MXPA99010704A - Composition and method for enhancing transport across biological membranes - Google Patents

Abstract

Se describen métodos y composiciones para transportar medicamentos y macromoléculas a través de membranas biológicas. En una modalidad, la invención incluye un método para mejorar el transporte de un compuesto seleccionado a través de una membrana biológica en donde la membrana biológica se pone en contacto con un conjugado que contiene un agente biológicamente activo que se une covalentemente a un polímero de transporte. En una modalidad, el polímero consiste de desde 6 hasta 25 subunidades al 50%del las cuales contiene un residuo de sidecaína guanidino o amidino. El polímero es efectivo para impartir al agente unido una velocidad de transporte de la transmembrana a través de una membrana biológica que es mayor que la velocidad de transporte de la transmembrana del agente en forma no conjugada. Los polímeros mejoradores del transporte se ejemplifican, en una modalidad preferida, por péptidos en los cuales los residuos de arginina constituyen las subunidades. Tal péptido de poliarginina puede componerse tanto de todas las D-, todas las L- o las D- y L-argininas mezcladas, cualquiera puede incluir aminoácidos adicionales. más preferentemente, al menos uno y más preferentemente todas las subunidades son residuos de D-arginina, para mejorar la estabilidad biológica del polímero durante el tránsito del conjugado de su objetivo biológico.

Description

COMPOSITION AND METHOD FOR IMPROVING TRANSPORTATION THROUGH BIOLOGICAL MEMBRANES FIELD OF THE INVENTION The present invention is directed to methods and compositions that are effective to improve the transport of biologically active agents, such as organic compounds, polypeptides, oligosaccharides, nucleic acids and metal ions, through biological membranes.

References Barsou et al., PCT Pub. No WO 94/04686 (1994). Bonifaci, N., et al., Aids 9: 995-1000. Brugidou, J., et al. Biochem, Biophys. Res. Comm. 214 (2): 685-93 (1995). Derossi, D., et al., J. Biol. Chem. 269: 10444-50 (1996). Egholm, M.O., et al., Nature 365: 566-568 (1993). Eberle and Nuninger, J. Org. Chem, 57: 2689 (1992). Fawell, S., et al. , Proc. Nati Acad. Sci. USA 91: 664-668 (1994). Fletcher, M.D. , et al., Chem. Rev. 98: 763 (1998) ^ Fran el et al., PCT Pub. No. WO 91/09958 (1991). Gennaro,. R. , Ed. , REMINGTON 's PHAR ACEUTICAL SCIE CES, 18TH ED. , Mack Publishing Co., Easton, PA (1990). Giannis, A., et al. , Advances Drug Res 29: 1 (1997). Kessler, H., Angew, Chem. Int. Ed. Engl. 32: 543 (1993).

Lam, K.S., et al. , Chem. Rev. 97: 411 (1997).

P1700 / 99MX Langston, S., DDT 2: 255 (1997). Rivas, A., et al., J. Immunol. 154: 4423-33 (1995). Ruegg, C, et al., J. Immunol. 154: 4434-43 1995). Ryser, H.J.P., PCT Pub. No. WO 79/00515 (1979). Simón et al., Proc. Nati Acad. Sci. 89: 9367 (1992).

Suffness, M., Ed., Taxol: Science and Applications, CRC Press, New York, NY, pp. 237-239 (1995). Shaheen et al., J. Virology 70: 3392 (1996). Tavladoraki et al., Nature 366: 469 (1993). Thompson, L.A., and Ellman, J.A. , Che. Rev. 96: 555 (1996). Wong, S. S. , Ed., Chemistry of Protein Conjugation and Cross-Linking, CRC Press. Inc., Boca Raton, FL (1991). Zuckermann, R.N. , Chemtracts-Macromol. Chem. 4:80 (1993). All references cited in this application are incorporated therein as references.

BACKGROUND OF THE INVENTION The plasma membranes of cells present a barrier to the passage of many useful therapeutic agents. In general, a drug must be freely soluble both in the body's aqueous compartments and in the lipid layers through which it must pass to enter the cells. The highly charged molecules particularly experience a difficulty in passing through the membranes. Many therapeutic macromolecules, such as peptides and oligonucleotides, are also P1700 / 99MX particularly refractory to transmembrane transport. In this way, while biotechnology has made available a large number of potentially valuable therapeutic agents, bioavailability considerations frequently hinder its medicinal utility. Therefore, there is a need for a reliable means for transport into the cells of drugs and, particularly, of macromolecules. Until today, several transporter molecules have been proposed to escort molecules through biological membranes. Ryser et al. (1979) teaches the use of polymers of high molecular weight of the Usin to increase the transport of several molecules through the cell membranes, where very high molecular weights are preferred. Although the authors contemplated polymers of other positively charged residues, such as ornithine and arginine, the operability of such polymers was not shown. Frankel et al. (1991) reported that the conjugation of selected molecules with the HIV protein rat can increase the cellular absorption of these molecules. However, the use of rat protein has certain disadvantages, which include unfavorable aggregation and insolubility properties. Barsoum et al. (1994) and Fawell et al. (1994) proposed using shorter fragments of the rat protein containing the basic tat region (residues 49-57 having the sequence RKKRRQRRR.) Barsoum et al., P1700 / 99 X noted that moderately long polyarginine polymers (molecular weight of 5000-15000 daltons) failed to enable the transport of β-galactosidase through the membranes of the cell (e.g., Barsoum on page 3), contrary to the suggestion of Ryser et al. (supra) Other studies have shown that a peptide-cholesterol conjugate of 16 amino acids, derived from the homeodomain of Antennapedia, is quickly internalized by cultured neurons (Brugidou et al., 1995). However, slightly shorter versions of this peptide (15 residues) are not effectively absorbed by the cells (Derossi et al., 1996). The present invention is partially based on the discovery of the applicants that the conjugation of certain polymers composed of contiguous and very basic subunits, particularly of subunits containing guanidyl or amidinyl entities, with macromolecules or small molecules is effective to significantly improve the transport of the attached molecule, through the biological membranes. In addition, transport occurs at a rate significantly greater than the transport rate or rate provided by a basic VTH tat peptide consisting of residues 49-57.

SUMMARY OF THE INVENTION The present invention includes, in one aspect, a method for improving the transport of a P1700 / 99MX selected compound, through a biological membrane. In the method, a biological membrane is contacted with a conjugate containing a biologically active agent, which is covalently bound to at least one transport polymer. The conjugate is effective to promote the transport of the agent through the biological membrane at a rate that is greater than the transmembrane transport rate of the biological agent in unconjugated form. In one embodiment, the polymer consists of from 6 to 25 subunits, of which at least 50% contains a guanidino or amidino side chain entity, wherein the polymer contains at least 6 and, more preferably, at least 7 entities side chain of guanidino or amidino. In another embodiment, the polymer consists of 6 to 20, 7 to 20 or 7 to 15 subunits. More preferably, at least 70% of the polymer subunits contain a guanidino or amidino side chain entity and, even more preferably, 90%. Preferably, no guanidino or amidino side chain entity is separated from another of these entities in more than one subunit other than guanidino or other than amidino. In a more specific embodiment, the polymer contains at least 6 contiguous subunits, each of which contains either a guanidino or an amidino group and, P1700 / 99MX preferably, at least 6 or 7 contiguous guanidino side chain entities. In another embodiment, the transport polymer contains from 6 to 25 contiguous subunits, from 7 to 25, from 6 to 20 or, preferably, from 7 to 20 contiguous subunits, each of which contains a guanidino side chain entity or of amidino and, with the optional condition that one of the contiguous subunits may contain a residue other than arginine to which the agent binds. In one embodiment, each contiguous subunit contains a guanidino entity, as exemplified by a polymer containing at least six contiguous arginine residues. Preferably, each transport polymer is linear. In a preferred embodiment, the agent is attached to a terminal end of the transport polymer. In another specific embodiment, the conjugate contains a single transport polymer. In a preferred embodiment, polymers that improve transport are employed by peptides, in which the arginine residues constitute the subunits. This polyarginine peptide may be composed of either only D-arginines, of only L-arginines or of D-arginines and mixed L-arginines and may include additional amino acids. More preferably, at least one of the subunits and, preferably all, are P1700 / 99MX D-arginine residues, to improve the biological stability of the polymer during the transit of the conjugate to its biological target. The method can be used to improve the transport of selected therapeutic agents, through any of several biological membranes including, but not limited to, eukaryotic cell membranes, prokaryotic cell membranes and cell walls. Membranes of prokaryotic cells including enzymes include membranes of bacteria. Membranes of eukaryotic and emplyficating cells of interest include, but are not limited to, membranes of dendritic cells, epithelial cells, endothelial cells, keratinocytes, muscle cells, fungal cells, bacterial cells, plant cells. and the similar. In accordance with a preferred embodiment of the invention, the transport polymer of the invention has an apparent affinity (Km) that is at least 10 times greater and, preferably, at least 100 times greater than the affinity measured for the tat peptide ( 49-57) by the procedure of Example 6 when measured at room temperature (23 ° C) or at 37 ° C. Biologically active agents (encompassing therapeutic agents) include, but are not limited to, metal ions, which are P1700 / 99MX normally supply as metal chelates; small organic molecules, such as anticancer (for example, taxane) and antimicrobial molecules (for example, against bacteria or fungi, such as yeast); and nucleic acid macromolecules, peptides, proteins and analogs thereof. In a preferred embodiment, the agent is a nucleic acid or a nucleic acid analog, such as a ribosome optionally containing one or more 2'-deoxy nucleotide subunits for an improvement in stability. Alternatively, the agent is a peptide nucleic acid (PNA). In another preferred embodiment, the agent is a polypeptide, such as a protein antigen and the biological membrane is a cell membrane of an antigen-presenting cell (APC). In another embodiment, the agent is selected to promote or elicit an immune response against a selected tumor antigen. In another preferred embodiment, the agent is a taxane or taxoid anticancer compound. In another embodiment, the agent is an agent that is not a polypeptide, preferably a therapeutic agent that is not a polypeptide. In a more general embodiment, the agent preferably has a molecular weight less than 10 kDa. The agent can be linked to the polymer by a binding entity, which can impart a conformational flexibility within the P1700 / 99 X conjugated and facilitate the interactions between the agent and its biological target. In one embodiment, the binding entity is a cleavable linker, for example, that contains a linking group that is cleavable by an enzyme or by solvent-mediated cleavage, such as an ester, amide or disulfide group. in another embodiment, the scissile linker contains a photocleavable group. In a more specific embodiment, the cleavable linker contains a first cleavable group that is distant from the biologically active agent and a second cleavable group that is close to the agent, such that cleavage of the first cleavable group produces a linker-agent conjugate that contains a nucleophilic entity capable of reacting intramolecularly to cleave the second cleavable group, thereby releasing the binder agent and the polymer. In another embodiment, the invention can be used to classify a plurality of conjugates for a selected biological activity, wherein the conjugates are formed from a plurality of candidate agents. The conjugates are contacted with a cell that exhibits a detectable signal with the absorption of the conjugate into the cell, such that the magnitude of the signal indicates the efficacy of the conjugate with respect to P1700 / 99MX the selected biological activity. This method is particularly useful for testing the activities of agents that by themselves are incapable or have a poor ability to enter cells that manifest biological activity. In another embodiment, the candidate agents are selected from a combinatorial library. The invention also includes a conjugated library that is useful for classification with the above method. In another aspect, the invention includes a pharmaceutical composition for the delivery of a biologically active agent through a biological membrane. The composition comprises a conjugate containing a biologically active agent covalently linked, ie, covalently to at least one transport polymer, as described above and, excipiently pharmaceutically acceptable. The polymer is effective to impart to the agent a transmembrane transport rate that is greater than the transmembrane transport rate of the agent in unconjugated form. The composition can be packaged further with instructions for using it. In another aspect, the invention includes a therapeutic method for treating a mammal, particularly a human subject, with a pharmaceutical composition according to the above.P1700 / 99MX These and other objects and features of the invention will be more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B are graphs of the cellular uptake of certain polypeptide-fluorescein conjugates containing the basic peptide tat (49-57, SEQ ID NO: 1), poly-Lys (K9, SEQ ID N0) : 2) and poly-Arg (R4-R9 and r4-r9, SEQ ID: 3-8 and 12-17, respectively), as a function of the concentration of the peptide; Figure IC is a histogram of the levels of absorption of the conjugates, measured for the conjugates at a concentration of 12.5 μM (Examples 2-3); Figures 2A-2F show computer-generated images of confocal micrographs (Example 4) showing the emitted fluorescence (2A-2C) and the transmitted light (2D-2F) of cells "Jurkat after incubation at 37 ° C for 10 days. minutes with 6.25 μM of conjugate tat (49-57) with fluorescein (panels A and D), a 7-mer of poly-L-arginine (R7) labeled with fluorescein (panels B and E) or a 7-mer poly -D-arginine (r7) labeled with fluorescein (panels C and F), Figure 3 shows the cellular uptake of P1700 / 99MX certain poly-Arg-fluorescein conjugates (r9, R9, R15, R20 and R25, SEQ ID NO: 17 and 8-11, respectively) as a function of the concentration of the conjugate (Example 5); Figure 4 shows a histogram of cellular uptake of conjugates fluorescein-tat (49-57) conjugated and poly-Arg-fluorescein (R9, R8 and R7, respectively) in the absence (four bars on the left) and in the presence ( four bars on the right) of 0.5% sodium azide (Example 7); Figures 5A-5C show graphs of the absorption levels of selected polymer conjugates (K9, R9, r4, r5, r6, r7, r8 and r9) in bacterial cells as a function of conjugate concentration; Figure 5A compares the absorption levels observed for the conjugates R9 and r9 as a function of the concentration of the conjugate, when incubated with HB 101 cells of E. coli; Figure 5B shows the absorption levels observed for the K9 and r4 to r9 conjugates, when incubated with HB 101 cells of E. col i; Figure 5C compares the absorption levels of the conjugates of r9 and K9 when incubated with Screp cells. Bovi s. Figures 6A-6E show exemplary conjugates of the invention, which contain cleavable linkers; Figures 6F and 6G show chemical structures and numbering Conventional P1700 / 99MX of the main chain constituent atoms of paclitaxel and "TAXOTERE"; Figure 6H shows a general chemical structure and the atomic numbering of the ring of taxoid compounds; And Figure 7 shows the inhibition of gamma-interferon (? -IFN) secretion by murine T cells as a function of the concentration of the PNA-r7-sense conjugate (SEQ ID NO: 18), of the PNA antisense conjugate. -r7 (SEQ ID NO: 19) and of unconjugated antisense PNA (SEQ ID NO: 20), wherein the PNA sequences are based on a gamma-interferon gene sequence.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS The term "biological membrane", as used herein, refers to a lipid-containing barrier, which separates cells or groups of cells from the extracellular space. Biological membranes include, but are not limited to, plasma membranes, cell walls, membranes of intracellular organelles, such as the mitochondrial membrane, nuclear membranes, and the like. The term "transmembrane concentration" refers to the concentration of a compound present on the side of a membrane that is opposite or "trans" to the side of the membrane to which a P1700 / 99MX particular composition. For example, when a compound is added to the extracellular fluid of a cell, the amount of the compound measured subsequently within the cell is the transmembrane concentration of the compound. With the terms "biologically active agent" or "biologically active substance", refer to a chemical substance, such as a small molecule, a macromolecule or a metal ion, which causes an observable change in the structure, function or composition of a cell with the absorption by the cell. The observable changes include an increase or decrease in the expression of one or more mRNAs, an increase or decrease in the expression of one or more proteins, the phosphorylation of a protein or other cellular component, the inhibition or activation of an enzyme, inhibition or activation of the union between members of a binding pair, an increase or decrease in the rate of synthesis of a metabolite, an increase or decrease in cell proliferation and the like. The term "macromolecule", as used herein, refers to large molecules (molecular weight greater than 1000 daltons), exemplified, enunciatively, by peptides, proteins, oligonucleotides and polynucleotides of biological or synthetic origin. The term "small organic molecule" is P1700 / 99MX refers to a carbon containing agent which has a molecular weight (MW) less than or equal to 1000 daltons. The terms "therapeutic agent", "therapeutic composition" and "therapeutic substance" refer, without limitation, to any composition that can be used to benefit mammalian species. These agents can take the form of ions, small organic molecules, peptides, proteins or polypeptides, oligonucleotides and oligosaccharides, for example. The terms "non-polypeptide agent" and "non-polypeptide therapeutic agent" refer to the portion of the transport polymer conjugate that does not include the polymer that improves transport and that is a biologically active agent other than a polypeptide. An example of an agent that is not a polypeptide is an antisense oligonucleotide, which can be conjugated to a poly-arginine peptide to form a conjugate for improved delivery through biological membranes. The term "polymer" refers to a linear chain of two or more identical or non-identical subunits linked by covalent bonds. A peptide is an example of a polymer that can be composed of identical or non-identical amino acid subunits, which are linked by peptide bonds.

P1700 / 99MX The term "peptide", as used herein, refers to a compound consisting of a single chain of D-amino acids or L-amino acids or a mixture of D-amino acids and L-amino acids, linked by linkages of peptide. In general, the peptides contain at least two amino acid residues and in length they are less than about 50 amino acids. The term "protein" as used herein, refers to a compound that is comprised of linearly arranged amino acids linked by peptide linkage but, in contrast to the peptides, has a well-defined conformation. Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids. The term "polypeptide" as used herein, refers to a polymer of at least two amino acid residues and containing one or more peptide bonds. The term "polypeptide" encompasses peptides and proteins, regardless of whether the polypeptide has a well-defined conformation. The terms "guanidil", "guanidinyl" and "guanidino" are used interchangeably to refer to an entity that has the formula HN = C (NH2) NH (non-protonated form). As an example, arginine contains a guanidyl entity (guanidino) and is also referred to as 2-amino-5- acid P1700 / 99MX guanidinovaler or a-amino-d-guanidinovaleric acid. The term "guanidinium" refers to the acid form of the positively charged conjugate. The terms "amidinyl" and "amidino" refer to an entity that has the formula C (= NH) (NH3). "Amidinium" refers to the positively charged form of conjugated acid. The term "poly-arginine" or "poly-Arg" refers to a polymer sequence composed of contiguous arginine residues; poly-L-arginine means that they are all L-arginines; poly-D-arginine refers to all are D-arginines. Poly-L-arginine is also abbreviated by a capital "R", followed by the number of L-arginines in the peptide (for example, R8 represents 8-mer residues of contiguous L-arginine); poly-D-arginine is abbreviated by a lowercase "r", followed by the number of D-arginines in the peptide (r8 represents residues of 8 contiguous D-arginine numbers). The amino acid residues are referred to herein by their full names or by the single-letter or three-letter notations: A, Ala, alanine; C, Cys, cysteine; D, Asp, aspartic acid; E, Glu, glutamic acid; F, Phe, phenylalanine; G, Gly, glycine; H, His, ~ his tidina; I, lie, isoleucine; K, Lys, lysine; L, Leu, leucine; M, Met, methionine; N, Asn, asparagine; P, Pro, proline; Q, Gln, glutamine; R, Arg, arginine; S, Ser, serine; P1700 / 99MX T, Thr, threonine; V, Val, valine; W, Trp, tryptophan; X, Hyp, hydroxyproline; And, Tyr, tyrosine.

II. Structure of the Polymer Entity _ In one embodiment, the transport polymers, in accordance with the present invention, contain short polymers of from 6 to 25 subunits, as described above. The conjugate is effective to improve the transport rate of the conjugate through the biological membrane, with respect to the transport rate of the unconjugated biological agent alone. Although the polymer compositions exemplified are peptides, the polymers may contain backbones that are not peptides and / or subunits, as discussed below below. In an important aspect of the invention, the conjugates of the invention are particularly useful for transporting biologically active agents through the cell or the membranes of the organelles, when the agents are of the type that require transmembrane transport to exhibit their biological effects and that they do not exhibit their biological effects mainly by binding to a surface receptor, that is, in such a way that the entry of the agent does not occur. In addition, the conjugates are particularly useful for transporting biologically active agents of the type that requires P1700 / 99MX transmembrane transport to exhibit its biological effects and that, by themselves (without conjugation with-a transport polymer or with any other modification) are incapable or only have a poor ability to enter the cells and manifest their biological activity . As a general matter, the transport polymer used in the conjugate preferably includes a linear backbone of subunits. The main chain will usually comprise heteroatoms selected from carbon, nitrogen, oxygen, sulfur and phosphorus, where most of the atoms in the main chain are usually carbons. Each subunit contains a side chain entity that includes a guanidino or amidino terminal group. Although the spacing between adjacent side chain entities will normally be consistent from one subunit to another, the polymers used in the invention may also include a variable spacing between side chain entities along the main chain. The side chain entities extend away from the main chain, such that the central carbon atom of guanidino or amidino (to which the NH groups are attached) is linked to the main chain by a side chain linker which preference contains at least 2 chain atoms P1700 / 99 X linker, more preferably, 2 to 5 chain atoms, such that the central carbon atom is the third to sixth atom of the chain away from the main chain. Atoms in the chain are preferably provided as methylene carbon atoms, although one or more other atoms, such as oxygen, sulfur or nitrogen, may also be present. Preferably, the side chain linker between the main chain and the central carbon atom of the guanidino or amidino group is 4 atoms long, as exemplified by the arginine side chain. The sequence of the transport polymer of the invention may be flanked by one or more subunits that are not guanidino / non-amidino or a linker such as an aminocaproic acid group, which does not significantly affect the transport rate in the membrane of the corresponding polymer-containing conjugate, such as glycine, alanine and cysteine, for example. Also, any amino-free terminal group can be closed at the end, i.e., quenched with a blocking group, such as an acetyl or benzyl group, to avoid ubiquitination in vivo. The agent that will be transported can be linked to the transport polymer, in accordance with various modalities. In a preferred embodiment, the agent binds to a single transport polymer, P1700 / 99MX either by linkage to a terminal end of the transport polymer or to an internal subunit within the polymer by a suitable linker group. In a second embodiment, the agent binds to more than one polymer, in the same manner as above. This modality is a little less preferred, since it can lead to the cross-linking of adjacent cells. In a third embodiment, the conjugate contains two agent entities attached to each terminal end of the polymer. For this embodiment, it is preferred that the agent have a molecular weight less than 10 kDa. With respect to the first and third embodiments just mentioned, the agent in general is not bound to any of the guanidino or amidino side chains, so that they are free to interact with the target membrane. The conjugates of the invention can be prepared by direct synthetic schemes. Furthermore, the conjugated products are normally practically homogeneous in length and composition, so that they provide a greater consistency and reproducibility in their effects than heterogeneous mixtures. In accordance with an important aspect of the present invention, applicants have P1700 / 99MX found that the binding of a single transport polymer to any of a variety of types of biologically active agents is sufficient to significantly improve the rate of absorption of an agent through biological membranes, even without requiring the presence of a large hydrophobic entity in the conjugate. In fact, the binding of a large hydrophobic entity can significantly impede or edit the transport through the membrane, due to the adhesion of the hydrophobic entity to the lipid bilayer. In accordance with the above, the present invention includes conjugates containing large hydrophobic entities, such as lipids and fatty acid molecules. In another embodiment, the method is used to treat a condition of the non-central nervous system (non-CNS) in a subject that does not require delivery through the blood brain barrier.

A. Amino Acid Polymer Components. In one embodiment, the transport polymer is composed of D or L amino acid residues. The use of L-amino acid residues of natural occurrence in transport polymers has the advantage that the products of decomposition must be relatively innocuous to the cell or the organism. The preferred amino acid subunits are arginine (a-amino acid) P1700 / 99MX d-guanidinovaleric acid) and -amino-e-amidinohexanoic acid (analog of the isothermal amidino). The guanidinium group in arginine has a pKa of approximately 12.5. More generally, it is preferred that each polymer subunit contains a very basic side-chain entity, which (i) has a pKa greater than 11, more preferably, 12.5 or greater and (ii) contains, in its protonated state, at least two terminal amino groups (NH2) which share a positive charge stabilized by resonance, which gives the entity a bidentate character. Other amino acids, such as a-amino-β-guanidinopropionic acid, -amino-β-guanidinobutyric acid or -amino-e-guanidinocaproic acid, can also be used (containing 2, 3 or 5 linking atoms, respectively, between the chain principal and guanidinium central coal). D-amino acids can also be used in the transport polymers. The compositions containing exclusively D-amino acids have the advantage of reduced enzymatic degradation. However, these can also remain mainly intact within the target cell. This stability is generally not problematic if the agent is biologically active when the polymer is still attached. For agents that are inactive in their conjugated form, a linker that is cleavable in the P1700 / 99MX site of action (eg, by enzyme-mediated or solvent-mediated cleavage, within a cell) must be included within the conjugate to promote the release of the agent in the cells or organelles. Other Subunits: Other subunits other than amino acids can also be selected for use in the formation of transport polymers. These subunits may include, but are not limited to, hydroxy amino acids, N-methyl amino acids, aminoaldehydes and the like, which result in polymers with reduced peptide bonds. Other types of subunits can be used, depending on the nature of the selected main chain, as discussed in the next section.

B. Main Chain Type A variety of main chain types can be used to order and place the guanidino and / or amidino side chain entities, such as, alkyl main chain entities linked by thioethers or sulfonyl groups, hydroxy acid esters (equivalent to replacing amide bonds with ester linkages), replacing alpha carbon with nitrogen to form an aza analog, alkyl main chain entities linked by carbamate groups, polyethylene imines (PEIs) and P1700 / 99MX aminoaldehydes, which result in polymers composed of secondary amines. A more detailed backbone includes amide N-substituted (CONR replaces links CONH), esters (C02), ketomethylene (COCH2) reduced or methyleneamino (CHNH), thioamide (CSNH), phosphinate (P02RCH2), phosphonamidate ester phosphonamidate (P02RNH), retropéptido (NHCO), transalqueno (CR = CH), fluoroalkene (CF = CH), dimethylene (CH2CH2), thioether (CH 2 S), hydroxyethylene (CH (OH) CH2), methyleneoxy (CH20), tetrazole (CN2), retrotioamida (NHCS) retroreducida (NHCH2), sulfonamido (S02NH), metilensulfonamido (CHRS02NH), retrosulfonamida (NHS0) and peptoids (wisteria N-substituted) and backbones with malonate and / or subassemblies gene-diaminoalkyl, by example, as reviewed by Fletcher et al. (1998) and is detailed in the references cited here. Main chains of peptoids (N-substituted glycines) can also be used (for example, Kessler, 1993, Zuckermann et al., 1992, and Simón et al., 1992.). Many of the foregoing substitutions result in approximately iso-meric polymer backbones with respect to the backbones formed from α-amino acids. Studies conducted in support of the present invention have used polypeptides (e.g., peptide backbones). Without P1700 / 99MX However, other backbones, such as those described above, can provide improved biological stability (e.g., resistance to enzymatic degradation in vivo).

C. Synthesis of Transport Molecules Polymeric __ _ The polymers were constructed by any method known in the art. Exemplary peptide polymers can be produced in synthetic form, preferably using a peptide synthesizer (Applied Biosystems Model 433) or can be synthesized recombinantly by methods well known in the art. The recombinant synthesis is used in general form when the transport polymer is a peptide that will be fused to a polypeptide or a protein of interest. The N-methyl and hydroxy amino acids can be substituted by conventional amino acids in the solid phase peptide synthesis. However, the production of polymers with reduced peptide bonds requires the synthesis of the dimer of amino acids containing the reduced peptide bond. These dimers are incorporated into the polymers using standard solid phase synthesis methods. Other synthesis methods are well known and can be found, for example, in Flecher et al. (998), Simón et al. (1992) P1700 / 99MX Wong, 1991 and in the references cited therein.

III. Union or Annexation of the Transport Polymers to the Biologically Active Agents The transport polymers of the invention can be covalently bound to the biologically active agents by chemical or recombinant methods.

TO . Chemical Links Biologically active agents, such as small organic molecules and macromolecules can be linked to the transport polymers of the invention by various methods known in the art (see, for example, Wong, 1991), either directly (e.g. , with carbodiimide) or by a binding entity. In particular, for most applications the carbamate, ester, thioether, disulfide and hydrazone bonds are generally easily formed and are suitable. Ester and disulfide bonds are preferred if the bonds will be readily degraded in the cytozole, after transport of the substance through the cell membrane. Various functional groups (hydroxyl, amino, halogen, etc.) can be used to bind the biologically active agent to the polymer of P1700 / 99MX transport. Groups which are not known to be part of an active site of the biologically active agent are preferred, particularly if the polypeptide or any portion thereof will remain bound to the substance after delivery. Polymers, such as the peptides produced according to Example 1, are generally produced with an amino terminal protection group, such as FMOC. For biologically active agents that can survive the conditions used for the cleavage of the polypeptide from the synthesis resin and to deprotect the side chains, the FMOC can be cleaved from the N-terminus of the polypeptide bound to the finished resin, so that the agent can be linked to the N-terminal free amine. In these cases, the agents to be bound are normally activated by means well known in the art to produce an active ester or active carbonate entity effective to form an amine or carbamate link, respectively, to the polymeric amino group. Of course, other binding chemistries can also be used. To help minimize side reactions, the guanidino and amidino entities can be blocked using conventional protecting groups, such as the carbonbenzyloxy (CBZ), di-t-BOC, PMC, Pbf, N-N02, and P1700 / 99MX the similar. The coupling reactions are carried out by known coupling methods in any of a solvent arrangement, such as N, N-dimethyl formamide (DMF), N-methyl pyrrolidinone, dichloromethane, water and the like. The following coupling reagents include O-benzotriazolyloxy tetramethyluronium hexafluorophosphate (HATU), dicyclohexyl carbodiimide, bromo-tris (pyrrolidino) phosphonium bromide (PyBroP), etc. Other reagents may be included, such as N, -dimethylamino pyridine (DMAP), 4-pyrrolidino pyridine, N-hydroxysuccinimide, N-hydroxybenzotriazole and the like. For biologically active agents that are inactive until the bound transport polymer is released, the binder is preferably an easily cleavable binder, which means that it is susceptible to enzymatic or solvent-mediated cleavage in vivo .. For this purpose , binder containing carboxylic acid esters and disulfide bonds are preferred, wherein the first groups are hydrolyzed enzymatically or chemically and the latter is cleaved by disulfide exchange, for example, in the presence of glutathione. In a preferred embodiment, the cleavable linker contains a first cleavable group that is distant from the agent and a second group P1700 / 99 X cleavable that is close to the agent, such that the cleavage of the first cleavable group produces a binder-agent conjugate that contains a nucleophilic entity capable of reacting intramolecularly to cleave the second cleavable group, thereby releasing the agent from the bonding and polymer. This embodiment is further illustrated by the various small molecule conjugates discussed below.

B. Fusion Polypeptides The transport polypeptide polymers of the invention can be linked to biologically active polypeptide agents by recombinant means by constructing fusion protein vectors comprising the polypeptide of interest and the transport peptide, in accordance with the methods well known in the art. In general, the transport peptide component will be joined at the C-terminus or N-terminus of the polypeptide of interest, optionally, by a short peptide linker.

IV. Improved Transport of Biologically Active Agents through Biological Membranes A. Measurement of Transport through Biological Membranes Model systems to evaluate the P1700 / 99MX ability of the polymers of the invention to transport biomolecules and other therapeutic substances through biological membranes, include systems that measure the ability of the polymer to transport a fluorescent molecule covalently bound through the membrane. For example, fluorescein (= 376 MW) can serve as a model for the transport of small organic molecules (Example 2). For the transport of macromolecules, a transport polymer can be fused to a large polypeptide, such as ovalbumin (molecular weight 45 kDa; for example, Example 14). The detection of macromolecule absorption can be facilitated by attaching a fluorescent label. Cell absorption can also be analyzed by confocal microscopy (Example 4).

B. Improved Transport Through Biological Membranes In experiments conducted in support of the present invention, transmembrane transport and concomitant cell uptake were evaluated by absorption of a transport peptide linked to fluorescein according to the methods described in the Examples 2 and 3. Briefly, the cell suspensions were coated with fluorescent conjugates suspended in a regulator during P1700 / 99MX variable times at 37 ° C, 23 ° C or 3 ° C. After incubation, the reaction was stopped and the cells were harvested by centrifugation and analyzed by fluorescence using fluorescence activated cell sorting (FACS). Under the conditions used, the cellular uptake of the conjugates was not saturable. Consequently, the ED50 values of the peptides could not be calculated. Instead, the data are presented as a histogram to allow direct comparisons of cellular uptake at single conjugate concentrations. Figures 1A-1C show the results of a study in which the ability of polymers of L-arginine (R; Figure 1A) or D-arginine (r; Figure IB) was tested, with a variable length of 4 to 9 arginine subunits, to transport fluorescein in Jurkat cells. For comparison, the transport levels of residues 49-57 ("49-57") HIV tat and an N-lysine (K9) nonamer were also tested. Figure 1C shows a histogram of absorption levels of the conjugates at a concentration of 12.5 μM. As shown in the figures, peptide polymers labeled in fluorescent form and compounds of 6 or more arginine residues more efficiently entered cells than the tat 49-57 sequence. In particular, it was improved P1700 / 99MX absorption at at least twice the absorption level of tat 49-57 and, as much as approximately 6 to 7 times the absorption level of tat 49-57. The absorption of only fluoroscein was negligible. Also, the lysine nonamer (K9) showed very little absorption, indicating that the short lysine polymers are ineffective as transmembrane transport, in contrast to polymers containing guanidinium of comparable length. With reference to Figure IB, D-arginine homopolymers exhibited an even greater transport activity than their L counterparts. However, the order of absorption levels was approximately the same. For the D-homopolymers, the peptides with 7 to 9 arginines exhibited in general equal activity. The hexamer (R6 or r6) was slightly less effective but still showed at least one transport activity about 2 to 3 times greater than that of the tat (49-57). The ability of D- and L-arginine polymers to improve transmembrane transport was confirmed by confocal microscopy (Figures 2A-2F and Example 4). Consistent with the FACS data described above, the cytosol of cells incubated with either R9 (Figures 2B and 2E) or r9 (Figures 2C and 2F) was more brightly fluorescent, indicating higher levels of conjugate transport to cells . By P1700 / 99MX contrast, the tat (49-57) at the same concentration showed only a weak staining (Figures 2A and 2D). The confocal micrographs also emphasized the point that the D-arginine polymer (Figure 2C) was more effective to enter the cells than the polymer composed of L-arginine (Figure 2F). From the foregoing, it is evident that the transport polymers of the invention are significantly more effective than HIV tat 47-59 peptide in transporting drugs through the plasma membranes of cells. In addition, poly-nonamer was ineffective as a transporter. To determine if there was an optimal length for continuous guanidinium-containing homopolymers, a set of longer arginine homopolymer conjugates (R15, R20, R25 and R30) was examined. To examine the effect of substantially longer polymers, a mixture of polymers of L-arginine with an average molecular weight of = 12.00 daltons (= 100 amino acids) was also tested (Example 5). However, to avoid precipitation problems, the serum level in the assay should be reduced to test conjugates with the polymeric material of ~ 12,000 MW. Cellular uptake was analyzed by FACS, as previously, and the mean fluorescence of live cells was measured. The cytotoxicity of each conjugate was also measured. With reference to Figure 3, the absorption P1700 / 99MX of L-arginine homopolymer conjugates with 15 or more arginines showed distinct cellular uptake patterns in a distinctive way from polymers containing nine arginines or less. The longer conjugate curves were flatter, crossing those of the conjugates of R9 and r9. At higher concentrations (> 3 μM), the absorption of R9 and r9 was significantly better than for longer polymers. However, at lower concentrations, cells incubated with longer peptides showed higher fluorescence. Based on these data, it appears that r9 and R9 enter cells at higher speeds than polymers containing 15 or more contiguous arginines. However, the biological half-life of R9 (L-peptide) was shorter than for longer conjugates, presumably because the proteolysis of longer peptides (due to serum enzymes) produces fragments that retain transport activity. In contrast, the D-isomer (r9) showed no evidence of proteolytic degradation, consistent with the high specificity of serum proteases of L-polypeptides. Thus, the overall transport efficiency of a transport polymer appears to depend on a combination of (i) the transmembrane absorption rate (a polymer with less than about 15 continuous arginines against the P1700 / 99MX susceptibility to proteolytic inactivation longer polymers are better). In accordance with the foregoing, polymers containing from 7 to 20 contiguous guanidinium residues and, preferably, from 7 to 15 are preferred. Notably, the high molecular weight polyarginine conjugate (12,000 MW) showed no absorption detectable This result is consistent with the observations of Barsoum et al. (1994) and suggests that arginine polymers have transport properties that are significantly different from those that can be exhibited by lysine polymers. In addition, it was found that the 12,000 polyarginine conjugate was very toxic (Example 5). In general, the toxicity of the polymers increased with length although only the 12,000 MW conjugate showed high toxicity at all concentrations tested. When the cellular uptake of the polymers of D- and L-arginine was analyzed by the Michaelis-Menten kinetics (Example 6), the absorption rate in Jukat cells was so efficient that only accurate Km values could be obtained when the assays were performed. performed at 3 ° C (on ice). Both the maximum transport velocity (Vma?) And the apparent affinity of the peptides for the putative receptor of the Michaelis constant (Km) were derived from Lineweaver-Burk plots of the P1700 / 99 X observed fluorescence of Jurkat cells after incubation with varying concentrations of the D- and L-arginine nonamers for 30, 60, 120 and 240 seconds. Kinetic analysis also revealed that arginine-rich polymers exhibit a better ability to bind and cross a putative cell transport site than, for example, the tat peptide (49-57), since the Km for transport of the poly L-arginine nonamérica (44 μM) was substantially less than the Km of the tat peptide (722 μM). In addition, the D-arginine nonamer showed the lowest Km (7 μM) of the polymers tested in this assay (Table 1), that is, an apparent affinity approximately 100 times greater. According to a preferred embodiment of the invention, the transport polymer of the invention has an apparent affinity (Km) that is at least 10 times larger and, preferably, at least 100 times larger than the affinity measured for tat by the procedure of Example 6, when measured at room temperature (23 ° C) or 37 ° C.

Table 1 KM (μM) VMAX (μM / sec) H3N-RRRRRRRRRR-COO '44. 43 0. 3 5 H N-rrrrrrrrr-COO "7 .21 0. 3 9 tat 49 - 57 722 0. 3 8 P1700 / 99MX The experiments carried out in support of the present invention indicate that the transport facilitated by polymer depends on the metabolic integrity of the cells. The addition of a toxic amount of sodium azide (0.5% w / v) to the cells resulted in the inhibition of the uptake of the conjugates by approximately 90% (Example 7). The results shown in Figure 4 demonstrate (i) the sensitivity of transmembrane transport to sodium azide, which suggests energy dependence (cellular absorption) and (ii) the superiority of the poly-guanidinium polymers of the invention (R9, R8, R7), with respect to HIV tat (49-57). Without being ascribed to any particular theory, the data suggest that the transport process is an energy-dependent process, mediated by the specific recognition of polymers containing guanidinium or amidinium by a molecular transporter present in cell plasma membranes. Other experiments in support of the invention have shown that the conjugates of the invention are effective in transporting biologically active agents through the membranes of a variety of cell types, including human T cells (Jurkar), B cells (murine CH27) , lymphoma T cells (murine EL-4), mastocystoma cells (P388) Murine P1700 / 99MX), several murine T-cell hybridomas, neuronal cells (PC-12), fibroblasts (murine RT), kidney cells (murine HELA), myeloblas toma (murine K562) and primary tissue cells, which include to all human blood cells (with the exception of erythrocytes), such as T and B lymphocytes, macrophages, dendritic cells and eosinophils; basophils, mast cells, endothelial cells, cardiac tissue cells, liver cells, spleen cells, lymph node cells and keratinocytes. The conjugates are also effective to pass through both gram-negative and gram-positive bacteria cells, as disclosed in Example 8 and Figures 5A-5C. In general, D-arginine subunit polymers were found to enter both gram-positive and gram-negative bacteria at significantly faster rates than the transport rates observed for L-arginine polymers. This is illustrated by Figure 5A, which shows much higher absorption levels for the conjugate r9 (D-arginines) than for the conjugate R9 (L-arginine), when incubated with (prokaryotic) cells HB 101 of E. This observation can be attributed to the proteolytic degradation of L-polymers by bacterial enzymes. Figure 5B shows the absorption levels of the D-arginine conjugates as a P1700 / 99MX function of length (r4 to r9) compared to a conjugate of poly-L-lysine (K9), when incubated with HB 101 cells of E. coli As can be seen, the polyarginine conjugates showed a tendency similar to that of Figure 2B observed with eukaryotic cells, so that the shorter polymers of r6 showed low levels of absorption, where the absorption levels increase as a function of the length. Gram-positive bacteria, as exemplified by Strep. bovis, were also efficiently stained with polymers of arginine but not with lysine, as shown in Figure 5C. More generally, the highest levels of absorption were observed in bacteria at 37 ° C. However, significant staining was observed when the incubation was performed either at room temperature or at 3 ° C. Confocal microscopy revealed that the pretreatment of the bacteria with 0.5% sodium azide, inhibited transport through the internal plasma membranes of both gram-positive and gram-negative bacteria but not the transport through the cell wall (bacteria gram positive) towards the periplasmic space. Thus, the invention includes conjugates containing antimicrobial agents, such as antibacterial and antifungal compounds, for use in the prevention or inhibition of P1700 / 99MX infection or microbial proliferation and to disinfect surfaces that improve medical safety. In addition, the invention can be used for transport to plant cells, particularly, in green leafy plants. Further studies in support of the invention have shown that translocation through bacterial membranes is dependent on both energy and temperature, consistent with the observations noted above for other cell types.

V. Therapeutic Compositions A. Small Organic Molecules The small organic molecule therapeutics can advantageously be linked to linear polymeric compositions, as described herein, to facilitate or enhance transport through biological membranes. For example, the delivery of highly charged agents, such as levodopa (L-3, 4-dihydroxy-phenylalanine; L-DOPA) can benefit by binding to polymeric transport molecules, as described herein. Peptide and peptidomimetic agents are also contemplated (eg Langston, 1997; Giannis et al., 1997). Also, the invention is advantageous for delivering small organic molecules that have bad P1700 / 99 X solubilities in aqueous liquids, such as serum and aqueous saline solution. Thus, compounds whose therapeutic efficacies are limited by their low solubilities can be administered in higher doses with the present invention and can be more effective on a molar basis in conjugated form, with respect to the unconjugated form, due to the higher levels of absorption by the cells. Since a significant portion of the topological surface of a small molecule is frequently involved and, therefore, is required, for biological activity, the small molecule portion of the conjugate in particular cases may need to be separated from the bound transport polymer and the binding entity (if any) for the small molecule agent to exert its biological activity after crossing the white biological membrane. For these situations, the conjugate preferably includes a cleavable linker to release the free drug after passing through a biological membrane. In one approach, the conjugate may include a disulfide bond, as illustrated in Figure 6A, which shows a conjugate (I) containing a transport polymer T which is linked to a cytotoxic agent, 6-mercaptopurine, by an N-acetyl-protected cysteine group which serves as P1700 / 99MX a linker. In this way, the cytotoxic agent is linked by a disulfide bond to the 6-mercapto group and the transport polymer is bonded to the cysteine carbonyl entity via an amide bond. Cleavage of the disulfide bond by reduction or disulfide exchange results in the release of the free cytotoxic agent. In Example 9A a method for synthesizing a conjugate containing disulfide is provided. The product contains a heptamer of Arg residues, which is linked to 6-mercaptopurine by an N-acetyl-Cys-Ala-Ala binder, where the Ala residues are included as an additional separator to make the disulfide more accessible to thiols and reducing agents for cleavage within a cell. The binder in this example also illustrates the use of amide bonds, which can be enzymatically cleaved within a cell. In another approach, the conjugate includes a photocleavable linker, in which it is cleaved by exposure to electromagnetic radiation. An exemplary linkage is illustrated in Figure 6B, which shows a conjugate (II) containing a transport polymer T, which is linked to 6-mercaptopurine by a meta-nor trobenzoate linker. The polymer T is linked to the nitrobenzoate entity by an amide bond P1700 / 99MX with the benzoate carbonyl group and the cytotoxic agent is linked through its 6-mercapto group to the p-methylene group. The compound can be formed by reacting 6-mercaptopurine with p-bromomethyl-m-nor trobenzoic acid in the presence of NaOCH / methanol under heating, followed by the coupling of benzoate carboxylic acid to a transport polymer, such as the amino group of a? -aminobutyric acid binder bonded to the polymer (Example 9B). The photoilumination of the conjugate causes the release of 6-mercaptopurine by virtue of the nitro group, which is ortho in the mercaptomethyl entity. This approach finds utility in phototherapy methods as is known in the art, particularly for the activation of the drug located in a selected area of the body. Preferably, the cleavable binder contains first and second cleavable groups that can cooperate to cleave the biologically active agent polymer, as illustrated by the following approaches. That is, the cleavable linker contains a first cleavable group that is distant from the agent and a second cleavable group that is close to the agent, such that cleavage of the first cleavable group produces a linker-agent conjugate that contains a nucleophilic entity capable of react P1700 / 99MX intramolecularly to cleave the second cleavable group, thereby releasing the binder and polymer agent. Figure 6C shows a conjugate (III) containing a transport polymer T bound to the anticancer agent, 5-fluorouracil (5FU). Here, the linkage is provided by a modified lysyl residue. The transport polymer is linked to the a-amino group and the 5-fluorouracil is linked by the α-carbonyl. The lysyl group e-a ino is modified to an o-hydroxymethyl nitrobenzene carbamate ester, which comprises a first cleavable photolabile group in the conjugate. The photo-illumination separates the conjugate from the nitrobenzene entity, leaving a carbamate that also decomposes rapidly to provide the free e-amino group, an effective nucleophile. The intramolecular reaction of the e-amino group with the amino bond of the 5-fluorouracil group leads to cyclization with the release of the 5-fluorouracil group ^ Figure 6D illustrates a conjugate (IV) containing a T transport polymer bound to the 2'-antigen of the anticancer agent, paclitaxel. The linkage is provided by a linking entity that includes (i) a nitrogen atom attached to the transport polymer, (ii) a phosphate monoester located for the nitrogen atom and (iii) a carboxymethyl group meta to the nitrogen atom, what P1700 / 99MX is linked to the 2'-paclitaxel oxygen by a carboxylate ester bond. Enzymatic cleavage of the phosphate group from the conjugate provides a free hydroxyl phenol group. This nucleophilic group then reacts intramolecularly with the carboxylate ester to liberate free paclitaxel, for binding to its biological target. Example 9C describes a synthetic protocol for preparing this type of conjugate. Figure 6E illustrates another approach, wherein a transport polymer is linked to a biologically active agent, for example, paclitaxel, by an aminoalkyl carboxylic acid. Preferably, the linking amino group is bonded to the carboxy-linking carbon by 3 to 5 chain atoms (n = 3 to 5), preferably 3 or 4 chain atoms, which are preferably provided as carbon atoms. methylene .. As seen in Figure 6E, the binding amino group is linked to the transport polymer by an amide bond, and is attached to the paclitaxel entity via an ester linkage. The enzymatic cleavage of the amide bond liberates the polymer and produces a free nucleophilic amino group. The free amino group can then react intramolecularly with the ester group to liberate the paclitaxel binder. Figures 6D and 6E are illustrative of another aspect of the invention, comprising conjugates P1700 / 99MX anticancer taxane and taxoid, which have improved transmembrane transport speeds, with respect to the corresponding unconjugated forms. The conjugates are particularly useful for inhibiting the growth of cancer cells. It is considered that taxanes and taxoids manifest their anticancer effects by promoting the polymerization of microtubules (and inhibiting depolymerization) to a degree that is detrimental to cellular function, inhibiting cell replication and ultimately leading to cell death. The term _ "taxane" refers to paclitaxel (Figure 6F, R '= acetyl, R "= benzyl) also known under the trademark" TAXOL- ") and analogs that occur naturally, in synthetic form or designed Genetically engineered, they have a main chain core containing rings A, B, C, and D of paclitaxel as illustrated in Figure 6G Figure 6F also indicates the structure of the "TAXOTERE ™" (R1 = H, R "= BOC), which is a slightly more soluble synthetic analogue of paclitaxel, sold by Rhone-Poulenc. The term "Taxoid" refers to paclitaxel analogues that occur naturally, synthetically or genetically engineered, which contain the basic A, B and C rings of paclitaxel, as shown in Figure 6H. Synthetic information and Important biological P1700 / 99MX is available in the synthesis and activities of a variety of taxane and taxoid compounds, as reviewed by Suffness (1995) particularly in Chapters 12 to 14, as well as in the subsequent literature on paclitaxel. In addition, a host of cell lines is available to predict anti-cancer activities of these compounds against certain types of cancers, as described, for example in Suffness in Chapters 8 and 13. The transport polymer is conjugated with the taxane or taxoid entity by any suitable binding site in the taxane or taxoid. Conveniently, the transport polymer is bonded by a C2 '-oxygen, C7-oxygen atom, using binding strategies as above. The conjugation of a transport polymer by C7-oxygen leads to taxane conjugates having anticancer and antitumor activity despite conjugation in that position. In accordance with the foregoing, the linker may be cleavable or non-cleavable. Conjugation by C2 '-oxygen significantly reduces the anticancer activity, so that a cleavable linker is preferred for conjugation at this site. Other binding sites, such as the CIO, can also be used. It will be appreciated that the taxane and P1700 / 99 X taxoid of the invention have an improved solubility in water with respect to taxol (= 0.25 μg / mL) and taxotere (6-7 μg / mL). Therefore, large quantities of solubilizing agents are not required, such as "CREMOPHOR EL" (polyoxyethylated risino oil), polysorbate 80 (polyoxyethylene sorbitan monoleate, also known as "TWEEN 80") and ethanol, so that they can be reduced the side effects normally associated with these solubilizing agents, such as anaphylaxis, dyspnea, hypotension and redness.

B. Metals Metals can be transported to eukaryotic or prokaryotic cells using chelating agents, such as texaphyrin or eZL diethylene triamine pentacetic acid (DTPA), conjugated to a transport membrane of the invention, as illustrated by Example 10. These conjugates are useful for supplying metal ions for imaging or therapy. The metal ions eg emplificativos include Eu, Lu, Pr, Gd, Tc99m, Ga67, Inlll, Y90, Cu67 and Co57. Preliminary membrane-transport studies can be performed with conjugate candidates, using cell-based assays, as described in the examples section below. For example, when using europium ions, cell absorption can be monitored P1700 / 99MX by means of fluorescence measurements in resolved time. For metal ions that are cytotoxic, absorption can be monitored by cytotoxicity.

C. Macromolecules The improved transport method of the invention is particularly adapted to improve transport through biological membranes for various macromolecules including, but not limited to, proteins, nucleic acids, polysaccharides and analogues thereof. Exemplary nucleic acids include oligonucleotides and polynucleotides formed by DNA and RNA and analogs thereof, which have selected sequences designed for hybridization with complementary targets (eg, antisense sequences for single- or double-stranded targets) or for express nucleic acid transcripts or proteins encoded by the sequences. Analogs include charged, and preferably uncharged, main chain analogs, such as phosphonates (preferably, methyl phosphonates), phosphoramidates, (N3 'or N5'), thiophosphates, uncharged morpholino-based polymers and protein nucleic acids. (PNAs). These molecules can be used in a variety of therapeutic regimens, which include enzyme replacement therapy, gene therapy and therapy P1700 / 99MX antisense, for example, By way of example, protein nucleic acids (PNA) are DNA analogues, since the backbone is structurally homomorphic to a deoxyribose backbone. It consists of N- (2-amino ethyl) glycine units to which the nucleobases are attached. The PNAs containing the four natural nucleobases hybridize with complementary oligonucleotides that obey Watson-Crick base pair rules and are a true DNA mimic in terms of base pair recognition (Egholm et al., 1993). The main chain of a PNA is formed by peptide bonds, instead of phosphate esters, making it very suitable for antisense applications. Since the main chain is unloaded, the PNA / DNA or the PNA / RNA duplicates that form shows a greater thermal stability than normal. PNAs have the additional advantage that they are not recognized by nucleases or proteases. In addition, PNAs can be synthesized in an automated peptide synthesizer, using the standard T-Boc chemistry. The PNA is then easily linked to a transport polymer of the invention. Examples of antisense oligonucleotides whose transport to the interior of cells can be improved using the methods of the invention are described in, for example, U.S. Patent No. 5,592,122. These oligonucleotides are P1700 / 99MX focused to treat the human immunodeficiency virus (HIV). The conjugation of a transport polymer with an antisense oligonucleotide can be effected, for example, by forming an amide bond between the peptide and the 5 'terminus of the oligonucleotide by means of a succinate linker, in accordance with well-established methods. The use of PNA conjugates is further illustrated in Example 11. Figure 7 shows the results obtained with a conjugate of the invention containing a PNA sequence to exhibit the secretion of gamma-interferon (? -IFN) by T cells, as described in Example 11. As can be seen, the antisense PNA conjugate was effective in blocking the secretion of? -IFN, when the conjugate was present at levels above about 10 μM. in contrast, no inhibition was observed with the sense PNA conjugate or with the unconjugated antisense PNA alone. Another class of macromolecules that can be transported through biological membranes is exemplified by proteins and, in particular, by enzymes. Therapeutic proteins include, but are not limited to, replacement enzymes. Therapeutic enzymes include, but are not limited to, alglucerase, for use in the treatment of lysozomal glucocerebrocidase deficiency P1700 / 99MX (Gaucher's disease), alpha-L-iduronidase, for use in the treatment of mucopolysaccharidosis I, alpha-N-acetylglucosamidase, for use in the treatment of syndrome B of sanfilipo, lipase, to be used in the treatment of pancreatic insufficiency, adenosine deaminase, to be used in the treatment of severe combined immunodeficiency syndrome and triosephosphate isomerase, to be used in the treatment of neuromuscular dysfunction associated with triose phosphate isomerase deficiency. In addition, and in accordance with an important aspect of the invention, protein antigens can be delivered to the cytosolic compartment of antigen-presenting cells (APCs), from where they are degraded into peptides. The peptides are then transported to the endoplasmic reticulum, where these they are associated with nascent HLA class I molecules and are stuck on the surface of the cell. These "activated" APCs can serve as inducers of restricted class I antigen-specific cytotoxic T-lymphocytes (CTLs), which then proceed to recognize and destroy the cells that display the particular antigen. APCs that have the ability to carry out this process include, but are not limited to, certain macrophages, B cells and dendritic cells. In one embodiment, the protein antigen is a tumor antigen to produce or P1700 / 99MX promote an immune response against tumor cells. The transport of proteins isolated or soluble in the cytosol of APC with the subsequent activation of CTL is an exception, since, with a few exceptions, the injection of isolated or soluble proteins does not result either in the activation of the APC nor in the induction of the CTLs. Thus, the antigens that are conjugated with the compositions that improve the transport of the present invention can serve to stimulate a cellular immune response in vi tro or in vivo. Example 14 provides details of the experiments carried out in support of the present invention, in which a protein antigen, eg, ovalbumin, was supplied to the APCs after conjugation with a polymer R7. The subsequent addition of APCs to cytotoxic T lymphocytes (CTLs) resulted in CD8 + albumin-specific cytotoxic T cells (stimulated CTLs). In contrast, APCs that have been exposed to unmodified ovalbumin failed to stimulate CTLs. In parallel experiments, the histocompatible dendritic cells (a specific type of APC) were exposed to the ovalbumin-R7 conjugates, then injected into mice. Subsequent blood analysis of these mice revealed the presence of albumin-specific CTLs. To the P1700 / 99MX control mice were given dendritic cells that had been exposed to unmodified albumin. The control mice did not show the albumin-specific CTL response. These experiments exemplify one of the specific utilities associated with the supply of macromolecules in general and, in particular, proteins inside cells. In another embodiment, the invention is useful for delivering immunospecific antibodies or fragments of antibodies to the cytosol to interfere with deleterious biological processes, such as microbial infection. Recent experiments have shown that intracellular antibodies can be effective antiviral agents in plant and mammalian cells for example, Tavladoraki et al., 1993; and Shaheen et al., 1996). These methods have typically used fragments of the single-chain variable region (scFv), in which the heavy and light chains of antibodies are synthesized as a single polypeptide. The variable heavy and light chains are usually separated by a flexible linker peptide (e.g., 15 amino acids) to produce a 28 kDa molecule that retains the high affinity ligand binding site. The main obstacle to the wide application of this technology has been the absorption efficiency in the infected cells. But when joining P1700 / 99MX transport polymers to HIV fragments, the degree of cellular uptake can be increased, allowing the inmnospecific fragments to bind and deactivate important microbial components, such as HIV Rev, HIV reverse transcriptase and integrase proteins.

D. Peptides __ Peptides that will be delivered by the improved transport methods described herein include but are not limited to, effector polypeptides, receptor fragments, and the like. Examples include peptides that have phosphorylation sites used by intracellular signal mediating proteins. Examples of these proteins include, but are not limited to, protein kinase C, RAF-1, p2lRas, NF-KB, C-JUN and cytoplasmic tails of membrane receptors, such as the receptor IL-4, CD28, CTLA-4, V7 and MHC Class I and Class II antigens. When the molecule that improves transport is also a peptide, synthesis can be achieved using either an automated peptide synthesizer or recombinant means, in which a polynucleotide encoding a fusion peptide is produced, as mentioned above. In the experiments carried out in support of the present invention (Example 15), a P1700 / 99MX 10-amino acid segment of the cytoplasmic tail region of the transmembrane protein V7 (also known as CD101) with a polymer sequence R7 at its C terminus. It is known that this tail region is physically associated and measured inactivation of the RAF-1 kinase, a critical enzyme in the pathway of cellular activation of MAP kinase. the conjugate V7-R7 was added to T cells, which subsequently was washed with detergent. Immunoprecipitation of the soluble fraction was tested by murine anti-V7 antibodies together with goat anti-mouse IgG. In the absence of peptide treatment, RAF-1, it is known that a kinase is associated and inactivated by association with V7, coprecipitated with -V7. In cells treated with peptide, the RAF-1 protein was removed from the immunocomplex or V7. The same peptide treatment did not disturb a complex consisting of RAF-1 and p21, eliminating any non-specific modification of RAF-1 by the V7 peptides. These results showed that a V7 peptide with cytoplasmic tail region, when conjugated with a peptide that improves transport in membranes of the present invention, enters a target cell and is specifically associated with a physiological effector molecule, RAF-1. This association can be used to alter the intracellular processes.

P1700 / 99 X In a second set of studies, the V7 portion of the conjugate was phosphoryl in vi tro, using protein kinase C. The anti-RAF-1 precipitates of the T cells that had been exposed to the phosphorylated V7 tail peptides but not to the non-phosphorylated V7 tail peptide, demonstrated the potent inhibition of RAF-kinase activity. These studies demonstrate two principles. First, the transport polymers of the invention can effect the transport of a much highly charged (phosphorylated) molecule through the membrane of the cell. Second, while the phosphorylated and unphosphorylated V7 tail peptides can bind to RAF-1, only the phosphorylated peptide modified the activity of the RAF-1 kinase.

SAW . Classification and Library Test Method In another embodiment, the invention can be used to classify one or more conjugates in terms of a selected biological activity, wherein the conjugates are formed from one or more candidate agents. The conjugates are contacted with a cell that exhibits a detectable signal with the absorption of the conjugate into the cell, such that the magnitude of the signal is indicative of the effectiveness of the conjugate with respect to the selected biological activity. An advantage of this modality is that it is P1700 / 99MX particularly useful for testing the activities of agents who, by themselves, are unable or have a poor ability to enter the cells and manifest biological activity. Thus, the invention provides a particularly efficient way to identify active agents that may otherwise not be accessible through large-scale classification programs, for lack of an effective and convenient way to transport agents within the cell or organelle Preferably, one or more candidate agents are provided as a combinatorial library of conjugates, which are prepared using any of several synthetic combinatorial methods known in the art. For example, Thompson and Ellman (1996) recognized at least five different general approaches to prepare combinatorial libraries on solid supports, namely (1) synthesis of discrete compounds, (2) synthesis by division (division and combination), (3) soluble library deconvolution, (4) structural determination by analytical methods and (5) coding strategies, in which chemical compositions of active candidates are determined by unique labels, after positive test of biological activity in the assay. The synthesis of libraries in solution includes at least (1) spatially separated syntheses and (2) synthesis of P1700 / 99 X combinations (Thompson, supra). Further description of the synthetic combinatorial methods can be found in Lam et al. (1997), which describes in a particular way in focus of a globule a compound. These approaches are readily adapted to prepare conjugates in accordance with the present invention, which include suitable protection schemes, as necessary. for example, for a library that is constructed on one or more solid supports, a transport peptide entity can first bind to the support or supports, followed by the construction or adhesion of candidate agents combinatorially on the polymers by suitable reactive functionalities. In an alternative example, a combinatorial library of agents is first formed into one more solid supports, followed by adhesion or annexation of a transport polymer to each immobilized candidate agent. Similar or different approaches can be used for synthesis in the solution phase. Libraries formed on a solid support are preferably separated from the support by a cleavable linker group by known methods (Thompson et al., And Lam et al., Supra). The one or more conjugated candidates can be tested with any of several assays based on cells that produce detectable signals in P1700 / 99MX proportion to the effectiveness of the conjugate. Suitably, the candidates are incubated with cells in multi-well plates and the biological effects are measured by a signal (eg, fluorescence, reflectance, absorption or chemiluminescence) which can be quantified using a plate reader. Alternatively, the incubation mixtures can be removed from the cavities for processing and / or further analysis. The structures of active and, optionally inactive compounds, if not yet known, are then determined and this information can be used to identify guiding compounds and to focus on additional synthesis and classification efforts. For example, the? -interferon secretion assay, detailed in Example 11, is easily adapted to the multi-cavity format, such that inhibitors of active secretion can be detected by europium-based fluorescence detection, using a cell-reading reader. license plate.

Anticancer agents can be classified using established cancer cell lines (eg, provided by the National Institutes of Health (NIH) and the National Cancer Institute (NCI)), the cytotoxic effects of anticancer agents can be determined by exclusion with gut staining, for example.

P1700 / 99 X Other examples include assays directed to inhibit cell signaling, such as inhibition of the IL-4 receptor; assays to block cell proliferation and gene expression assays. In a typical gene expression assay, a gene of interest is placed under the control of a suitable promoter and is followed downstream by a gene to produce reporter species such as β-galactosidase or firefly luciferse. Or an inhibitory effect can be detected as a basis for a decrease in the reporter's signal. The invention also includes a library of conjugates that is useful for classification in the above method. The library includes a plurality of candidate agents for one or more selected biological activities, each of which is conjugated with at least one transport polymer, according to the invention. Preferably, the conjugate library is a combinatorial library. In another embodiment, the invention includes a regular array of different polymer-agent conjugates, distributed in an indexed or indexable plurality of sample cavities, to test and identify the active agents of interest.

SAW . Utility The compositions and methods of this P1700 / 99MX invention have particular utility in the area of human and veterinary therapeutics. In general, the doses administered will be effective to deliver picomolar to micromolar concentrations of the therapeutic composition towards the effector site. The appropriate doses and concentrations will depend on factors such as the therapeutic composition or drug, the site of the intended delivery and the route of administration, all of which may be derived empirically, in accordance with methods well known in the art. Additional guidance may be obtained from studies using experimental animal models to evaluate the dosage, as is known in the art. The administration of the compounds of the invention with a pharmaceutically suitable excipient, as necessary, can be effected by any of the accepted administration nodes. Thus, the administration can be, for example, intravenous, topical, subcutaneous, transcutaneous, intramuscular, oral, intra-articular, parenteral, peritoneal, intranasal or by inhalation. The formulation can take the form of solid, semi-solid, lyophilized powder or liquid dosage forms, such as, for example, tablets, pills, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions. , aerosols or what P1700 / 99MX similar, preferably in unit dosage forms, suitable for the simple administration of precise doses. The compositions will normally include a conventional pharmaceutical carrier or vehicle and additionally may include other medicinal agents, vehicles, adjuvants and the like. Preferably, the composition will be from about 5% to 75% by weight of a compound or compounds of the invention, wherein the remainder consists of suitable pharmaceutical excipients. Suitable excipients can be tailored to the particular composition and route of administration by methods well known in the art, for example, (Gennaro, 1990). For oral administration, these excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate and the like. The composition can take the form of a solution, suspension, tablet, pill, capsule, powder, prolonged release formulation and the like. In some embodiments, the pharmaceutical compositions may take the form of a pill, a tablet or a capsule and, thus, the composition may contain, together with the biologically active conjugate, any of the following: a diluent, such as lactose, sucrose, phosphateD1600 / 99MX dicalcium and the like; a disintegrant, such as starch or derivative thereof; a lubricant, such as magnesium stearate and the like; and a binder, such as a starch, acacia gum, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. The active compounds of the formulas can be formulated in a suppository comprising, for example, from about 0.5% to about 50% of a compound of the invention, located in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96% ] and PEG 4000 [4%]). Liquid compositions can be prepared by dissolving or dispersing the compound (from about 0.5% to about 20%) and optional pharmaceutical adjuvants in a vehicle, such as, for example, aqueous saline solution (eg, 0.9% w / v sodium chloride). ), aqueous dextrose, glycerol, ethanol and the like, to form a solution or suspension, for example, for intravenous administration. The active compounds can also be formulated in a retention enema. If desired, the composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH regulating agents, such as, for example, P1700 / 99MX sodium, sorbitan monoalurate or triethanolamine oleate. For topical administration, the composition is administered in any suitable format, such as a lotion or a transdermal or transdermal patch. For delivery by inhalation, the composition can be supplied as a dry powder (for example, inhalation therapy) or in liquid form by a nebulizer. The methods for preparing these dosage forms are known or will be apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (1980). The composition to be administered will, in any case, contain a certain amount of a prodrug and / or active compounds in a pharmaceutically effective amount for the release or alleviation of the condition to be treated when administered in accordance with the teachings of the present invention. invention In general, the compounds of the invention are administered in a therapeutically effective amount, that is, in a dose sufficient to effect the treatment, which may vary, depending on the individual and the condition to be treated. Normally, a therapeutically effective daily dose is 0.1 to 100 mg / kg of P1700 / 99MX body weight per day of the drug. Most conditions respond to the administration of a total dose of between about 1 and about 30 mg / kg of body weight per day or between about 70 mg and 2100 mg per day for a 70 kg person. The stability of the conjugate can be further controlled by the composition and stereochemistry of the main chain and side chains of the polymer. For polypeptide polymers, the D-isomers are generally resistant to endogenous proteases and, therefore, have longer half-lives in the serum and within the cells. The D-polypeptide polymers are therefore suitable when a longer duration of action is desired. L-polypeptide polymers have shorter half-lives, due to their susceptibility to proteases and, therefore, are chosen to impart shorter acting effects. This allows the side effects to be avoided more easily by withdrawing the therapy as soon as the side effects are observed. The polypeptides comprising mixtures of D and L-residues have intermediate stabilities. In general, homo-D-polymers are preferred. It is intended that the following examples illustrate, in an enunciative manner, the present invention.

P1700 / 99MX Example 1 Peptide synthesis Peptides were synthesized using solid-phase techniques on an Applied Biosystems peptide synthesizer using FastMOC ™ chemistry and Wang resins obtained in commercial form and Fmoc-protected amino acids, in accordance with well-established methods. known in the art (Bonifaci). Peptides were purified using C4 or C18 reverse phase HPLC columns and their structures were confirmed using amino acid analysis and mass spectrometry.

Example 2 Fluorescence Assays Fluorescent peptides were synthesized by modifying the amino terminus of the peptide with aminocaproic acid, followed by the reaction with isothiocyanate of f luoroscens in the presence of (2-IH-benzotriazol-1-yl) -l hexafluorophosphate, 1, 3,3-tetramethyl uronium / N-hydroxybenzotriazole dissolved in N-methylpyrrolidone. The products were purified by gel filtration. Cell suspensions (106 / mL) were incubated for variable times, at 30 ° C, 23 ° C or 4 ° C, with a range of concentrations of peptides or conjugates in PBS at pH 7.2, containing 2% of P1700 / 99MX fetal calf serum (PBS / FCS) in 96-well plates. After a 15 minute incubation, the cells were pelleted by centrifugation, washed three times with PBS / FCS, containing 1% sodium azide, incubated with trypsin / EDTA (Gibco) at 37 ° C for five minutes and , then, they were washed twice more with PBS / FCS / NaN3. The pelleted cells were resuspended in PBS containing 2% FCS and 0.1% propidium iodide and analyzed in a FACScan (Becton Dickenson, Mountain View, CA). Positive cells for propidium iodide were excluded from the analysis. For the analysis of the arginine polymers, the voltage of the photomultiplier was reduced by an order of magnitude to allow a more accurate measurement.

Example 3 Tat Basic Peptide against Poly-Arg Peptides The absorption levels of the following polypeptides were measured by the method of Example 2: (1) a polypeptide comprising HIV tat 49-57 residues (rg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg = SEQ ID N0: 1), (2) a nonamer of L-Lys residues (K9, SEQ ID N0: 2) and (3) homo-L or homo-polypeptides that contain four to nine Arg residues (SEQ ID NO: 3-8 and 12-17). The results are shown in the Figures 2A-2C.

P1700 / 99 X Example 4 Confocal Cell Microscopy Cells incubated with fluorescent polyarginine peptides were prepared as described above for the binding assays and analyzed in the Cell Sciences Imaging Facility.

(Stanford University, Stanford, CA), using a confocal scanning microscope of a single beam "laser, with an excitation wavelength of 488 nm (Argon-ion laser) and an emission bandwidth of 510-550, using a bandpass filter. The conjugates (6.25 μM) containing tat (49-57), R7 or r7 coupled to fluorescein, were incubated with Jurkat cells at 37 ° C for 10 minutes. Figures 2A-2F show the results for the emitted fluorescence (Figures 2A-2C) and the transmitted light (2D-2F) for tat (49-57) (Figures 2A and 2C), R7 (Figures 2B and 2E) and r7 (Figures 2C and 2F).

Example 5 Length Interval Studies The following polyarginine homopolymers were tested by the fluorescence assay of Example 2, with incubation at 37 ° C for 15 minutes before cell pelletization: r9, R9, R15, R20, R25 and R30. In addition, a mixture of L-arginine polymers having an average molecular weight of 12,000 daltons was also tested.

P1700 / 99MX (approximately 100 amino acids) (Sigma Chem. Co.), After being labeled with fluorescein isothiocyanate and purified by gel filtration ("SEPHADEX" G-25). The cells were analyzed by FACS and the mean fluorescence of the living cells was measured. The cytotoxicity of each conjugate was also measured by calculating the percentage of cells stained with propidium iodide, which is characteristic of cell death. The absorption results for the conjugates r9, R9, R15, R20 and R25 are shown in Figure 3. The precipitated proteins of polyarginine (12,000 MW) available commercially, in serum, most likely, l-glycoprotein acid. Therefore, the level of fetal calf was reduced 10 times in the assay for conjugates prepared from this material. The 12,000 MW poly-Arg composition was toxic at concentrations from 800 nM to 50 μM and was excluded from Figure 3. Poly-L-Arg conjugates containing 20 or more arginine residues were toxic at concentrations greater than 12 μM, so that toxicity increased with length.

Example 6 Kinetics of Absorption To measure the Vmax and Km parameters of cell uptake, the assay method of the P1700 / 99MX Example 2 with the following modifications. Peptides were incubated with cells for 0.5, 1, 2, and 4 minutes at 4 ° C in triplicate, in 50 μl of PBS / FCS in 96-well plates. At the end of the incubation, the reaction was stopped by diluting the samples in 5 mL of PBS / FCS, centrifuging and washing once with PBS / FCS, trypsin / EDTA and finally, again with PBS / FCS and removing the pellets in PBS / FCS containing propidium iodide for analysis in a FACScan. The FACS data were adjusted with the Lineweaver-Burk equation for the Michaelis-Menten kinetics. The kinetics data for the fluorescent conjugates of tat (49-57), R9 and r9 are shown in Table 1 above.

Example 7 - Effects of the Metabolic Inhibitor on Transport The suspension cells (106 / mL) were incubated for 30 minutes with 0.5% sodium azide in PBS containing 2% FCS. At the end of the incubation, the fluorescent peptides (tat (49-57)), R7, R8 or R9) were added to a final concentration of 12.5 μM. After incubation for 30 minutes, the cells were washed according to Example 2, except that all the washing regulators contained 0.1% sodium azide. The results are shown in Figure 5.

P1700 / 99MX Example 8 Transport to Bacterial Cells Gram-negative bacteria (E. coli strain HB101) and gram-positive bacteria (Strep. Bovis) were grown in the appropriate logarithmic phase medium. Cell cultures (4 x 108 per mL) were incubated for 30 minutes at 37 ° C with varying concentrations of fluorescent conjugates containing linear polymers of L-arginine (R4 to R9), D-arginine (from r4 to r9) or L-lysine (K9) at conjugate concentrations of 3 to 50 μM. Cells were washed and extracted in propidium iodide containing PBS (to distinguish dead cells) and analyzed by FACS and by fluorescent microscopy. The results are shown in Figures 5A-5C, as discussed above.

Example 9 Conjugates with Slicing Bindings Ex emplificativos __ __ A. Conjugate of 6-Mercaptopurine Cysteine Disulfide Al. Activation with Tiol. The N-acetyl-Cys (SH) -Ala-Ala- (Arg) 7-C02H (12.2 mg, 0.0083 mmol) was dissolved in 3 mL of 3: 1 AcOH: H20 with stirring at room temperature. To this solution was added dithio-bis (5-nitropyridine) (DTNP) (12.9 mg, 0.0415 mmol, 5 eq). The solution was allowed to stir for 24 hours at room temperature, after P1700 / 99MX which the mixture took a bright yellow color. The solvent was removed in vacuo and the residue redissolved in 5 mL of H20 and extracted 3 times with ethyl acetate to remove excess DTNP. The aqueous layer was lyophilized and the product was used without further purification. A2. Drug Union. The N-acetyl-Cys (SH) -Ala-Ala- (Arg) 7-C02H (0.0083 mmol) was dissolved in 1 mL of degassed H20 (pH = 5) in argon at room temperature, with stirring. The 6-mercaptopurine (1.42 mg, 0.0083 mmol, 1 eq) in 0.5 mL DMF was added to the mixture. The reaction was allowed to stir for 18 hours in an inert atmosphere at room temperature. After 18 hours, a bright yellow color developed, indicating the presence of free 5-nitro-2-thiopyridine. The solvent was removed under reduced pressure and the residue was purified by HPLC to provide the desired product (I, Figure 6A) in an overall yield of 50%.

B. Taxol Conjugate Execistable 3-nitro-4- (bromomethyl) benzoic acid (ÍOOmg, 0.384 mmol) was dissolved in anhydrous methanol (5 mL) in a nitrogen atmosphere. To this solution was added sodium methoxide (88 μ, 25% (w / w) in methanol, 0.384 mmol, 1 eq) followed by the addition of 6-mercaptopurine (58.2 mg, 0.384 mmol, 1 eq). The mixture was heated to reflux and left in P1700 / 99MX shaking for 3 hours. The reaction mixture was then cooled, filtered and stopped by acidification with 6N HCl. The reaction volume was then reduced by half, at which point the product precipitates and is collected by filtration. The residue is redissolved in methanol, filtered (if necessary) and concentrated under reduced pressure to provide the desired sulfide (II, Figure 6B) in 50% yield as a yellow powdery solid.

C. Phosphate-Taxol Conjugate Cleavable _ Cl. To a suspension of o-hydroxy phenylacetic acid (15.0 g, 0.099 mol) in H20 (39 mL) at 0 ° C was added a solution of nitric acid (12 mL of 65% in 8 mL of H0) slowly by means of a pipette. The solution was stirred for an additional 1.5 hours at 0 ° C. The mixture was then heated to room temperature and allowed to stir for an additional 0.5 hours. The heterogeneous solution was emptied on ice (10 g) and filtered to remove the insoluble ortho-nitro isomer. The reddish solution was concentrated under reduced pressure and the thick residue was redissolved in 6N HCl and filtered through celite. The solvent was removed again under reduced pressure to provide the desired 2-hydroxy-4-nitro-phenylacetic acid as a light red-white partum solid. (40% yield). The product (IV-a) was used P1700 / 99MX in the next step without further purification. C2. The product IV-a (765 mg, 3.88 mmol) was dissolved in THF (5 mL) freshly distilled under an argon atmosphere. The solution was cooled to 0 ° C and borane-THF (1.0 M in THF, 9.7 mL, 9.7 mmol, 2.5 eq) was added dropwise by syringe with evident evolution of hydrogen. The reaction was allowed to stir for an additional 16 hours, slowly heating to room temperature. The reaction was stopped by the slow addition of 1M HCl (with strong bubbling) and 10 mL of ethyl acetate. The layers were separated and the aqueous layer was extracted five times with ethyl acetate. The combined organic layers were washed with brine and dried over magnesium sulfate. The solvent was evaporated in vacuo and the residue was purified by flash column chromatography (1: 1 hexane: ethyl acetate) to provide the desired nitro-alcohol (IV-b) as a light yellow solid (85% yield). C3. Nitro-alcohol (IV-b) (150 mg, .819 mmol) was dissolved in dry DMF (5 mL) containing di-t-butyldicarbonate (190 mg, 1.05 eq) and 10% Pd-C (10 mg). The mixture was placed in a Parr apparatus and pressurized / purged five times. The solution was then pressurized to 47 psi and left in agitation for 24 hours. The reaction was stopped by filtration through celite and the solvent was removed under reduced pressure. The residue was purified P1700 / 99MX by column chromatography (1: 1 hexane: ethyl acetate) to afford the protected aniline product (IV-c) as a dark crystalline solid in a 70% yield. C. TBDMS-C1 (48 mg, 0.316 mmol) was dissolved in freshly distilled dichloromethane (4 mL) under an argon atmosphere. To this solution was added imidazole (24 mg, 0.347 mmol, 1.1 eq) and a white precipitate immediately formed. The solution was stirred for 30 minutes at room temperature, at which point the product IV-c (80 mg, 0.316 mmol, 1.0 eq) was added rapidly as a solution in dichloromethane / THF (1.0 mL). The resulting mixture was allowed to stir for an additional 18 hours at room temperature. The reaction was stopped by the addition of saturated aqueous ammonium chloride. The layers were separated and the aqueous phase was extracted 3 times with ethyl acetate and the combined organic layers were washed with brine and dried over sodium sulfate. The organic phase was concentrated to give the silyl ether phenol product (IV-d) as a light yellow oil (90% yield). C5 Silyl ether-phenol IV-d (150 mg, 0.408 mmol) was dissolved in freshly distilled THF (7 mL) in argon and the solution was cooled to 0 ° C. Then dropwise n-BuLi (2.3 M in hexane, 214 'μL) was added via syringe. Immediately a change in color from light yellow to deep red was observed.

P1700 / 99MX After 5 minutes to the stirring solution under an argon atmosphere, tetrabenzyl pyrophosphate (242 mg, 0.45 mmol, 1.1 eq) was added rapidly, the solution was stirred for an additional 18 hours in an inert atmosphere, slowly warmed to room temperature , during this time formed a white precipitate. The reaction was stopped by the addition of saturated aqueous ammonium chloride and 10 mL of ethyl acetate. The layers were separated and the aqueous layer was extracted 5 times with ethyl acetate. The combined organic phases were washed with brine and dried over magnesium sulfate. The solvent was removed by evaporation and the residue was purified by flash column chromatography (1: 1 hexane: ethyl acetate) to provide the desired phosphate-silyl ether (IV-e), like a light orange oil (90% yield). C6 The phosphate-silyl ether (IV-e) (10 mg, 0.0159 mmol) was dissolved in 2 mL of dry ethanol at room temperature. To the stirring solution was added 20 μL conc. HCl. (1% v: v solution) and the mixture was allowed to stir until TLC analysis indicated that the reaction had been completed. Solid potassium carbonate was added to stop the reaction and the mixture was filtered rapidly through silica gel and concentrated to give the crude dibenzyl alcohol-phosphate product (IV-f) as a light yellow oil (100% strength). performance) .

P1700 / 99MX C7. The alcohol IV-f (78 mg, 0.152 mmol) was dissolved in freshly distilled dichloromethane (10 mL) under an argon atmosphere. To the solution was added Dess-Martin periodium (90 mg, 0.213 mmol, 1.4 eq). The solution was allowed to stir and the progress of the reaction was monitored by TLC analysis. Once the TLC indicated completion, the reaction was stopped by the addition of saturated aqueous sodium dicarbonate: saturated aqueous sodium thiosulfate, 1: 1 / The biphasic mixture was allowed to stir for 1 hour at room temperature. The layers were separated and the aqueous phase was extracted 3 times with ethyl acetate. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was removed under reduced pressure to provide the aldehyde product (IV-g) as a light dark oil (100% yield). C8. The aldehyde IV-g (78 mg, 0.152 mmol) was dissolved in t-butanol / water (3.5 mL) under an inert atmosphere. To the solution under rapid stirring was added 2-methyl-2-butene (1.0 M in THF, 1.5 L), sodium phosphate-monobasic (105 mg, 0.76 mmol, 5 eq) and sodium chlorite (69 mg, 0.76 mmol, 5 eq). The solution was allowed to stir for an additional 8 hours at room temperature. The solution was concentrated and the residue was acidified and extracted with ethyl acetate 3 times. The combined organic phases were dried over magnesium sulfate. The solution is again P1700 / 99MX concentrated under reduced pressure and the residue was purified by column chromatography (2: 1, ethyl acetate: hexane) to provide the desired carboxylic acid-dibenzylphosphate (IV-h) as a light yellow oil (65% strength). performance) . C9 The IV-h acid (8.0 mg, 0.0152 mmol, 1.1 eq) was dissolved in freshly distilled dichloromethane (2 mL) under argon at room temperature. To this mixture was added paclitaxel (12 mg, 0.0138 mmol, 1 eq) followed by DMAP (2 mg, 0.0138 mmol, 1 eq) and DCC (3.2 mg, 0.0152, 1.1 eq). The mixture was allowed to stir at room temperature for an additional four hours, during which time a light precipitate formed. Once the TLC analysis indicated that the reaction had been terminated, the solvent was removed or removed under reduced pressure and the residue was purified by flash column chromatography (1: 1, hexane: ethyl acetate) to provide pacli taxel-C2 '-carboxylate ester (IV-i) as a white crystalline solid 65% yield). CIO. The ester IV-1 (5.0 mg) was dissolved in clean formic acid (1.0 mL) under an argon atmosphere at room temperature and allowed to stir for 30 minutes. Once the TLC indicated that the reaction was over, the solution was concentrated under reduced pressure and the residue was purified by filtration through silica gel to provide the desired aniline-taxol compound.

P1700 / 99MX (IV-j) in a 50% yield as a white powder. Cll. To a solution of (poly di-CBZ) -protected AcHN-RRRRRRRR-C02H (1.2 eq, 0.1 to 1.0 M) in dry DMF was added O-benzotriazolyloxy tetramethyluronium hexafluorophosphide (HATU, 1.0 eq) and a catalytic amount of DMAP (0.2 eq). The solution was stirred under an inert atmosphere for 5 minutes at room temperature. To this mixture was then added Taxol-aniline derivative (IV-j) as a solution in dry DMF (the minimum volume to dissolve). The resulting solution was stirred for an additional 5 hours at room temperature. The reaction was terminated by concentrating the reaction mixture under reduced pressure. The crude reaction mixture was then purified by HPLC to provide the desired material (IV, Figure 6D).

Example 10 Metallic Ion Transport 3.93 g of DTPA were dissolved in 100 ml of regulator HEPES and 1.52 ml of atomic standard solution of europium chloride (Aldrich) were dissolved in 8 ml of the HEPES regulator was added and stirred for 30 minutes at room temperature. Chromatographic separation and lyophilization provide a Eu-DTPA chelate complex. This complex is then conjugated with the amino terminus of a polypeptide P1700 / 99MX by peptide chemistry in solid phase. The cellular absorption of the europium ion can be monitored by resolved fluorescence over time.

EXAMPLE 11 Absorption of PNA-Peptide Conjugates The PNA peptide conjugates were synthesized using solid phase chemistry with commercially available Fmoc reagents (PerSeptive Biosystems, Cambridge, MA) either on the Applied Biosystems 433A peptide synthesizer or in a synthetic synthesis system. Millipore Expedite nucleic acid. The polymers of D or L-arginine bound to the amino or carboxyl termini of the PNAs, which are analogous to the 5 'and 3' ends of the nucleic acids, respectively. The conjugates were also modified to include fluorescein or biotin by the addition of an amino caproic acid spacer to the amino terminus of the conjugate and then to bind biotin or fluorescein. The PNA-peptide conjugates were excised from the resin in solid phase using 95% TFA, 2.5% triisopropyl silane and 2.5% aqueous phenol. The resin was removed by filtration and the residual acid was removed by evaporation. The product was purified by HPLC using a C-18 reverse phase column and the product was lyophilized. The desired PNA-polymer conjugates were identified using P1700 / 99MX mass spectrometry by laser desorption.

A. Inhibition of Cellular Secretion of Gamma-IFN 1. PNA-Peptide Conjugates. The following PNA-sense and antisense peptide conjugates were prepared to inhibit gamma-IFN production, where r = D-arginine and R = L-arginine: Sense: NH2-rrrrrrr-AACGCTACAC-COOH (SEQ ID N0: 18) Antisense: NH2-rrrrrrr-GTGTAGCGTT-COOH (SEQ ID NO: 19; Fluorescent antisense: X-rrrrrrr-GTGTAGCGTT-COOH (X SEQ ID NO: 19! Where X = fluorescein-aminocaproate Biotinylated antisense Z-rrrrrrr-GTGTAGCGTT-COOH (Z-SEQ ID NO: 19) where Z = biotin-aminocaproate 2. T Cell Absorption. To show that the PNA-polyarginine conjugates enter the cell effectively, the above fluorescent antisense conjugate (X-SEQ ID NO: 19) was synthesized by conjugation of fluorescein isothiocyanate to the amino terminus of SEQ ID NO: 18, using an aminocaproic acid separator.

P1700 / 99MX Cellular uptake was assayed by incubation of the Jurkat human T cell line (5 x 103 cells / well), either pretreated for 30 minutes with 0.5% sodium azide or phosphate buffered saline, with varying amounts (100 nM to 50 μM) of the sense and antisense PNA-r7 conjugate labeled with fluorescein, as well as the antisense PNA alone (without the r7 segment). The amount of antisense PNA that entered the cells was analyzed by confocal microscopy and FACS. In both cases, the fluorescent signals were presented only in cells not exposed to the azide and the fluorescent signal was dose dependent of the fluorescent conjugate and the temperature and duration of the incubation. 3. Range-IFM test. The amount of inferred gamma secreted by a murine T cell line (clone 11.3) was measured by incubating 105 T cells with varying amounts of antigen (peptide consisting of residues 110-121 of whale sperm myoglobin) and Histocompatible spleens from DBA / 2 mice (H-2d, 5 x 105), which act as antigen-presenting cells (APCs), in 96-well plates. After incubation for 24 hours at 37 ° C, 100 μL of the supernatants were transferred to microtiter plates coated with monoclonal antibodies (MAbs) anti-gamma-IFN obtainable in the form Commercial P1700 / 99MX (Pharmingen, San Diego, CA). After incubation for one hour at room temperature, the plates were washed with PBS containing 1% fetal calf serum and 0.1% Tween 20, then a second biotinylated IFN gamma-IFN was added. After a second hour of incubation, the plates were washed as above and europium (Eu) -streptavidin (Delphia-Pharmacia) was added. Again, after one hour of incubation, an acid buffer was added to release Eu, which was measured by time-resolved fluorometry in a Delphia plate reader. The amount of fluorescence was proportional to the amount of gamma-IFN that had been produced and could be quantified accurately using known amounts of gamma-IFN to generate a standard curve. 4. Inhibition of Gama-IFN Production by Conjugates. The ability of PNA-polyarginine conjugates to inhibit gamma-IFN secretion was tested by adding various concentrations of the above gamma-IFN conjugates with suboptimal doses of the peptide antigen (0.5 μM), to a mixture of T cells from clone 11.3 and histocompatible spleen cells. PNA sequences lacking polyarginine and non-conjugated D-arginine heptamer entities were also tested. After 24 hours, aliquots of the cultured supernatants were taken and the amount was measured P1700 / 99MX gamma-IFN, using the fluorescent binding assay described in the previous section 3. The treatment of the cells with the PNA-r7 antisense conjugate resulted in a reduction of more than 70% in the secretion of IFN, while that equivalent molar amounts of sense PNA-r7, antisense PNA lacking r7 or r7 alone none showed inhibition (Figure 7).

Example 12 Transportation of Large Protein Antigen to APCs An ovalbumin conjugate coupled to a poly-L-arginine heptamer was formed by reacting a polypeptide polymer containing cysteine (Cys-Ala-Ala-Ala-Arg, SEQ ID NO: 21) with ovalbumin (45 kDa) in the presence of sulfo-MBS, a heterobifunctional crosslinker (Pierce Chemical Co., Rockford, IL). The molar ratio of the peptide conjugate to ovalbumin was quantified by amino acid analysis. The conjugate was designated OV-R7. The conjugate was added (final concentration = 10 μM) to B cells, also referred to as antigen presenting cells (APCs), which were isolated according to standard methods. The APCs were incubated with OV-R7 and then added to a preparation of isolated cytotoxic T-lymphocytes by standard methods. The exposure of CTLs P1700 / 99MX to APCs that had been incubated with 0V-R7 produced albumin-specific CTLs CD8 +. In contrast, APCs that had been exposed to unmodified ovalbumin failed to stimulate CTLs. In another experiment, histocompatible dendritic cells (a specific type of APC) were exposed to albumin-R7 conjugates and then injected into mice. Subsequent analysis of the blood of these mice revealed the presence of albumin-specific CTLs. Control mice were given dendritic cells that had been exposed to unmodified albumin. The control mice did not show the albumin-specific CTL response.

Example 13 _ Enhanced Absorption of V7 Derived Peptide A conjugate was synthesized consisting of a portion of the C-terminal cytoplasmic tail region of V7 (a leukocyte surface protein) having the sequence KLSTLRSNT (SEQ ID NO: 22; Ruegg et al., 1995) with 7 arginine residues attached to their C terminus, in accordance with standard methods, using a peptide synthesizer (Applied Biosystems Model 433). The conjugate was added (final concentration = 10 μM) to T cells which had been isolated by standard methods and incubated at 37 ° C for several hours until the entire P1700 / 99MX night. The cells were lysed using detergents (1% Triton X-100). The DNA was removed and the soluble fraction (containing protein) was subjected to immunoprecipitation with an anti-V7 murine monoclonal antibody in combination with goat anti-mouse IgG. RAF-I is a kinase that associates with V7 and is inactivated by association with V7. In the absence of treatment with the peptide, the RAF-1 protein co-precipitated with V7. In cells treated with peptide, the RAF-1 protein was removed from the immunocomplex or V7. The same peptides were unable to disturb or alter a complex consisting of RAF-1 and p21 Ras, eliminating the non-specific modification of RAF-1 by the V7 peptide. In a second study, the V7 peptide portion of the V7-poly-arginine conjugate was subjected to phosphorylation, i.e., phosphorylated, in vitro, using protein C kinase. The anti-RAF-1 precipitates of the T cells that are had exposed to phosphorylated V7 tail peptides but not V7 non-phosphorylated tail peptide, demonstrated a potent inhibition of RAF kinase activity. While the invention has been described with reference to specific methods and modalities, it will be appreciated that various modifications and changes may be made without departing from the spirit of the invention.

P1700 / 99MX

Claims (55)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property; A conjugate comprising a biologically active agent that covalently binds to a carrier having a non-peptide backbone and comprises sufficient guanidino or amidino side chain entities to increase the delivery of the conjugate through a biological membrane , compared to the delivery of the biologically active agent in unconjugated form.
2. 2. The conjugate according to claim 1, wherein the vehicle comprises from 5 to 25 guanidino or amidino side chain entities. The conjugate according to claim 2, wherein the vehicle includes between 6 and 20 contiguous guanidino sidechain entities. The conjugate according to claim 1, wherein the carrier comprises at least 6 contiguous guanidino and / or amidino side chain entities. The conjugate according to claim 1, wherein the carrier comprises from 6 to 25 subunits, each of which comprises a side chain, wherein at least 50% of the side chains contain a guanidino or amidino entity. P1700 / 99MX 6. The conjugate according to claim 1, wherein the delivery rate of the conjugate is increased through the biological membrane. The conjugate according to claim 1, wherein the amount of the conjugate is increased through the biological membrane. The conjugate according to claim 1, wherein the delivery of the conjugate through the biological membrane is significantly greater than that of the biologically active agent conjugated with a basic HIV tat peptide consisting of LOS residues 49-57. The conjugate according to claim 8, wherein the delivery of the conjugate through the biological membrane is at least about 2 times greater than that of the biologically active agent conjugated with the tat HIV basic peptide. The conjugate according to claim 9, wherein the rate of transport of the conjugate through the biological membrane is at least about 6 times greater than that of the biologically active agent conjugated with the tat HIV basic peptide. The conjugate according to claim 1, wherein the non-peptide backbone comprises atoms selected from the group consisting of carbon, oxygen, sulfur, phosphorus and nitrogen. 12. The conjugate according to claim 1, P1700 / 99MX wherein the non-peptide backbone is selected from the group consisting of alkyl entities linked by thioethers, sulfonyl groups, carbamate groups, polyethylene imines or amino aldehydes, esters of hydroxy acids and aza analogs in which the alpha carbon is it replaces with nitrogen. The conjugate according to claim 12, wherein the non-peptide backbone is selected from the group consisting of an N-substituted amide, an ester, a keto-methylene, a reduced or methyleneamino, a thioamide, a phosphinate , a phosphonated idate, a phosphonamidate ester, a retropeptide, a trans-alkene, a fluoroalkene, a dimethylene, a thioether, a hydroxyethylene, a methyleneoxy, a tetrazole, a retrothioamide, a sulfonamido, a methylenesulfonamido, a retrosulfonamide and a peptoid . The conjugate according to claim 12, wherein the non-peptide backbone comprises subunits of malonate and / or gem-diaminoalkyl. The conjugate according to claim 1, wherein the guanidino or amidino side chain entities are joined to the backbone by a side chain linker comprising at least 2 linker chain atoms. 16. The conjugate according to claim 15, wherein the side chain linker comprises P1700 / 99MX 2 to 5 linker chain atoms. The conjugate according to claim 15, wherein the side chain linker comprises atoms selected from the group consisting of carbon, oxygen, sulfur, phosphorus and nitrogen. 18. The conjugate according to claim 1, wherein the biologically active agent is a small organic molecule. 19. The conjugate according to claim 1, wherein the biologically active agent is linked to at least two vehicles. The conjugate according to claim 1, wherein at least two biologically active agents are attached to the vehicle. 21. A method for increasing the transport of a selected compound, through a biological membrane, the method comprises: contacting a biological membrane with a conjugate according to claim 1, whereby the contact is effective to increase the supply of the conjugate through the biological membrane, compared to the delivery of the biologically active agent in unconjugated form. 22. The method according to claim 21, wherein the biological membrane is a eukaryotic cell membrane. 23. The method according to claim 21, in P1700 / 99MX where the biological membrane is a prokaryotic cell membrane. 24. The method according to claim 21, wherein the contact is made in vi tro. 25. The method according to claim 21, wherein the vehicle comprises from 6 to 25 guanidino or amidino side chain entities and includes at least 6 contiguous guanidino and / or amidino side chain entities. 26. The method according to claim 21, wherein the vehicle includes between 7 and 20 contiguous guanidino sidechain entities. The method according to claim 21, wherein the transmembrane transport rate of the conjugate is significantly greater than the transport rate of the biologically active agent conjugated to a tat HIV basic peptide consisting of residues 49-57. The method according to claim 27, wherein the transport speed of the conjugate is at least about 2 times higher than the transport rate of the biologically active agent conjugated to the tat HIV basic peptide. 29. The method according to claim 28, wherein the transport speed of the conjugate is at least about 6 times greater than the transport rate of the biologically active agent conjugated to the tat HIV basic peptide. P1700 / 99MX 30. A conjugate comprising a biologically active agent that is covalently linked by a linker cleavable to a carrier comprising sufficient guanidino or amidino side chain entities to increase the delivery of the conjugate through the biological membrane, in comparison with the supply of the biologically active agent in unconjugated form, wherein the binder is cleaved in vi. 31. The conjugate according to claim 30, wherein the vehicle comprises from 6 to 25 guanidino or amidino side chain entities. 32. The conjugate according to claim 30, wherein the carrier comprises at least 6 contiguous guanidino and / or amidino side chain entities. The conjugate according to claim 31, wherein the vehicle includes between 7 and 20 contiguous guanidino sidechain entities. 34. The conjugate according to claim 30, wherein the carrier comprises D-arginine or L-arginine residues. 35. The conjugate according to claim 30, wherein the cleavable linker is enzymatically cleavable. 36. The conjugate according to claim 30, wherein the cleavable binder is chemically cleavable. P1700 / 99MX 37. The conjugate according to claim 30, wherein the cleavable binder is photocleavable. 38. The conjugate according to claim 30, wherein the cleavable binder comprises an ester. 39. The conjugate according to claim 30, wherein the cleavable binder comprises a disulfide bond. 40. The conjugate according to claim 30, wherein the cleavable linker contains a first cleavable group that is distant from the agent and a second cleavable group that is close to the agent, whereby the cleavage of the first cleavable group produces a linker-agent conjugate. which contains a nucleophilic entity that reacts intramolecularly to cleave the second cleavable group, thereby releasing the binder agent and the vehicle. 41. The conjugate according to claim 30, wherein the delivery of the conjugate through the biological membrane is significantly greater than that of the biologically active agent conjugated with a tat HIV basic peptide consisting of residues 49-57. 42. The conjugate according to claim 41, wherein the delivery of the conjugate through the biological membrane is at least about 2 times greater than that of the biologically active agent conjugated with the tat HIV basic peptide. P1700 / 99MX 43. The conjugate according to claim 42, wherein the delivery of the conjugate through the biological membrane is at least about 6 times greater than that of the biologically active agent conjugated with the tat HIV basic peptide. 44. A method to increase the transport of a selected compound, through a biological membrane, the method comprises: contacting a biological membrane with a conjugate according to claim 30, whereby the contact is effective to increase the delivery of the conjugate through the biological membrane, in comparison with the supply of the biologically active agent in unconjugated form. 45. The method according to claim 44, wherein the cleavable binder is cleaved to release the free biologically active agent after passing through the biological membrane. 46. The method according to claim 45, wherein the cleavable binder is an enzymatically cleavable binder and the free biologically active agent is released by contacting the conjugate with an enzyme that cleaves the binder. 47. The method according to claim 45, wherein the cleavable binder is a photocleavable binder and the free biologically active agent is released by contacting the conjugate with P1700 / 99 X light. 48. The method according to claim 45, wherein the binder is a chemically cleavable binder. 49. The method according to claim 48, wherein the cleavable binder comprises a disulfide bond and cleavage of the binder is effected by reduction or disulfide exchange. 50. The method according to claim 45, wherein the vehicle comprises from 6 to 25 guanidino or amidino side chain entities. 51. The method according to claim 45, wherein the vehicle comprises at least 6 contiguous guanidino and / or amidino side chain entities. 52. The method according to claim 45, wherein the vehicle includes between 7 and 20 contiguous guanidino sidechain entities. 53. The method according to claim 45, wherein the delivery of the conjugate through the biological membrane is significantly greater than the transport rate of the biologically active agent conjugated with a tat HIV basic peptide consisting of residues 49-57. 54. The method according to claim 53, wherein the delivery of the conjugate through the biological membrane is at least about 2 times greater than that of the biologically active agent conjugated with the tat HIV basic peptide. P1700 / 99MX 55. The method according to claim 54, wherein the delivery of the conjugate through the biological membrane is at least about 6 times greater than that of the biologically active agent conjugated with the tat HIV basic peptide. P1700 / 99MX