nosocomial infections.
  • the subject presenters will be useful in the treatment of states caused by gram positive organisms (e.g., pneumococcal infections, staphylococcal infections, streptococcal infections, corynebacterial infections, listeria infections, tetanus, botulism clostridial infections, and anthrax), and states caused by gram negative bacteria (e.g., meningococcal infections, gonoccoccal infections, Moraxella and Kingella infections, Haemophilus infections, Legionaella infections, pertussis, infections with enteric bacilli, Pseudomonas infections, salmonellosis, shigellosis, Campylobacter infections, cholera and other vibrioses, brucellosis, tularemia, Yersinia infections, bartonellosis, and donovanosis.
  • gram positive organisms e.g., pneumococcal infections, staphylococcal infections, streptococcal infections, coryn
  • the subject presenters will also be useful in the treatment of nocardiosis, actinomycosis, mixed anaerobe infections, and mycobacterial and spiral bacterial (H. pylori) infections (e.g., tuberculosis, leprosy, and Mycobacterium avium infection).
  • Polyvalent presenters can also be used in the treatment of spirochetal diseases (e.g., syphilis, treponematoses, leptospirosis, relapsing fever, and lyme borreliosis).
  • Rickettsia, Mycoplasma, and Chlamydia infections will, likewise, benefit from treatment with the subject polyvalent presenters.
  • Viral infections will also benefit by treatment with the subject polyvalent presenters.
  • DNA viruses e.g., herpes simplex, varicella-zoster, Epstein-Barr, cytomegalovirus infection, poxvirus infection, parvovirus, and human papillomavirus
  • RNA viruses e.g., retroviruses, influenza, gastroenteritis, enteroviruses and reoviruses, rubeola, rubella, mumps, rabies, rhabdoviruses, and marburg-like agents, arbovirus infections, and arenavirus infections
  • retroviruses e.g., retroviruses, influenza, gastroenteritis, enteroviruses and reoviruses, rubeola, rubella, mumps, rabies, rhabdoviruses, and marburg-like agents, arbovirus infections, and arenavirus infections
  • retroviruses e.g., retroviruses, influenza, gastroenteritis, enter
  • the subject presenters can be used to treat fungal infections, e.g., histoplasmosis, coccidioidomycosis and paracoccidioidomycosis, blastomycosis, cryptococcosis, candidiasis, aspergillosis, mucormycosis, among others can be treated.
  • fungal infections e.g., histoplasmosis, coccidioidomycosis and paracoccidioidomycosis, blastomycosis, cryptococcosis, candidiasis, aspergillosis, mucormycosis, among others can be treated.
  • polyvalent presenters can be used to treat protozoal infections (e.g., amebiasis, malaria and babesiiosis, leishmaniasis, trypanosomiasis, toxoplasma, pneumocyctis cariniii, giardiasis, cryptosporidiosis, and trichomoniasis).
  • protozoal infections e.g., amebiasis, malaria and babesiiosis, leishmaniasis, trypanosomiasis, toxoplasma, pneumocyctis cariniii, giardiasis, cryptosporidiosis, and trichomoniasis.
  • the subject presenters can be used to treat helminthic infections (e.g., trichinosis, tissue nematodes, intestinal nematodes, filariasis, loiasis, onchocerciasis, dracunculiasis, schistosomiasis and other trematode infections, or cestodes).
  • helminthic infections e.g., trichinosis, tissue nematodes, intestinal nematodes, filariasis, loiasis, onchocerciasis, dracunculiasis, schistosomiasis and other trematode infections, or cestodes.
  • the subject presenters can be used to treat ecotparasite infestations.
  • the subject presenters are also useful in modulating the immune response, both by upregulating and downregulating that response. Accordingly, the subject polyvalent presenters will be useful both in the treatment of immunodeficiency diseases, (regardless of the underlying cause) as well as in the treatment of autoimmune disease and resulting immune-mediated injury. In certain embodiments the subject presenters will be useful in inhibiting graft rejection.
  • the subject polyvalent presenters are also useful in the treatment of disorders of coagulation and thrombosis and in anticoagulant, fibrinolytic, and antiplatelet therapy.
  • the subject presenters are also useful in the treatment of neoplasia.
  • the subject presenters are used to inhibit metastasis of primary tumors.
  • the initial event in the biological action of certain plant-derived and bacterial toxins involves their multivalent adhesion to the surface of a cell; that is, their binding by the simultaneous interaction of multiple toxin receptors to multiple cellular ligands (Lord, J. M.; Roberts, L. M.; Robertus, J. D. FASEB 1994, 8, 201-208; Montecucco, C; Papini, E.; Schiavo, G. Experientia 1996, 52, 1026-1032; Kuziemko, G.
  • polyvalent polymeric galactosides can be used to block the adhesion of the cytotoxic lectins from Ricinus communis, commonly referred to as ricins, to the surface of mammalian cells. These polymers act by preventing the ricins from interacting with galactoside residues on the cell surface. The number of receptor-ligand interactions involved in ricin-cell adhesion is small.
  • Ricins, Ricinus communis agglutinins belong to a class of cytotoxic lectins, referred to as ribosomal-inactivating proteins (RIPs).
  • ribosomal-inactivating proteins RIPs.
  • One ricin subtype, RCA_ ⁇ o (an AlBi covalent complex; MW ⁇ 65 kDa) is composed of two subunits: an A-chain — an N-glycosidase that cleaves a specific adenine base of ribosomal R ⁇ A hydrolytically and inhibits protein synthesis — and a B-chain — a sugar- binding lectin (Kuziemko, G. M.; Stroh, M.; Stevens, R. C.
  • a second ricin subtype, RCA ⁇ 20 (2 x A'lB' i; MW ⁇ 130 kDa) is related closely to RCA ⁇ o in many aspects of its structure and function; it is composed of two, non-covalently associated A'lB' i units (Endo, Y.; Mitsui, K.; Motiguki, M.; Tsurugi, K. J. Biol. Chem. 1987, 262, 5908-5912).
  • the A-chain- catalyzed inactivation of rR ⁇ A is the major cause of the toxicity of ricins. This action cannot, however, occur without cellular internalization of the toxins; this internalization occurs via receptor (B-chain)-mediated endocytosis.
  • Ricins inhibit biosynthesis of proteins strongly in mammalian cells.
  • the molecular action of ricins requires cellular internalization, which begins with adhesion of ricin to the surface of the cell: this strong toxin-cell attachment occurs by the simultaneous binding of multiple galactoside-recognition sites of ricin to multiple galactoside (Gal) groups of cell-surface glycoproteins and glycolipids.
  • Ricins attach to the cellular surface using multivalent, selective binding of Gal-binding sites of the B chain ( ⁇ 3 sites per B chain) to multiple ⁇ -D-galactose ( ⁇ - Gal)- and ⁇ -N-acetyl-D-galactosamine ( ⁇ -Gal ⁇ Ac)-te ⁇ ninated oligosaccharides present on the surface of cells (Roberts, L. M.; Lamb, F. I.; Pappin, D. J. C; Lord, J. M. J. Biol. Chem. 1985, 260, 15682-15686) (The number of Gal-binding sites per B subunit has been controversial.
  • RCA ⁇ o and RCA 120 have ⁇ 3 (> 2) and ⁇ 6 (> 4) Gal-sites, respectively).
  • Binding studies of 125 I- labeled ricins indicated that ricins (and the isolated B-chain) attached to human erythrocytes with dissociation constant K ⁇ t ⁇ 0.1-0.01 ⁇ M.
  • Simple monovalent analogs of ⁇ -D-galactosides have relatively low affinity for RCA 12 o, RCAeo and the isolated B-chain, when studied by various techniques (equilibrium dialysis, fluorescence, and NMR) (Kj ⁇ lO ⁇ -lO ⁇ ⁇ M).
  • polyvalent presenters can be produced to achieve relatively tight binding of ricins to the surface of the cell.
  • Ricins are, in many aspects of structure and modes of action, related closely to bacterial toxins (anthrax; cholera; shiga; verotoxin) and other plant-derived cytotoxic lectins (ribosomal-inactivating proteins; abrin). These toxins are the objects of current research as the cytotoxic components of immunotoxins in development as anticancer drugs, and have been classified as threat agents in biological warfare.
  • the subject presenters are useful in preventing adhesion of ricins to erythrocytes.
  • the polyvalent presenters may be used to block the toxicity of ricins based on polyvalency by blocking ricin-cell adhesion. In this manner, the first step in their entry into the cell is blocked, using polyvalent polymeric inhibitors capable of competing with the multiple copies of the ligands presented on the surface of the cell.
  • the synthesis and bioassay of polymeric polyvalent molecules designed to inhibit adhesion of ricins (RCA ⁇ 20 and RCA ⁇ o) to chicken red blood cells are discussed herein.
  • the inhibitors are derivatives of poly(acrylic acid) and poly(butadiene-co-maleic acid); they present multiple copies of galactoside-containing side chains as amide groups: pAA(Gal) and pBMA(Gal).
  • this polymeric polyvalent galactoside is ⁇ 1500 times more active than monovalent Gal- ⁇ -OMe, and 270 times more active than monovalent Gal- ⁇ o-LlNH2.
  • the polymeric polyvalent inhibitors are significantly more active in blocking hemagglutination induced by RCA 120 (with ⁇ 6 Gal receptor sites) than that by RCAeo (with ⁇ 3 Gal receptor sites).
  • the molecular basis of the enhanced activities of the polymeric polyvalent galactosides in blocking hemagglutination is ascribed to multivalent (that is, multiple, simultaneous) interactions of the Gal receptor sites of ricin and Gal moieties on the polymers.
  • synthetic giycopolymers can inhibit ricin-cell adhesion. These polymers are, thus, a new class of synthetic antitoxins.
  • these polymers may mimic the action of anti-toxin antibodies by preventing the approach of the ricin Gal-binding site to Gal moieties on the surface of the cell, they also introduce the possibility of fundamentally new mechanisms of inhibition: entropically-enhanced occupancy of the ricin Gal-binding sites; steric stabilization of unoccupied Gal binding sites by the surrounding polymer.
  • entropically-enhanced occupancy of the ricin Gal-binding sites steric stabilization of unoccupied Gal binding sites by the surrounding polymer.
  • a scheme summarizing ricin-cell interactions (for both RCA120 and RCA ⁇ O), and a strategy for the neutralization of ricins using a polymeric polyvalent agent is set forth in Figure 3 and involves the following: (a) Receptor-ligand mediated adsorption of ricins to a mammalian cell: multivalent binding of receptor sites (B-chain) of ricins to multiple, specific cellular ligands ( ⁇ -galactosides) results in tight toxin-cell association; the attachment of ricins to the cell constitutes an initial step in their toxic action, (b) The mechanism of toxicity of ricins: after entry of toxins into the interior of the cell (via endocytosis), and reductive cleavage of the A-chain from the B-chain, monomeric A-chain efficiently catalyzes hydrolytic N-glycosidation of a specific adenine base of ribosomal R ⁇ A; this damage interferes with protein biosynthesis, (c)
  • Gal-presenting polymers of the invention may be used as inhibitors that prevent the attachment of ricins to mammalian cells; these compounds, or derivatives of these compounds, are potential leads as therapeutics and as protective materials against other exposures to ricins.
  • Multivalent binding of toxin receptors to specific cell-surface ligands is also important in other situations.
  • abrins contain multiple receptor sites per B-chain of toxin molecule; this toxin binds selectively to galactoside residues (Lord, J. M.; Roberts, L. M.; Robertus, J. D. FASEB 1994, 8, 201-208; Sandvig, K.; Olsnes, S.; Pihl, A. J. Biol. Chem. 1976, 251, 3977-3984).
  • Cholera toxin utilizes pentameric B (sugar-binding) subunits, each of which recognizes GMi pentasaccharide (Gal( ⁇ 1 ,3)Gal ⁇ Ac( ⁇ 1 ,4)(NeuAc( ⁇ 2,3))Gal( ⁇ 1 ,4)Glc( ⁇ 1,1)- ceramide) residues on the cellular surface (Kuziemko, G. M.; Stroh, M.; Stevens, R. C. Biochem.
  • shiga toxin and verotoxin utilize five B- subunits for binding to Gal-containing glycosphingolipid (globoside, Gal( ⁇ l,4)Gal( ⁇ l,4)Glc( ⁇ l,l)-ceramide) (Richardson, J. M.; Evans, P. D.; Homans, S. W.; Donohue-Rolfe, A. Nature Struct. Biol. 1997, 4, 190-193).
  • the present invention finds use in designing inhibitors from pathogen (cell)-cell association (characterized by high valency: about 100-1000 sites are potentially available for interaction) to toxin-cell interactions (characterized by low valency: that is, ⁇ 10 Gal receptor sites are involved).
  • the subject polyvalent presenters can be used to inhibit egg-sperm interactions such that fertilization is inhibited, e.g. the acrosomal reaction can be interfered with using a polyvalent presenter.
  • the subject presenters are also useful in preventing conception, e.g., by inhibiting the sperm-egg interaction, e.g., inhibition of attachment of sperm to eggs or by inducing the acrosomal reaction prior to interaction between sperm and egg, e.g., premature activation of the reaction.
  • Fertilization is initiated by species-specific adhesion of sperm to the extracellular coat of egg.
  • the sperm-egg interaction causes the sperm to undergo a series of acrosomal reactions (acrosomal process): the early reaction appears as exocytosis of acrosomal vesicles of sperm to egg coat.
  • This acrosomal reaction leads eventually to fusion of the plasma membranes of sperm and egg, and to entry of a nucleus of the sperm into the cytoplasm of the egg.
  • a surface protein ⁇ -l,4-galactosyltransferase (Gal- transferase) on the plasma membrane of the sperm is the receptor that recognizes specifically GlcNAc residues of zona pellucida (ZP) glycoproteins of egg coat ( Figure 4).
  • GlcNAc zona pellucida glycoproteins of egg coat
  • Adhesion of a mouse sperm to an egg is mediated by receptor-ligand interactions: Gal-transferase on the surface of sperm head and terminal N-acetylglucosamine (Glc ⁇ Ac) residues of ZP3 glycoprotein on zona pellucida (ZP) of egg coat: (a) Multivalent binding of the Gal-transferase receptors to multiple Glc ⁇ Ac ligands of ZP3 results in aggregation of the Gal-transferase, and (b) subsequently in induction of acrosomal exocytosis of sperm via intracellular signal transduction.
  • Glc ⁇ Ac N-acetylglucosamine
  • the acrosomal reaction of sperm occurs through an intracellular signal transduction mechanism, triggered by activation of certain G-protein coupled receptors.
  • the activation of the G-protein receptors requires spatial aggregation of Gal-transferase ( Figure 4).
  • Figure 4 In the normal process of sperm-egg fertilization, ZP3 glycoproteins in ZP of the egg coat bind to and aggregate Gal-transferases: the aggregation is a result of multivalent interactions of multiple Glc ⁇ Ac ligands of ZP3 and sperm surface Gal-transferases.
  • polyvalent forms of Glc ⁇ Ac a mimic of multivalent Glc ⁇ Ac of ZP3 — induce acrosomal reaction of sperm ( Figure 4).
  • Polymeric polyvalent Glc ⁇ Ac induces acrosomal exocytosis of mouse sperm, and furthermore inhibits the binding of sperm to egg in vitro.
  • the polyvalent presenters of the present invention may be combined with other methods of contraception or can be used to also prevent disease while being used in the prevention of contraception.
  • the groups A can be ligands useful for treating herpes, HIV, chlamydia, human papillomavirus, gonorrhea and syphilis.
  • the subject presenters are also useful for treating or modulating cardiogenic shock, angina, thrombus formation within the circulatory system, heart failure, arteriosclerosis, hypertension, stroke, restenosis, failure of the kidney to secrete erythropoeitin, secretory disease, allergic reaction, psoriasis, sickle cell anemia, secondary diseases of the mematopoetic system secondary to treatment (chemotherapy, side effect of a drug) or another disease (metastasis, autoimmune disease), renal cancer, prostate cancer, brain cancer, lung cancer, thyroid cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, tumor resistance to chemotherapy, adult onset diabetes, growth deficiencies, C ⁇ S trauma, neurodegenerative disease, schizophrenia, migraines, anxiety, depression, obsessive compulsive disorders, idiopathic disease, (diseases whose causes are unknown but have etiologies which are amenable to polyvalent drugs including fever of unknown origin, multi-organ failure), ophthalmic indication
  • Even further embodiments include a condition or disease involving the modulation, e.g., promotion, of cell death, the modulation, e.g., promotion, inhibition, or augmentation, of a metabolic pathway (e.g., via an enzyme) or the targeting of a particular receptor type, e.g., clustered, seven member transmembrane receptor, or an enzyme, e.g., intracellular, extracellular, or membrane bound.
  • a condition or disease involving the modulation, e.g., promotion, of cell death the modulation, e.g., promotion, inhibition, or augmentation, of a metabolic pathway (e.g., via an enzyme) or the targeting of a particular receptor type, e.g., clustered, seven member transmembrane receptor, or an enzyme, e.g., intracellular, extracellular, or membrane bound.
  • the present invention also provides for methods of assaying for polyvalent presenters which can be used, e.g., in identifying desirable useful groups A or in testing the efficacy of presenters.
  • assays can be either in vitro or in vivo.
  • in vitro assays can be designed which test for the ability of a polyvalent presenter to modulate the interaction between A and B, or a biological response which results from the interaction of A with B (e.g., cell adhesion assays, agglutination assays, platelet aggregation assays, ELISA assays, as well as muscle contractility assays, infectivity assays, or lymphocyte stimulation assays, and the like).
  • in vitro assays can be used to test the effectiveness of polyvalent presenters which assay the ability of a polyvalent presenter to interact with target binding sites B or to inhibit the interaction of Groups C with binding sites B.
  • assays which test the ability of Group A of a presenter to interact with binding sites B on a polyvalent surface can be used in screening presenters (Charych, D. et al. Chem. & Biol 1996, 3, 113- 120).
  • capillary electrophoresis can be used.
  • CE is a convenient high resolution analytical technique requiring only femtomoles of material.
  • CE allows separation of mixtures of molecules (ions, small molecules, polymers, proteins, micelles) on the basis of their charge and hydrodynamic drag.
  • groups A By adding groups A to the buffer solution at varying concentration, and by monitoring the influence of this concentration of the mobility of injected binding sites B, it is possible to quantitate accurately the binding constant of the group for the binding site.
  • This technique is referred to as affinity capillary electrophoresis (ACE).
  • affinity capillary electrophoresis ACE
  • the affinity of a presenter for a whole virus expressing binding sites B can be determined using ACE. It has also been shown that ACE forms the basis of a very efficient library search.
  • CE is also useful, as are GPC and light scattering, in the analysis of presenters, especially when they carry charge.
  • SPR Surface plasmon resonance
  • Model Surfaces Based on Self-Assembled Monolayers can also be used to assay the subject presenter molecules.
  • SAM's of alkanethiolates on gold and silver is another model system for studying adso ⁇ tion, or other molecular events occurring at interfaces.
  • agglutination can be tested, for example, using synthetic beads mixed with pathogens or cells (equivalent to hemagglutination inhibition assays) for the quantitative analysis of polyvalent molecules.
  • Agglutination is a highly convenient method of probing cell-cell and pathogen-cell contacts and 96-well microtiter plates are an especially suitable format for such an assay. Automation of this assay is also possible.
  • a bead can be constructed that presents a group that interacts specifically with the surface of the pathogen or cell. The construction of such a bead can consider steric access to the group relative to the background.
  • appropriate attachment point to the group can be considered and may be based on crystal structures where available.
  • a target cell or surrogate target cell
  • influenza virus binds to erythrocytes, and erythrocytes, rather than beads, can be used.
  • auxiliary groups e.g., A2- A ⁇
  • A2- A ⁇ auxiliary groups
  • One mechanism whereby such enhancement is possible is by randomly locating hydrophobic pockets on the surface of the pathogen (or cell) using a small mole fraction of short hydrophobic side chains.
  • Important properties of the polymer that are expected to control its potency include: mole fraction of active group, charge, hydrophobicity, persistence length, randomness, physical dimensions, and number of associated water molecules.
  • Inhibition assays can also be used, such as those that measure the extent to which a molecule prevents a biological surface, e.g., a virus, from binding to another biological surface, e.g., a cell.
  • the molecule may do so by binding competitively to the receptor, and prevent binding of the surface bound groups to the same binding site.
  • One exemplary assay is the hemagglutination inhibition (HAI) assay, which is described in more detail in the appended examples and appendices.
  • HAI assay is based on molecules inhibiting viral agglutination (gel formation) of a solution of erythrocytes. The lower limit of effectiveness that HAI can conveniently measure is ⁇ 1 nM.
  • Another exemplary assay is the optical collision (OPTOCOL) assay, and is based on the inhibition of attachment upon collision of one erythrocytes and one virus-coated microsphere in the presence of inhibitor.
  • This assay is performed under an inverted microscope using parallel optical tweezers, with each of the tweezers holding one of the two colliding species.
  • the OPTOCOL assay allows quantitation of effectiveness of inhibitors that are active at « 1 nM.
  • Both the HAI and OPTOCOL assays yield a concentration at which the inhibition is partially effective, i.e., a concentration at which approximately half the interactions between groups A and binding sites B are inhibited.
  • the inhibition constant is KHA or KOPTOCOL.
  • KHA ⁇ KOPTOCOL. Both these inhibition constants can be referred to as KINH.
  • OPTOCOL is based on the manipulation of biological particles using parallel dual optical tweezers. (Mammen, et al, 1996 Chem. Biol. 3 :757) This technique enables study of interactions between, e.g., a single erythrocyte and a single microsphere presenting influenza virus. This technique is especially useful for very tight-binding systems and can also be used for studying the mechanisms of polyvalent inhibition.
  • Most preferred assays weight the characteristics of the polyvalent group that are important for achieving the desired goal.
  • an effective polyvalent molecule for the pu ⁇ ose of aggregating a polyvalent binding site might be composed of "n" greater than 10 groups linked through a long flexible linker. The assay, therefore, must reflect the ultimate pu ⁇ ose of the polyvalent species.
  • thermodynamically i.e., to obtain a binding constant
  • the relative proportions of uncomplexed and complexed group (or binding site) must be measured (directly or indirectly).
  • different techniques can be used. .
  • Aggregation assays can be used to measure the ability of a polyvalent group to aggregate a polyvalent binding site (precipitation, gel formation, aggregation).
  • a polyvalent presenter can precipitate polyvalent binding sites B on a surface in immunoprecipitation assays.
  • affinity of the polyvalent entity is important in determining the ability of the polyvalent presenter to precipitate, other characteristics can be important. For example, at low concentrations, the presenter may not bind to polyB; at some optimal concentration zone, precipitation occurs; while at higher concentrations, each binding site B is bound by a group A and precipitation again does not occur. In this example, which is analogous to antibody precipitation reactions, affinity alone does not determine the pattern of precipitation.
  • In vivo assays can be conducted which, for example, measure the polyvalent presenter therapeutic effect for a disease or condition in an animal, for example, protection against infection could be measured.
  • Such assays include, but are not limited to, a measure of the inhibitor to prevent polyvalent interaction.
  • the molecule may, for example, not only slow the rate of infection by blocking attachment to host receptors, but may slow the rate of clearance by blocking the clearance mechanisms.
  • One exemplary assay is phage infection of bacteria. Chu, Y.-H., et al, Acc. Chem. Res. 1995, 28, 461-468.
  • Table 8 sets forth numerous assays which can used to screen the polyvalent presenters of the present invention for useful properties.
  • Influenza-Erythrocyte labeling of the interaction polyvalent species in general limited to dissociation constants greater than 10-50 nM
  • Shear Flow T Neutrophil yes yes, in principle no based on counting Springer and interaciton with large number of events others) surfaces derivatized (stuck, non-stuck) non-covalently with under an optical different selectins microscope; probability of adhesion is measured as a function of flow rate of solution past surface
  • the present invention also provides for polyvalent presenters for ex vivo applications, e.g., materials and systems for passive protection against biological threats, components for systems for decontamination and new diagnostic and characterization systems.
  • the subject polyvalent presenters are designed, ultimately to block infection or intoxication of a subject.
  • the subject presenters may additionally comprise one or more antimetabolites.
  • passive protection can be provided by the use of protective masks comprising polyvalent presenters that are effective against infectious agents or toxic gases, as well as protective wraps, gowns, dressings and washes for hospitals.
  • the polyvalent presenters of the present invention can be inco ⁇ orated into solutions and processes for decontamination systems.
  • the subject polyvalent presenters can be used in, for example, panning assays, or optical assays based on adso ⁇ tion at surfaces.
  • panning assays or optical assays based on adso ⁇ tion at surfaces.
  • optical assays based on adso ⁇ tion at surfaces.
  • the subject polyvalent presenters exhibit enhanced affinity for binding sites B over that seen for monoA.
  • the subject polyvalent presenters have a greater specificity than monovalently presented A.
  • specificity it is meant that the nonspecific interaction of poly A with binding sites nonB is reduced over than observed with monoA.
  • the subject polyvalent presenters produce a biological effect at a lower concentration than that observed for A presented in monovalent form.
  • the polyvalent presenters of the present invention can function by one or more of the mechanisms described below.
  • binding sites B e.g., N receptor sites
  • groups A e.g., N ligands
  • an interaction between N ligands and N receptors distributed on two entities will be referred to as an Nth-order polyvalent interaction (illustrates a third-order polyvalent interaction). It occurs with free energy of association AG ⁇ oly .
  • the average free energy of interaction, ⁇ G ° ⁇ between a single ligand moiety and a single receptor moiety in the polyvalent interaction shown is equal to
  • Equations 3-5 give the values of the corresponding association constants, K.
  • the average free energy of interaction between a ligand moiety and receptor moiety in a polyvalent interaction can be greater than, equal to, or less than the free energy in the analogous monovalent interaction ( ⁇ G mono ) (eqs. 6-8).
  • these classes of polyvalent interactions will be referred to as being positively cooperative (synergistic), non-cooperative (additive) or negatively cooperative (interfering), respectively.
  • the convention of defining ⁇ as the degree of cooperativity will be followed (Connors, K. A. Binding Constants: The Measurement of Molecular Complex Stability;John Wiley 8c Sons: New York, 1987).
  • the units of ⁇ depend on the order of the polyvalent interaction.
  • ⁇ > 1 a positively cooperative polyvalent interaction
  • GMj a positively cooperative polyvalent interaction
  • Cholera toxin consists of five subunits (AB5).
  • the binding of the toxin to monomeric GMj (GMI that has been cleaved from the ceramidyl moieties on the cellular surface) provides a well-studied example of positive cooperativity through purely enthalpically enhanced binding.
  • the entropy of the first binding event of a monomeric GMj derivative to pentameric cholera toxin is equal to the entropy of each subsequent binding event. All differences in the free energies of binding of monomeric GM j to the toxin are therefore due to differences in enthalpy. Schoen et al. performed calorimetric studies of the binding of pentameric B5 to five independent GMj oligosaccharide units in solution (Schoen, A.; Freire, E. Biochemistry 1989, 28, 5019-24). The binding constant of the first ligand was less than the binding constant of the second by a factor of 4.
  • the first example is the binding of a bivalent antibody (Ab) to ligands found densely packed on a biological surface (such as a mammalian cell, a virus, or a solid support during an ELISA assay), or immobilized in a polymeric matrix. Tanaka, T.; Suzuno, R; Nakamura, K.; Kuwahara, A.; Takeo, K. Electrophoresis 1986.
  • Enhanced affinity in polyvalent interactions the magnitude of ⁇
  • N the number of ligand-receptor interactions
  • SA sialic acids
  • the binding of this polymeric polyvalent inhibitor to the surface of the virus using an ELISA-like assay can be measured.
  • the surface-bound virus was incubated with varying concentrations of polymer containing SA, and the amount of bound polymer was measured indirectly through an enzyme-linked streptavidin-biotin interaction.
  • the measurable quantities in constructing a binding isotherm were the concentration of SA, and the amount of S A contained on polymer bound to the surface of the virus.
  • a polyvalent presenter of the invention where group A is a saccharide usually has a ⁇ value that is greater than or equal to about 10 8 , preferably.
  • a polyvalent presenter of the invention where group A is a non-saccharide, particularly where group A is a non-natural ligand usually has a ⁇ values that is greater than or equal to about 10, preferably, greater than about 10 2 , more preferably, greater than about 10 4 .
  • Non-natural ligands generally are not polyvalent although the receptor to which it binds may have multi-valency.
  • the ⁇ G ⁇ y is comprised of enthalpic ( ⁇ H ⁇ y ) and entropic ( ⁇ S ⁇ o y ) components (eq 12).
  • ⁇ H ⁇ y enthalpic
  • ⁇ S ⁇ o y entropic
  • the value of ⁇ H ⁇ y is the sum of the enthalpies of N monovalent interactions, N ⁇ H 1110110 . This value may be made either larger or smaller by other interactions around the active site.
  • the binding of one ligand to a receptor with a given enthalpy may cause the next ligand to bind to its receptor with greater enthalpy; that is, the value of ⁇ H ⁇ ° y is in this case more negative (more favorable) than the value of ⁇ H 1110110 .
  • Enthalpically Diminished Binding If the binding of one ligand to its receptor interferes with the next binding event, the enthalpy of the polyvalent interaction is less favorable than that expected for N equivalent monovalent interactions. Such binding is enthalpically diminished. Enthalpically diminished binding can occur when formation of multiple ligand-receptor interactions between two polyvalent entities requires energetically unfavorable molecular conformations. As a rule of thumb, the more conformationally rigid the polyvalent entity, the more likely that spatial mismatches between ligand and its receptor will result in enthalpically diminished binding (unless the geometric fit between ligand and receptor is exact).
  • Entropy of interaction Understanding the entropy of polyvalent interactions is essential in understanding the relationship of monovalent to polyvalent binding. Incomplete understanding of entropy in the design of polyvalent inhibitors has resulted in many synthetic polyvalent molecules that are only marginally more effective than their monovalent counte ⁇ arts.
  • the total entropy of a polyvalent interaction ⁇ Sj ⁇ y should be considered in terms of contributions from changes in translational ( ⁇ S ⁇ N ), rotational ( ⁇ S j ° t y N ) and
  • the translational entropy of a molecule arises from its freedom to translate independently through space; the value of ⁇ St rans is related to the logarithm of its mass, M ( ⁇ St rans ex ln(M)), and inversely to the logarithm of its concentration ( ⁇ St ⁇ -ans oc ln[L]"l).
  • the rotational entropy, ⁇ S rolJ arises from the freedom of the particle to rotate around all three of its principle axes, and is related logarithmically to the product of its three principle moments of inertia, I ⁇ , I ⁇ and I z ( ⁇ S ro t ⁇ ln ⁇ yl ⁇ ).
  • concentrations of molecules can vary over more than nine orders of magnitude (mM to pM). Even though the translational entropy is only logarithmically dependent on concentration, such wide range makes knowledge of the concentration of interacting particles essential to estimating the importance of translational entropy: this cost increases with decreasing concentration.
  • Entropy and enthalpy can have partly compensating effects on the affinity of polyvalent interactions: where conformational flexibility increases the conformational entropic cost of association, the same flexibility increases the likelihood that all ligand- receptor interactions can occur without energetic strain.
  • This loss in conformational entropy on association of a polyvalent ligand with a polyvalent receptor has been notoriously difficult to quantitate.
  • the change in entropy on freezing a single, rotating carbon-carbon bond is approximately 0.5 kcal/mol (ref).
  • the range of values for other single bonds is 0.1 - 1.0 kcal/mol.
  • the maximum loss in conformational entropy would occur if all bonds that were initially rotating freely lost all degrees of torsional freedom on complexation.
  • a final contribution to the total entropy of association in aqueous system is the change in the entropy of the surrounding molecules of water
  • the entropic cost of association between N ligands freely diffusing in 2D (such as ligands tethered to the surface of a cell) with N receptors also freely diffusing in 2D (such as trans-membrane proteins on the surface of another cell) is less than for the interaction of these species diffusing independently in 3D.
  • the subject polyvalent presenters function by having extremely low off-rates, such that they are effectively "permanently” bound with regard to the time frame of a biological event relevant to therapy.
  • This difference in kinetics between polyA and monoA, i.e., the lower off-rate for the polyvalent form is another mechanism by which the subject presenters are different from mono A.
  • thermodynamic cost of the first ligand-receptor interaction between two polyvalent entities is approximately the same as the thermodynamic cost of the analogous monovalent interaction; it is therefore plausible that the rates of association might be similar.
  • Dissociation of species interacting polyvalently requires breaking N ligand-receptor interactions; it is therefore plausible that dissociation occurs more slowly in the polyvalent interaction than in the corresponding monovalent one.
  • polyvalent presenters of the present invention function by "steric inhibition".
  • Steric inhibition is a new strategy in the design of effective pharmaceutical agents.
  • polyvalent inhibitors of attachment may be designed that involve any molecule that binds tightly to the surface of the infectious agent, i.e., the polymers that present molecules need not be directly involved in attachment.
  • NA neuraminidase
  • NA site is commonly regarded by those of skill in the art as not mediating adhesion, thus the anti-adhesive effect observed with a polymer directed toward it may occur as a result of the attachment of the polymeric gel layer to the viral surface.
  • This effect may be "pure" steric inhibition, i.e., with no entropically enhanced occupancy of the active site of hemagglutinin, the protein that the virus normally uses in adhesion.
  • polyvalent presentation of a drug may change the original mechanism of action for that drug.
  • drugs There are currently no known examples of such drugs.
  • the mechanism of steric inhibition is believed to be more related to colloidal stabilization than to receptor-mediated events, although it does depend on receptor-directed specificity to target the polymer to the appropriate binding sites B.
  • Bringing two moieties or groups together when one or both is coated by a gel layer is unfavorable both entropically (because the conformational mobility of the water-swollen polymer is decreased on approach to another surface) and enthalpically (because of unfavorable osmotic effects).
  • Polymeric presenters are unique in that they function by this type of mechanism, although there are other, related mechanisms that may appear with liposomes (Kingery-Wood, et al. J. Am. Chem. Soc.
  • the subject polyvalent presenters function by mediating the adso ⁇ tion of binding sites B on a surface.
  • binding sites B e.g., a viral particle
  • the polyvalent presenters function by being involved in precipitation or aggregation.
  • the framework that is chosen and its inherent properties will influence the pharmacodynamics of the polyvalent presenter.
  • two of the properties that are considered when designing polyvalent presenters of the present invention are solubility and size.
  • Solubility in most embodiments the polyvalent presenters of the present invention will be more water soluble than conventional pharmaceutical agents, (e.g., in the mg/ml range or higher). Solubility (and size as described infra) can influence the pharmacodynamics of the polyvalent presenter. For example, solubility of the presenters may influence one or more of the related characteristics described below. For example, solubility can influence the clearance profile of the subject polyvalent presenters. Clearance can be dramatically increased as the solubility of a molecule increases. The kidney tends to filter water soluble molecules more rapidly. Also, the rate of drug clearance is directly proportional to the frequency of drug administration.
  • the water solubility of the subject presenters can also influence the duration of action of the presenters.
  • the subject presenters have a longer duration of action than does monovalently presented A.
  • the solubility of poly A can influence therapeutic index.
  • therapeutic index refers to the (LD50/ED50) as can be determined by methods well known in the art.
  • the therapeutic index as used herein is meant to be calculated on a per-group A basis. The therapeutic index is inversely proportional to the frequency of drug administration. Owing to the lower clearance rates of the subject presenters, polyA will be able to be administered to a subject in at less frequent doses than mono A.
  • polyA will exhibit a lower concentration variance over time in a subject at the site of interest than mono A, since the rate of clearance of these drugs can be very slow, they can stay at a more even concentration in the plasma, i.e., polyvalent drugs will have a reduced difference between maximum and minimum concentration at the site of interest (e.g., lower trough-peak variance). Since polyvalent molecules are large they have the distinct advantage that their lifetimes can be significantly longer than those observed for small molecules. Owing to the lower clearance rates of the subject presenters, polyA will be able to be administered to a subject in at less frequent doses than monoA. These longer half- lives are advantageous for a variety of reasons. For example: (i) patient compliance and patient happiness would increase as frequency of drug administration is decreased; (ii) patients can be discharged earlier from hospitals than is currently possible; (iii) drugs can be administered that have lower therapeutic indices than is currently possible.
  • the solubility of the subject polyvalent presenters can also influence compartmentalization of polyvalent presenters.
  • Other factors besides solubility also may be responsible for the compartmentalization and all forms of compartmentalization, e.g., inclusion or exclusion, are intended to be part of this invention.
  • the distribution coefficient between water and an organic solvent is important.
  • Polymeric, polyvalent species having high molecular weight will, in general, not cross biological membranes effectively. This characteristic can, in certain embodiments, make it preferable that they be administered by direct delivery into the compartment of interest. Alternatively, this property means that the polyvalent presenters can be excluded from the undesirable compartments.
  • intravenous injection accesses the vascular compartment; intrathecal injection accesses the cerebrospinal fluid and the central nervous system; the oral route accesses the gastrointestinal tract; eye drops access the ocular compartment, creams and ointments access the epithelium; catherization accesses the biliary tree and the pancreas and the gall bladder, as well as the cystourethral system, and the vagino-tuboovarian system; and finally inhalation accesses the bronchioalveolar compartment.
  • Molecules may also be designed to keep them out of particular compartments. Examples of areas where this concept may be useful include obstetrics (keeping agents out of the fetal circulation), and agents that are toxic to the kidney (keep agents from being taken up by the kidneys by keeping them in circulation), or to keep presenters out of the central nervous system.
  • Polyvalent presenters can also have a tendency to stay localized at a site of interest, with the advantage of reducing systemic toxicity and maximizing local concentrations.
  • molecules can be designed that are not confined to a compartment when that property is desirable. For example, in the acute setting, it may be undesirable to have a long-acting agent, but it may be important to have the increased potency that a polyvalent agent can bring.
  • polymers may be designed to be of intermediate size, or may comprise cleavable connectors. These molecules will be potent but sufficiently small to be filtered by the kidney and thereby cleared.
  • compartments include: the eye (e.g., agonists and antagonists of tearing during surgical procedures or antibiotics), the Gl tract (e.g., agonists and antagonists of peristalsis (cholinergic agonists) (cathartic) agonists and antagonists of muscle tone (glucagon) (prior to double contrast barium studies).
  • the CNS the urogenital system (e.g., kidney, ureter, or bladder, vagina, uterus, fallopian tubes (e.g., contraceptives), and the bronchial tree (e.g., antiasthmatic medication, or cystic fibrosis therapies).
  • compartments include surface (e.g., skin and mucus membranes for topical applications); the ear canal and middle ear (e.g., antibiotics, antivirals), the blood (e.g., intravenous injection, as well as transdermal and transmucosal delivery vehicles).
  • compartments include intravasular, extravasular, cerebrospinal fluid, the bronchoalveolar space, the pleural space, the peritoneal space, the ocular compartment, the topical compartment, the urinary tract, and the reproductive tract, e.g., vaginal, uteral, and fallopian tubes. Size
  • the size of the polyvalent presenter can influence the same properties of duration of action, therapeutic index, compartmentalization, and clearance profile as can solubility (as described supra).
  • size is intended to encompass both to molecular size in terms of carbohydrates (i.e., Stokes radius) and molecular weight (kD) in terms of proteins.
  • Polyvalent presenters of molecular size above 60 kD or greater than 5 ⁇ A mean hydrodynamic diameter are more likely to be compartmentalized than are molecules of smaller molecular size.
  • polyvalent presenters of the present invention are greater than 2kD and /or 5nM 5 ⁇ A mean hydrodynamic diameter.
  • the size of polyvalent presenter selected may vary with the "state" to be treated.
  • low molecular weight compounds (less than about 10,000 MW) will generally be cleared more rapidly.
  • larger compounds that contain cleavable linkages that link units small enough to be cleared when released can be used.
  • Molecules of size greater than that of a 60-70 kDa protein may not be filtered effectively by the kidney, which is important in instances where the polyvalent presenters are to be used in the bloodstream.
  • the materials When used for oral, lung or topical applications, the materials may not need to be cleared or degradable in vivo.
  • clearance when desirable, can be induced by a number of mechanisms.
  • small molecular weight pieces (of a size can easily be cleared by the kidney) can be joined by connectors that hydrolyze at a significant rate in the serum.
  • such low molecular weight pieces can be joined by connectors that are hydrolyzed by agents (e.g., enzymes) naturally present in the plasma.
  • agents e.g., enzymes
  • such small molecular weight pieces can be joined by connectors that can be cleaved by agents (e.g., a second drug, either polyvalent or monovalent) that is taken at the time that clearance of the polyvalent presenter is desirable.
  • An example of such a second agent may be a tbiol or a chelating agent, and examples of linkages susceptible to these agents may be disulfides and organometallic links).
  • high molecular weight species cannot access the blood by oral administration.
  • High molecular weight systems are, nonetheless, useful for other application.
  • they will be made available to the subject by transmembrane permeation (across nasal or pulmonary membranes following administration as aerosols).
  • Some polyvalent species may be taken up through cells in the gut, e.g., by formulation to survive the digestive process or administration as suppositories. Large polyvalent agents, because they will not pass from the lung, gut, or respiratory passages into the systemic circulation, can be advantageous in that they have limited side effects.
  • a subject with one or more polyvalent presenters and, in addition, with a monovalent inhibitor.
  • Such monovalent inhibitors may or may not interact with the same binding site B as does group A on the polyvalent presenter.
  • monovalent inhibitors of influenza neuraminidase (NA) a hydrolytic enzyme present on the surface of influenza virus, enhance the ability of polyacrylamide presenting HA inhibitors to prevent hemagglutination (Choi, S.-K.; Mammen, M.; Whitesides, G. M. Chem. & Bio. 1996, 3, 97-104).
  • NA sites on the surface of virus act as secondary binding sites for SA. Adding monovalent inhibitors of NA prevents the secondary binding of SA leading to an increase in the effectiveness of these polymeric inhibitors, probably due to increased steric stabilization.
  • the subject presenters can be used in conjunction with any other method of treatment.
  • the time to onset of action for a polyvalent presenter also may be important.
  • Multivalent hapten-carrier conjugates typically require a long period of time to elicit a useful biological response.
  • the polyvalent presenters of the present invention can elicit a useful biological response or a therapeutic effect within a day, or preferably within twelve hours, within five hours, within one hour or in some situations a substantially immediate response or effect, e.g., within several minutes down to a second, e.g., when asthma is being treated.
  • Example 1 The Preparation of Polyvalent Presenters for Facilitating the Treatment of Influenza
  • the method is based on the conversion of poly(acrylic acid anhydride) (pAAn) to derivatives of pAA by reaction with various amines RNH2, in water.
  • pAAn poly(acrylic acid anhydride)
  • RNH2 amines
  • These derivatized polymers were prepared by ultrasonication of the reactants directly in the wells of a microtiter plate; then assayed in the same plate with no further manipulation.
  • Influenza initiates infection by adso ⁇ tion to the surface of mammalian cells through the multiple interactions of the viral protein hemagglutinin (HA) and clusters of NeuAc moieties expressed on the cellular surface.
  • HA hemagglutinin
  • Monomeric sialosides are, in general, weak inhibitors of the adso ⁇ tion of virus (as measured by inhibition of hemagglutination) (see, e.g., Sauter, N. K. et al, Biochemistry 1989, 28, 8388).
  • pAA was synthesized having multiple R groups as side chains, pAA(R) by sonicating a suspension (0.12 mg/ ⁇ L) of poly(acrylic acid anhydride) (pAAn) (see, e.g., Jones, J. F. J. Polymer Sci. 1958, 33, 15.; Brotherton, T. K.; Smith, Jr, J.; Lynn, J. W. J. Org. Chem.
  • Solutions of co-polymers pAA(NeuAc-L) were prepared by reacting of NeuAc-L-NH2 (1, 2, 3 or 4) with poly(acrylic acid anhydride) (pAAn) using different numbers of molar equivalents (mol eq.) of NeuAc-L- NH2 to pAAn (eq 1). The polymer for which mol eq.
  • Each solution of generated co-polymers (pH ⁇ 3) in a well was neutralized to pH ⁇ 7 by adding 60 ⁇ L of 1.0 M NaOH, and adjusted to 100 or 200 ⁇ L (total volume) with PBS, pH 7 before the HAI assay.
  • the above protocol was easily extended to the preparation of ter-polymers pAA(NeuAc-L; R); here, a three-component mixture (pAAn (6 mg), 50 ⁇ L of 0.1 M NeuAc- L-NH2 (1 or 3) and 30 ⁇ L of 0.2 M RNH2) was sonicated.
  • D2O 1H-NMR
  • the 'HNMR signals of R from pAA(R) were distinguished readily from those of free unreacted RNH 2 by their shape (the lines due to polymer-attached species are relatively broad) and by their chemical shift (the ⁇ valure of CH 2 or CH groups next to the amide group are shifted downfield).
  • RNH2 (optimal pH ⁇ 7 and 12 for aromatic and aliphatic amines, respectively) and to the number of molar equivalents of RNH2 to pAAn (optimal mol eq. ⁇ 0.2).
  • Co-polymeric derivatives of pAA were generated presenting NeuAc-L as a side chain (pAA(l) - pAA(4)) by using this quasi-solid-phase synthetic method with derivatives of NeuAc having different linking groups L (1 - 4; NeuAc-L n -NH2; see Figure 1 or Scheme 1) (for synthesis of NeuAc-L-NH2 1 and 2, see Sparks, M. A.; Williams, K. W.;
  • NeuAc-L-NH 2 (3, 4), were prepared as described below. 3 and 4 were used for inco ⁇ oration into side chains of the polymer, because aromatic moieties in the middle of the linkage may enhance the binding affinity of monomeric NeuAc-L to HA site: Watowich, S. J.; Skehel, J. J.; Wiley, D. C. Structure 1994, 2, 719).
  • NeuAc R OH; N-Acetylneuraminic Acid or Sialic Acid
  • Section A was extended (eq 1) to generate libraries of ter- polymers, pAA(NeuAc-L; R) which polyvalently present both NeuAc-L and one other R group, by sonicating a three-component mixture of NeuAcL-NH2, RNH2 and pAAn.
  • the best of these pAA(NeuAc-L; R) belongs to a new class of hemagglutination inhibitors that have unusually high activities at relatively modest mole fractions of NeuAc-L (-5%) and R ( ⁇ 6%): each 1% in mole fraction of NeuAc-L or R is equivalent to - 6 side chains (per polymer molecule). This finding emphasizes the importance of combinations of side chains in modulating the activities of these polyvalent presenters.
  • Sections A & B streamline the generation of derivatives of pAA and the evaluation of the biological activities of these polymers by carrying out both synthesis and assay in the wells of microtiter plates.
  • the methods allow convenient screening of libraries of polymers presenting multiple combinations of side chains at controlled mole fractions. Since microtiter plates assays are routine in biology and medical sciences, this method can serve generally for screening and obtaining leads for a range of agglutination interactions and other processes that might be influenced by polyvalent inhibitors. This procedure is a rapid, economical method for synthesizing and screening polyvalent, polymeric inhibitors for bioactivity.
  • Example 2- The Preparation of A Polyvalent Presenter for Facilitating the Inhibition of Platelet Aggregation
  • RGD (L-Arg-L-Gly-L-Asp) is a recognition sequence for the binding of fibrinogen to platelets. Analogs of RGD inhibit the interaction between fibrinogen and the platelet membrane glycoprotein Ilb/IIIa complex, thus acting as platelet aggregation inhibitors (see Phillips, D. R, and Cell, 65:359 (1991)).
  • a polyvalent presenter that presents RGD as a ligand is prepared as follows:
  • RGD immobilized on a solid support at the C-terminus is prepared according to known methods of solid-phase peptide synthesis (SPPS), e.g., on Wang resin.
  • SPPS solid-phase peptide synthesis
  • Solid- supported RGD is reacted with succinic anhydride to provide immobilized succinylated RGD, which is then coupled with mono-BOC-protected 1,6-diaminohexane (e.g., with DCC/HOBt).
  • Cleavage of the derivatized RGD from the resin and concomitant removal of the BOC group is achieved by acid treatment, followed by purification (e.g., by HPLC) to provide RGD functionalized with a linker moiety having a terminal amino group.
  • Polysuccinimide (prepared according to known methods; see, e.g., U.S. Patent No. 5,484,878 for preparation of polysuccinimide from aspartic acid) is suspended in a solvent such as dimethylformamide (DMF) or dimethylacetamide, and the linker- fiinctionalized RGD is added to the suspension with stirring. After the aminolysis of the polysuccinimide is complete, the reaction mixture is diluted with aqueous base to hydrolyze the unaminolysed polysuccinimide, and then dialyzed to remove impurities, providing a poly(aspartic acid) backbone functionalized with a linker bearing RGD.
  • a solvent such as dimethylformamide (DMF) or dimethylacetamide
  • the degree of substitution of the backbone with RGD, and the size of the poly(aspartic acid) polymer produced by the aminolysis is controlled by adjusting the pH of the reaction mixture, the amount of RGD-linker present in the reaction mixture, the time of reaction, and the like.
  • the resulting RGD-poly(aspartic acid) polyvalent presenter is screened to determine the effect of the polyvalent presenter on fibrinogen binding to platelet membrane glycoprotein Ilb/IIIa complex according to methods known in the art, e.g., platelet aggregation assays.
  • Example 3 Polyvalent Polymeric Galactosides in Prevention of Adhesion of Ricins to Erythrocytes Abbreviations.
  • PBS phosphate buffered saline; Et - ethyl; MeOH - methanol; h - hour; i-Pr - isopropyl;
  • pBMA poly(butadiene-co-maleic acid);
  • RCA Risinus Communis Agglutinin; BHA - Bromelain-cleaved Hemagglutinin; LDH - lactate dehydrogenase.
  • Ricins (RCA120, RCA60) including fluorescein isothiocyanate (FITC)- labeled ricins (FITC-labeled RCA120, FITC-labeled RCA ⁇ O) were purchased from Sigma Co.
  • FITC fluorescein isothiocyanate
  • FITC-labeled RCA120, FITC-labeled RCA ⁇ O fluorescein isothiocyanate
  • Gal- ⁇ o-LlNH2 To a solution of methylene chloride (180 mL) containing ⁇ -D- galactose pentaacetate (7.8 g, 19.98 mmol) and allyl alcohol (5.6 ml, 58.82 mmol) cooled in ice bath BF3 » Et2 ⁇ (4.0 mL, 32.52 mmol) was added dropwise. After stirring (4 h, 0 °C; then 30 h, about 20 °C), the mixture was poured into cold saturated NaHCO3 (200 mL) in a separatory funnel.
  • pAA(Gal- ⁇ ) the method described below for synthesis of pAA(Gal- ⁇ ; 0.4) is a protocol for general synthesis of pAA(Gal).
  • DMF NN-dimethylformamide
  • p ⁇ AS 500 mg, equivalent to 3 mmol of ⁇ AS
  • the mixture was transferred into a dialysis bag (MW cutoff about 12-14 kDa; Spectrum® from Sprcturm Medical Industries, Inc.), and was dialyzed at about 20 °C over 3 d: H2O (2 x 4L), 0.05 M NaOH (4L), 0.5 M NH4CI (4L), and H2O (2 x 4L).
  • pBMAn An aliquot of pBMAn, which was provided as a solution in acetone (Polysciences, Inc.), was dried in vacuo and redissolved in DMF before use.
  • % S calcd for pAA(Gal- ⁇ ; 0.05) 1.62, found 1.55; calcd for pAA(Gal- ⁇ ; 0.09) 2.62, found 2.69; calcd for pAA(Gal- ⁇ ; 0.17) 4.11, found 4.12; calcd for pAA(Gal- ⁇ ; 0.22) 4.81, found 4.85.
  • the incubated mixture was centrifuged for 2 min at 2000 rpm. Red pellets, obtained after removing the supernatant, were washed with 1.0 mL of PBS, and resuspended in 0.2 mL of PBS before being examined with an optical microscope.
  • HA hemagglutination
  • HAI is the last well in which an agglutinated pellet is observed. This end point
  • K HAI ( i ) is defined as the lowest concentration of galactoside in solution that inhibited
  • K HAI the ricin-induced agglutination of erythrocytes.
  • the values of » were calculated on the basis of at least 5 independent trials.
  • Gal- ⁇ o-L 1 NH 2 Two derivatives of D-galactosides (Gal- ⁇ o-L 1 NH 2 , Gal- ⁇ c-L 2 NH 2 ) were synthesized as monomeric precursors to polymeric polyvalent D-galactosides: Gal- ⁇ o-L ⁇ NH 2 contains a ⁇ -O-linkage between the galactoside (Gal) group and the amine-terminated linker (Scheme 1); Gal- ⁇ c-L 2 NH 2 contains an ⁇ -C-glycoside. As a Ci-epimeric analog of ⁇ -O-galactosides, the Gal- ⁇ c- L 2 NH 2 was chosen because it could be prepared easily in high stereoselectivity and large scale.
  • the C-glycosidic linkage of Gal- ⁇ c- L 2 NH provides the additional advantage of resistance to chemical and enzymatic hydrolysis.
  • Both epimers of D-galactoside were used to compare their binding affinities to ricins.
  • These polymers were referred to as pAA(Gal- ⁇ ), pAA(Gal- ⁇ ), pBMA(Gal- ⁇ ), and pBMA(Gal- ⁇ ).
  • the pAA-based polymers were chosen with the expectation that they would be relatively flexible, at least at high ionic strength; the pBMA-derived polymers were expected to be less flexible.
  • pAA(Gal- ⁇ ) was achieved using methods described previously by allowing poly(N- acryloyloxysuccinimide) (p ⁇ AS) (Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Med. Chem. 1995, 38, 4179-4190) to react with Gal- ⁇ o L ⁇ H 2 in DMF (about 20 °C, 2 d), and quenching with excess 1.0 M NaOH.
  • p ⁇ AS poly(N- acryloyloxysuccinimide)
  • Red blood cells (RBCs) from 2-week-old chick were used as a model system of mammalian cells. Erythrocytes lack a nucleus, and do not synthesize proteins. Nevertheless, they provide a good model of the cells targeted by ricin, and provide a system with which to study the adhesion of ricin to the cell surface: the surface of erythrocytes presents a variety of ⁇ -galactoside-containing glycoconjugates (about 2-3 x 10 ⁇ Gal residues per human RBC) (Sandvig, K.; Olsnes, S.; Pihl, A. J. Biol Chem. 1976, 257, 3977-3984).
  • chick erythrocytes (0.5% by volume) as a suspension in phosphate buffered saline (PBS) solution, pH 7.2 and ricins (RCA120 ⁇ 16 nM; RCA60 ⁇ 1.9 ⁇ M), the activities of polymeric galactosides in inhibiting ricin-mediated agglutination were assayed.
  • PBS phosphate buffered saline
  • Table 10 summarizes the hemagglutination inhibition (HAI) activity, K H ' AI (defined as the lowest concentration of an inhibitor required to prevent hemagglutination) of purified polymers with various mole fractions of carbohydrate-containing side chains ( ⁇ carbohydrate)
  • ⁇ carbohydrate carbohydrate-containing side chains
  • ⁇ ' refers to the lowest concentration of carbohydrate-containing side chains of the polymer that could inhibit ricin-induced agglutination of chick erythrocytes.
  • Each value represents an average of five independent measurements; the experimental uncertainty in each value is approximately ⁇ 50%. 6 No inhibition was observed at the indicated concentrations. This value represents the concentration of carboxylic acid side chains of the polymer in solution that could inhibit ricin-induced agglutination of chick erythrocytes.
  • Gal- ⁇ -Ome 200 42 pBMA(Gal- ⁇ ; 0) >30000 b - c >30000 b - c
  • Gal- ⁇ -Ome 400 50 pBMA(Gal- ⁇ ; 0.05) 2.0 1.0
  • GlcNAc- ⁇ 0 -L ⁇ NH 2 >9000 b >9000 b
  • pBMA(Gal- ⁇ ; 0.05) >160 ⁇ 160
  • PAA (Gal- ⁇ ; 0.6)
  • pBMA (Gal- ⁇ ; 0.22)
  • KHAI pAA(Gal- ⁇ ; 0.2 to 1.0) showed HAI activity, » , against RCA120- induced agglutination at submicromolar concentrations of Gal moieties in solution.
  • the same polymers had HAI activities against RCA60 that were about 50- to about 300-fold lower than those against RCA120 . the activities against RCA60 were, at best, 50 times better than those of monomeric Gal derivatives.
  • pAA(Gal- ⁇ ; 0.4) had an inhibitory activity (against RCA120) that was about 1500 times higher than that of monovalent Gal- ⁇ -OMe, and about 270 times higher than that of Gal- ⁇ o L1NH2.
  • pBMA(Gal- ⁇ ; 0.05 to 0.22) showed HAI activities at (sub)micromolar concentrations against RCA120 and RCA ⁇ O; the activities against RCA120 were better than those against RCA60- pBMA(Gal- ⁇ ; 0.05 to 0.22) had, however, HAI activities against RCA60 approximately 4 times better than those against RCAi20- Of the polyvalent galactosides built on pBMA, pBMA(Gal- ⁇ ; 0.22) and pBMA(Gal- ⁇ ; 0.05) were the most active inhibitors against RCA120 and RCA ⁇ O, respectively.
  • pBMA(Gal)s are relatively small polymers with molecular weight about 10-15 kDa, and have a polymer backbone (pBMA) that is biocompatible (Conroy, C. W.; Wynns, G. C; Maren, T. H. Bioorg. Chem. 1996, 24, 262-272).
  • Figure 6 summarizes the HAI activity of the polymers against ricins.
  • K HAI Figure 6a shows that values of i of polymeric galactosides are a non-linear function of the mole fraction of Gal ( ⁇ G l) of the polymers.
  • the relationship between activity and ⁇ Gal of the polymers in inhibiting hemagglutination by RCA ⁇ O seem to be related closely to that describing inhibition by RCA120-.
  • K HAI r . i _ ⁇ ra i relationship of the polymers depended also on the type of polymer backbone: a smooth curve with large, flexible pAA(Gal- ⁇ ); a sha ⁇ , partly quasi-linear relationship with small, extended pBMA(Gal).
  • K HAI Gals per polymer chain still showed an activity ( » ' - 2.0 ⁇ M), that was greater than that of monovalent Gal- ⁇ -L ⁇ NH2 by a factor of 19.
  • the titer of RCA120 a minimal concentration that could agglutinate RBCs (200 ⁇ L of 0.25% by volume, suspended in PBS solution) — was 4 nM, and that of RCA60 was 480 nM. This difference indicated that RCA60 (one B-chain; about 3 Gal receptor sites) was more weakly agglutinating than is RCA120 (two B-chains; about 6 Gal receptor sites). Monovalent galactosides were more effective against RCA60 than against RCA120 in preventing the ricin-induced agglutination of erythrocytes.
  • RCA ⁇ O relative to RCA 120 may be related to the number (n) of blocked Gal receptor sites of ricins required for inhibition of agglutination relative to the total number of Gal receptor sites of ricins: n/3 (RCA ⁇ O) . n/6 (RCAi2 ⁇ )- That is, blocking of the same number of Gal sites on RCA60 and RCAi 20 results in a larger fraction of Gal sites on RCA60 being blocked, and is more effective in abolishing the agglutination ability of RCA60 than of RCA120-
  • K HAI moieties on polyvalent presentation that is, the value of * for the polymer relative to that of the monomer was higher for RCA120 than for RCA60, as well.
  • K HAI receptor sites determine » . Since each polymer presents multiple Gal moieties, the binding of polymer to ricin may, of course, be relatively tight, even if individual Gal groups bind relatively weakly. The experiments presented here do not directly measure the occupancy of the Gal receptor sites on ricin, and the inhibition of hemagglutination may therefore be due either to entropically-enhanced binding of these Gal-binding receptor sites, or to other, non-receptor directed effects such as steric inhibition.
  • glycoproteins and oligosaccharides bind tightly to B-chains of ricins.
  • Glycopeptides derived enzymatically from fetuins are one example: these compounds have dissociation constants to ricins (and to B-chains) of Kd - 10 to 0.1 ⁇ M (Baenziger, J. U.; Fiete, D. J. Biol. Chem. 1979, 254, 9795-9799).
  • glycopeptides are multi-branched oligosaccharides and oligopeptides that present multiple copies of galactosides and N- acetylgalactosamine moieties.
  • the galactoside-containing glycoconjugates are, thus, structurally multivalent in terms of the number and intramolecular distribution of galactosides per molecule, and we believe that these structural features may account for the high binding affinities of these glycopeptides to ricins relative to monomeric galactosides.
  • Our galactoside-presenting polymers, pAA(Gal) and pBMA(Gal) are synthetically well-characterized, polyvalent species with the hemagglutination
  • the above results demonstrate that synthetic polymers presenting multiple copies of a simple derivative of ⁇ -D-galactose as side chains effectively inhibited adhesion of ricins to chicken erythrocytes as examined with optical/fluorescence microscopy, and as measured quantitatively by the hemagglutination inhibition (HAI) assay.
  • HAI hemagglutination inhibition
  • the activities of the polymeric galactosides were affected by a number of factors such as the mole fraction of Gal-containing side chains of the polymer, the C-l epimeric configurations ( ⁇ > ⁇ ) of galactosides, and the polymer backbone.
  • Gal-transferase ⁇ -l,4-GalactosylTransferase
  • ZP zona pellucida
  • GlcNAc N- acetylglucosamine
  • Gal galactose
  • pA poly(acrylamide)
  • pAA poly(acrylic acid)
  • pAA(Glc ⁇ Ac- ⁇ ) poly(acrylic acid) presenting multiple copies of ⁇ -O-linked GlcNAc as amide side chains
  • pAA(Gal- ⁇ ) poly(acrylic acid) presenting multiple copies of ⁇ -O-linked Gal as amide side chains.
  • Polymeric polyvalent N-acetylglucosamine (Glc ⁇ Ac) and galactoside (Gal): Poly(acrylic acid) presenting multiple copies of Glc ⁇ Ac or Gal as amide side chains — pAA(Glc ⁇ Ac; ⁇ ) and pAA(Gal; ⁇ ): ⁇ 0.2, 0.4, 0.6, 0.8, 1.0 — ere prepared according to procedures similar to those described above in Example 3 for the preparation of the polymeric polyvalent compounds.
  • Figure 7 depicts the structure of polyvalent polymeric N- acetylglucosamine (Glc ⁇ Ac) and galactoside (Gal).
  • the pAA(Glc ⁇ Ac) and pAA(Gal) refer to poly(acrylic acid) presenting multiple copies of GlcNAc- ⁇ -L ⁇ -NH 2 and Gal- ⁇ -L ⁇ -NH 2 , respectively, as amide side chains.
  • mice sperm were capacitated for 30 min at 35°C, and incubated at 37°C in the presence or absence of polymers. Each experiment had the following controls: 1) phosphate buffer used only to assess the degree of "spontaneous" acrosome reactions (usually less than 5% during the assay and was subtracted from all other determinations); 2) polymer backbone — poly(acrylyl amide), or pA — used at the same mass as that used to generate the desired molarity of GlcNAc in pAA(GlcNAc); 3) zona pellucida glycoproteins (ZP), the "native” inducer; and 4) calcium ionophore A23187 used to drive the reaction mixture at time zero, 15 min, 30 min, and sometimes 60 min.
  • phosphate buffer used only to assess the degree of "spontaneous" acrosome reactions (usually less than 5% during the assay and was subtracted from all other determinations)
  • polymer backbone poly(acrylyl amide
  • the values (%) of acrosome-reacted sperm by ZP and the ionophore were 27.1 ( ⁇ 0.10) and 53.7, respectively.
  • Poly(acrylamide), or pA was tested as a negative control: the values of the x-axis indicate the concentrations of carboxamide groups of pA.
  • the sperm sample was dried onto slides, stained for the intact acrosome, and the number of stained vs. unstained cells were determined in 200 sperm for each sample.
  • Figure 8 summarizes the activities (acrosome-reacted sperm, %) of a monovalent GlcNAc and a polyvalent GlcNAc in induction of acrosome reaction.
  • the pAA(GlcNAc; 0.2) induced significant amounts (9-17%) of acrosome reaction at concentrations of 100 ⁇ M ([GlcNAc]), while monovalent GlcNAc or polymer backbone (pA) did not induce meaningful amounts of acrosome reaction ( ⁇ 5%) under the identical experimental conditions.
  • This example illustrates that the acrosome reaction of sperm appears to require multivalent binding (spatial aggregation on the surface) of Gal-transferase to GlcNAc residues of egg coat, and can be induced by a synthetic polymer presenting multiple copies of a simple O- linked derivative of GlcNAc.
  • Polymers of pAA(GlcNAc) showed weak to moderate activities (4-10%) at 0.1-100 ⁇ M ([GlcNAc]). Above 100 ⁇ M, they showed increased activities (17-27%).
  • Figure 9a also shows the activities of poly(acrylic acid) presenting O-linked galactoside, pAA(Gal). The low (no) activities were probably due to low (no)-affinity binding of Gal residues of pAA(Gal) to Gal-transferase.
  • the plot is derived from Figure 9a and 14% for pAA(NeuAc; 1.0) is an estimated value.
  • the plot utilizes the concentration of 100 ⁇ M ([GlcNAc]), because it is a threshold concentration leading to large increases in the activities of pAA(GlcNAc).
  • Figure 10 summarizes the activities of inhibition of sperm-egg binding by polyvalent polymeric GlcNAc.
  • the results suggest that the polymeric polyvalent GlcNAc induced acrosome reaction in mouse sperm and inhibit sperm-egg binding.
  • the polymer backbone, pA was used as a control at 100 ⁇ M ([-CONH 2 ]).
  • Each solution of co-polymers (pH - 3) generated in a well was neutralized to pH - 7 by adding 30 mL of 1.0 M NaOH and adjusted to 100 or 200 mL (total volume) with PBS, pH 7.2, before the inhibition assay of influenza virus-induced agglutination of chicken red blood cells.
  • Example 6 Generation of pMVMA(NeuAc;R) using quasi-solid phase reaction (see. Figure l ib)
  • the protocol used in Example 5 was extended to the preparation of ter-polymers pMVMA(Neu Ac-Li ; R).
  • Each solution of ter-polymers (pH - 3) generated in a well was neutralized to pH - 7 by adding 30 mL of 1.0 M NaOH and adjusted to 100 or 200 mL (total volume) with PBS, pH 7.2, before the inhibition assay of influenza virus-induced agglutination of chicken red blood cells.
  • Example 7 Generation of pAA(Gal using quasi-solid phase reaction (see. Figure l ie) Solutions of co-polymers of pAA(Gal) were prepared by reacting of RNH 2
  • Co-polymeric pAA(Gal) for which mol equiv is >0 was generated in microtiter plates with 96 conically-bottomed wells as follows: (i) placing 6 mg of pAAn into a well; (ii) soaking the powder with a variable amount (10-100 mL) of 0.1 M of RNH 2 (Gal-b-L 2 NH 2 , or Gal-a-L 3 NH 2 ) in PBS buffer, pH 12; (iii) immediately sealing the plate, and then ultrasonicating the mixture for 0.5 h.
  • Example 7 The protocol used in Example 7 was extended similarly to the preparation of co-polymers of poly(butadiene-c ⁇ -maleic acid)(Gal), or pBMA(Gal). Solutions of co-polymers of pBMA(Gal) were prepared by reacting of RNH 2 (Gal-b-L 2 NH ;
  • Co-polymeric pBMA(Gal) for which mol equiv is >0 was generated in microtiter plates with 96 conically-bottomed wells as follows: (i) placing 6 mg of pBMAn into a well; (ii) soaking the polymer with a variable amount (10-100 mL) of 0.1 M of RNH 2 (Gal-b-L 2 NH 2 , or Gal-a-L 3 NH 2 ) in PBS buffer, pH 12; (iii) immediately sealing the plate, and then ultrasonicating the mixture for 0.5 h.
  • Example 9 Generation of pAA(SLe x ) using quasi-solid phase reaction (see. Figure 12a) The protocol used in Example 7 is extended similarly to the preparation of co-polymers of pAA(SLe x ).
  • Each reaction mixture of co-polymers (pH - 3) generated in a well is neutralized to pH - 7 by adding 60 mL of 1.0 M NaOH and adjusted to 100 or 200 mL (total volume) with PBS, pH 7.2, before the inhibition assay of adhesion of neutrophils to endothelial cells.
  • Example 6 The protocol used in Example 6 is extended to similarly to the preparation of ter-polymers pAA(Bacitracin;R).
  • a three-component mixture including pAAn, Bacitracin and R 2 NH 2 (aliphatic amines, aromatic amines, amino acids, aminosugars, or peptides) is sonicated.
  • Each solution of ter-polymers (pH - 3) generated in a well is neutralized to pH - 7 by adding 30 mL of 1.0 M NaOH and adjusted to 100 or 200 mL (total volume) with PBS, pH 7.2, before the inhibition assay of bacterial growth.

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Abstract

La présente invention concerne des compositions polyvalentes permettant de présenter de façon polyvalente un agent thérapeutique. Ces compositions pharmaceutiques contiennent un agent de présentation polyvalent. Selon une réalisation, l'agent de présentation est formulé comme (Y)-(X-A)n, Y étant un squelette, X une liaison directe ou un lien, A un groupe fonctionnel à fonction de présentation, et n un entier supérieur à 10 choisi de façon que les groupes à fonction de présentation puissent entrer en interaction avec une pluralité de sites cibles de liaison. La composition peut également comporter un vecteur pharmaceutiquement acceptable. Selon une autre réalisation, l'agent de présentation peut constituer lui-même son propre vecteur pharmaceutiquement acceptable. L'invention concerne également le traitement d'affections ou d'états. Ce traitement consiste en l'administration à un sujet d'une pluralité de groupes A de façon que le traitement se produise. Le traitement se produit du fait de l'interaction d'un agent de présentation polyvalent avec une pluralité de sites cibles de liaison B. Sous d'autres aspects, l'invention concerne, d'une part des agents de présentation polyvalents emballés avec un mode d'emploi destiné aux traitements considérés, et d'autre part des procédés de conception, d'agents de présentation polyvalent convenant particulièrement dans le cadre des procédés et traitements de l'invention. Les agents de présentation polyvalents de l'invention assurent une liaison spécifique, ce qui est relativement avantageux. En outre, ces agents de présentation polyvalents permettent des interactions positives et négatives.
EP98918079A 1997-04-11 1998-04-09 Molecules presentant une pluralite de groupes fonctionnels actifs Withdrawn EP0973551A2 (fr)

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PCT/US1998/007171 WO1998046270A2 (fr) 1997-04-11 1998-04-09 Molecules presentant une pluralite de groupes fonctionnels actifs

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ATE473759T1 (de) 1998-05-22 2010-07-15 Univ Leland Stanford Junior Bifunktionelle moleküle sowie darauf basierende therapien.
JP3468149B2 (ja) * 1999-02-19 2003-11-17 松下電器産業株式会社 微細物体の操作装置および操作方法
AU4995700A (en) * 1999-05-10 2000-11-21 Glaxo Group Limited High throughput crystal form screening workstation and method of use
AU2044001A (en) 1999-11-19 2001-05-30 Board Of Trustees Of The Leland Stanford Junior University Targeted bifunctional molecules and therapies based thereon
US7220552B1 (en) 1999-11-19 2007-05-22 The Board Of Trustees Of The Leland Stanford Junior University Bifunctional molecules and their use in the disruption of protein-protein interactions
US6887842B1 (en) 1999-11-19 2005-05-03 The Board Of Trustees Of The Leland Stanford Junior University Modulating a pharmacokinetic property of a drug by administering a bifunctional molecule containing the drug
EG24716A (en) 2002-05-17 2010-06-07 Novartis Ag Combination of organic compounds
KR100543171B1 (ko) * 2002-05-31 2006-01-20 한국과학기술원 분자수준에서 배향성을 조절하면서 항체 단분자막을 제조하는 방법
WO2006044577A1 (fr) * 2004-10-13 2006-04-27 Ilypsa, Inc. Préparations pharmaceutiques comprenant un oligosaccharide complexant les toxines et une particule polymère
PL1951279T3 (pl) 2005-10-08 2017-12-29 Apellis Pharmaceuticals, Inc. Kompstatyna i jej analogi w zaburzeniach oczu
US8580735B2 (en) 2007-02-05 2013-11-12 Apellis Pharmaceuticals, Inc. Local complement inhibition for treatment of complement-mediated disorders
WO2008116293A1 (fr) * 2007-03-23 2008-10-02 The Governors Of The University Of Alberta Polymères hétérobifonctionnels multivalents et leurs procédés d'utilisation
CA2698108A1 (fr) * 2007-08-27 2009-03-12 Massachusetts Institute Of Technology Inhibiteurs du virus de la grippe couples a un polymere bifonctionnel
EP4105224A1 (fr) 2011-05-11 2022-12-21 Apellis Pharmaceuticals, Inc. Analogues de la compstatine à longue durée d'action et leurs utilisations
AU2012272706B2 (en) 2011-06-22 2017-07-06 Apellis Pharmaceuticals, Inc. Methods of treating chronic disorders with complement inhibitors
EP3660033B9 (fr) 2012-11-15 2022-06-22 Apellis Pharmaceuticals, Inc. Analogues de la compstatine à longue durée d'action et compositions et méthodes associées
US20160010077A1 (en) * 2013-02-28 2016-01-14 The University Of Southern Mississippi Covalent Attachment of Bacteriophages (Phages) to Polymeric Surfaces
BR112018006810A2 (pt) 2015-10-07 2018-10-23 Apellis Pharmaceuticals Inc regimes de dosagem
US11564995B2 (en) 2018-10-29 2023-01-31 Wisconsin Alumni Research Foundation Peptide-nanoparticle conjugates
AU2019369299A1 (en) 2018-10-29 2021-05-20 Wisconsin Alumni Research Foundation Dendritic polymers complexed with immune checkpoint inhibitors for enhanced cancer immunotherapy
CN115087465A (zh) 2019-10-29 2022-09-20 威斯康星校友研究基金会 肽-纳米颗粒缀合物

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DE19640791A1 (de) * 1996-10-02 1998-04-16 Syntesome Ges Fuer Medizinisch Glycokonjugate als Inhibitoren der viralen Zelladhäsion

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CN1269013A (zh) 2000-10-04
BR9808521A (pt) 2000-05-23
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