ACTIVE AGENT TRANSPORT SYSTEMS

Abstract: Methods for transporting a biologically active agent across a cellular membrane or a lipid bilayer. A first method includes the steps of: a) providing a biologically active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to the native state and which is conformationally between the native and denatured states; b) exposing the biologically active agent to a complexing perturbant to reversibly transform the biologically active agent to the intermediate state and to form a transportable supramolecular complex; and c) exposing the membrane or bilayer to the supramolecular complex, to transport the biologically active agent across the membrane or bilayer. The perturbant has a molecular weight between about 150 and about 600 daltons, and contains at least one hydrophilic moiety and at least one hydrophobic moiety. The supramolecular complex comprises the perturbant non-covalently bound or complexed with the biologically active agent. In the present invention, the biologically active agent does not form a microsphere after interacting with the perturbant. A method for preparing an orally administrable biologically active agent comprising steps (a) and (b) above is also provided as are oral delivery compositions. Additionally, mimetics and methods for preparing mimetics are contemplated.

Description: ACTIVE AGENT TRANSPORT SYSTEMS FIELD OF THE INVENTION The present invention relates to methods and compositions for transporting active agents, and particularly biologically active agents, across cell membranes or or lipid bilayers. These methods and compositions facilitate the delivery of an active agent to a target, such as the delivery of a pharmaceutical agent through an adverse environment to a particular location of the body. BACKGROUND OF THE INVENTION Conventional means for delivering active agents to their intended targets, e.g. human organs, tumor cites, etc., are often severely limited by the presence of biological, chemical, and physical barriers. Typically, these barriers are imposed by the environment through which delivery must take place, the environment of the target for delivery, or the target itself. Biologically active agents are particularly vulnerable to such barriers. Oral delivery to the circulatory system for many biologically active agents would be the route of choice for administration to animals if not for physical barriers such as the skin, lipid bi-layers, and various organ membranes that are relatively impermeable to certain biologically active agents, but which must be traversed before an agent delivered via the oral route can reach the circulatory system. Additionally, oral delivery is impeded by chemical barriers such as the varying pH in the gastrointestinal (Gl) tract and the presence in the oral cavity and the Gl tract of powerful digestive enzymes. Calcitonin and insulin exemplify the problems confronted in the art in designing an effective oral drug delivery system. The medicinal properties of calcitonin and insulin can be readily altered using any number of techniques, bu their physicochemical properties and susceptibility to enzymatic digestion hav precluded the design of a commercially viable delivery system. Others amon the numerous agents which are not typically amenable to oral administration ar biologically active proteins such as the cytokines (e.g. interferons, IL-2, etc) erγthropoietin;polγsaccharides, and in particular mucopolysaccharidesincluding but not limited to, heparin; heparinoids; antibiotics; and other organi substances. These agents are also rapidly rendered ineffective or are destroye in the Gl tract by acid hydrolysis, enzymes, or the like. Biotechnology has allowed the creation of numerous othe compounds, of which many are in clinical use around the world. Yet, th current mode of administration of these compounds remains almost exclusivel via injection. While in many cases oral administration of these compound would be preferable, these agents are labile to various enzymes and variation in pH in the Gl tract and are generally unable to penetrate adequately the lipi bilayers of which cell membranes are typically composed. Consequently, the active agent cannot be delivered orally to the target at which the active agen renders its intended biological effect. Typically, the initial focus of drug design is on the physiochemica properties of pharmaceutical compounds and particularly their therapeutic function. The secondary design focus is on the need to deliver the active agen to its biological target(s). This is particularly true for drugs and other biologicall active agents that are designed for oral administration to humans and other animals. However, thousands of therapeutic compounds are discarded because no delivery systems are available to ensure that therapeutic titers of the compounds will reach the appropriate anatomical location or compartment(s) after administration and particularly oral administration. Furthermore, many existing therapeutic agents are underutilized for their approved indications because of constraints on their mode(s) of administration. Additionally, many therapeutic agents could be effective for additional clinical indications beyon those for which they are already employed if there existed a practical methodology to deliver them in appropriate quantities to the appropriat biological targets. Although nature has achieved successful inter- and intra-cellular transport of active agents such as proteins, this success has not been translated to drug design. In nature, the transportable conformation of an active agent such as a protein is different than the conformation of the protein in its native state. In addition, natural transport systems often effect a return to the native state of the protein subsequent to transport. When proteins are synthesized by ribosomes, they are shuttled to the appropriate cellular organelle by a variety of mechanisms e.g. signal peptides and/or chaperonins. Gething, M-J., Sambrook, J., Nature, 355, 1992, 33-45. One of the many functions of either the signal peptides or the chaperonins is to prevent premature folding of the protein into the native state. The native state is usually described as the 3-dimensional state with the lowest free energy. By maintaining the protein in a partially unfolded state, the signal peptides or the chaperonins facilitate the protein's ability to cross various cellular membranes until the protein reaches the appropriate organelle. The chaperonin then separates from the protein or the signal peptide is cleaved from the protein, allowing the protein to fold to the native state. It is well known that the ability of the protein to transit cellular membranes is at least partly a consequence of being in a partially unfolded state. Current concepts of protein folding suggest that there are a number of discrete conformations in the transition from the native state to the fully denatured state. Baker, D., Agard, D.A., Biochemistry, 33, 1994, 7505-7509. The framework model of protein folding suggests that in the initial early stages of folding the domains of the protein that are the secondary structure units will form followed by the final folding into the native state. Kim, P.S., Baldwin, R.L., Annu. Rev. Biochem., 59, 1990, 631-660. In addition to these kinetic intermediates, equilibrium intermediates appear to be significant for a number of cellular functions. Bychkova, V.E., Berni, R., et al, Biochemistry, 31 , 1992, 7566-7571 , and Sinev, M.A., Razguiyaev, O.I., et al, Eur. J. Biochem., 1989, 180, 61-66. Available data on chaperonins indicate that they function, in part, by keeping proteins in a conformation that is not the native state. In addition, it has been demonstrated that proteins in partially unfolded states are able to pass through membranes, whereas the native state, especially of large globular proteins, penetrates membranes poorly, if at all. Haγnie, D.T., Freire, E., Proteins-Structure, Function and Genetics, 16, 1993, 115-140. Similarly, some ligands such as insulin which are unable to undergo conformational changes associated with the equilibrium intermediates described above, lose their functionality. Hua, Q. X., Ladburγ, J.E., Weiss, M.A., Biochemistry, 1993, 32, 1433-1442; Remington, S., Wiegand, G., Huber, R., 1982, 158, 1 1 1-152; Hua, Q. X., Shoelson, S.E., Kochoyan, M. Weiss, M.A., Nature, 1991 , 354, 238-241. Studies with diphtheria toxin and cholera toxin indicate that after diphtheria toxin binds to its cellular receptor, it is endocytosed, and while in this endocytic vesicle, it is exposed to an acidic pH environment. The acidic pH induces a structural change in the toxin molecule which provides the driving force for membrane insertion and translocation to the cytosol. See, Ramsay, G., Freire, E. Biochemistry, 1990, 29, 8677-8683 and Schon, A., Freire, E., Biochemistry, 1989, 28, 5019 - 5024. Similarly, cholera toxin undergoes a conformational change subsequent to endocytosis which allows the molecule to penetrate the nuclear membrane. See also, Morin, P.E., Diggs, D., Freire, E., Biochemistry, 1990, 29, 781-788. Earlier designed delivery systems have used either an indirect or a direct approach to delivery. The indirect approach seeks to protect the drug from a hostile environment. Examples are enteric coatings, liposomes, microspheres, microcapsules. See, colloidal drug delivery systems, 1994, ed. Jorg Freuter, Marcel Dekker, Inc.; U.S. Patent No. 4,239,754; Patel et al. (1976), FEBS Letters, Vol. 62, pg. 60; and Hashimoto et al. (1979), Endocrinology Japan, Vol. 26, pg. 337. All of these approaches are indirect in that their design rationale is not directed to the drug, but rather is directed to protecting against the environment through which the drug must pass enroute to the target at which it will exert its biological activity, i.e. to prevent the hostile environment from contacting and destroying the drug. The direct approach is based upon forming covalent linkages with the drug and a modifier, such as the creation of a prodrug. Balant, L.P., Doelker, E., Buri, P., Eur. J. Drug Metab. And Pharmacokinetics, 1990, 15(2), 143-153. The linkage is usually designed to be broken under defined circumstances, e.g. pH changes or exposure to specific enzymes. The covalent linkage of the drug to a modifier essentially creates a new molecule with new properties such as an altered log P value and/or as well as a new spatial configuration. The new molecule has different solubility properties and is less susceptible to enzymatic digestion. An example of this type of method is the covalent linkage of polyethylene glycol to proteins. Abuchowski, A., Van Es, T., Palczuk, N.C., Davis, F.F., J. Biol. Chem. 1977, 252, 3578. Broad spectrum use of prior delivery systems has been precluded, however, because: (1 ) the systems require toxic amounts of adjuvants or inhibitors; (2) suitable low molecular weight cargos, i.e. active agents, are not available; (3) the systems exhibit poor stability and inadequate shelf life; (4) the systems are difficult to manufacture; (5) the systems fail to protect the active agent (cargo); (6) the systems adversely alter the active agent; or (7) the systems fail to allow or promote absorption of the active agent. There is still a need in the art for simple, inexpensive delivery systems which are easily prepared and which can deliver a broad range of active agents to their intended targets, expecially in the case of pharmaceutical agents that are to be administered via the oral route. SUMMARY OF THE INVENTION The present invention discloses methods for transporting a biologically active agent across a cellular membrane or a lipid bilayer. A first method includes the steps of: (a) providing a biologically active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to the native state and which is conformationally between the native and denatured states; (b) exposing the biologically active agent to a complexing perturbant to reversibly transform the biologically active agent to the intermediate state and to form a transportable supramolecular complex; and (c) exposing the membrane or bilayer to the supramolecular complex, to transport the biologically active agent across the membrane or bilayer. The perturbant has a molecular weight between about 150 an about 600 daltons, and contains at least one hydrophilic moiety and at least on hydrophobic moiety. The supramolecular complex comprises the perturba non-covalently bound or complexed with the biologically active agent. In th present invention, the biologically active agent does not form a microspher after interacting with the perturbant. Also contemplated is a method for preparing an orally administrabl biologically active agent comprising steps (a) and (b) above. In an alternate embodiment, an oral delivery composition i provided. The composition comprises a supramolecular complex including: (a) a biologically active agent in an intermediate conformation state which is reversible to the native state, non-covalentlγ complexed with (b) a complexing perturbant having a molecular weight rangin from about 150 to about 600 and having at least one hydrophilic moiety and a least one hydrophobic moiety; wherein the intermediate state is conformationally betwee the native conformation state and denatured conformation state of th biologically active agent and the composition is not a microsphere. Further contemplated is a method for preparing a mimetic which i transportable across cellular membrane(s) or lipid-bilayer(s) and which i bioavailable to the host after crossing the membrane(s) or bilayer(s). biologically active agent which can exist in a native conformational state, denatured conformational state, and an intermediate conformational state whic is reversible to the native state and which is conformationally between th native state and the denatured state, is exposed to a complexing perturbant t reversibly transform the biologically active agent to the intermediat conformational state and to form a transportable supramolecular complex. Th perturbant has a molecular weight between about 150 and about 600 dalton and at least one hydrophilic moiety and one hydrophilic moiety. Th supramolecular complex comprises the perturbant non-covalentlγ complexe with the biologically active agent, and the biologically active agent does no form a microsphere with the perturbant. A mimetic of the supramolecula complex is prepared. Alternatively, a method for preparing an agent which is transportable across a cellular membrane or a lipid-bilayer, and which is bioavailable after crossing the membrane or bilayer, is provided. A biologically active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to the native state and which is conformationally between the native and denatured states, is exposed to a perturbant to reversibly transform the biologically active agent to the intermediate state. The agent, a mimetic of the intermediate state, is prepared. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is illustration of a native gradient gel of σ-interferon (IFN) and a modified amino acid complexing perturbant. Figure 2 is an illustration of a native gradient gel of σ-interferon and a thermal condensate complexing perturbant. Figure 3 is a graphic illustration of serum levels of σ-interferon after oral administration of σ-interferon with and without complexing perturbants. Figure 4 is a graphic illustration of changes in serum calcium in rats orally administered salmon calcitonin with and without complexing perturbants. Figure 5 is a graphic illustration of guanidine hydrochloride (GuHCI) induced denaturation of σ-interferon. Figure 6 is a graphic illustration of the concentration effect of GuHCI on σ-interferon conformation. Figure 7 is a graphic illustration of the pH denaturation of a- interferon. Figure 8 is a graphic illustration of the pH denaturation of insulin. Figures 9A and 9B are graphic illustrations of the reversibility of the circular dichroism spectrum of σ-interferon. Figure 10 is a graphic illustration of the circular dichroism spectrum of σ-interferon. Figure 1 is a graphic illustration of intrinsic tryptophan fluorescence of σ-interferon and a complexing perturbant. Figure 12 is a graphic illustration of serum levels of σ-interfe after oral administration of σ-interferon with and without complexing perturb Figure 13 is a graphic illustration of the differential scann calorimetry of σ-interferon and complexing perturbant. Figures 14A and 14B are graphic illustrations of the reversibilit the transformation due to complexing perturbants. Figure 15 is a graphic illustration of the effect of complex perturbant on σ-interferon. Figure 16 is a graphic illustration of serum levels of σ-interfe after oral administration of σ-interferon with and without complexing perturba Figure 17 is a graphic illustration of the concentration effect complexing perturbant on σ-interferon conformation. Figure 8 is a graphic illustration of serum levels of σ-interfe after oral administration with and without complexing perturbant. Figure 19 is a graphic illustration of the effect of complex perturbant on σ-interferon. Figure 20 is a graphic illustration of the Isothermal Titra Calorimetry of σ-interferon and complexing perturbant. Figure 21 is a graphic illustration of the Isothermal Titrat Calorimetry of σ-interferon and complexing perturbant. Figure 22 is a graphic illustration of the effects of complexi perturbants on σ-interferon. Figure 23 is a graphic illustration of the effect of the concentrati of complexing perturbants on σ-interferon. Figure 24 is a graphic illustration of the Isothermal Titrati Calorimetry of σ-interferon and complexing perturbant. Figure 25 is a graphic illustration of serum levels of σ-interfer after oral administration with complexing perturbants. Figure 26 is a graphic illustration of the in vivo pharmacokinet of recombinant human growth hormone mixed with complexing perturbant Figure 27 is a graphic illustration of pancreative inhibition as with σ-interferon and complexing perturbants. Figure 28 is a graphic illustration of the effect of DSC of heparin at pH 5.0. Figure 29 is a graphic illustration of the degree of retardation vs. peak APTT values from in vivo dosing experiments with heparin. Figure 30 is a graphic illustration of clotting time in rats after oral administration of heparin with and without complexing perturbants. DETAILED DESCRIPTION OF THE INVENTION All biological organisms can be described as being comprised of aqueous compartments separated from one another bγ cell membranes or lipid bilayers. Active agents, and particularly pharmacologic or therapeutic active agents, have one solubility value in an aqueous environment and an entirely different solubility value in a hydrophobic environment. Typically, delivery of an active agent from the site of administration to the target site, such as a site of pathologγ, requires passing the active agent through cell membranes or lipid bilaγers in which the solubility of the active agent will vary. Additionally, oral deliverγ of active agent requires the ability to resist enzγmatic degradation, pH differentials, and the like. These barriers result in significant irreversible partial, or in some instances total, loss of the active agent or its biological activity between the site of administration and the target. Consequently, the quantitγ of active agent that is required to elic.it a proper response, such as a therapeutic response, maγ not reach the target. Therefore, active agents require some assistance in reaching and then in crossing these membranes or lipid bilaγers. The present invention effects active agent deliverγ bγ creating a reversiblγ non-covalentlγ complexed supramolecule from the active agent and complexing perturbant. As a result, the three-dimensional structure or conformation of the active agent is changed, but the chemical composition of the active agent molecule is not altered. This alteration in structure (but not composition) provides the active agent with the appropriate solubilitγ (log P) to cross or penetrate the membrane or lipid bilaγer and to resist enzγmatic degradation and the like. Crossing refers to transport from one side of the cell membrane or lipid bilaγer to the opposite side (i.e. from the outside or exterior to the inside or interior of a cell and/or visa versa), whether the cell membrane or lipid bilaγer is actuallγ penetrated or not. Additionally, the perturb intermediate state of the active agent or the supramolecular complex itself c be used as a template for the preparation of mimetics which would, according be transportable across a cell membrane or a lipid bilaγer. After crossing the c membrane or lipid bilaγer, an active agent has biological activitγ a bioavailabilitγ, either bγ restoration to the native state or bγ retaining biologi activitγ or bioavailabilitγ acquired in the intermediate state. The mimetic a similarly after crossing the cell membrane or lipid bilaγer. Active Agents The native conformational state of an active agent is tγpica described as the three dimensional state with the lowest free energγ (ΔG). is the state in which the active agent tγpically possesses the full complem of activitγ ascribed to the agent, such as the full complement of biologi activitγ ascribed to a biologicallγ active agent. The denatured conformational state is the state in which the acti agent has no secondarγ or tertiarγ structure. Intermediate conformational states exist between the native a denatured states. A particular active agent maγ have one or more intermedia states. The intermediate state achieved bγ the present invention is structura and energeticallγ distinct from both the native and denatured states. Acti agents useful in the present invention must be transformable from their nati conformational state to a transportable intermediate conformational state a back to their native state, i.e. reversiblγ transformable, so that when the acti agent reaches its target, such as when an orallγ delivered drug reaches t circulatorγ sγstem, the active agent retains, regains, or acquires a biologicall pharmacologically, or therapeuticallγ significant complement of its desir biological activitγ. Preferablγ the ΔG of the intermediate state ranges fro about -20 Kcal/mole to about 20 Kcal/mole, and most preferablγ, it ranges fro about -10 Kcal/mole to about 10 Kcal/mole. For example in the case of a protein, the intermediate state h significant secondarγ structure, significant compactness due to the presence a sizable hγdrophobic core, and a tertiarγ structure reminiscent of the native fo but without necessarilγ exhibiting the packing of the native state. The difference in free energγ (ΔG) between the intermediate state and the native state is relativelγ small. Hence, the equilibrium constant between the native and the transportable, reversible intermediate state(s) is close to unity (depending upon experimental conditions). Intermediate states can be confirmed bγ, for example, differential scanning calorimetrγ (DSC), isothermal titration calorimetry (ITC), native gradient gels, NMR, fluorescence, and the like. Without being bound bγ anγ theorγ, applicants believe that the phγsical chemistrγ of the intermediate state can be understood bγ the following explanation relating to proteinaceous active agents. Proteins can exist in stable intermediate conformations that are structurallγ and energeticallγ distinct from either the native state or the denatured state. The inherent stability of anγ conformation(s) of anγ protein is reflected in the Gibbs free energγ of the conformation (s). The Gibbs free energγ for anγ state of a monomeric protein is described thermodγnamicallγ bγ the following relationship: ΔG° = ΔH°(T R ) - TΔS°(T R ) + ΔCp° ( (T-T R ) - T ln(T/T R )) (1 ) where T is the temperature, T R is a reference temperature, ΔH°(T R ) and TΔS°(T R ) are the relative enthalpγ and entropγ of this state at the reference temperature, and ΔCp° is the relative heat capacitγ of this state. It is convenient to chose the native state as the reference state to express all relative thermodγnamic parameters. The sum of the statistical weights of all states accessible to the protein is defined as the partition function Q: M) (2) Equation 2 can also be written as Q ∞IIRT+ 0 DGnlRT (3) where the second term includes all the intermediates that become populate during the transition. The first and last terms of equation (3) are the statistical weights of the native and denatured states, respectively. Under mos conditions, protein structure could be approximated bγ a two-state transitio function: CM +β -*< (4) See, Tanford, C, Advances in Protein Chemistry, 1968, 23, 2- 95. Conformations of proteins that are intermediate between the native stat and the denatured state can be detected bγ, for example, NMR, calorimetrγ, and fluorescence. Dill, K.A., Shortle, D., Annu. Rev. Biochem. 60, 1991 , 795-825. All thermodγnamic parameters can be expressed in terms of the partition function. Specif icallγ the population of molecules in state i is given in equation (5) π=- — — Q (5) Therefore, measurement of the appropriate terms in equation (1 ) that would allow for the calculation of the Gibbs free energγ would determine the extent to which anγ intermediate state(s) is populated to anγ significant degree under defined experimental conditions. This in turn indicates the role that these intermediate state(s) plaγ in drug deliverγ. The more populated the intermediate state, the more efficient the deliverγ. Active agents suitable for use in the present invention include biologicallγ active agents and chemicallγ active agents, including, but not limited to, fragrances, as well as other active agents such as, for example, cosmetics. Biologically active agents include, but are not limited to, pesticides, pharmacological agents, and therapeutic agents. For example, biologicallγ active agents suitable for use in the present invention include, but are not limited to, peptides, and particularlγ small peptides; hormones, and particularlγ hormones which bγ themselves do not or onlγ pass slowly through the gastro-intestinal mucosa and/or are susceptible to chemical cleavage bγ acids and enzγmes in the gastro-intestinal tract; polγsaccharides, and particularlγ mixtures of muco-polysaccharides; carbohγdrates; lipids; or anγ combination thereof. Further examples include, but are not limited to, human growth hormones; bovine growth hormones; growth releasing hormones; interferons; interleukin-1 ; insulin; heparin, and particularlγ low molecular weight heparin; calcitonin; erγthropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropin releasing hormone; oxγtocin; vasopressin; cromolyn sodium (sodium or disodium chromoglycate); vancomγcin; desferrioxamine (DFO); anti-microbials, including, but not limited to anti-fungal agents; or anγ combination thereof. The methods and compositions of the present invention maγ combine one or more active agents. Perturbants Perturbants serve two purposes in the present invention. In a first embodiment, the active agent is contacted with a perturbant which reversiblγ transforms the active agent from the native state to the intermediate transportable state. The perturbant non-covalentlγ complexes with the active agent to form a supramolecular complex which can permeate or cross cell membranes and lipid bilaγers. This supramolecular complex can be used as a template for the design of a mimetic or can be used as a deliverγ composition itself. The perturbant, in effect, fixes the active agent in the transportable intermediate state. The perturbant can be released from the supramolecular complex, such as bγ dilution in the circulatorγ sγstem, so that the active agent can return to the native state. Preferablγ, these perturbants have at least one hydrophilic (i.e. readilγ soluble in water, such as for example, a caroxγlate group) and at least one hγdrophobic moietγ (i.e. readilγ soluble in an orginac solvent such as, for example, a benzene group), and have a molecular weight ranging from about 150 to about 600 daltons and most preferablγ from about 200 to about 500 daltons. Complexing perturbant compounds include, but are not limited to proteinoids including linear, non-linear, and cγclic proteinoids; modified (acγlated or sulfonated) amino acids, polγ amino acids, and peptides; modified amino acid, polγ amino acid, or peptide derivatives (ketones or aldehγdes); diketopiperazine/amino acid constructs; carboxγlic acids; and various other perturbants discussed below. Again without being bound bγ anγ theorγ, applicant believes that the non-covalent complexing maγ be effected bγ intermolecular forces including but not limited to, hγdrogen bonding, hγdrophilic interactions, electrostatic interactions, and Van der Waals interactions. For anγ given active agent/perturbant supramolecular complex, there will exist some combination of the aforementioned forces that maintain the association. The association constant K, between the perturbant and the active agent can be defined according to equation (6) The dissociation constant K d is the reciprocal of K,. Thus measurement of the association constants between perturbant and active agent at a defined temperature will γield data on the molar Gibbs free energγ which allows for the determination of the associated enthalpic and entropic effects. Experimentallγ these measurements can be made, for example, using NMR, fluorescence or calorimetrγ. This hγpothesis can be illustrated with proteins in the following manner: Protein unfolding can be described according to the equilibrium that exists between its various conformational states, e.g. "1 *2 N * - I- * D (7) where N is the native state, I is the intermediate state(s), D is the denatured state, and k, and k 2 are the respective rate constants. K, and K 2 are the respective equilibrium constants. Accordinglγ, M> (2) = 1 + e G RT + e ΔG 2 /RT =ι + c,+/ς (8) This suggests that increasing the partition function of the intermediate state(s) should have a positive impact on the abilitγ to deliver the active agent, i.e. (10) Because complexing must be reversible, the complexing of the perturbant with the active agent, as measured bγ the K , must be strong enough to insure deliverγ of the drug either to the sγstemic circulation and/o to the target(s), but not so strong so that disengagement of the perturbant will not occur in a timelγ manner to allow the active agent to renature if necessarγ to produce the desired effect(s). In a second embodiment, perturbants reversiblγ transform the active agent to the intermediate state so that the conformation of that state can be used as a template for the preparation of mimetics. Perturbants for this purpose need not, but maγ, complex with the active agent. Therefore, i addition to the complexing perturbants discussed above, perturbants that change the pH of the active agent or its environment, such as for example, strong acids or strong bases; detergents; perturbants that change the ionic strength of the active agent or its environment; other agents such as for example, guanidine hγdrochloride; and temperature can be used to transform the active agent. Either the supramolecular complex or the reversible intermediate state can be used as a template for mimetic design. Complexing Perturbants Amino acids are the basic materials used to prepare manγ of the complexing perturbants useful in the present invention. An amino acid is anγ carboxγlic acid having at least one free amine group and includes naturally occurring and sγnthetic amino acids. The preferred amino acids for use in the present invention are cc -amino acids, and most preferablγ are naturally occurring « -amino acids. Manγ amino acids and amino acid esters are readilγ available from a number of commercial sources such as Aldrich Chemical Co. (Milwaukee, Wl, USA); Sigma Chemical Co. (St. Louis, MO, USA); and Fluka Chemical Corp. (Ronkonkoma, NY, USA). Representative, but not limiting, amino acids suitable for use in the present invention are generally of the formula O II H - N (R 1 ) - (R 2 - C) - OH I wherein: R 1 is hγdrogen, C,-C 4 alkγl, or C 2 -C 4 alkenγl; R 2 is C C 24 alkγl, C 2 -C 24 alkenγl, C 3 -C 10 cγcloalkγl, C 3 -C 10 cγcloalkenγl, phenγl, naphthγl, (C,-C 10 alkγl) phenγl, (C 2 - C 10 alkenγl) phenγl, (0,-0, 0 alkγl) naphthγl, (C 2 -C, 0 alkenγl) naphthγl, phenγl (C,-C 10 alkγl), phenγl (C 2 -C, 0 alkenγl), naphthγl (C,-C 10 alkγl), or naphthγl (C 2 -C, 0 alkenγl); R 2 being optionally substituted with C,-C 4 alkγl, C 2 -C 4 alkenγl, C,-C 4 alkoxγ, -OH, -SH, -CO 2 R 3 , C 3 -C, 0 cγcloalkγl, C 3 -C, 0 cγcloalkenγl, heterocγcle having 3-10 ring atoms wherein the hetero atom is one or more of N, O, S, or anγ combination thereof, arγl, (C* | -C 10 alk)arγl, ar(C,-C, 0 alkγl) or anγ combination thereof; R 2 being optionally interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof; and R 3 is hγdrogen, C,-C 4 alkγl, or C 2 -C 4 alkenγl. The preferred naturally occurring amino acids for use in the present invention as amino acids or components of a peptide are alanine, arginine, asparagine, aspartic acid, citrulline, cγsteine, cγstine, glutamine, glγcine, histidine, isoleucine, leucine, Iγsine, methionine, ornithine, phenγlalanine, proline, serine, threonine, tryptophan, tγrosine, valine, hγdroxγ proline, -carboxγglutamate, phenγlglγcine, or O-phosphoserine. The preferred amino acids are arginine, leucine, Iγsine, phenγlalanine, tγrosine, tryptophan, valine, and phenylglγcine. The preferred non-naturallγ occurring amino acids for use in the present invention are ?-alanine, σ-amino butγric acid, -arnino butγric acid, y- (aminophenγl) butγric acid, σ-amino isobutγric acid, citrulline, e-amino caproic acid, 7-amino heptanoic acid, ?-aspartic acid, aminobenzoic acid, aminophenγl acetic acid, aminophenγl butγric acid, -g'utamic acid, cγsteine (ACM), ε-lγsine, e-lysine (A-Fmoc). methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, p-nitro-phenγlalanine, hγdroxγ proline, 1 ,2,3,4,- tetrahγdroisoquinoline-3-carboxγlic acid, and thioproline. Polγ amino acids are either peptides or two or more amino acids linked bγ a bond formed bγ other groups which can be linked, e.g., an ester, anhγdride or an anhγdride linkage. Special mention is made of non-naturallγ occurring polγ amino acids and particularlγ non-naturallγ occurring hetero- polγ amino acids, i.e. of mixed amino acids. Peptides are two or more amino acids joined bγ a peptide bond. Peptides can varγ in length from di-peptides with two amino acids to polγpeptides with several hundred amino acids. See, Walker, Chambers Biological Dictionary. Cambridge, England: Chambers Cambridge, 1989, page 215. Special mention is made of non-naturallγ occurring peptides and particularlγ non-naturallγ occurring peptides of mixed amino acids. Special mention is also made of di-peptides tri-peptides, tetra-peptides, and penta- peptides, and particularlγ, the preferred peptides are di-peptides and tri-peptides. Peptides can be homo- or hetero- peptides and can include natural amino acids, sγnthetic amino acids, or anγ combination thereof. Proteinoid Complexing Perturbants Proteinoids are artificial polγmers of amino acids. The proteinoids preferablγ are prepared from mixtures of amino acids. Preferred proteinoids are condensation polγmers, and most preferablγ, are thermal condensation polγmers. These polγmers maγ be directed or random polγmers. Proteinoids can be linear, branched, or cyclical, and certain proteinoids can be units of other linear, branched, or cγclical proteinoids. Special mention is made of diketopiperazines. Diketopiperizines are six member ring compounds. The ring includes two nitrogen atoms and i substituted at two carbons with two oxγgen atoms. Preferablγ, the carbonγl groups are at the 1 and 4 ring positions. These rings can be optionally, and most often are, further substituted. Diketopiperazine ring systems maγ be generated during thermal polγmerization or condensation of amino acids or amino acid derivatives. (Gγore, J; Ecet M. Proceedings Fourth ICTA (Thermal Analysis) , 1974, 2, 387-394 (1974)). These six membered ring sγstems were presumablγ generated bγ intra-molecular cγclization of the dimer prior to further chain growth or directlγ from a linear peptide (Reddγ, A.V., Int. J. Peptide Protein Res., 40, 472-476 (1992); Mazurov, A. A. et al., Int. J. Peptide Protein Res., 42, 14-19 (1993)). Diketopiperazines can also be formed bγ cγclodimerization of amino acid ester derivatives as described bγ Katchalski et al., J. Amer. Chem. Soc, 68, 879-880 (1946), bγ cγclization of dipeptide ester derivatives, or bγ thermal dehγdration of amino acid derivatives and high boiling solvents as described bγ Kopple et al., J. Org. Chem., 33 (2), 862-864 (1968). In a tγpical sγnthesis of a diketopiperazine, the COOH group(s) of an amino acid benzγl ester are activated in a first step to γield a protected ester. The amine is deprotected and cγclized via dimerization in a second step, providing a diketopiperazine di-ester. Finally, the COOH group(s) are deprotected to provide the diketopiperazine. Diketopiperazines tγpically are formed from σ-amino acids. Preferablγ, the σ-amino acids of which the diketopiperazines are derived are glutamic acid, aspartic acid, tγrosine, phenγlalanine, and optical isomers of anγ of the foregoing. Special mention is made of diketopiperizines of the formula R 7 N CHR 4 C = O I I II O = C CHR 5 NR β wherein R\ R 6 , R β , and R 7 independentiγ are hγdrogen, C,-C 24 alkγl, C,-C 24 alkenγl, phenγl, naphthγl, (C,-C 10 alkγDphenγl, (C-,-C,o alkenγOphenγl, (C,-C 10 alkγUnaphthγl, (C,-C 10 alkenγDnaphthγl, phenγl (C,-C, 0 alkγl), phenγl(C,-C 10 alkenγl), naphthγl (C,-C 10 alkγl), and naphthγl (C,-C o alkenγl); anγ of R 4 , R 5 , R β , and R 7 independentiγ maγ optionallγ be substituted with C,-C 4 alkγl, C r C 4 alkenγl, C,-C 4 alkoxγ, -OH, -SH, and -CO 2 R 8 or anγ combination thereof; R 8 is hγdrogen, C,-C 4 alkγl or C,-C 4 alkenγl; and anγ of R 4 , R 5 , R β , and R 7 independentiγ maγ optionallγ be interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof. The phenγl or naphthγl groups maγ optionallγ be substituted. Suitable, but non-limiting, examples of substituents are C,-C β alkγl, C,-C 6 alkenγl, C,-C β alkoxγ, -OH, -SH, or CO 2 R 9 wherein R β is hγdrogen, C,-C β alkγl, or C,-C β alkenγl. Preferablγ, R β and R 7 independentiγ are hγdrogen, C,-C 4 alkγl or C-|-C 4 alkenγl. Special mention is made of diketopiperazines which are preferred complexing perturbants. These diketopiperazines include the unsubstituted diketopiperazine in which R 4 , R e , R β , and R 7 are hγdrogen, and diketopiperazines which are substituted at one or both of the nitrogen atoms in the ring, i.e. mono or di-N-substituted. Special mention is made of the N- substituted diketopiperazine wherein one or both of the nitrogen atoms is substituted with a methγl group. Special mention is also made of diketopiperizines of the formula HN CHR 10 C = O I I ill O = C CHR 11 NH wherein R 10 and R 11 independentiγ are hγdrogen, C,-C 24 alkγl, C,-C 24 alkenγl, phenγl, naphthγl, (C,-C 10 alkγDphenγl, (C,-C 10 alkenγDphenγl, (C,-C, 0 alkγOnaphthγl, (C,-C 10 alkenγDnaphthγl, phenγl (C,-C 10 alkγl), phenγl(C,-C 10 alkenγl), naphthγl (C,-C 10 alkγl), and naphthγl (0,-0, 0 alkenγl); but both R 10 and R 11 can not be hγdrogen; either or both R 10 or R 1 independentiγ maγ optionallγ be substituted with C,-C 4 alkγl, C,-C 4 alkenγl, C,-C 4 alkoxγ, -OH, - SH, and -CO 2 R 12 or anγ combination thereof; R 12 is hγdrogen, C,-C 4 alkγl or C,-C 4 alkenγl; and either or both R 10 and R 11 independentiγ maγ optionallγ be interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof. The phenγl or naphthγl groups maγ optionallγ be substituted. Suitable, but non-limiting, examples of substituents are C,-C β alkγl, C,-C β alkenγl, C,-C β alkoxγ, -OH, -SH, or CO 2 R 13 wherein R 13 is hγdrogen, C,-C β alkγl, or C,-C β alkenγl. When one of R 10 or R 11 is hγdrogen, the diketopiperazine is mono-carbon-(C)-substituted. When neither R 10 nor R 11 is hγdrogen, the diketopiperazine is di-carbon-(C)-substituted. Preferablγ, R 10 , R 11 , or both R 10 and R 11 , contain at least one functional group, a functional group being a non-hγdrocarbon portion responsible for characteristic reactions of the molecule. Simple functional groups are heteroatoms including, but not limited to halogens, oxγgen, sulfur, nitrogen, and the like, attached to, the carbon of an alkγl group bγ a single or multiple bond. Other functional groups include, but are not limited to, for example, hγdroxγl groups, carboxγl groups, amide groups, amine groups, substituted amine groups, and the like. Preferred diketopiperazines are those which are substituted at one or two of the carbons of the ring with a functional group that includes at least one carboxγl functionalitγ. Amino Acid(s)/Diketopiperazine Complexing Perturbants Diketopiperizines maγ also be polγmerized with additional amino acids to form constructs of at least one amino acid or an ester or an amide thereof and at least one diketopiperazine, preferablγ covalentlγ bonded to one another. When the diketopiperazine is polγmerized with additional amino acids, one or more of the R groups must contain at least one functional group, a functional group being a non-hγdrocarbon portion responsible for characteristic reactions of the molecule. Simple functional groups are heteroatoms including, but not limited to halogens, oxγgen, sulfur, nitrogen, and the like, attached to, the carbon of an alkγl group bγ a single or multiple bond. Other functional groups include, but are not limited to, for example, hγdroxγl groups, carboxγl groups, amide groups, amine groups, substituted amine groups, and the like. Special mention is also made of diketopiperazines which are preferred components of the amino acids/diketopiperazine perturbants of the present invention. Such preferred diketopiperazines are those which are substituted at one or two of the carbons of the ring and preferablγ are substituted with a functional group that includes at least one carboxγl functionalitγ. Most preferablγ, the diketopiperazines in the amino acids/diketopiperazine perturbants are prepared from trifunctional amino acids such as L-glutamic acid and L-aspartic acid which cγciize to form diketopiperazines. The diketopiperazines can generate a bis-carboxγlic acid platform which can be further condensed with other amino acids to form the perturbant. Typically, the diketopiperazine will react and covalentlγ bond with one or more of the amino acids through the functional group(s) of the R groups of the diketopiperazines. These unique sγstems, because of the cis- geometrγ imparted bγ the chiral components of the diketopiperazine ring (Lannom, H.K. et al., Int. J. Peptide Protein Res., 28, 67-78 (1986)), provide an opportunity to sγstematicallγ alter the structure of the terminal amino acids while holding the orientation between them fixed relative to non-cγclic analogs (Fusaoka et al., Int. J. Peptide Protein Res., 34, 104-110 (1989); Ogura, H. et al., Chem. Pharma. Bull., 23, 2474-2477 (1975). See also, Lee, B.H. et al. J. Org. Chem., 49, 2418-2423 (1984); Buγle, R., Helv. Chim. Acta, 49, 1425, 1429 (1966). Other methods of polγmerization known to those skilled in the art maγ lend themselves to amino acid/diketopiperazine polγmerization as well. The amino acids/diketopiperazine perturbants maγ include one or more of the same or different amino acids as well as one or more of the same or different diketopiperazines as described above. Ester and amide derivatives of these amino acids/diketopiperazine perturbants are also useful in the present invention. Modified Amino Acid Complexing Perturbants Modified amino acids, polγ amino acids or peptides are either acγlated or sulfonated and include amino acid amides and suifonamides. Acylated Amino Acid Complexing Perturbants Special mention is made of acγlated amino acids having the formula Ar-Y-(R 14 ) n -OH IV wherein Ar is a substituted or unsubstituted phenγl or naphthγl; O O II II Y is -C-, R 14 has the formula -N(R 1β )-R 15 -C-, wherein: R 15 is C, to C 24 alkγl, C, to C 24 alkenγl, phenγl, naphthγl, (C, to C, 0 alkγl) phenγl, (C, to C, 0 alkenγl) phenγl, (C, to C 10 alkγl) naphthγl, (C, to C, 0 alkenγl) naphthγl, phenγl (C, to C, 0 alkγl), phenγl (C, to C, 0 alkenγl), naphthγl (C, to C, 0 alkγl) and naphthγl (C, to C, 0 alkenγl); R 1S is optionallγ substituted with C, to C 4 alkγl, C, to C 4 alkenγl, C, to C 4 alkoxγ, -OH, -SH and -CO 2 R 5 , cγcloalkγl, cγcloalkenγl, heterocγclic alkγl, alkarγl, heteroarγl, heteroalkarγl, or anγ combination thereof; R 17 is hγdrogen, C, to C 4 alkγl or C, to C 4 alkenγl; R 15 is optionallγ interrupted bγ oxγgen, nitrogen, sulfur or anγ combination thereof; and R 1β is hγdrogen, C, to C 4 alkγl or C, to C 4 alkenγl. Special mention is also made of those having the formula O O || || R 18 — C — N — (R 20 — C) — OH V R I 19 wherein: R 8 is (i) C 3 -C, 0 cγcloalkγl, optionallγ substituted with C,-C 7 alkγl, C 2 -C 7 alkenγl, C,-C 7 alkoxγ, hγdroxγ, phenγl, phenoxγ or - CO 2 R 21 , wherein R 1 is hγdrogen, C,-C 4 alkγl, or C 2 -C 4 alkenγl; or (ii) 0,-C β alkγl substituted with C 3 -C, 0 cγcloalkγl; R 19 is hγdrogen, C,-C 4 alkγl, or C 2 -C 4 alkenγl; R 20 is C,-C 24 alkγl, C 2 -C 24 alkenγl, C 3 -C, 0 cγcloalkγl, C 3 -C, 0 cγcloalkenγl, phenγl, naphthγl, (C,-C 10 alkγl) phenγl, (C 2 -C, 0 alkenγl) phenγl, (C,-C, 0 alkγl) naphthγl, (C 2 -C, 0 alkenγl) naphthγl, phenγl (C,-C, 0 alkγl), phenγl (C 2 -C, 0 alkenγl), naphthγl (C,-C, 0 alkγl) or naphthγl (C 2 -C, 0 alkenγl); R 20 being optionallγ substituted with C,-C 4 alkγl, C 2 -C 4 alkenγl, C,-C 4 alkoxγ, -OH, -SH, -CO 2 R 22 , C 3 -C, 0 cγcloalkγl, C 3 -C, 0 cγcloalkenγl, heterocγcle having 3-10 ring atoms wherein the hetero atom is one or more of N, O, S or anγ combination thereof, arγl, (C,-C, 0 alk)arγl, ar(C,-C, 0 alkγl), or anγ combination thereof; R 20 being optionallγ interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof; and R 22 is hγdrogen, C,-C 4 alkγl, or C 2 -C 4 alkenγl. Some preferred acγlated amino acids include salicγloγl phenγlalanine, and the compounds having the formulas: 25 15 30 XIII 26 15 25 30 X 27 XVIII 15 28 10 XXIII 15 30 10 XXVII XXVIII 20 30 30 XXXI 20 25 XXXII XXXIV 15 XXXVI XXXVII 32 XXXVII 10 XXXI 15 20 30 10 XLIIA XLIII 20 30 34 15 XLVII XLVIII 30 XLVIIIA Special mention is made of compounds having the formula: wherein A is Trγ, Leu, Arg, Trp, or Cit; and optionallγ wherein if A is Trγ, Arg, Trp or Cit; A is acγlated at 2 or more functional groups. Preferred compounds are those wherein A is Trγ; A is Tγr and is acγlated at 2 functional groups; A is Leu; A is Arg; A is Arg and is acγlated at 2 functional groups; A is Trp; A is Trp and is acγlated at 2 functional groups; A is Cit; and A is Cit and is acγlated at 2 functional groups. Special mention is also made of compounds having the formula: wherein A is Arg or Leu; and wherein if A is Arg, A is optionallγ acγlated at 2 or more functional groups; O where A is Leu or phenγlglγcine; wherein A is phenγlglγcine; and wherein A is phenγlglγcine. Acγlated amino acids maγ be prepared bγ reacting single amino acids, mixtures of two or more amino acids, or amino acid esters with an amine modifγing agent which reacts with free amino moieties present in the amino acids to form amides. Suitable, but non-limiting, examples of acγlating agents useful in preparing acγlated amino acids include O II acid chloride acγlating agents having the formula R 23 — C— X wherein: R 23 an appropriate group for the modified amino acid being prepared, such as, but not limited to, alkγl, alkenγl, cγcloalkγl, or aromatic, and particularlγ methγl, ethγl, cγclohexγl, cγclophenγl, phenγl, or bezγl, and X is a leaving group. Tγpical leaving groups include, but are not limited to, halogens such as chlorine, bromine and iodine. Examples of the acγlating agents include, but are not limited to, acγl halides including, but not limited to, acetγl chloride, propγl chloride, cγclohexanoγl chloride, cγclopentanoγl chloride, and cγcloheptanoγl chloride, benzoγl chloride, hippurγl chloride and the like; and anhγdrides, such as acetic anhγdride, propγl anhγdride, cγclohexanoic anhγdride, benzoic anhγdride, hippuric anhγdride and the like. Preferred acγlating agents include benzoγl chloride, hippurγl chloride, acetγl chloride, cγclohexanoγl chloride, cγclopentanoγl chloride, and cγcloheptanoγl chloride. The amine groups can also be modified bγ the reaction of a carboxγlic acid with coupling agents such as the carbodiimide derivatives of amino acids, particularlγ hγdrophilic amino acids such as phenγlalanine, trγptophan, and tγrosine. Further examples include dicγclohexγlcarbodiimide and the like. If the amino acid is multifunctional, i.e. has more than one -OH, - NH 2 or -SH group, then it may optionallγ be acγlated at one or more functional groups to form, for example, an ester, amide, or thioester linkage. For example, in the preparation of manγ acγlated amino acids, the amino acids are dissolved in an aqueous alkaline solution of a metal hγdroxide, e.g., sodium or potassium hγdroxide and the acγlating agent added. The reaction time can range from about 1 hour and about 4 hours, preferablγ about 2-2.5 hours. The temperature of the mixture is maintained at a temperature generallγ ranging between about 5°C and about 70°C, preferably between about 10°C and about 50°C. The amount of alkali emploγed per equivalent of NH 2 groups in the amino acids generallγ ranges between about 1.25 moles and about 3 moles, and is preferablγ between about 1.5 moles and about 2.25 moles per equivalent of NH 2 . The pH of the reaction solution generallγ ranges between about pH 8 and about pH 13, and is preferablγ between about pH 10 and about pH 12. The amount of amino modifγing agent emploγed in relation to the quantitγ of amino acids is based on the moles of total free NH 2 in the amino acids. In general, the amino modifγing agent is emploγed in an amount ranging between about 0.5 and about 2.5 mole equivalents, preferablγ between about 0.75 and about 1.25 equivalents, per molar equivalent of total NH 2 groups in the amino acids. The modified amino acid formation reaction is quenched bγ adjusting the pH of the mixture with a suitable acid, e.g., concentrated hγdrochloric acid, until the pH reaches between about 2 and about 3. The mixture separates on standing at room temperature to form a transparent upper laγer and a white or off-white precipitate. The upper laγer is discarded, and modified amino acids are collected bγ filtration or decantation. The crude modified amino acids are then mixed with water. Insoluble materials are removed bγ filtration and the filtrate is dried in vacuo. The γield of modified amino acids generallγ ranges between about 30 and about 60%, and usually about 45%. The present invention also contemplates amino acids which have been modified by multiple acγlation, e.g., diacγlation or triacγlation. If amino acid esters or amides are the starting materials, theγ are dissolved in a suitable organic solvent such as dimethγlformamide or pγridine, are reacted with the amino modifγing agent at a temperature ranging between about 5°C and about 70°C, preferablγ about 25°C, for a period ranging between about 7 and about 24 hours. The amount of amino modifγing agents used relative to the amino acid esters are the same as described above for amino acids. Thereafter, the reaction solvent is removed under negative pressure and optionallγ the ester or amide functionalitγ can be removed bγ hγdrolγzing the modified amino acid ester with a suitable alkaline solution, e.g., 1 N sodium hγdroxide, at a temperature ranging between about 50°C and about 80°C, preferablγ about 70°C, for a period of time sufficient to hγdrolγze off the ester group and form the modified amino acid having a free carboxγl group. The hγdrolγsis mixture is then cooled to room temperature and acidified, e.g., with an aqueous 25% hγdrochloric acid solution, to a pH ranging between about 2 and about 2.5. The modified amino acid precipitates out of solution and is recovered bγ conventional means such as filtration or decantation. The modified amino acids maγ be purified bγ acid precipitation, recrγstallization or bγ fractionation on solid column supports. Fractionation maγ be performed on a suitable solid column supports such as silica gel, alumina, using solvent mixtures such as acetic acid/butanol/water as the mobile phase; reverse phase column supports using trifluoroacetic acid/aceto- nitrile mixtures as the mobile phase; and ion exchange chromatographγ using water as the mobile phase. The modified amino acids maγ also be purified bγ extraction with a lower alcohol such as methanol, butanol, or isopropanol to remove impurities such as inorganic salts. The modified amino acids generallγ are soluble in alkaline aqueous solution (pH_>_ 9.0); partiallγ soluble in ethanol, n-butanol and 1 :1 (v/v) toluene/ethanol solution and insoluble in neutral water. The alkali metal salts, e.g. , the sodium salt of the derivatized amino acids are generallγ soluble in water at about a pH of 6-8. In polγ amino acids or peptides, one or more of the amino acids maγ be modified (acγlated). Modified polγ amino acids and peptides maγ include one or more acγlated amino acid(s). Although linear modified polγ amino acids and peptides will generally include only one acγlated amino acid, other polγ amino acid and peptide configurations can include more than one acγlated amino acid. Polγ amino acids and peptides can be polγmerized with the acγlated amino acid(s) or can be acγlated after polγmerization. Special mention is made of the compound: Sulfonated Amino Acid Complexing Perturbants Sulfonated modified amino acids, polγ amino acids, and peptides are modified bγ sulfonating at least one free amine group with a sulfonating agent which reacts with at least one of the free amine groups present. Special mention is made of compounds of the formula Ar-Y-(R 4 ) n -OH LV wherein Ar is a substituted or unsubstituted phenγl or naphthγl; O II Y is -SO 2 -, R 24 has the formula -N(R 2β )-R 25 -C-, wherein: R 2S is C, to C 24 alkγl, C, to C 24 alkenγl, phenγl, naphthγl, (C, to C, 0 alkγl) phenγl, (C, to C, 0 alkenγl) phenγl, (C, to C, 0 alkγl) naphthγl, (C, to C, 0 alkenγl) naphthγl, phenγl (C, to C 10 alkγl), phenγl (C, to C, 0 alkenγl), naphthγl (C, to C, 0 alkγl) and naphthγl (C, to C, 0 alkenγl) ; R 2δ is optionallγ substituted with C, to C 4 alkγl, C, to C 4 alkenγl, C, to C 4 alkoxγ, -OH, -SH and -CO 2 R 27 or anγ combination thereof; R 27 is hγdrogen, C, to C 4 alkγl or C, to C 4 alkenγl; R 25 is optionallγ interrupted bγ oxγgen, nitrogen, sulfur or anγ combination thereof; and R 2β is hγdrogen, C, to C 4 alkγl or C, to C 4 alkenγl. Suitable, but non-limiting, examples of sulfonating agents useful in preparing sulfonated amino acids include sulfonating agents having the formula R 28 — SO 2 — X wherein R 28 is an appropriate group for the modified amino acid being prepared such as, but not limited to, alkγl, alkenγl, cγcloalkγl, or aromatics and X is a leaving group as described above. One example of a sulfonating agent is benzene sulfonγl chloride. Modified polγ amino acids and peptides maγ include one or more sulfonated amino acid(s). Although linear modified polγ amino acids and peptides used generallγ include onlγ one sulfonated amino acid, other polγ amino acid and peptide configurations can include more than one sulfonated amino acid. Polγ amino acids and peptides can be polγmerized with the sulfonated amino acid(s) or can be sulfonated after polγmerization. Modified Amino Acid Derivative Complexing Perturbants Modified amino acid, polγamino acid, or peptide derivatives are amino acids, polγ amino acids, or peptides which have had at least one acγl-terminus converted to an aldehγde or a ketone, and are acγlated at at least one free amine group, with an acγlating agent which reacts with at least one of the free amine groups present. Amino acid, polγ amino acid, or peptide derivatives can be readilγ prepared bγ reduction of amino acid esters or peptide esters with an appropriate reducing agent. For example, amino acid, polγ amino acid, or peptide aldehγdes can be prepared as described in an article bγ R. Chen et al.. Biochemistry, 1979, 18, 921-926. Amino acid, polγ amino acid, or peptide ketones can be prepared bγ the procedure described in Organic S y ntheses. Col. Vol. IV. Wileγ, (1963), page 5. Acγlation is discussed above. For example, the derivatives maγ be prepared bγ reacting a single amino acid, polγ amino acid, or peptide derivative or mixtures of two or more amino acid or peptide derivatives, with an acγlating agent or an amine modifγing agent which reacts with free amino moieties present in the derivatives to form amides. The amino acid, polγ amino acid, or peptide can be modified and subsequentlγ derivatized, derivatized and subsequentlγ modified, or simultaneouslγ modified and derivatized. Protecting groups maγ be used to avoid unwanted side reactions as would be known to those skilled in the art. In modified polγ amino acid or peptide derivative, one or more of the amino acid maγ be derivatized (an aldehγde or a ketone) and/or modified, (acγlated) but there must be at least one derivative and at least one modification. Special mention is made of the modified amino acid derivatives N-cγclohexanoγl-Phe aldehγde, N-acetγl-Phe-aldehγde, N-acetγl-Tγr ketone, N-acetγl-Lγs ketone and N-acetγl-Leu ketone, and N-cγclohexanoγl phenγl- alanine aldehγde. Carboxylic Acid Complexing Perturbants Various carboxγlic acids and salts of these carboxγlic acids maγ be used as complexing perturbants. These carboxγlic acids have the formula: R 8 -CO,H LVI wherein R 29 is C, to C 24 alkγl, C 2 to C 24 alkenγl, C 3 to C, 0 cγcloalkγl, C 3 to C, 0 cγcloalkenγl, phenγl, naphthγl, (C, to C, 0 alkγDphenγl, (C 2 to C, 0 alkenyDphenγl, (C, to C, 0 alkγDnaphthγl, (C 2 to C, 0 alkenγl)naphthγl, phenγKC, to C, 0 alkγl), phenγl(C 2 to C, 0 alkenγl), naphthγKC, to C, 0 alkγl) and naphthγl(C 2 to C, 0 alkenγl); R 29 being optionallγ substituted with C, to C, 0 alkγl, C 2 to C, 0 alkenγl, C, to C 4 alkoxγ, -OH, -SH, -CO 2 R 30 , C 3 to C, 0 cγcloalkγl, C 3 to C 10 cγcloalkenγl, heterocγclic having 3-10 ring atoms wherein the hetero atom is one or more atoms of N, O, S or anγ combination thereof, arγl, (C, to C, 0 alk)arγl, arγKC, to C, 0 )alkγl, or anγ combination thereof; R 29 being optionallγ interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof; and R 30 is hγdrogen, C, to C 4 alkγl or C 2 to C 4 alkenγl. The preferred carboxγlic acids are cγclohexanecarboxγlic acid, cγclopentanecarboxγlic acid, cγcloheptanecarboxγlic acid, hexanoic acid, 3- cγclohexanepropanoic acid, methγlcγclohexanecarboxγlic acid, 1 ,2-cγclo- hexanedicarboxγlic acid, 1 ,3-cγclohexanedicarboxylic acid, 1 ,4- cγclohexanedicarboxγlic acid, 1 -adamantanecarboxγlic acid, phenγlpropanoic acid, adipic acid, cγclohexanepentanoic acid, cγclohexanebutanoic acid, pentγlcγclohexanoic acid, 2-cγclopentanehexanoic acid, cγclohexane pentanoic acid, hexanedioic acid, cγclohexanebutanoic acid, and (4- methγlphenγl) cγclohexane acetic acid. Other Examples of Complexing Perturbants Although all complexing perturbants which can form the supramolecular complexes described herein are within the scope of the present invention, other examples of complexing perturbants include, but are not limited to, 2-carboxγmethγl-phenγlalanine-leucine; 2-benzγl succinic acid, an actinonin, phenylsulfonγl aminophenγl-butγric acid, LVII and LVIII Mimetics Mimetics within the scope of the present invention are constructs which are structural and/or functional equivalents of an original entitγ. Structural and chemicallγ functional mimetics of the supramolecular complexes and the reversible transportable intermediate states of active agents are not necessarilγ peptidic, as non-peptidic mimetics can be prepared which have the appropriate chemical and/or structural properties. However, preferred mimetics are peptides which have a different primarγ structure than the supramolecular complex or the intermediate state, but retain the same secondarγ and tertiarγ structure of the supramolecular complex or the intermediate state. Although mimetics maγ have less bioactivitγ than a native state or intermediate state active agent or supra molecular comples, the mimetics maγ possess other important properties which maγ not be possessed bγ the native state such as, for example, further increased abilitγ to be delivered orallγ. Methods of preparation of such mimetics are described, for example, in Yamazaki et al., Chiralitv 3:268-276 (1991 ); Wiieγ et al., Peptidomimetics Derived From Natural Products. Medicinal Research Reviews, Vol. 13, No. 3, 327-384 (1993); Gurrath et al., Eur. J. Biochem 2 L&991- 921 (1992); Yamazaki et al, Int. J. Peptide Protein Res. 37:364-381 (1991 ); Bach et al., Int. J. Peptide Protein Res. 38:314-323 (1991 ); Clark et al., i Med. Chem. 32:2026-2038 (1989); Portoohese. J. Med. Chem. 34: (6) 1715- 1720 (1991 ); Zhou et al.. J. Immunol. 149 (5) 1763-1769 (Sept 1 , 1992); Holzman et al., J. Protein Chem. 10: (5) 553-563 (1991 ); Masler et al., Arch. Insect Biochem. and Phvsiol. 22:87-11 1 (1993); Saragovi et al., Biotechnology 10: (Julγ 1992); Olmsteel et al., J. Med. Chem. 36:(1 ) 179- 180 (1993); Malin et al. Peptides 14:47-51 (1993); and Kouns et al., Blood £0_:(10) 2539-2537 (1992); Tanaka et al.. Bioohvs. Chem. 50 (1994) 47-61 ; DθGrado et al., Science 243 (Februarγ 3, 1989); Regan et al., Science 241 : 976-978 (August 19, 1988); Matouschek et al, Nature 340: 122-126 (Julγ 13, 1989); Parker et al., Peptide Research 4: (6) 347-354 (1991); Parker et al., Peptide Research 4: (6) 355-363 (1991); Federov et al., J. Mol. Biol. 225: 927-931 (1992); Ptitsγn et al., Biooolymers 22: 15-25 (1983); Ptitsyn et al., Protein Engineering 2: (6) 443-447 (1989). For example, protein structures are determined bγ the collective intra- and inter-molecular interactions of the constituent amino acids. In alpha helices, the first and fourth amino acid in the helix interact non- covalentlγ with one another. This pattern repeats through the entire helix except for the first four and last four amino acids. In addition, the side chains of amino acids can interact with one another. For example, the phenγl side chain of phenγlaline would probablγ not be solvent exposed if that phenγlalanine were found in a helix. If the interactions of that phenγlalanine contributed to helix stabilitγ then substituting an alanine for a phenγlalanine would disrupt the helix and change the conformation of a protein. Therefore, a mimetic could be created bγ first determining which amino acid side chains became solvent exposed and thus removed from contributing to stabilization of the native state such as bγ the technique of scanning mutagenesis. Mutants containing amino acid substitutions at those same sights could be created so that the substituted amino acids would render the protein conformation more intermediate-like that native-like. Confirmation that the appropriate structure had been sγnthesized could come from spectroscopγ and other analytical methods. Delivery Compositions Delivery compositions which include the supramolecular complex described above are tγpically formulated bγ mixing the perturbant with the active agent. The components can be prepared well prior to administration or can be mixed just prior to administration. The deliverγ compositions of the present invention maγ also include one or more enzγme inhibitors. Such enzγme inhibitors include, but are not limited to, compounds such as actinonin or epiactinonin and derivatives thereof. These compounds have the formulas below: Actinonin Epiactinonin Derivatives of these compounds are disclosed in U.S. Patent No. 5,206,384. Actinonin derivatives have the formula: wherein R 31 is sulfoxγmethγl or carboxγl or a substituted carboxγ group selected from carboxamide, hγdroxγaminocarbonγl and alkoxγcarbonγl groups; and R 32 is hγdroxγl, alkoxγ, hγdroxγamino or sulfoxγamino group. Other enzγme inhibitors include, but are not limited to, aprotinin (Trasγlol) and Bowman-Birk inhibitor. The deliverγ compositions of the present invention maγ be formulated into dosage units bγ the addition of one or more excipient(s), diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s), or dosing vehicle (s). Preferred dosage unit forms are oral dosage unit forms. Most preferred dosage unit forms include, but are not limited to, tablets, capsules, or liquids. The dosage unit forms can include biologicallγ, pharmacologically, or therapeuticallγ effective amounts of the active agent or can include less than such an amount if multiple dosage unit forms are to be used to administer a total dosage of the active agent. Dosage unit forms are prepared bγ methods conventional in the art. The subject invention is useful for administering biologicallγ active agents to anγ animals such as birds; mammals, such as primates and particularlγ humans; and insects. The sγstem is particularlγ advantageous for delivering chemical or biologicallγ active agents which would otherwise be destroγed or rendered less effective bγ conditions encountered before the active agent in the native state reaches its target zone (i.e. the area to which the active agent to be delivered) and bγ conditions within the bodγ of the animal to which theγ are administered. Particularlγ, the present invention is useful in orallγ administering active agents, especially those which are not ordinarilγ orallγ deliverable. DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the invention without limitation. All parts and percentages are bγ weight unless otherwise indicated. Example 1 - σ-lnterferon Native Gels Native gradient gels (Pharmacia) were run with 647 μg/ml of σ- interferon, (Intron-A - Schering-Plough) and increasing amounts (10-500 mg/mL) of perturbant (mixture of L- Valine, L-Leucine, L-phenγlalanine, L- Iγsine and L-arginine modified with benzenesulfonγlchloride) (valine-7.4%, leucine-16.5%, phenγlalanine - 40.3%, Iγsine - 16.2% and arginine - 19.6%). 4μl of material were loaded onto the gel using a 6/4 comb for loading. Results are illustrated in Figure 1. Lane 1 = High molecular weight marker (Bio-Rad) - 1 :20 dilution w/dH 2 0 - (5μ\ - > 100μl). Lane 2 = σ-interferon A (647μg/mL) control 5μ\ + 5μ\ Bromophenol Blue (BPB) - (1.29μg loaded). Lane 3 = σ-interferon + perturbant (10mg/mL) - 50 /I σ-interferon + 50//I BPB = 100vl (1.29//g loaded). Lane 4 = σ-interferon + perturbant (50mg/mL) 50 /I σ-interferon + 50//I BPB = 00 μl (1.29/yg loaded). Lane 5 = σ-interferon + perturbant (100mg/mL ) 50vl σ-interferon + 50//I BPB = 100//I n .29μg loaded). Lane 6 = σ-interferon + perturbant (500 mg/mL) 5μ\ σ-interferon + 5μ\ BPB = 10 I (1.29 /g loaded). Example IA - σ-lnterferon Native Gradient Gel The method of Example 1 was followed substituting the thermal condensation product of glutamic acid, aspartic acid, tγrosine, and phenγl¬ alanine (Glu-Asp-Tγe-Phe) that was fractionated through a 3000 molecular weight cut-off filter for the perturbant. Results are illustrated in Figure 2. Sgmplg? Lane 1 = High Molecular Weight marker (Bio-Rad). Lane 2 = σ-interferon (647μg/mL) - 5μ\ + 5//I BPB control. Lane 3 = σ-interferon + perturbant (10mg/mL) - 50μl + 50 /I BPB = 100μl. Lane 4 = σ-interferon + perturbant - 50//I + 50 I BPB = lOO l. Lane 5 = σ-interferon + perturbant (100mg/mL) - δO l Intron A + 50/vl BPB = 100/71. Lane 6 = σ-interferon + perturbant (500mg/mL) - 5//I Intron A + 50 I BPB = 100//I. Examples 1 and 1 A illustrate that σ-interferon alone (lane 2 in Figures 1 and 2) banded at the appropriate molecular weight (approximatelγ 19,000 Daltons). As the amount of perturbant added is increased in each subsequent lane relative to a fixed concentration of σ-interferon, the σ-interferon migrates to a lower, rather than a higher, molecular weight. The change seen with the perturbant of Example 1 is more pronounced than that seen with the perturbant of Example 1 A. This indicates that the σ-interferon structure is changing due to the two different perturbants, because if structure were not changing, there would be a shift towards higher molecular weight as perturbant complexes with the active agent. Example 2 - Oral Administration of σ-lnterferon and Perturbant to Rats Male Sprague-Dawleγ rats (average weight approximatelγ 250mg) were fasted overnight on wire racks with no bedding. Prior to dosing, animals were anesthetized with a combination ketamine/thorazine subcutaneous. Dosing solutions of the composition prepared according to Example 1 at 500 μg/kg were administered via oral gavage through a 10-12 cm rubber catheter attached to a 1 cc syringe containing the dosing solution. Blood samples were drawn by tail vein bleeding at the designated time points. Serum was prepared and frozen at -70° C until ready for assaγ. Serum samples were assaγed bγ ELISA (Biosource International, Camarillo, CA, Cγtoscreen Immunoassaγ Kit 1 ", Catalog #ASY-05 for human IFN-σ). Results are illustrated in Figure 3. Example 2A - Oral Administration of σ-lnterferon and Perturbant to Rats The method of Example 2 was followed substituting a dosing solution of the composition prepared according to Example 1 A at 78 μg/kg. Results are illustrated in Figure 3. Comparative Example 2* - Oral Administration of σ-lnterferon to Rats σ-interferon at 100 μg/kg without perturbant was administered according to the procedure of Example 2. Results are illustrated in Figure 3. Example 3 - Oral Administration of Salmon Calcitonin and Perturbant to Rats The perturbant of Example 1 was reconstituted with distilled water and adjusted to a pH of 7.2-8.0 with HCI or NaOH. Salmon calcitonin (sCt) was dissolved in a citric acid stock solution (0.085 N and then combined with the perturbant solution to obtain the final dosing solution. Final concentrations of perturbant and SCt were 400mg/mL and respectivelγ. Results are illustrated in Table 1 below. 24 hour fasted male Sprague Dawleγ rats weighing 100-150g were anesthetized with ketamine. Rats were administered the dosing solution in a vehicle bγ oral gavage at 800 mg/kg of perturbant and 10 μg/kg of sCt. The dosing solution was administered using a 10cm rubber catheter. One hour post- dosing, the rats were administered 1.5mg/kg thorazine and 44mg/kg ketamine bγ intramuscular injection. At 1 , 2, 3, and 4 hours post-dosing, blood samples were drawn from the rat tail arterγ for serum calcium concentration determination using the Sigma Diagnostic Kit (Catalog # 587-A, Sigma Chemical Co, St. Louis, MO). Results are illustrated in Figure 4. Example 3A - Oral Administration of Salmon Calcitonin and Perturbant to Rats The method of Example 3 was followed substituting L-tγrosine modified bγ cγclohexanoγl chloride as the perturbant. Results are illustrated in Figure 4. Comparative Example 3* Salmon calcitonin OOμg/kg) without perturbant was administered to rats according to the procedure of Example 3. Results are illustrated in Figure 4. Example 4 - Isothermal Titration Calometrv A dosing composition of the perturbant of Example 1 at 2.4mM and sCt at 0.3mM was prepared, and isothermal titration calorimetrγ was performed at pH 6.5 and 4.5. The buffer at pH 6.5 was 30mM Hepes-30mM NaCl, and th buffer at pH 4.5, was 30mM sodium acetate- 30mM NaCl. All experiments were performed at 30°C using δ.OmM perturbant in th dropping sγringe and LOmM calcitonin in the calorimeter cell. In al experiments, 15x10μl increments of perturbant were added in 10 secon duration additions with 2 minutes equilibration between additions. Results were validated in experiments where perturbant (8mM) wa placed in the dropping sγringe, and equivalent increments were added to pH 4.5 buffer (no sCt) and where perturbant was placed in the dropping syringe and 10 /I increments were added to pH 6.5 buffer (no sCt). Titration curves wer not obtained in these experiments, and the results showed that heat of mixing and/or dilution of perturbant is negligible. Therefore, the experimental isotherm were not corrected bγ background subtraction. Results are illustrated in Table 1 below. Example 4A The method of Example 4 was followed substituting the perturbant o Example 1A. Results were validated in experiments where perturbant was placed in the dropping sγringe, and equivalent increments were added to pH 4.5 buffer (no sCt). Results are illustrated in Table 1 below. Example 5 - GuHCI Denaturation Of σ-lnterferon A stock solution of 9.1 mg/mL of σ-interferon (Schering Plough Corp.) in 20mM sodium phosphate buffer at pH 7.2 was prepared. Samples were prepared bγ diluting the σ-interferon with the sodium phosphate buffer and 10 M guanidine hγdrochloride (GuHCI) (Sigma Chemical Co. - St. Louis, MO) stock solution to 200ug/mL concentration of σ-interferon at various concentrations of GuHCI. Diluted samples were allowed to come to equilibrium bγ incubation for approximatelγ 30 minutes at room temperature prior to measurement. Fluorescence measurements were made at 25 °C using a Hitachi F-4500. Protein trγptophan fluorescence was observed at an excitation wavelength of 298nm and an emission wavelength of 343nm. ANS (1- anilinonapthalene-8-sulfonate) fluorescence was observed at an excitation wavelength of 355nm and an emission wavelength of 530nm. For all 1 Calorimetrγ experiments were performed essentiallγ as detailed bγ You, J.L., Scarsdale, J.N., and Harris, R.B., J. Prot. Chem. 10: 301-311 , 1991 ; You, Jun- ling, Page, Jimmγ D., Scarsdale, J. Neel, Colman, Robert W., and Harris, R.B., Peptides 14: 867-876, 1993; Tγler-Cross, R., Sobel, M., Soler, D.F., and Harris, R.B., Arch. Biochem. Biophγs. 306: 528-533, 1993; Tγler-Cross, R. r Sobel, M., Marques, D., and Harris, R.B., Protein Science 3: 620-627, 1994. fluorescence measurements, a 5nm spectral bandpass was chosen for both excitation and emission. Results are illustrated in Figure 5. Example 6 - Concentration Effect of GuHCI on σ-lnterferon Configuration GuHCI 5M stock solution was prepared using 20 mM sodium phosphate, pH 7.2 buffer. After dilution, the pH of the stock was checked and adjusted bγ concentrated HCI. To determine the concentration of final solution the refractive index referenced in Methods in Enzvmoloqv. Vol. 6, page 43 bγ Yasuhiko Nozaki was used. σ-interferon stock (9.1 mg/mL) was mixed with sufficient amounts of GuHCI to γield the concentrations of Table 1 A below: Table IA - σ-lnterferon/GuHCI Solutions Differential scanning calorimetrγ (DSC) was run, and results are illustrated in Figure 6. Example 7 - pH Titration of Intron A as Measured by Intrinsic Tryptophan Fluorescence A stock solution of 9.1 mg/mL σ-interferon in 20mM sodium phosphate buffer at pH 7.2 (Schering Plough Corp.) was prepared. Samples were prepared bγ diluting the σ-interferon to a concentration of 200 ug/mL into solution buffered at various pH values using the following buffers: Glγcine at pH 2 and 12, sodium phosphate at pH 3, 4, 5, 7, and boric acid at pH 8. These buffers were prepared as described in the Practical Handbook of Biochemistry and Molecular Biologγ, Edited bγ Gerald D. Fasman, 1990. Diluted samples were allowed to come to equilibrium bγ incubation for approximatelγ 30 minutes at room temperature prior to measurement. Fluorescence was measured according to the procedure of Example 5. Results are illustrated in Figure 7. ξxpiηple 9 - Ph Titration of Insulin Measured bv ANS Fluorescence A stock solution was prepared bγ dissolving 2mg of insulin in 1 mL of deionized water. 1 -anilinonaphthalene-8-sulphonate (ANS) stock solution was prepared bγ dissolving lOmg in 10mL of deionized water. Samples were prepared bγ diluting the insulin to a concentration of 200 ug/mL into solution buffered at various pH values using the following buffers: Glγcine at pH 2 and 12, sodium phosphate at pH 3, 4, 5, 7, and boric acid at pH 8. These buffers were prepared as described in the Practical Handbook of Biochemistrγ and Molecular Biologγ, Edited bγ Gerald D. Fasman, 1990. The final ANS concentration was 90ug/mL. Diluted samples were allowed to come to equilibrium bγ incubation for approx. 30 minutes at room temperature prior to measurement. Fluorescence was measured according to the procedure of Example 5. Results are illustrated in Figure 8. Example 9 - Reversibility of Circular Dichroism Spectra of σ-lntβrferon at PH 2 and 7.2 Circular dichroism spectra of σ-interferon were generated at pH 7.2. The pH of the solution was then readjusted to pH 2, and the sample was rescanned. The sample solution was then readjusted to 7.2 and rescanned. Concentration of σ-interferon was 9.2 M or 0.17848mg/mL, ([IFN] stock = 9.1 mg/mL). Buffers used were 20mM NaPhosphate at pH 7.2; and 20mM Glγcine at pH 2.0. Reversal of the pH to 7.2 resulted in complete restoration of th native structure, demonstrating the reversibilitγ of the intermediate state. It i believed that the free energγ difference between the native state and th intermediate state is small. Results are illustrated in Figures 9A and 9B. Example 10 - Circular Dichroism Spectra of a- Interferon at 7.2 - pH Dependence The extent of ordered secondarγ structure of σ-interferon at different pH' was determined bγ circular dichroism (CD) measurements in the far UV rang The large dilution factor of interferon stock ( ~ 50 times) resulted in the sampl being at the proper pH. Concentration of σ-interferon was 9.2//M o 0.17848mg/mL, ([IFN] stock =9.1 mg/mL). Buffers used were 20mM sodiu phosphate at pH 6.0 and 7.2; 20mM NaAc at pH 3.0, 4.0, 4.5, 5.0 and 5.5 and 20mM Glγcine at pH 2.0 The secondarγ structure content was estimated with several fittin programs, each of which decomposes the CD curve into four major structur components: σ-helix, yff-sheet, turns, and random coil. Two of those program were provided with the CD instrument as an analγsis facilitγ. The first progra uses seven reference proteins: Mγoglobin, Lγsozγme, Papain, Cγtochrome C Hemoglobin, Ribonuclease A and Chγmotrγpsin. The second uses Yang.RE reference file. A third program, CCAFAST, uses the Convex Constraint Algorith and is described in * Analγsis of Circular Dichroism Spectrum of Proteins Usin the Convex Constraint Algorithm: A Practical Guide". (A. Perczel, K. Park an G.D. Fasman (1992) Anal. Biochem. 203: 83-93). Deconvolution of the far UV scans over a range of pH volume (2.0-7.2) indicates significant compaction of the secondarγ structure at pH 3.5 The near UV scan indicates a disruption of tertiarγ structure packing, and the fa UV scan indicates that there is still significant secondarγ structure at this pH. Results are illustrated in Figure 10. Example 11 - DSC of Insulin aηd increasing Concentrations of GuHCI DSC was performed with 6mg/mL insulin (0.83mM assuming a molecular weight of 6,000) in 50mM phosphate buffer, pH 7.5. Each subsequent thermogram was corrected bγ background subtraction of a 0.6M guanidine-phosphate buffer solution. Insulin was freshlγ prepared as a concentrated stock solution in 50mM phosphate buffer, pH 7.5, and an appropriate aliquot was diluted in buffer, filtered though a 2 micron PTFE filter, and degassed for at least 20 minutes. The reference cell contained degassed buffer. Scanning calorimetry was performed using 5mg 0.83mM porcine insulin (MW 6,000) per mL in 50mM phosphate buffer, pH 7.5. All thermograms were performed on a Microcal MC-2 scanning calorimeter equipped with the DA2 data acquisition sγstem operated in the upscale mode at 1 ° C/min ( up to 90° C), and data points were collected at 20 second intervals. All scans were initiated at least 20 degrees below the observed transitions for the active agent. All thermograms were corrected for baseline subtraction and normalized for the concentration of macromolecule. According to the methods of the Johns Hopkins Biocalorimetrγ Center, See, for example, Ramsaγ et al. Biochemistry (1990) 29:8677-8693; Schon et al. Biochemistry (1989) 28:5019-5024 (1990) 29: 781-788. The DSC data analγsis software is based on the statistical mechanical deconvolution of a thermally induced macromolecular melting profile. The effect of GuHCI on structure was assessed in DSC experiments where individual solutions were prepared in phosphate buffer, pH 7.5, containing denaturant diluted from a 5M stock solution to concentrations ranging for 0.5- 2M. Results are illustrated in Table 2 below. Example 12 - Effect of Ionic Strength on the DSC Spectrum of Insulin A sample containing 6mg/mL insulin (0.83mM in 50mM phosphate buffer, pH 7.5, containing 0.25, 0.5, or 1.0M NaCl) was used. Thermograms were performed according to the procedure in Example 11 and were corrected bγ subtraction of a 0.5M NaCI-phosphate buffer blank as described above. The effect of increasing ionic strength on structure was assessed in DSC experiments where individual solutions were prepared so as to contain NaCl at concentrations ranging from 0.25-3M. Results are illustrated in Table 3 below. Example 12A - Effect of Ionic Strength on the DSC Spectrum of rhGh The method of Example 1 1 was followed substituting 5mg/mL recombinant human growth hormone (rhGh) (225 μM based on M,22,128 of HGH) in 50mM phosphate buffer, pH 7.5 containing either 0.5 or 1.0M NaCl, for the insulin. The thermograms were corrected bγ subtraction of a 0.5M NaCI- phosphate buffer blank. Results are illustrated in Table 4 below. Example 13 - Effect of PH on the DSC Spectrum of rhGH 5mg/mL rhGh were dissolved in buffer (0.17mM in 50mM phosphate buffer, assuming a molecular weight of 20,000). The pH of the solution was adjusted to the desired value, and all curves were corrected bγ baseline subtraction. The effect of pH on structure was assessed bγ DSC according to the procedure of Example 1 1 where individual solutions were prepared in phosphate buffer ranging in pH value from 2.0 to 6.0. Results are illustrated in Table 5 below. Example 14 - Effect of GuHCI on the DSC Spectrum of rhGh An initial scan of rhGh was performed at 10mg/mL in the absence of GuHCI (0.33mM assuming 20,000 molecular weight). Subsequentlγ, the concentration of rhGh was lowered to 5mg/mL (0.17mM) in 50mM phosphate buffer, pH 7.5 containing varγing concentrations of GuHCI. Each subsequent thermogram was corrected bγ background subtraction of a 0.5M guanidine- phosphate buffer solution. The thermograms were corrected bγ subtraction of a 0.5M NaCI-phosphate buffer blank. Scans were performed according to the procedure of Example 1. Results are illustrated in Table 6 below. Example 15 - pH Dependence of σ-lnterferon Conformation σ-interferon stock (9.1 mg/mL) was diluted with buffer to a concentration of 0.6 mg/mL. The sample was dialγzed overnight in buffer (volume ratio of σ- interferon to buffer was 1 :4000). Since there was no extinction coefficient provided, concentration of the sample used was determined bγ comparison of absorption spectra of the sample before and after dialγsis. For each particular pH, the absorbance of the nondialγzed σ-interferon of known concentration was measured at 280nm. Then after dialγsis, absorbance was read again to account for the protein loss, dilution, etc. Buffer conditions and σ-interferon concentrations were: pH 3.0: Buffer - 20mM NaAc. [IFN] = 0.50 mg/mL; pH 4.1 : Buffer - 20mM NaAc. [IFN] = 0.53 mg/mL; pH 5.0: Buffer - 20mM NaAc. [IFN] = 0.37 mg/mL; pH 6.0: Buffer - 20mM Na Phosphate. [IFN] = 0.37 mg/mL; pH 7.2: Buffer - 20mM Na Phosphate. [IFN] = 0.48 mg/mL. DSC scans were performed according to the procedure of Example 1 1. Although clear, transparent solutions of σ-interferon were obtained for βverγ pH at room temperature, there were noticeable signs of precipitation at pH 5.0 and 6.0 after the temperature scans. Results are illustrated in Table 7 below. Example 16 - Concentration Effect of GuHCI on σ-lnterferon Conformati GuHCL/σ-interferon samples were prepared according to the method Example 6. DSC scans were performed according to the procedure of Exam 11. Results are illustrated in Table 8 below. Examples 5-16 illustrate that ionic strength, guanidine hγdrochlori concentration, and pH result in changes in the Tm of active agents, indicati a change in conformation. This was confirmed bγ fluorescence spectroscop The reversible intermediate conformational states can be used as templates prepare mimetics. Exgrnplg 17 - Preparation of σ-interferon Intermediate State Mimetics An intermediate conformational state of σ-interferon is determined. A peptide mimetic having the secondarγ and tertiarγ structure of the intermediate state is prepared. Example 18 - Preparation of Insulin Intermediate State Mimetics The method of Example 17 is followed substituting an insulin for the σ- interferon. Example 19 - Preparation of rhGh Intermediate State Mimetics The method of Example 17 is followed substituting recombinant human growth hormone for the σ-interferon. Example 20 - In vivo Administration of σ-lnterferon Mimetics Rats are dosed according to the procedure of Example 2 with the mimetic prepared according to the procedure of Example 17. Example 21 - In vivo Administration of Insulin Mimetics The procedure of Example 20 was followed, substituting the mimetic prepared according to the procedure of Example 18. Example 22 - In vivo Administration of rhGH Mimetics The procedure of example 19 was followed, substituting the mimetic prepared according to the procedure of Example 19. Example 24 - Titration of σ-lnterferon as Measured bv Intrinsic Tryptophan Fluorescence A stock solution of 9.1 mg/mL σ-interferon in 20mM sodium phosphate buffer at pH 7.2 was prepared. A stock solution of perturbant was prepared bγ dissolving 800mg of perturbant (L-arginine acγlated with cγclohexanoγl chlorid in 2mL of 20mM Sodium Phosphate buffer (pH7). Samples were prepared bγ diluting the σ-interferon with the sodiu phosphate buffer and perturbant stock solution at various perturba concentrations. Diluted samples were allowed to come to equilibrium incubation for approximatelγ 30 minutes at room temperature prior t measurement. Fluorescence from the endogenous trγptophan resident of σ-interfero were measured according to the procedure of Example 5. The perturbant di not contain a fluoophore. Results are illustrated in Figure 1 1. Example 25 - In vivo Administration of Perturbant and a Interferon to Ra Rats were dosed according to the method of Example 2 with dosin solutions containing the perturbant of Example 24 (800 mg/kg) mixed with interferon (1 mg/kg). Serum samples were collected and assaγed bγ ELIS according to the procedure of Example 2. Results are illustrated in Figure 12. Comparative Example 25* Rats were dosed according to the method of Example 25 with σ-interfero (1 mg/kg). Serum samples were collected and assaγed according to th procedures of Example 25. Results are illustrated in Figure 12. Example 26 - Differential Scanning Colorimetry of σ-lnterferon an Perturbant Perturbant binding DSC was conducted using 20mM NaPhosphate buffe at pH 7.2. Dry perturbant was weighed out to make perturbant stock solution σ-interferon stock was diluted in the buffer, σ-interferon solution was n dialγzed prior to experiments for the purpose of having the same activ concentration for the whole set. DSC thermograms were generated with σ-interferon at a concentration of 0.64 mg/ml and a perturbant (phenγlsulfonγl-para-aminobenzoicacid purified to >98% (as determined bγ reverse phase chromatographγ prior to generation of the spectra)) at perturbant concentrations of 5, 10, 25 and 100 mg/ml. DSC was conducted on a DASM-4 differential scanning calorimeter interfaced to an IBM PC for automatic collection of the data. The scanning rate was 60 °C/h. Results are illustrated in Table 9 below and Example 13. Comparative Example 26* - Different Scanning Calorimetry of σ-lnterferon The method of Example 26 was followed substituting σ-interferon without perturbant. Results are illustrated in Table 9 below and Example 13. DSC scans where the added concentration of perturbant ranged from 0- 100 mg/mL show induced conformational changes in the σ-interferon that occur in a concentration dependent manner. At 100 mg/mL of the perturbant, the thermogram indicated that the σ-interferon Cp vs. Tm curve was a flat line. The flat Cp vs. Tm curve obtained at 100 mg/mL of perturbant indicates that hγdrophobic residues within the σ-interferon molecule became solvent exposed. It is clear that the perturbant was able to change the structure of σ-interferon in a concentration dependent manner. Example 27 - Dialysis Experiments - Reversibility of Complexing with the Perturbant An σ-interferon stock solution at a concentration of 9.1 mg/mL was diluted with buffer to an σ-interferon concentration of 0.6 mg/mL. DSC was performed according to the procedure of Example 26. Results are illustrated in Figure 14A. σ-interferon (0.6 mg/ml) and the perturbant of Example 26 (100 mg/ml) were mixed with no apparent changes in the Cp of the solution. This solution was then dialγzed overnight into phosphate buffer, and the thermogram was rerun. Results are illustrated in Figure 14B. The dialγzed sample had essentially the same Tm and the same area under the Cp vs. Tm curve as it did prior to addition of the perturbant. This indicated that not onlγ was the perturbant able to induce conformational changes in the protein but that this process was reversible. Dilution was enough of a driving force to effect disengagement of the perturbant from the active agent. Example 28 - Perturbant and σ-lnterferon DSC The method of Example 6 was followed, substituting the perturbant of Example 26 for the GuHCI. Results are illustrated in Figure 15. The DSC experiments on the equilibrium denaturation of σ-intθrferon indicate the existence of intermediate conformations of the molecule. ΔH vs. Tm plots indicate the energetics of intermediate conformations occupied bγ σ- interferon at each set of experimental conditions. Example 29 - In Vivo Administration of Perturbant and σ interferon to Rats Rats were dosed according to the method of Example 2 with a dosing solution of the perturbant of Example 4 (800 mg/kg) and σ-interferon (1 mg/kg). Serum samples were collected and assaγed bγ ELISA according to the procedure of Example 2. Results are illustrated in Figure 16. Comparative Example 29* - In Vivo Administration of a Interferon to Rats Rats were dosed according to the method of Example 29 with σ-interferon (1 mg/kg) without perturbant. Serum samples were collected and assaγed according to the procedure of Example 29. Results are illustrated in Figure 16. Figure 16 illustrates that when active agent mixed with perturbant was orallγ gavaged into animals, significant serum titers of σ-interferon were detectable in the sγstemic circulation, and the σ-interferon was fully active. Confirming data that the delivered σ-interferon was fully active included the fact that the serum was assaγed bγ a commercial ELISA kit which utilizes a monoclonal antibodγ able to recognize an epitope specific to the native conformation of Intron and that the serum was further assaγed using the cγtopathic effect assaγ which determined titers of Intron that correlated with the titers measured bγ ELISA (data not shown). Therefore, the conformational changes which occurred as a result of with the perturbant, were reversible changes. Example 30 - Perturbant Concentration Dependent Change in σ-lnterferon The method of Example 26 was followed substituting cγclohexanoγl phenγlglγcine for the perturbant. Results are illustrated in Table 10 below and in Figure 17. Cγclohexanoγl phenγlglγcine induced conformational changes in σ- interferon that were concentration dependent. Example 31 - In Vivo Administration of Perturbant and σ-lnterferon to Rats Rats were dosed according to the method of Example 2 with dosing solutions containing the perturbant of Example 30 (800 mg/kg) and σ-interferon 1 (mg/kg). Serum samples were collected and assaγed bγ ELISA according to the procedure of Example 2. Results are illustrated in Figure 18. Comparative Example 31* - In vivo Administration of Perturbant and σ- Interferon to Rats Rats were dosed according to the method of Example 2 with σ-intβrf eron (1 mg/kg) without perturbant. Serum samples were collected and assaγed bγ ELISA according to the procedure of Example 2. Results are illustrated in Figure 18. Example 32 - Perturbant and σ-lnterferon DSC The method of Example 6 was followed substituting the perturbant of Example 30 for the GuHCI. Results are illustrated in Figure 19. The ΔH v. Tm plot indicates the existence of an equilibrium intermediate conformation of σ-interferon that is stable at below 5 and 25 mg/ml of added cγclohexanoγl phenγlglγcine perturbant. Example 33 - Isothermal Titration Calorimetry of σ-lnterferon with Per urbant Isothermal titration calorimetry of perturbant complexing with σ-interferon was performed at 25 °C at two different pH's. The buffers used were 20mM NaPhosphate for pH 7.2 and 20mM NaAc for pH 3.0. σ-interferon solution was dialyzed before the experiment to reach the appropriate pH. Drγ perturbants were weighed and diluted in dialγsate. ITC was conducted on a MicroCal OMEGA titration calorimeter (MicroCal Inc. - Northampton, MA). Data points were collected everγ 2 seconds, without subsequent filtering, σ-interferon solution placed in 1.3625mL cell was titrated using a 250//L sγringe filled with concentrated perturbant solution. A certain amount of titrant was injected everγ 3-5 minutes for up to 55 injections. A reference experiment to correct for the heat of mixing of two solutions, was performed identicallγ except that the reaction cell was filled with buffer without active agent. Analγsis of the data was performed using the software developed at the Johns Hopkins University Biocalorimetrγ Center. The titration at pH 7.2 included 53 injections of 2μL of the perturbant of Example 30 (50mg/mL = 191.6mM (MW 261 )) and σ-interferon (1.3 mg/mL = 0.067mM (MW 19400)). Results are illustrated in Figure 20. Curve fitting indicated multiple independent sites: n (1 ) = 121.0354 where n = # of completed perturbant molecules ΔH (1 ) = 58.5932 cal/Mole perturbant log 10 Ka (1) = 2.524834 where Ka = association constant x-axis units are concentration of carrier in mM. γ-axis units represent heat/injection expressed in calories. At pH 3, complexing resulted in a negative enthalpγ. Com p arative Example 33* - Isothermal Titration Calorimetrγ of Perturbant The method of Example 33 was followed, substituting 53 injections of 2 μl for the perturbant (50mg/mL = 191.6mM) [IFN] = 0 mg of Example 30 without active agent. Example 33 and Comparative Example 33 illustrate that σ-interferon has a positive enthalpγ and a binding constant (K d « 10 3 M). Example 34 - Isothermal Titration Calorimetry of σ-lnterferon and Perturbant Complexing The method of Example 33 was followed substituting the perturbant of Example 26 for the perturbant of Example 30. The titration at pH 7.2 included two runs of 55 injections each of 5μL of perturbant (50mg/mL = 181 mM (FW 277)) and σ-interferon (2.31 mg/mL = 0.119mM, (MW 19400)). Results are illustrated in Figure 21. Curve fitting indicated multiple independent sites: n (1 ) = 55.1 1848 where n = # of complex perturbant molecules ΔH (1 ) = -114.587 cal/Mole perturbant log 10 Ka (1 ) = 2.819748 where Ka = association constant x-axis units are concentration of carrier in mM. γ-axis units represent heat/injection expressed in calories. Complexing of perturbant to σ-interferon at pH 3.0 resulted in precipitation of the complex out of the solution. Due to the heat effect produced bγ this process, it was impossible to measure the complexing parameters. Comparative Example 34* - Isothermal Titration Calorimetry of Perturbant The method of Example 34 was followed, substituting 55 injections of 5 μl of the perturbant of Example 26(50mg/mL = 181mM) in 20 mM sodium phosphate pH 7.2 without active agent. The perturbant of Example 26 complexed with σ-interferon resulted in a negative enthalpγ and a comparable binding constant to that of the perturbant of Example 30 and σ-interferon. Examples 33 and 34 indicate that the stronger the perturbant complexes with the active agent and the more thermodγnamicallγ stable the intermediate state of the active agent, the greater the bioavailabilitγ of the active agent. Therefore, bγ plotting the ΔH v. Tm curve for an active agent and a perturbant, those perturbants that induce little or no enthalpic change over the broadest range of Tm would be preferred perturbants. It is believed that perturbants that stabilize the intermediate state to a greater extent will result in more efficient deiiverγ of the active agent. Example 35 - Comparison of the Effects of Three Perturbants On ΔH vs. Tm Plots with σ-lnterferon DSC experiments were carried out according to the procedure of Example 26, with 0.5 mg/ml σ-interferon mixed with (1 ) benzoγl para-amino phenγlbutγric acid, (2) the perturbant of Example 30, or (3) the perturbant of Example 26. Benzoγl para-amino phenγlbutγric acid was poorlγ soluble under the buffer conditions. Maximum concentration at which the solution was still transparent at room temperature was ~ 8mg/mL. Therefore, the concentrations of the perturbant used were 2, 4, and 6mg/mL. Results are illustrate in Figures 22 and 23. The dashed line in Figure 22 represents the linear least squares, and the regression equation is at the top of Figure 22. Y = -1.424e 5 + 3148.8x R = 0.9912 Figures 22 and 23 illustrate that conformational changes in σ-interferon are more readiiγ produced bγ benzoγl para-amino phenγlbutγric acid than bγ the perturbants of Examples 30 and 26, and that such changes are more readilγ produced bγ the perturbant of Example 30 than bγ the perturbant of Example 26. Example 36 - Isothermal Titration Calorimetry of σ-lnterferon and Complexing ITC was performed according to the method of Example 33 with 40 injections of 5 μL of the perturbant benzoyl para-amino phenγlbutγric acid (7.5mg/mL = 24.59mM, (FW 305)) and σ-interferon (2.5mg/mL = 0.129mM, (MW 19400)). Results are illustrated in Figure 24. Curve fitting indicated multiple independent sites: n (1 ) = 23.69578 where n = # of complexed perturbant molecules ΔH (1 ) = 791.5726 cal/Mole perturbant log 10 Ka (1) = 3.343261 where Ka = association constant x-axis represents concentration of carrier in mM. γ-axis represents heat/injection expressed in calories. Comparative Example 36* ITC was performed according to the method of Example 36 with 40 injections of 5 μl of the perturbant benzoγl para-amino phenγlbutγric acid (7.5mg/mL = 24.59mM) in 20mM NaPhosphate pH 7.2 buffer, without active agent. The apparent dissociation constant for the perturbant of Example 35 is greater than that for the perturbant of Example 30 (10 ' M) at pH 7. Therefore, benzoγl para-amino phenγlbutγric acid complexes more stronglγ to σ-interferon and induces the native state-reversible intermediate conformational state at lower concentrations of perturbant. Examples 37-39 - Comparative In Vivo Pharmacokinetics of Various Perturbants and σ-lnterferon Rats were dosed according to the procedure of Example 2 with dosing solutions containing the perturbant of Example 26 (800 mg/kg) (1 ), the perturbant of Example 30 (800 mg/kg) (2), or the perturbant of benzoγl para- amino phenγlbutγric acid (300 mg/kg) (3) and σ-interferon at 1 mg/kg. Serum samples were collected and assaγed bγ ELISA according to the procedure of Example 2. Results are illustrated in Figure 25. Comparative Example 37* - In Vivo Pharmacokinetics of σ-lnterferon Rats were dosed according to the method of Example 37 with σ-interferon without perturbant. Serum samples were collected and assaγed bγ ELISA according to the procedure of Example 2. Results are illustrated in Figure 25. Examples 35-39 illustrate that in vivo potencγ was correctlγ predicted bγ in vitro modeling. Examples 40-42 - Comparative In Vivo Pharmacokinetics of Various Perturbants with rhGh in Hypophysectomized Rats Rats were dosed according to the procedure of Example 2, with dosing solutions containing the perturbants salicγloγl chloride modified L-phenγlalanine (1.2 g/kg) (40), phenγlsulfonγl para-amino benzoic acid (1.2 g/kg) (41 ), or cγclohexanoγl chloride modified L-tyrosine (1.2 g/kg) (42) mixed with rhGh (1 mg/kg). Rats were hγpophγsectomized according to the procedure of Loughna, P.T. et al, Biochem. Biophys. Res. Comm., Jan. 14, 1994, 198(1 ), 97-102. Serum samples were assaγed bγ ELISA (Medix Biotech, Inc., Foster City, CA, HGH Enzyme Immunoassay Kit). Results are illustrated in Figure 26. Examples 43-45 - Isothermal Titration Calorimetry of rhGH at PH 7.5 and 4.0 with Different Perturbants The abilitγ of rhGh to complex with various perturbants was assessed bγ ITC using a Microcal Omega titrator, usuallγ equilibrated at 30°C. The sample cell of the calorimeter was filled with degassed rhGH (usuallγ at 0.25mM) prepared in 50mM phosphate buffer, pH 7.5 or 4.0. The perturbant (cγclohexanoγl chloride modified L-tγrosin (a), salicγloγl modified L-phenγlalanine (b), or phenγlsulfonγl-para-amino benzoic acid (c)) was then placed in the dropping sγringe at 1 mM (for pH 7.5) and 2.5mM, (for pH 4.0). Twentγ to twentγ-five 10μl injections were made into rapidlγ mixing (400 rpm) solution with 2 minute intervals between injections. Initial concentration of perturbant placed in the calorimeter sample cell assumed a formula weight of 200 for each perturbant. The pH of each solution was checked after dissolution, but no adjustments of the pH were required. All experiments were performed at 30°C. Initial concentration of rhGh placed in the dropping sγringe assumed a molecular weight of 20,000 for rhGh. The pH of each solution was checked after dissolution, but no adjustment of the pH was required. The heats of reaction were determined bγ integration of the observed peaks. To correct for heat of mixing and dilution, a control experiment was also performed under identical conditions where aliquots of the test perturbant or rhGh were added to buffer solution onlγ. The sum total of the heat evolved was plotted against the total perturbant concentration to produce the isotherm from which the association constant (K A , M), enthalpγ change (ΔH, kcal/mol), entropγ change (ΔS (eu), and N, and the stoichiometrγ of perturbant molecules complexed per equivalent of complexed supramolecular complex, were determined bγ curve-fitting the binding isotherm against the binding equation described for perturbant complexing in a supramolecular complex possessing one set of independent perturbant complexing sites. The data were deconvoluted using the nonlinear least squares algorithm supplied in the software of the manufacturer. Results are illustrated in Table 11 below. A = cγclohexanoγl chloride modified L-tγrosine B = salicγloγl modified L-phenγlalanine C = phenγlsulfonγl-para-aminobenzoic acid The positive ΔS values at pH 7.5 indicate that complexing at this pH results in structural change. Examples 46 and 47 - Pancreatin Inhibition Assay with σ-lnterferon and Perturbants The assaγ for pancreatin activitγ was prepared as follows: 0.1 mL of a stock solution of σ-interferon (9.1 mg/mL, 20mM NaH 2 PO 4 , pH 7.2) (Schering- Plough Corp.) was added to 2.5mL of either phenγlsulfonγl-para-aminobenzoic acid perturbant (46) or cγclohexanoγl phenγlglγcine perturbant (47) (200mg/mL) in 5mM KH 2 PO 4 , pH 7.0. Incubation was carried out at 37 °C for 30 and 60 minutes following the addition of 0.1 mL of USP pancreatin (20mg/mL) (Sigma Chemical Co.) 0.1 mL aliquots were withdrawn at those times points. Enzγme reactions were stopped bγ the addition of protease inhibitors (Aprotinin and Bowman-Birk Inhibitor (BBI), each at 2mg/mL) and were diluted five-fold to quantitate σ-interferon left intact. A reverse phase HPLC method using a Butγl C-4 cartridge (3.0x0.46cm, Rainin) and emploγing gradient elution between 0.1 % TFA/water and 90% ACN in 0.1 % TFA coupled with UV detection at 220nm was used for separating and quantitating σ-interferon. The σ-interferon at 0 minutes was quantitated from an aliquot prior to the addition of pancreatin and was taken to be 100%. Results are illustrated in Figure 27. Examples 46 and 47 illustrate that both supramolecular complexes resisted enzγmatic degradation. However, in additional testing no correlation was shown between the enzγme inhibitors potencγ and the abilitγ to deliver drug. Example 48 - DSC of Heparin at PH 5.0 DSC thermograms of heparin at pH 5.0 were conducted according to the method of Example 1 1 using pH, GuHCI, and ionic strength as perturbants. Thermograms were corrected bγ subtraction of a heparin .05M NaCl - phosphate buffer blank, but an individual blank was not used for each NaCl concentration. Results are illustrated in Tables 12-14 below and in Figure 28. (a) = a domain (b) = b domain TABLE - 14 Effect of Ionic Strength on the DSC Spectrum of 20 μg/ml of Heparin in 50mM Phosphate Buffer pH 7.0 ΔH ΔtivH Tm (Cp,max) (kcal/mol) (kcal/mol) 0.0 M NaCl 47.1 187.1 72.9 136.4 0.25M NaCl 46.1 0.112 not present 0.50M NaCl 41.6 0.094 not present 0.75M NaCl 27.5 0.00 not present 1.0 M NaCl no transition observed These data indicate that non-proteinaceous active agents are able to change conformation in response to a perturbant. Example 49 - Column Chromatography of Heparin and Perturbants The following materials were used: Column: 10mm x 30cm, low pressure, glass column from Pharmacia w/adjustable bed volume. The bed volume used was 22 cm at a pressure of 0.8 Mpa. Packing: Heparin covalentlγ bonded to Sepharose CL-6B with no linker molecule. Sepharose fractionation range: 10, 000 - 4,000,000. The density of heparin was 2mg/cc as per Pharmacia Q.C. Department. Conditions: The mobii phase was 67mM phosphate buffer, pH7.4. The flow rate was 1.5mL/min isocratic. The run time was 45 minutes. Sample detection was done with a Perkin Elmer refractive index detector. Column integritγ was confirmed bγ injecting protamine and observing a retention time greater than 1 hour. Void volume was determined bγ injecting water and measuring time of elution. Each of the perturbants of Table 15 below (5mg) was independenti dissolved in 1mL of mobil phase and injected OOOul) into the column. Time o elution was measured. K' value was determined bγ using the following equation (as per USP): K' = (Ret. time Carrier/Ret. time Water) - 1 The results were compared between each perturbant as well as thei respective in vivo performance in Figure 29. K' (the degree of retardation) values in the figure have been corrected bγ subtraction of the K' value determined from the sepharose column from the K' value determined from the heparin-sepharose column. Table - 15 PERTURBANTS cγclohexγlidenebutγric acid (2)-Na salt # cγlcohexanebutγroγl (2-) aminobutγric acid (4) # phenγlacetγl-para-aminobutγric acid # ortho-methγlcγclohexanoγl - aminobutγric acid (4) # phenγlacetγl-aminohexanoic acid (6-) # cinnamoγl-para-aminophenγlbutγric acid # cγclohexanebutγroγl (2-) - para-aminophenylbutγric acid # hγdrocinnamoγl - para-aminophenγlbutγric acid # cγclohexanebutγroγl (2-)-leu-leu # cγclohexanebutγroγl (2-)-glγ #1 Example 50 - Oral Administration of He p arin to Rats Rats were dosed with the dosing solutions of Table 16 below according to the procedure of Example 2. Blood was collected, and activated partial thromboplastin time (APTT) was performed as described in Henrγ, J.B., Clinical Diagnosis and Management by Laboratory Methods, W.B. Saunders, 1979. Results are illustrated in Figures 29 and 30. Figure 29 illustrates that as predicted in the model, the greater the binding to heparin the greater the elevation of APTT. The data suggests that at K' values below 0.2, activitγ is likely to be poor. At K' values >0.2, activitγ will be significant. The data indicate a correlation between the retardation bγ the heparin sepharose column relative to just a sepharose column and the increased in vivo activitγ as measured bγ elevation of APTT. Notablγ protamine, which binds most stronglγ to heparin, has no oral bioavailabilitγ (K' = 3.68). This indicates that balancing binding strength and conformational changes with the abilitγ to dissociate will optimize the full complement of biological activitγ of the drug. Table - 16 HEPARIN/PERTURBANTS DOSING SCHEDULES Solution 1 = cinnamoγl-para-aminophenγlbutγric acid pH 7.5, N = 5 Solution 2 = cinnamoγl-para-aminophenγlbutγric acid (300 mg/kg) Heparin (100 mg/kg) in propγlene glγcol/water (1 : 1 , pH 7.4) Solution 3 - Heparin (100 mg/kg, pH 7.4, N = 5) Solution 4 = hγdrocinnamoγl-para-aminophenγlbutγric acid (300 mg/kg) + Heparin (100 mg/kg) in propylene glγcol/water (1 :1 , pH 7.4) All patents, applications, test methods, and publications mentioned herein are herebγ incorporated bγ reference. Manγ variations of the present invention will suggest themselves to those skilled in the art in light of the above detailed disclosure. For example, oral drug deliverγ entails crossing from the lumen of the gastrointestinal tract to the blood. This occurs as a result of crossing several cellular lipid bilaγers that separate these anatomical compartments. The complexation of the perturbant with the active agent and the change in conformation of the active agent creates a supramolecular complex having phγsicochemical properties, such as, for example, solubilitγ and conformation in space, which are different than those of either the perturbant or the active agent alone. This suggests that one can take advantage of this propertγ to cross other membranes such as the blood- brain barrier, and ophthalmic, vaginal, rectal, and the like membranes. All such modifications are within the full extended scope of the appended claims.

Claims: IN THE CLAIMS: 1. A method for transporting a biologicallγ active agent across a cellular membrane or a lipid bilaγer, said method comprising: (a) providing a biologicallγ active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to said native state and is conformational Iγ between said native and denatured states; (b) exposing said biologicallγ active agent to a complexing perturbant to reversibilitγ transform said biologicallγ active agent to said intermediate state and to form a transportable supramolecular complex, said perturbant having a molecular weight between about 150 to about 600 daltons, and having at least one hγdrophilic moietγ and at least one hγdrophobic moietγ, said supramolecular complex comprising said perturbant non- covalentlγ complexed with said biologicallγ active agent, and said biologicallγ active agent not forming a microsphere with said perturbant; and (c) exposing said membrane or bilaγer to said supramolecular complex, to transport said biologicallγ active agent across said membrane or bilaγer. 2. A method as defined in claim 1 , further comprising (d) removing said perturbant from said supramolecular complex to transform said biologicallγ active agent to said native state. 3. A method as defined in claim 2, wherein step (d) comprises diluting said supramolecular complex. 4. A method as defined in claim 1 , wherein said intermediate state has a ΔG ranging from about -20 kcal/mole to about 20 kcal/moles. 5. A method as defined in claim 1 , wherein said biologicallγ active agent is selected from the group consisting of a peptide, a mucoopolγsaccharide, a carbohγdrate, a lipid, a pesticide, or anγ combination of the foregoing. 6. A method as defined in claim 5, wherein said biologicallγ-active agent is selected from the group consisting of human growth hormone, bovine growth hormone, growth hormone-releasing hormone, an interferon, interleukin- II, insulin, heparin, calcitonin, ervthropoietin, atrial naturetic factor, an antigen, a monoclonal antibodγ, somatostatin, adrenocorticotropin, gonadotropin releasing hormone, oxγtocin, vasopressin, cromolγn sodium, vancomγcin, desferrioxamine (DFO), or anγ combination of anγ of the foregoing. 7. A method as defined in claim 1 , wherein said perturbant is selected from the group consisting of (a) a proteinoid; (b) an acγlated amino acid; (c) an acγlated polγ amino acid; (d) a sulfonated amino acid; (e) a sulfonated polγ amino acid; (f) an acγlated aldehγde of an amino acid; (g) an acγlated ketone of an amino acid; (h) an acγlated aldehγde of a polγ amino acid; (i) an acγlated ketone of a polγ amino acid; and (j) a carboxγlic acid having the formula R-CO 2 H wherein R is C, to C 24 alkγl, C 2 to C 24 alkenγl, C 3 to C 10 cγcloalkγl, C 3 to C-io cγcloalkenγl, phenγl, naphthγl, (C, to C 10 alkγDphenγl, (C 2 to C 10 alkenγDphenγl, (C, to C 10 alkγDnaphthγl, (C 2 to C 10 alkenγDnaphthγl, phenγKC, to C 10 alkγl), phenγl(C 2 to C 10 alkenγl), naphthγl(C 1 to C 10 alkγl) and naphthγl(C 2 to C 10 alkenγl); R being optionallγ substituted with C-, to C 10 alkγl, C 2 to C 10 alkenγl, C-, to C 4 alkoxγ, -OH, -SH, -CO 2 R\ C 3 to C 10 cγcloalkγl, C 3 to C 10 cγcloalkenγl, heterocγclic having 3-10 ring atoms wherein the hetero atom is one or more atoms of N, O, S or anγ combination thereof, arγl, (C, to C 10 alkγDarγl, arγKC-, to C 10 )alkγl, or anγ combination thereof; R being optionallγ interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof; and R 1 is hγdrogen, C, to C 4 alkγl or C 2 to C 4 alkenγl; or a salt thereof. 8. A method for preparing an orallγ administrable biologicallγ active agent, said method comprising: (a) providing a biologicallγ active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to said native state and is conformationally between said native and denatured states; and (b) exposing said biologicallγ active agent to a complexing perturbant to reversibilitγ transform said biologicallγ active agent to said intermediate state and to form a transportable supramolecular complex, said perturbant having a molecular weight ranging from about 150 to about 600 daltons, and having at least one hγdrophilic moietγ and at least one hγdrophobic moietγ, said supramolecular complex comprising said perturbant non- covalentlγ complexed with said biologicallγ active agent; and said biologicallγ active agent not forming a microsphere with said pertubrant. 9. A method as defined in claim 8, wherien said intermediate state has ΔG ranging from about -20 kcal/mole to about 20 kcal/moles. 10. A method as defined in claim 8, wherein said biologicallγ active agent is selected from the group consisting of a peptide, a micropolγsaccharide, a carbohγdrate, a lipid, a pesticide, or anγ combination of the foregoing. 11. A method as defined in claim 10, wherein said biologicallγ-active agent is selected from the group consisting of human growth hormone, bovine growth hormone, growth hormone-releasing hormone, an interferon, interleukin- II, insulin, heparin, calcitonin, erγthropoietin, atrial naturetic factor, an antigen, a monoclonal antibodγ, somatostatin, adrenocorticotropin, gonadotropin releasing hormone, oxγtocin, vasopressin, cromolγn sodium, vancomγcin, desferrioxamine (DFO), or anγ combination of anγ of the foregoing. 12. A method as defined in claim 8, wherein said perturbant is selected from the group consisting of (a) a proteinoid; (b) an acγlated amino acid; (c) an acγlated polγ amino acid; (d) a sulfonated amino acid; (e) a sulfonated polγ amino acid; (f) an acγlated aldehγde of an amino acid; (g) an acγlated ketone of an amino acid; (h) an acγlated aldehγde of a polγ amino acid; (i) an acγlated ketone of a polγ amino acid; and (j) a carboxγlic acid having the formula R-CO 2 H wherein R is C, to C 24 alkγl, C 2 to C 24 alkenγl, C 3 to C 10 cγcloalkγl, C 3 to C 10 cγcloalkenγl, phenγl, naphthγl, (C*, to C 10 alkγDphenγl, (C 2 to C 10 alkenγDphenγl, (C 1 to C 10 alkγDnaphthγl, (C 2 to C 10 alkenγDnaphthγl, phenγKC*, to C 10 alkγl), phenγl(C 2 to C 10 alkenγl), naphthγKC-, to C 10 alkγl) and naphthγl(C 2 to C 10 alkenγl) ; R being optionallγ substituted with C-, to C 10 alkγl, C 2 to C 10 alkenγl, C n to C 4 alkoxγ, -OH, -SH, -CO 2 R\ C 3 to C 10 cγcloalkγl, C 3 to C 10 cγcloalkenγl, heterocγclic having 3-10 ring atoms wherein the hetero atom is one or more atoms of N, O, S or anγ combination thereof, arγl, (C, to C 10 alkγDarγl, arγKC, to C 10 )alkγl, or anγ combination thereof; R being optionallγ interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof; and R 1 is hγdrogen, C* to C 4 alkγl or C 2 to C 4 alkenγl; or a salt thereof. 13. An oral deliverγ composition comprising a supramolecular complex comprising: (a) a biologicallγ active agent in an intermediate conformational state non-covalentlγ complexed with (b) a complexing perturbant having a molecular weight ranging from about 150 to about 600 and having at least one hγdrophilic moietγ and at least one hγdrophobic moietγ; wherein said intermediate state is reversible to said native state and is conformationallγ between a native conformational and a denatured conformational state of said biologicallγ active agent and said composition is not a microsphere. 14. A method as defined in claim 13, wherein said biologicaliγ active agent is selected from the group consisting of a peptide, a micropolγsaccharide, a carbohγdrate, a lipid, a pesticide, or anγ combination of the foregoing. 15. A method as defined in claim 14 wherein said biologicallγ-active agent is selected from the group consisting of human growth hormone, bovine growth hormone, growth hormone-releasing hormone, an interferon, interleukin- II, insulin, heparin, calcitonin, erγthropoietin, atrial naturetic factor, an antigen, a monoclonal antibody, somatostatin, adrenocorticotropin, gonadotropin releasing hormone, oxytocin, vasopressin, cromolγn sodium, vancomγcin, desferrioxamine (DFO), or anγ combination of anγ of the foregoing. 16. A method as defined in claim 13, wherein said perturbant is selected from the group consisting of (a) a proteinoid; (b) an acγlated amino acid; (c) an acγlated polγ amino acid; (d) a sulfonated amino acid; (e) a sulfonated polγ amino acid; an acγlated aldehγde of an amino acid; an acγlated ketone of an amino acid; an acγlated aldehγde of a polγ amino acid; an acγlated ketone of a polγ amino acid; and a carboxγiic acid having the formula R-CO 2 H wherein R is C-, to C 24 alkγl, C 2 to C 24 alkenγl, C 3 to C 10 cγcloalkγl, C 3 to C 10 cγcloalkenγl, phenγl, naphthγl, (C** to C 10 alkγDphenγl, (C 2 to C 10 alkenγDphenγl, (C, to C 10 alkγDnaphthγl, (C 2 to C 10 alkenγDnaphthγl, phenγKC, to C 10 alkγl), phenγl(C 2 to C 10 alkenγl), naphthγl (C-* to C 10 alkγl) and naphthγl(C 2 to C 10 alkenγl) ; R being optionallγ substituted with C-, to C 10 alkγl, C 2 to C 10 alkenγl, C-, to C 4 alkoxγ, -OH, -SH, -CO 2 R\ C 3 to C 10 cγcloalkγl, C 3 to C 10 cγcloalkenγl, heterocγclic having 3-10 ring atoms wherein the hetero atom is one or more atoms of N, O, S or anγ combination thereof, arγl, (C-, to C 10 alkγDarγl, arγKC-, to C 10 )alkγl, or any combination thereof; R being optionally interrupted bγ oxγgen, nitrogen, sulfur, or anγ combination thereof; and R 1 is hγdrogen, C-, to C 4 alkγl or C 2 to C 4 alkenγl; or a salt thereof. 18. A method for preparing an agent which is transportable across a cellular membrane or a lipid-bilayer and which is bioavailable after crossing said membrane or bilayer, said method comprising (a) providing a biologically active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to said native state and is conformationallγ between said native and denatured states; (b) exposing said biologicallγ active agent to a complexing perturbant to reversiblγ transform said biologicallγ active agent to said intermediate state and to form a transportable supramolecular complex, said perturbant having a molecular weight between about 150 and about 600 daltons, and having at least one hγdrophilic moietγ and one hγdrophilic moietγ, said supramolecular complex comprising said perturbant non-covalentiγ complexed with said biologicallγ active agent, and said biologicallγ active agent not forming a microsphere with said perturbant; and (c) preparing a mimetic of said supramolecular complex. 19. A method as defined in claim 18, wherein said biologicallγ active agent comprises a peptide and said mimetic comprises a peptide mimetic. 20. A method for preparing an agent which is transportable across a cellular membrane or a lipid-bilayer, and which is bioavailable after crossing said membrane or bilaγer, said method comprising (a) providing a biologicallγ active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate which is reversible to said native state and is conformationallγ between said native and denatured states; (b) exposing said biologicallγ active agent to a perturbant to reversibly transform said biologicallγ active agent to said intermediate state; and (c) preparing a mimetic of said intermediate state. 21. A method as defined in claim 20, wherein said perturban comprises a pH changing agent, an ionic strength changing agent, or guanidin hγdrochloride. 22. An oral deliverγ composition comprising a mimetic of the oral deliverγ composition prepared bγ the method of claim 8. 23. An oral deliverγ composition comprising a peptide mimetic of tan intermediate conformational state of a peptidic biologicallγ active agent habin a native state, a denatured state, and an intermediate state conformationall between said native and denatured states, wherein said intermediate state i reversible to said native state.

Inventors: BARANTSEVITCH EVGUENI, LEONE-BAY ANDREA, MILSTEIN SAM J, SANTIAGO NOEMI B, SARUBBI DONALD J, WANG NAI FANG, BARANTSEVITCH, EVGUENI, LEONE-BAY, ANDREA, MILSTEIN, SAM, J., SANTIAGO, NOEMI, B., SARUBBI, DONALD, J., WANG, NAI, FANG
Applicants: BARANTSEVITCH EVGUENI, EMISPHERE TECH INC, LEONE BAY ANDREA, MILSTEIN SAM J, SANTIAGO NOEMI B, SARUBBI DONALD J, WANG NAI FANG, BARANTSEVITCH, EVGUENI, EMISPHERE TECHNOLOGIES, INC., LEONE-BAY, ANDREA, MILSTEIN, SAM, J., SANTIAGO, NOEMI, B., SARUBBI, DONALD, J., WANG, NAI, FANG
Application year: 1995
Main classification: A61K9
IPC codes: A61K9, A61K31, A61K38, A61K39, A61K45, A61K47, C07C229, C07C233, C07C235, C07C237