MXPA00003109A - Active agent transport systems - Google Patents

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

TRANSPORTATION SYSTEM OF ACTIVE AGENTS FIELD OF THE INVENTION The present invention relates to methods and compositions for transporting active agents, in particular to biologically active agents, through cell membranes or lipid bilayers. These methods and compositions facilitate the distribution of an active agent to an objective, such as the distribution of a pharmaceutical agent through an adverse environment to a particular location of the body.

BACKGROUND OF THE INVENTION Conventional methods for active agents to their intended purposes, for example, human organs, tumor sites, etc., are usually severely limited by the presence of biological, chemical, and physical barriers. Typically, these barriers are imposed by the environment through which the REF .: 119028 distribution must take place, the environment of the target for distribution, or the objective itself.

Biologically active agents are particularly vulnerable to such barriers. In the distribution of these agents to animals, which include, but are not limited to pharmacological and therapeutic agents, barriers are imposed by the body. The subcutaneous, nasal, or sublingual distribution to the circulatory system for many biologically active agents is the route of choice for administration to animals if not for physical barriers such as the skin, the lipid bilayers, various organic membranes that are relatively impermeable. certain biologically active agents, but one or more of which must be traversed before an agent is delivered through these routes can reach the circulatory system. In addition, the distribution such as, for example, sublingual distribution can be prevented by chemical barriers such as variable pH in the gastrointestinal tract (Gl) and the presence of effective and powerful enzymes.

While in many cases different methods of administering these compounds may be preferred, many of these agents can not be delivered by these routes directed to the target to which the active agent supplies its intended biological effect.

Typically, the initial approach to the design of the drug is found in the physiochemical properties of the pharmaceutical compounds and in particular, in their therapeutic function. The secondary focus of the design is on the need to deliver the active agent to its biological objectives (s). This is particularly true for drugs and other biologically active agents that are designed for oral administration in humans and other animals. However, thousands of therapeutic compounds are discarded because distribution systems are not available to ensure that the therapeutic titrations of the compounds reach the anatomical location or appropriate compartments after administration and in particular oral administration. In addition, many existing therapeutic agents with their approved indications are not used enough due to restrictions in their modes of administration. In addition, many therapeutic agents may be effective for additional clinical indications beyond which they are already used, if a practical methodology exists to deliver them in amounts appropriate to the appropriate biological objectives.

Although nature has achieved a successful inter-intracellular transport of active agents such as proteins, this success has not been translated into drug design. By nature, the transportable conformation of an active agent such as a protein, different in the conformation of the protein in its natural state. In addition, natural transport systems normally effect a return to the natural state of the protein subsequent to transport. When proteins are synthesized by ribosomes, they are released into the appropriate cellular organelle through a variety of mechanisms, for example, signaling peptides and / or chaperonins. Gething, MJ., Sambrook, "., Nature, 355, 1992, 33-45 One of the many functions, either of the signaling peptides, or of the chaperonins, is to prevent the premature folding of the protein to its state The natural state is usually described as a three-dimensional state with the lowest free energy possible.With the protein in a partially unfolded state, signaling peptides or chaperonins facilitate the ability of the protein to cross several cell membranes up to the protein reaches the appropriate organelle.The chaperonin is then separated from the protein due to the signaling peptide that cleaves the protein, which allows the protein to fold to the natural state.The ability of the protein to transit through cell membranes is, at least in part, as a consequence of being in a partially unfolded state.

Current concepts of protein folding suggest that there are several discrete conformations in the transition from the natural state to the totally denatured state. Baker, D., Agard, D.A., Biochemistry, 33, 1994, 7505-7509. The framing pattern of protein splitting suggests that in the early initial stages of doubling, the protein domains that are the secondary natural units are formed after the final doubling to the natural state. Kim, P. S., Bald in, R. L., Annu. Rev. Biochem. , 59, 1990, 631-660. In addition to these kinetic intermediates, equilibrium intermediates appear to be significant for a variety of cellular functions. Bychkova, V.E., Berni, R., et al, Biochemistry, 31, 1992, 7566-7571, and Sinev, M.A., Razgulyaev, O. I., et al., Eur. J. Biochem. , 1989, 180, 61-66. The data available from the chaperonins indicate that they work, in part, by keeping the proteins in a conformation that is not a natural state. In addition, it has been shown that proteins in partially split states are able to pass through the membranes, while in the natural state, especially the large globular proteins, penetrate poorly into the membranes, if at all. Haynie, D. T., Freire, E., Proteins: Structure, Function and Genetics, 16, 1993, 115-140.

In a similar way. Some ligands such as insulin, which are incapable of undergoing conformational changes associated with the equilibrium intermediates described above, lose their functionality. Hua, Q. X., Ladbury, J.E., eiss, M.A., Biochemistry, 1993, 32, 1433-1442; Remington, S., Wiegand, G., Huber, R., 1982, 158, 111-152; 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 the diphtheria toxin binds to its cell receptor, it is endocytic, and while it remains in this endocytic vesicle, it is exposed to an acid pH environment. The acid pH induces a structural change of a molecule of the toxin that provides the driving force for incersion and membrane translocation to the cytosol. See, Ramsay, G., Freire, E. Biochemistry, 1990, 29, 8677-8683 and Schon, AA., Freire, E. Biochemistry, 1989, 28, 5019-5024. Similarly, the cholera toxin undergoes a conformational change subsequent to endocytosis that allows the molecule to penetrate the nuclear membrane. See also, Morin, P. E., Diggs, D., Freire, E., Biochemistry, 1990, 29, 781-788.

Previously designed distribution systems use either a direct or indirect approach for distribution. The direct approach seeks to protect the drug from a hostile environment. Examples are enteric coatings, liposomes, microspheres, microcapsules. See, "Colloidal drug distribution systems", 1994, ed. Jorg Freuter, Marcel Dekker, Inc .; Patent of E. U. A. No. 4,239,754; Patel et al. (1976), FEBS Letters, Vol. 62, page 60; and Hashimoto et al., (1979), Endocrinology Japan, Vol. 26, page 337. All these approaches are indirect since their design basis is not directed to the drug, but rather is directed to protect it against the environment by which the drug must pass and go to the target to which it will exercise its biological activity, that is, prevent the hostile environment from contacting it and destroying the drug.

The direct approach is based on the formation of covalent bonds with the drug and a modifier, as in the creation of a prodrug. Balant, L. P., Doelker, E., Buri, P., Eur. J. Drug Metab. And Pharmacokinetics, 1990, 15 (2), 143-153. The bond is normally designed to break under defined circumstances, for example, changes in pH or exposure of specific enzymes. The covalent binding of a 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.

However, the use of the broad spectrum of the above distribution systems has been made impossible, because: (1) the systems require toxic amounts of adjuvants or inhibitors; (2) suitable low molecular weight fillers, ie, 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 (load); (6) the systems adversely alter the active agent; or (7) the systems fail to allow or promote the absorption of the active agent.

There is still a need in the art for simple and inexpensive distribution systems to be easily prepared and able to deliver a wide range of active agents to their intended purposes, especially in the case of pharmaceutical agents that are to be administered by means of the oral route BRIEF DESCRIPTION OF THE INVENTION The present invention discloses methods of administering, either by the subcutaneous, nasal or sublingual route, a biologically active agent to a subject in need of this agent. A first method includes the steps of: (a) providing a biologically active agent that can exist in a natural conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to its natural state and which is conformational between the natural and denatured states. (b) exposing the biologically active agent to a disturbing agent of complex formation to reversibly transform the biologically active agent to its intermediate state and to form a transportable supramolecular complex wherein the disrupting agent is in an effective amount for subcutaneous distribution , nasal or sublingual of the biologically active agent; Y (c) administering the supramolecular complex to the subject either subcutaneously, nasally or sublingually.

The disturbing agent has a molecular weight of between about 150 and about 600 daltons, and contains at least one hydrophilic portion and at least one hydrophobic portion. The supramolecular complex comprises the disturbing agent bound non-covalently or complexed with the biologically active agent. In the present invention, the biologically active agent does not form a microsphere after interacting with the disrupting agent.

A method for preparing a biologically active agent administrable subcutaneously, nasally or sublingually comprising the steps (a) and (b) above is also contemplated.

In alternative embodiments, subcutaneous, nasal or sublingual distribution compositions are provided. The compositions comprise a supramolecular complex that includes: (a) a biologically active agent of an intermediate conformational state, which is reversible to its natural state, complexed non-covalently with (b) a disturbing complexing agent having a molecular weight with a range that goes from about 150 to about 600 and having at least one hydrophilic portion and at least one hydrophobic portion wherein the disrupting agent is in an effective amount for the subcutaneous, nasal or sublingual distribution of the biologically active agent; Y wherein the intermediate state is conformationally between the state of natural conformation and the denatured state of the biologically active agent and the composition is not a microsphere.

Also contemplated is the method for preparing the agent which is a mimetic agent which is capable of being administered, by the subcutaneous, nasal or sublingual route, to a subject in need of this agent. A biologically active agent that can exist in its natural conformational state, a denatured conformational state, and an intermediate conformational state, which is reversible to its natural state and which is conformationally between the natural state and the denatured state, and which is exposed to a disturbing agent of complex formation in an effective amount for subcutaneous, nasal or sublingual distribution of such an agent to reversibly transform the biologically active agent to its intermediate conformational state and to form a transportable supramolecular complex. The disturbing agent has a molecular weight of between about 150 and about 600 daltons and at least one hydrophilic portion and a hydrophobic portion. The supramolecular complex comprises the disturbing agent formed in complexes non-covalently with the biologically active agent, and the biologically active agent does not formulate microspheres with the disturbing agent. A mimetic agent of the supramolecular complex is prepared.

Otherwise, a method for preparing an agent that is capable of being administered with either the subcutaneous, nasal, or sublingual routes is provided. A biologically active agent that can exist in its natural conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to its natural state that is conformational between natural and denatured states, is exposed to a disturbing agent to reversibly transform the biologically active agent at its intermediate state. The disturbing agent is in a respective effective amount for subcutaneous, nasal or sublingual distribution of the agent.

The agent, a mimetic agent of the intermediate state is prepared.

The administration by these routes of the methods and compositions of the present invention results in the distribution of the agent in bioavailable and bioactive form.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the illustration of a natural gradient gel of α-interferon (IFN) and a modified amino acid disturbing agent of complex formation.

Figure 2 is the illustration of a natural a-interferon gradient gene and a thermal perturbation agent of complex formation.

Figure 3 is the graphic illustration of guanidine hydrochloride (GuHCl) that induced the denaturing of α-interferon.

Figure 4 is a graphic illustration of the effect of GuHCl concentration on an a-interferon conformation.

Figure 5 is a graphic illustration of the pH denaturation of α-interferon.

Figure 6 is a graphic illustration of the pH denaturation of insulin.

Figures 7 A and 7B are graphic illustrations of the reversibility of the circular dichroism specter of α-interferon.

Figure 8 is a graphic illustration of the circular spectrum of dichroism of α-interferon.

Figure 9 is a graphic illustration of the intrinsic fluorescence of a-interferon tryptophan and a disturbing complexing agent.

Figure 10 is a graphic illustration of the differential scanning calorimetry of α-interferon and a disturbing agent of complex formation.

Figures 11 A and 11B are graphic illustrations of the reversibility of the transformation due to disturbing complex formation agents.

Figure 12 is a graphic illustration of the effect of the interfering complexing agent of α-interferon.

Figure 13 is a graphic illustration of the concentration effect of the disturbing agent of complex formation in the conformation of α-interferon.

Figure 14 is a graphic illustration of the effect of the disturbing agent of complex formation on α-interferon.

Figure 15 is a graphic illustration of the Isothermal Calorimetry of Titration of α-interferon and the disturbing agent of complex formation.

Figure 16 is a graphic illustration of the Isothermal Calorimetry of Titration of α-interferon and the disturbing agent of complex formation.

Figure 17 is a graphic illustration of the effects of interfering complexing agents of α-interferon.

Figure 18 is a graphical illustration of the effect of disturbance concentration of complex formation on α-interferon.

Figure 19 is a graphic illustration of the Isothermal Calorimetry of Titration of α-interferon and the disturbing agent of complex formation.

Figure 20 is a graphic illustration of the assessment of pancreatic inhibition with α-interferon and the disrupting agent of complex formation.

Figure 21 is a graphic illustration of the effect of the DSC of heparin at a pH of 5.0.

Figure 22 is a graphic illustration of the DSC of the DPPC with compounds of disturbing agents at various concentrations (units TM, ° C vs. concentration).

Figure 23 is a graphic illustration of the effect of the concentration of the disturbing agent of complex formation of compound L in the DPPC conformation.

Figure 24 is a graphical illustration of the effect of the concentration of the disturbing agent of complex formation of compound L in the rhGH conformation.

Figure 25 is a graphical illustration of the effect of concentration of the disturbing agent of complex formation of compound Ll in the rhGH conformation.

Figure 26 is a graphic illustration of the effect of the concentration of the disturbing agent of complex formation of compound XI in the rhGH conformation.

Figure 27 is a graphic illustration of the differential light scattering of the disrupting agent of compound L in 10 M of a phosphate buffer at a pH of 7.0.

Figure 28 is a graphic illustration of the results of subcutaneous injection of the rhGH composition in rats.

Figure 29 is a graphic illustration of the results of Sublingual (SL), intranasal (IN), and intracolonic (IC) dosing of rhGH in rats.

Figure 30 is a graphic illustration of the results of the intracolonic dosage of heparin with a carrier of compound XXXI.

DETAILED DESCRIPTION OF THE INVENTION Subcutaneous, sublingual and intranasal coadministration of an active agent such as recombinant human growth hormone (rhGH), as well as distribution agents, particularly proteins, describe their results in an invrementated bioavailability of the active agent as compared to the administration of the agent active only A similar result is obtained with the co-administration of salmon calcitonin with the distribution agents, in rats. The data shows these findings in the examples.

The present methods effect the distribution of the active agent by creating a non-covalent supramolecule of reversible complex formation from the active agent and an amount of the disrupting agent of complex formation appropriate for the distribution route. 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 the structure (but not in composition) provides the active agent with the appropriate properties, such as, for example, solubility (log P) to cross or penetrate a physical or chemical barrier, membrane, or lipid bilayer, to resist the Enzymatic degradation and the like. Crossing refers to the transport of one side of the cell membrane or lipid bilayer to the opposite side (ie, from the outside to the inside or inside of a cell and / or vice versa), whether the cell membrane or the bilayer lipid is really penetrated or not. In addition, the disturbed intermediate state of the active agent or the supramolecular complex itself can be used as a template for the preparation of mimetics which, consequently, can be delivered to a target by the appropriate route, i.e., subcutaneous, nasal or sublingual. After crossing the cell membrane or lipid bilayer, an active agent has biological activity and bioavailability, either by restoration to its natural state by retaining its biological activity or acquired bioavailability in the intermediate state. The mimetic agent acts similarly after crossing the cell membrane or lipid bilayer.

Active Agents The natural conformational state of an active agent is typically described as a three-dimensional state with the lowest free energy (? G). It is the state in which the active agent typically possesses the total complement of the activity ascribed to the agent, such as the total complement of the biological activity ascribed to a biologically active agent.

The denatured conformational state is the state in which the active agent does not have a secondary or tertiary structure.

The intermediate conformational states exist between the natural and denatured states. A particular active agent may have one or more intermediate states. The intermediate state achieved by the present invention is structurally and energetically intermediate both of the natural and denatured states. The natural agents useful in the present invention must be transformable from their natural conformational state to an intermediate conformational state that can be administered via the route of choice back to their natural state., that is, transformably reversible, so that when the active agent reaches its objective, such as when the delivered drug reaches the circulatory system, the active agent contains, recovers, or acquires a pharmacologically or therapeutically significant complement of its desired biological activity. Preferably the? G of the intermediate state has a range of about -20Kcal-moles to about 20Kcal-moles, and most preferably, has a range from about -10 Kcal-moles to about 10 Kcal-moles, all relative to its natural state.

For example, in the case of a protein, the intermediate state has a significant secondary structure, a significant compaction due to the presence of a large hydrophobic core, a tertiary structure reminiscent of the natural fold but without necessarily exhibi the packing of the natural state. The difference in free energy (? G) between the intermediate state and the natural state is relatively small. Consequently, the equilibrium constant between the transportable natural reversible intermediate states is close to unity (depending on the experimental conditions). The intermediate states can be confirmed by, for example, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), natural gradient gels, NMR, fluorescence, and the like.

Without being bound by any theory, the applicants believe that the physical chemistry of the intermediate state can be understood by the following explanation related to the active proteinaceous agents. Proteins can exist in stable intermediate conformations that are structurally and energetically dist from either the natural state or the denatured state. The inherent stability of any conformation (s) of any protein is reflected in the Gibbs free energy of the conformation (s). The Gibbs free energy for any state of a monomeric protein is described thermodynamically by the following relationship: ? G ° =? H ° (TR) - T? S ° (TR) +? CP ° ((T-TR) - T ln (T / TR)) (1) where T is the temperature, TR is the Reference temperature,? H ° (TR) and T? S ° (TR) are the relative enthalpy and entropy of this state at the reference temperature, and? CP ° is the relative heat capacity of this state. It is convenient to choose the natural state as the reference state to express all relative thermodynamic parameters.

The sum of the statistical weights of all the states accessible to the protein is defined as the partition function Q: = S -? Gi / RT (2) i = 0 Equation 2 can also be written as Q = 1 + n-l -D Gi / RT + -D Gn / RT o; where the second term includes all the intermediates that are populated during the transition. The first and last terms of equation (3) are the statistical weights of the natural and denatured states, respectively. Under most conditions, the protein structure can be approximated by a transition function in two states: Q (X 1 + e -? Gn / RT (4) See, Tanford, C, Advances in Protein Chemistry, 1968, 23, 2-95. The conformations of the proteins that are intermediate between the natural state and the denatured state can be detected by, for example, NMR, calorimetry, and fluorescence. Dill, K.A., Shortle, D., Annu. Rev. Biochem. , 60, 1991, 795-825.

All thermodynamic parameters can be expressed in terms of the partition function. Specifically, the population of molecules in state i is given in equation (5) PÍ = (5) Q Therefore, the measurement of the appropriate terms in equation (1) that allow the calculation of Gibbs free energy determines the extent to which any intermediate state is populated to any significant degree under the defined experimental conditions. This in turn indicates the role that these intermediate states play in the distribution of the drug. The more populated the intermediate state, the more efficient the distribution.

Active agents suitable for use in the present invention include biologically active agents and chemically 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, biologically active agents suitable for use in the present invention include, but are not limited to, peptides, particularly small peptides, hormones, and in particular hormones that by themselves do not pass or pass through slowly. the gastrointesl mucosa and / or are susceptible to chemical cleavage by acids and enzymes in the gastrointesl tract; polysaccharides, and particularly mixtures of muco-polysaccharides; carbohydrates; lipids; or any combination of these. Other examples include, but are not limited to, human growth hormones; bovine growth hormones; growth vibration hormones; interferons; interleukin-1; insulin; heparin, and particularly low molecular weight heparin; calcitonin; erythropoietin; atrial naturético factor; antigens; monoclonal antibodies; somatostatin; adenocortinotropin; gonadotropin-releasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium or disodium cromolyte); vancomycin: deferriloxamine (DFO); anti-microbial agents, including, but not limited to, antifungal agents; or any combination of these.

The methods and compositions of the present invention can be combined with one or more active agents.

Perturbants Agents Disturbing agents serve two purposes in the present invention. In a first embodiment, the active agent is contacted with a quantity of disturbing agent that reversibly transforms the active agent from its natural state into an intermediate state suitable for administration. The disrupting agent, in the appropriate amount forms non-covalent complexes with the active agent to form a supramolecular complex that can be administered by a selected route. This supramolecular complex can be used as a template for the design of a mimic agent or can be used as a distribution composition itself. The disturbing agent, in effect, fixes the active agent in the intermediate transportable state. The disturbing agent can be released from the supramolecular complex, such as by dilution in the circulatory system, so that the active agent can return to the appropriate natural state for the distribution route. Preferably, these disrupting agents have at least one hydrophilic (i.e., readily water soluble, such as, for example, a carboxylate group) moiety at least one hydrophobic portion (i.e., readily soluble in an organic solvent such as, for example, a benzene group) and has a molecular weight ranging from about 150 to about 600 daltons and more preferably from about 200 to about 500 daltons.

Compounds of disrupting complexing agents include, but are not limited to, proteinoids that include linear, non-linear, and cyclic proteinoids; modified amino acids (acylated or sulphonated), polyamino acids, and peptides; derivatives of modified amino acids, polyamino acids, or peptides (ketones or aldehydes); diketopiperazine structures / amino acids; carboxylic acids; and different disturbing agents that are discussed below.

Again without being bound by any theory, the applicants believe that the formation of non-covalent complexes can be effected by intermolecular forces including, but not limited to, hydrogen bonding, hydrophilic interactions, electrostatic interactions, and Van der Waals interactions. For any supramolecular complex of agents / active disturbing agent, there will be some combination of the above-mentioned forces that maintain the association.

The association constant Ka between the disturbing agent and the active agent can be defined according to equation (6) Ka = e-? Gn / RT (6) The dissociation constant Ká is the reciprocal of Ka. This measure of the association constants between the disturbing agent and the active agent at a defined temperature will produce data of the molar Gibbs free energy that allows the determination of the associated enthalpic and entropic effects. These measurements can be made experimentally, for example, using NME, fluorescence or calorimetry. This hypothesis can be illustrated with proteins as follows: Protein unfolding can be described according to the balance that exists between its various conformational states, for example: N - I ~ D (1) where N is the natural state, I is the intermediate state (s), D is the denatured state, and ki and k2 are the respective rate constants. Ki and K2 are the respective equilibrium constants. Consequently, i = 0 _ = 11 +. e? -? G1 / R +, e -? G2 / RT = 1 + K? + K2 (8) l + k? + k? k2 (9) This suggests that increasing the partition function of the intermediate states should have a positive impact on the ability to deliver the active agent, ie: Ki Pi = (10) (1 + K1 + K2) Because the formation of complexes must be reversible, the formation of complexes of the disturbing agent with the active agent, as measured by Ka, must be strong enough to ensure the distribution of the drug either to the systemic circulation and / or to the targets, but not so strong that the release of the disturbing agent does not occur in a timely manner so that it allows the active agent to become naturalized if this is necessary to produce the effects desired.

In a second embodiment, the appropriate quantities of the perturbing agents for the distribution path reversibly transform the active agent to its intermediate state so that the conformation of that state can be used as a template for the preparation of the mimetics. Disturbing agents for this purpose do not need, but can, complex with the active agent. Therefore, in addition to the disturbing complexing agents discussed above, the disrupting agents that change the pH of the active agent or its environment such as, for example, strong acids or strong bases; detergents; disturbing agents that change the ionic strength of the active agent or its environment; other agents such as, for example, guanidine hydrochloride; and the temperature can be used to transform the active agent. Both the supramolecular complex or the reversible intermediate state can be used as a template for the design of mimetic agents.

Disturbing Complex Formation Agents Amino acids are the basic materials used to prepare many of the disturbing complex formation agents useful in the present invention. An amino acid is any carboxylic acid having at least one free amino group and including natural and synthetic amino acids. Preferred amino acids for use in the present invention are cc-amino acids, and most preferably natural cc-amino acids. Many amino acids and amino acid esters are readily available from various commercial sources such as Aldrich Chemical Co. (Milwaukee, WI, 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 0 H-NÍR ^ -d ^ -O-OH where: R 1 is hydrogen, C 1 -C 4 alkyl, or C 2 -C alkenyl; R2 is C?-C24 alkyl, C2-C24 alkenyl, C2-C? Cycloalkyl or C3-C10 cycloalkenyl, phenyl, naphthyl, phenyl- (C1-C10 alkyl), phenyl- (C2-C? Alkenyl), naphthyl - ((C 1 -C 10 alkyl), naphthyl- (C 2 -C 8 alkenyl), phenyl- (C 1 -C 10 alkyl), phenyl- (C 2 -C 0 alkenyl), naphthyl- (C 1 -C 10 alkyl), naphthyl - (C2-C2 alkenyl); R is optionally substituted with C? -C alkyl, C2-C alkenyl, C1-C4 alkoxy, -OH, -SH, -C02R3, C3-C10 cycloalkyl / C3-C10 cycloalkenyl, a heterocycle having 3-10 ring atoms wherein the heteroatom is one or more of N, O, S, or any combination thereof, aryl, (C 1 -C 10, alkyl) aryl, ar (C 1 -C 10 alkyl) or any combination thereof; and R2 is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; Y R3 is hydrogen, C1-C4 alkyl, or C2-C4 alkenyl; 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, cystine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine , ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, hydroxyproline,? -carboxyglutamate, phenylglycine, or O-phosphoserine. Preferred amino acids are arginine, leucine, lysine, phenylalanine, tyrosine, tryptophan, valine, and phenylglycine.

Preferred amino acids that do not occur naturally for use in the present invention are β-alanine, α-amino isobutyric acid, citrulline, e-amino caproic acid, 7-amino heptanoic acid, β-aspartic acid, aminobenzoic acid, aminophenyl acetic acid , aminophenyl butyric acid, β-glutamic acid, cysteine (ACM), e-lysine, e-lysine (A-Fmoc), methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, p-nitro-phenylalanine, hydroxyproline, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and thioproline.

The polyamino acids are either peptides or two or more amino acids linked via a bond formed by other groups that can be linked, for example, an anhydride ester or an anhydride linkage. Special mention is made of polyamino acids that do not occur naturally and particularly of hetero.polyamino acids that do not occur naturally, that is, of mixed amino acids.

The peptides are two or more amino acids joined by a peptide bond. The peptides may vary in length from dipeptides with two amino acids to polypeptides with several hundred amino acids. See, Walker, Chambers Biological Dictionary, Cambridge, England: Chambers Cambridge, 1989, page 215.

Special mention is made of peptides that do not occur naturally and particularly to peptides that do not naturally occur from mixed amino acids. Special mention is also made of dipeptides, tripeptides, tetrapeptides, and pentapeptides, and particularly, the preferred peptides are dipeptides and tripeptides. The peptides may be homo- or heteropeptides which may include natural amino acids, synthetic amino acids, or any combination thereof.

Proteinoids as Perturban Complexing Formation Agents Proteinoids are artificial polymers of amino acids. Preferably the proteinoids are prepared from mixtures of amino acids. Preferred proteinoids are condensation polymers, and more preferably, thermal condensation polymers. These polymers can be targeted or random polymers. Proteinoids can be linear, branched, or cyclic, certain proteinoids can be units of other linear, branched, or cyclic proteinoids.

Special mention is made of diketopiperazines. Dystopiperazines are 6-membered ring compounds. The ring includes two nitrogen atoms and is replaced in two carbons by two oxygen atoms. Preferably the carbonyl groups are in the ring positions 1 and 4. These rings can optionally, and more often are, further substituted.

Annular diketopiperazine systems can be generated during the thermal polymerization or condensation of amino acids or amino acid derivatives.

(Gyore, J; Ecet M. Proceedings Fourth ICTA (Thermal Analysis), 1974, 2, 387-394 (1974)). These 6-membered ring systems were presumably generated by intramolecular cyclization of the dimer before another chain growth or directly from a linear peptide (Reddy, AV, Jn. J., Peptide Protein Res., 40, 472-476 ( 1992), Mazurov, AA et al., Jnt. Peptide Protein Res., 42, 14-19 (1993).) Diketopiperazines can also be formed by the cyclodimerization of the amino acid ester derivatives as described by Katchalski et al. , J. Amer. Chem. Soc., 68, 879-880 (1946), by cyclization of the dipeptide derivatives of the ester or by thermal dehydration of the amino acid derivatives and high boiling point solvents as described by Kopple. et al., J. Org. Chem., 33 (2), 862-864 (1968).

In a typical synthesis of a diketopiperazine, the COOH group of an amino acid benzyl ester is activated in a first step to produce a protected ester. The amine is deprotected and cyclized by dimerization in a second step, providing a diester of diketopiperazine. Finally, the COOH groups are deprotected to provide diketopiperazine.

Diketopiperazines are typically formed from a-amino acids. Preferably, the a-amino acids from which the diketopiperazines are derived are glutamic acid, aspartic acid, tyrosine, phenylalanine, and optical isomers of any of the foregoing.

Special mention is made of the diketopiperazines of the formula R7N-CHR-C = 0 II 0 = C-CHR5-NR6 wherein R4, R5, R6 and R7 are each hydrogen, C? -C2 alkyl, C? -C2 alkenyl, phenyl, naphthyl, (Ci-C? alkyl) phenyl, (Ci-Cio alkenyl) phenyl, (alkyl) C? -C? O) naphthyl, phenyl (Ci-Cio alkyl), phenyl (Ci-Cio alkenyl), naphthyl (Ci-Cio alkyl), and naphthyl (C-C? O alkenyl); any of R 4, R 5, R 6 and R 7 independently may optionally be substituted with C 1 -C 4 alkyl, Ci-C alkenyl, C 1 -C 4 alkoxy, -OH, -SH, and -C0 2 R 8 or any combination thereof; R 8 is hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 alkenyl, and any of R 4, R 5, R 6 and R 7 independently may optionally be interrupted by oxygen, nitrogen, sulfur or any combination thereof.

The phenyl and naphthyl groups can be optionally substituted. Suitable but not limiting examples of the substituents are Ci-Cß alkyl, Ci-Cß alkenyl, Ci-Cß alkoxy, -OH, -SH or C02R9 wherein R9 is Ci-C6 alkyl, or Ci-Cß alkenyl.

Preferably, R6 and R7 are each hydrogen, C1-C4 alkyl, or C_-C_ alkenyl. Special mention is made of the diketopiperazines which are the disturbing agents of formation of preferred complexes. These diketopiperazines include the unsubstituted diketopiperazine wherein R4, R5, R6 and R7 are hydrogen, and diketopiperazines are substituted on one or both of the ring nitrogen atoms, i.e., mono or di-N-substituted. Special mention is made of the N-substituted diketopiperazine wherein one or both nitrogen atoms are replaced by a methyl group.

Special mention is made of the diketopiperazines of the formula: III 0 = C-CHR11-NH wherein R10 and R11 are each hydrogen, C? -C24 alkyl, C? -C2 alkenyl, phenyl, naphthyl, (Ci-Cio alkyl) phenyl, (C? -C10 alkenyl) phenyl, (Ci-Cio alkyl) naphthyl (alkenyl Ci-Cio) naphthyl, phenyl (Ci-Cio alkyl), phenyl (Ci-Cio alkenyl), Ci-Cio alkenyl, naphthyl (Ci-Cio alkyl), and naphthyl (Ci-Cio alkenyl); but both R10 and R11 can not be hydrogen; one or both of R 10 or R 11 each may be optionally substituted with C 1 -C 4 alkyl, C 1 -C 4 alkenyl, C 1 -C 4 alkoxy, -OH, -SH, and -C02R 12 or any combination thereof; R 12 is hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 alkenyl; and one or both of R10 and R11 each may optionally be interrupted by an oxygen, nitrogen, sulfur, or any combination thereof.

The phenyl or naphthyl groups can optionally be substituted. Suitable, but not limiting, examples of the substituents are Ci-Ce alkyl, Ci-Cβ alkenyl. C6-C6 alkoxy, -OH, -SH or C02R13 wherein R13 is hydrogen, C6-C6 alkyl, or Ci-C3 alkenyl. When an R10 or R11 is hydrogen, diketopiperazine is mono-carbon- (C) -substituted. When none of R10 or R11 is hydrogen, diketopiperazine is di-carbon- (C) -substituted.

Preferably, R10, R11, or R10 and R11, contain at least one functional group, a functional group is a non-hydrocarbon portion responsible for the characteristic reactions of the molecule. Simple functional groups are heteroatoms that include, but are not limited to, halogens, oxygen, sulfur, nitrogen, and the like, attached to the carbon of an alkyl group by a single or multiple bond. Other functional groups include, but are not limited to, for example, hydroxyl groups, carboxyl groups, amido groups, amino groups, substituted amino groups, and the like.

Preferred diketopiperazines are those substituted on one or two of the ring carbons by a functional group that includes at least one carboxyl functionality.

To Ozácidos / Dicetopiperazinas As Perturban Complexing Formation Agents The diketopiperazines can also be polymerized with additional amino acids to form structures of at least one amino acid or an ester or an amide thereof and at least one diketopiperazine preferably covalently linked to one another.

When diketopiperazine is polymerized with additional amino acids, one or more of the R groups must contain at least one functional group, a functional group is a non-hydrocarbon portion responsible for the characteristic reactions of the molecule. Simple functional groups are heteroatoms that include but are not limited to halogens, sulfur, oxygen, nitrogen, and the like, attached to the carbon of an alkyl group by a single or multiple bond. Other functional groups include, but are not limited to, for example, hydroxyl groups, carboxyl groups, amide groups, amino groups, substituted amino groups and the like.

Special mention is made of the diketopiperazines which are preferred components of the disturbing amino acid / diketopiperazine agents of the present invention. These diketopiperazines are those preferred which are substituted on one or two of the ring carbons and are preferably replaced by a functional group including at least one carboxyl functionality.

More preferably, the diketopiperazines in the disturbing amino acids / diketopiperazines are prepared from trifunctional amino acids such as L-glutamic acid and L-aspartic acid which are cyclized to form the diketopiperazines.

The diketopiperazines can generate a bis-carboxylic acid platform that can also be condensed with other amino acids to form the disturbing agent. Typically, diketopiperazines react and covalently link to one or more of the amino acids through the functional groups of the R groups of the diketopiperazines. These unique systems, due to the -cis geometry distributed by the chiral components of the diketopiperazine ring (Lannom, H. K. et al., Jnt. J.

Peptide Protein Res. , 28, 67-78 (1986)), provide an opportunity to systematically alter the structure of terminal amino acids while maintaining the orientation between them fixed relative to non-cyclic analogues (Fusaoka et al., Jnt. J. Peptide Protein Res., 34, 104-110 (1989); Ogura, H. et al., Chem. Pharma Bull., 23, 2474-2477 (1975) See also Lee, BH et al., J. Org. Chem., 49, 2418-2423 (1984), Buyle, R., Helv. Chim. Acta, 49, 1425, 1429 (1966) Other polymerization methods known to those skilled in the art can be given for polymerization of amino acids / diketopiperazines.

The disrupting amino acids / diketopiperazines may include one or more of the same or different amino acids as well as one or more different diketopiperazines as described above.

The ester and amide derivatives of these disturbing amino acid / diketopiperazine agents are also useful in the present invention.

Co-Amino Acids or Modified Complex Formation Agents The modified amino acids, polyamino acids or peptides are either acylated or sulphonated and include amino acid amides and sulfonamides.

Modified amino acids are typically prepared by modifying the amino acid or an ester thereof.

Many of these compounds are prepared by acylation or sulfonation with agents having the formula: X-Y-R4 wherein: R4 is the appropriate radical to produce the modification indicated in the final product, OR And it is C or C02, and X is a residual group. Typical residual groups include, but are not limited to, halogens, such as chlorine, bromine, and iodine. In addition, the corresponding anhydrides are modifying agents.

Acylated Amino Acids As Disturbing Complex Formation Agents Special mention is made of acylated amino acids having the formula: wherein Ar is a substituted or unsubstituted phenyl or naphthyl; O O Y is -C-, R14 has the formula -N (R16) -R15-C-, where: R 15 is C 1 to C 2 alkyl, Ci to C 24 alkenyl, phenyl, naphthyl, (C 1 to C 1 alkyl) phenyl, (C to C alkenyl) phenyl, (C 1 to C 1 alky) naphthyl, (C to C al alyl) naphthyl, phenyl ( Ci to Cι alkyl), phenyl (Ci to Cι alkenyl), Cι alkenyl, naphthyl (Ci to Cι alkyl), and naphthyl (C to C alkenyl); R) is optionally substituted with Ci to C4 alkyl, Ci to C4 alkenyl, Ci to C4 alkoxy, -OH, -SH, and -C02R5, cycloalkyl, cycloalkenyl, heterocyclic alkyl, alkaryl, heteroaryl, heteroalkyl, or any combination of these; R17 is hydrogen, Ci to C4 alkyl or Ci to C4 alkenyl; R15 is optionally interrupted by oxygen, sulfur, or any combination thereof; and R16 is hydrogen, Ci to C4 alkyl or Ci to C4 alkenyl.

Special mention is made for those who have the formula: O O R18-C-N- (R20-C) -OH V, 19 wherein: R18 is (i) C3-C10 cycloalkyl, optionally substituted with C1-C7 alkyl. C2-C7 alkenyl, C1-C7 alkoxy, hydroxyl, phenyl, phenoxy or -C02R21, wherein R21 is hydrogen, C1-C4 alkyl, or C2-C alkenyl; or (ii) CI-CT alkyl substituted with C3-Cyclocycloalkyl; R19 is hydrogen, C1-C4 alkyl, or C2-C4 alkenyl; R 2o is C 1 -C 24 alkyl, C 2 -C 24 alkenyl, cycloalkyl C 3 -C 10, C 3 -C 0 cycloalkenyl, phenyl, naphthyl, (C 1 -C 10 alkyl) phenyl, (C 2 -C 8 alkenyl) phenyl, (C 1 -C 4 alkyl) naphthyl, (C 2 -C 8 alkenyl) naphthyl , phenyl (Ci-Cι alkyl), phenyl (C 2 -C y alkenyl), naphthyl (Ci-Cι alkyl). or naphthyl (C2-C? alkenyl); .20 is optionally substituted with C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 1 -C 4 alkoxy, -OH, -SH, and -C 0 2 R) C 3 -C 10 cycloalkyl, C 3 -C 10 cycloalkenyl, a heterocycle having 3 -10 ring atoms wherein the heteroatom is one or more of N, O, S, or any combination thereof, aryl, (C1-C10 alkyl) aryl, ar (CL-CIO alkyl). or any combination of these; R '20 is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; and R22 is hydrogen, C4-C4 alkyl, or C2-C alkenyl. Some preferred acylated amino acids include salicyloyl phenylalanine, and the compounds having the formulas: fifteen 30 fifteen XIV twenty XVIII fifteen twenty XXIII twenty XXVII XXVIII 30 XXXII XXXIII 10 XXXIV XXXVI 30 XXXVII XXXVIII XXXIX 30 XLIIA XLIII 25 30 XLVII XLVIII 10 15 twenty Lll LIV 11-1 ill-1 3 IV-1 V-1 10 Vi-1 VII-1 VIII-1 IX-1 X-1 fifteen XI-1 XII-1 XIII-1 XIV-1 XV-1 XVI-1 XVII-1 XVlil-1 XIX-1 XX-1 XXI-1 10 XXII-1 XXIII-1 XXIV-1 XXV-1 XXVI-1 XXVII-1 XXVIH-1 XXIX-1 XXX-1 XXXI-1 XXXII-1 XXX1I1-1 XXXIV-1 XXXV- 1 XXXVI-1 Compound n m X XXXVIIA 0 0 4-C1 XXXVI11A 3 0 H XXIXA 3 1 4-CH3 XLA 3 1 2-F XLIA 3 1 2- CH3 XLIIA 3 or 3- CF3 XLIIIA 3 4 H XLIVA 3 0 3-C1 XLVA 3 0 3-F XLVIA 3 0 3- CH3 XLVIIA 0 or 2- CF3 XLVIIIA 1 2 H XLIXA 3 2 2-F LA 3 0 3, 4-0 CH20 LIA 3 0 2 -COOH LILA 1 0 2 -OH LIIIA 3 0 2, 6-dihydroxyl LIVA 2 0 2 -OH LVA 0 0 2, 4-dif luoro LVIA 2 0 2, 6-dihydroxyl LVII 0 0 4- CF3 LVIIIA 3 0 3-NMe2 LIXA 2 0 3-NMe2 LXA O 0 2, 6-dimeti LXIA 3 0 2-N02 LXIIA 3 0 2- CF3 LXIIIA 3 0 4-n-Pr LXIVA 3 0 2- NH2 LXVA 3 0 2-OCH3 LXVIA 3 0 3-NO2 LXVIIA 3 0 3- NH2 LXVIIIA 2 0 2- N02 LXIXA 2 0 2- NH2 LXXIA 2 0 2- OCH3 LXXIIA 2 0 2- OCF3 B Compound n X LXXIIIB 4-CF3 LXXIVB 1 2-F LXXVB 1 4- CF3 LXXVIB 3 3, 4-dimethoxy LXXVIIB 0 3-OCH3 LXXVIIIB 3 3-OCH3 LXXIXB 3 2, 6-dif luror LXXXB 3 4- CH LXXXIB 1 4-OCH3 LXXXIIB 2 2-F LXXXIIIB 0 2-F LXXXIVB 2 4-OCH3 LXXXVB 0 2-OCH3 LXXXVIB 2 2-OCH3 LXXXVIIB 0 4-CF3 LXXXVIIIB 3 3-F LXXXIXB 3 2-OCH3 X Compound n m X VII-C 3 0 2- carboxycyclohexyl VIII-C 3 3 Ciciohexyl IX-C 3 0 2-adamantyl X-C 3 0 1-morpholino Compound m xi-D 0 XI I -D 3 Compound X XIII-E OH XIV-E = 0 Compound n XV-F 0 XVI-F 2 C-1 CI-1 CII-1 CHI-1 CIV-1 CV-1 CVI-1 CVII-1 CVIII-1 CtX-1 Compound n m X CXI-G 6 0 2 -OH CXII-G 7 3 H CXI I I -G 7 0 2-1 CXIV-G 7 0 2-Br CXV-G 7 0 3-N02 CXVI-G 7 0 3-N (CH 3) 2 CXVII-G 7 0 2-N02 CXVIII-G 7 0 4-N02 CXIX-G 9 0 2 -OH H Compound X CXX-H 1-morpholino CXXI-H O-t-butyl CXXII-H CH (CH 2 P h) NC (O) O-t-Bu CXXIII-H 2-hydroxyphenyl of the organic acid compounds, and their salts, which have an aromatic amido group, which have a substituted hydroxyl group in the ortho position of the aromatic ring, and a lipophilic chain with about 4 carbon atoms up to about 20 atoms in the chain which they are as useless as disturbing agents. In a preferred manner, the lipophilic chain can have from 5 to 20 carbon atoms.

Disturbing agents that are also useful also include those that have the formula: 2-HO-Ar-CONR -R'-COOH wherein Ar is a substituted or unsubstituted phenyl or naphthyl; R7 is selected from the group consisting of C4 to C2o alkyl. C4 to C20 alkenyl. phenyl, naphthyl, (alkyl Ci to Cío) phenyl, (alkenyl Ci to Cío) phenyl, (alquil) Ci a Cio) naphthyl, (alkenyl Ci to Cio) naphthyl, phenyl (C 1 to C 1 alkyl), phenyl (C to C alkenyl). naphthyl (Ci to Cι alkyl), and naphthyl (Ci to Cι alkenyl); R8 is selected from the group consisting of hydrogen, Ci to C4 alkyl, Ci to C4 alkenyl, hydroxyl and Ci to C4 alkoxy; R7 is optionally substituted with Ci to C4 alkyl, Ci to C4 alkenyl, Ci to C4 alkoxy, -OH, -SH, and -C02R9, or any combination thereof; R9 is hydrogen, C1 to C4 alkyl, Ci to C4 alkenyl; R is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof; with the proviso that the compounds are not substituted with an amino group in the alpha position of the acid group, or salts thereof.

Preferred R6 groups are C4 to C2o alkyl, and C4 to C20 alkenyl. The most preferred groups Rd are C5 to C2o alkyl. and C5 to C2o alkenyl.

A preferred carrier compound may have the formula: where R7 is defined previously. Special mention is made of the compounds having the formula: where A is Tri, Leu, Arg, Trp, or Cit; and optionally where A is Tri, Arg, Trp, or Cit; A is acylated in two or more functional groups.

Preferred compounds also include those wherein A is Tri; A is Tri and is acylated into two functional groups; A is Tir; A is Tir and is acylated into two functional groups; A is Leu; A is Arg; A is Arg and is acylated into two functional groups; A is Trp; A is Trp and is acylated into two functional groups; A is Cit; and A is Cit and is acylated into two functional groups.

Special mention is also made of the compounds having the formula: where A is Arg or Leu; and wherein if A is Arg, A is optionally acylated in two or more functional groups; wherein A is Leu or Phenylglycine; LVIII wherein A is Phenylglycine; Y where A is Phenylglycine.

The acylated amino acids can be prepared by reacting the individual amino acids, mixtures of two or more amino acids, or amino acid esters with a modifying amino agent that reacts with the free amino moieties present in the amino acids to form the amides.

Suitable but not limiting examples of the acylating agents useful in the preparation of the acylated amino acids include acid chloride acylating agents having the 0 formula R¿ -C-X where; R23 is prepared as an appropriate group for the modified amino acid, such as, but not limited to, alkyl, alkenyl, cycloalkyl, or aromatic, and particularly ethyl, methyl, cyclohexyl, iclphenyl, phenyl or benzyl, and X is a residual group. Typical residual groups include, but are not limited to, halogens such as chlorine, bromine and iodine.

Examples of the acylating agents include, but are not limited to, acyl halides including, but not limited to, acetyl chloride, propyl chloride, cyclohexanoyl chloride, cyclopentanoyl chloride, and cycloheptanoyl chloride, benzoyl chloride , hipuril chloride, and the like; and anhydrides, such as acetic anhydride, propyl anhydride, cyclohexanoic anhydride, benzoic anhydride, hippuric anhydride, and the like. Preferred acylating agents include benzoyl chloride, hipuryl chloride, acetyl chloride, cyclohexanoyl chloride, cyclopentanoyl chloride, and cycloheptanoyl chloride.

Amino groups can also be modified by the reaction of a carboxylic acid with coupling agents such as the carbodiimide derivatives of the amino acids, particularly the hydrophilic amino acids such as phenylalanine, tryptophan, and tyrosine. Other examples include dicyclohexylcarbo-diimide and the like.

If the amino acid is multifunctional, ie it has more than one group -OH, -NH2 or -SH, then it can be acylated optionally in one or more functional groups to form, for example, an ester, amide or thioester of ligation For example, in the preparation of many acylated amino acids, the amino acids are dissolved in an aqueous alkaline solution of a metal hydroxide, for example, sodium or potassium hydroxide, and a acylating agent is added. The reaction time may vary from about one hour to about 4 hours, preferably about 2-2.5 hours. The temperature of the mixture is maintained at a temperature that generally ranges from about 5 ° C to about 70 ° C, preferably between 10 ° C to about 50 ° C. The amount of alkali employed by an NH2 group equivalent in the amino acids generally ranges from about 1.25 moles to about 3 moles, and is preferably between about 1.5 moles and about 2.25 moles per NH2 equivalent. The pH of the reaction solution generally ranges from about a pH of 8.0 to about a pH of 13 and is preferably between about pH 10 and about pH 12. The amount of the modifying amino agent used in relation to the amount of amino acids is based on the moles of total free NH2 in the amino acids. In general, the amino modifying agent is employed in an amount ranging from about 0.5 to about 2.5 moles equivalents, preferably from about 0.75 to about 1.25 equivalents, per molar equivalent of the total NH2 groups in the amino acids.

The modified reaction in amino acid formation is quenched by adjusting the pH of the mixture with a suitable acid, for example, concentrated hydrochloric acid, until the pH reaches from about 2 to about 3. The mixture is separated upon standing at room temperature to form a transparent top layer or an objective or off-white precipitate. The top layer is discarded, and the modified amino acids are collected by filtration or decantation. The crude modified amino acids are then mixed with water. The insoluble materials are removed by filtration and the filtrate is dried under vacuum. The production of the modified amino acids generally has a range of between about 30 and about 60%, and usually about 45%. The present invention also contemplates amino acids that have been modified by multiple acylation, for example, diacylation or triacylation.

If desired, the amino acid esters, such as, for example, benzyl, methyl, or ethyl esters of the amino acid compounds, can be used to prepare the modified amino acids of the invention. The amino acid ester, which is dissolved in a suitable organic solvent such as dimethylformamide, pyridine, or tetrahydrofuran, is reacted with the appropriate modifying amino agent at a temperature ranging from 5 ° C to about 70 ° C, preferably about 25 ° C, during a period ranging from approximately 7 to approximately 24 hours. The amount of amino acid modifier used relative to the amino acid ester is the same as described above for amino acids. This reaction can be carried out with or without a base such as, for example, triethylamine or diisopropylethylamine.

If the amino acid or amide esters are the raw material, they are dissolved in a suitable organic solvent such as dimethylformamide or pyridine, they are reacted with the amino modifying agent at a temperature ranging from about 5 ° C to about 70 ° C, preferably about 25 ° C, for a period ranging from about 7 to about 24 hours. The amount of amino modifying agents used relative to the amino acid esters is the same as that described for the above amino acids.

After this, the reaction solvent is removed under negative pressure and optionally the ester or amide functionality can be removed by hydrolyzing the modified amino acid ester with a suitable alkaline solution., for example, sodium hydroxide IN at a temperature ranging from about 50 ° C and about 80 ° C, preferably about 70 ° C, for a period of time sufficient to hydrolyse out the ester group and form the modified amino acid having a free carboxyl group. The hydrolysis mixture is then cooled to room temperature and acidified, for example, with an aqueous solution of 25% hydrochloric acid, to a pH ranging from about 2 to about 2.5. the modified amino acid is precipitated out of the solution and recovered by conventional methods such as filtration or decantation.

The modified amino acids can be purified by acid precipitation, by recrystallization, or by fractionation on solid column supports. Fractionation can be carried out on suitable column solid supports sas silica gel, alumina, using solvent mixtures sas acetic acid / butanol / water as the mobile phase; the reversible phase column supports utilize trifluoroacetic acid / acetonitrile as the mobile phase; and ion exchange chromatography using water as the mobile phase. The modified amino acids can also be purified by the fraction with a reduced alcohol sas methanol, butanol, or isopropanol to remove impurities sas inorganic salts.

The modified amino acids are generally soluble in aqueous alkaline solutions (pH > 9.0); partially soluble in ethanol, n-butanol and a 1: 1 (v / v) solution of toluene / ethanol and insoluble in neutral water. The alkali metal salts, for example, the sodium salt of the amino acid derivatives are generally soluble in water at a pH of about 6-8.

In polyamino acids or peptides, one or more of these amino acids can be modified (acylated). The modified polyamino acids and modified peptides may include one or more acylated amino acids. Although the modified linear peptides and polyamino acids generally include only one acylated amino acid, other configurations of polyamino acids and peptides may include more than one acylated amino acid. The polyamino acids and peptides can be polymerized with the acylated amino acids or can be acylated after the polymerization.

Special mention is made of the compound: where A and B are each Arg or Leu.

Sulfonated Amino Acids As Disturbing Complex Formation Agents The modified sulfonated amino acids, polyamino acids, and peptides are modified by sulfonating at least one free amino group with a sulfonating agent that reacts at least one of the free amino groups present.

Special mention is made to the compounds of the formula: wherein Ar is a substituted or unsubstituted phenyl or naphthyl; O Y is -S02-, R2 has the formula -N (R26) -R -C-, where: R is Ci to C24 alkyl, Ci to C24 alkenyl, phenyl, naphthyl, (Ci to Cio alkyl) phenyl, (Ci to Cio alkenyl) phenyl, (Ci to Cio alky) naphthyl, (Ci to Cio alkenyl) naphthyl, phenyl ( Ci to Cι alkyl), phenyl (Ci to Cι alkenyl), naphthyl (Ci to Cι alkyl), and naphthyl (C to C alkenyl); R 2'5 is optionally substituted with Ci-alkyl a C4, alkenyl Ci to C, alkoxy Ci to C, -OH, -SH, and -C02R27, or any combination thereof; R is hydrogen, Ci to C4 alkyl, Ci to C4 alkenyl; R is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof; Y R is hydrogen, alkyl Ci to C, alkenyl Ci to C4 Suitable, but not limiting examples of the sulfonating agents useful for preparing sulfonated amino acids include the sweetening agents having the formula R28-S02-X wherein R28 is a group appropriate for the amino acid modified, but not limited to, alkyl, alkenyl, cycloalkyl or aromatics, and X is a residual group as described above. A group of a sulfonating agent is sulfonylbenzene chloride.

The modified polyamino acids and peptides may include one or more sulfonated amino acids. Although the linearly modified polyamino acids and peptides that are generally used include only one sulfonated amino acid, other amino acid and peptide configurations may include more than one sulfonated amino acid. The polyamino acids and peptides can be polymerized with the sulfonated amino acids or can be sulfonated after the polymerization. Special mention is made to the compound: 0 NHS02P HO Modified Derived Amino Acids As Disturbing Complex Formation Agents Derivatives of the modified amino acids, polyamino acids or peptides are amino acids, polyamino acids or peptides which have at least one term -acyl converted to an aldehyde or ketone and are acylated in at least one free amino group, with a reactive acylating agent with at least one of the free amino groups present.

Amino acid derivatives, polyamino acids or peptides can be easily prepared by reducing the amino acids or ester peptides with an appropriate reducing agent. For example, amino acid aldehydes, polyamino acids, or peptides can be prepared as described in the article by Chen et al. , Biochemistry, 1979, 18, 921-926. Amino acid ketones, polyamino acids or peptides can be prepared by the procedure described in Organic Syntheses, Col. Vol. IV, Wiley, (1963), page 5. Acylation is discussed above.

For example, derivatives may be prepared by reacting an individual branch of an amino acid or peptide inoácido polia, or mixtures of two or more amino acids or derivatives pé'ptidos with acylating agent or an amine modifying agent which reacts with free portions amino acids present in the derivatives to form the amides. The amino acid, polyamino acid or peptide can be modified and subsequently derivatized, derivatized and subsequently modified, or simultaneously modified and derivatized. Protective groups can be used to avoid undesired side reactions as known to those skilled in the art.

In modified derivatives of polyamino acids or peptides, one or more amino acids can be derived (an aldehyde or ketone) and / or modified (acylar) but there must be. at least one derivative and at least one modification.

Special mention is made of the modified amino acid derivatives N-cyclohexanoyl-Fe-aldehyde. N-acetyl-Tir-ketone, N-acetyl-Lis-ketone and N-acetyl-Leu-ketone, and N-cyclohexanoyl phenylalanine aldehyde.

Carboxylic Acid as a Disturbing Complex Formation Agent Various carboxylic acids and salts of these carboxylic acids can be used as disturbing complexing agents. These carboxylic acids have the formula: R29-C02H LXII wherein R29 is Ci to C24 alkyl, C2 to C24 alkenyl, C3 to Cio cycloalkyl. cycloalkenyl C3 to Clo, phenyl, naphthyl, (alkyl Ci to Clo) phenyl, (alkenyl C2 to Clo) phenyl, (alkyl da Clo) naphthyl, (alkenyl C2 to Clo) naphthyl, phenyl (alkyl Ci to Clo), phenyl ( C 2 to Cι alkenyl), naphthyl (O. to Cι alkyl), and naphthyl (C 2 to Cι alkenyl); R is optionally substituted with Ci to Cio alkyl, C2 to Cio alkenyl, Ci to C4 alkoxy, -OH, -SH, and -C02R30, C3 to Cio cycloalkyl. C3 cycloalkenyl to Cio. a heterocyclic having from 3 to 10 ring atoms wherein the heteroatom is one or more atoms of N, O, S, or any combination thereof; aryl, (C 1 to Cι) aryl, aryl (Ci to Cι) alkyl or any combination thereof; R > 2"9 is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; R is hydrogen, Ci to C4 alkyl, or C2 to C4 alkenyl; Preferred carboxylic acids are cyclohexanedicarboxylic 2-cyclohexanecarboxylic acid, acid, cyclopentanecarboxylic, cycloheptanecarboxylic acid, hexanoic acid, 3-ciclohexanopropanoico, methylcyclohexanecarboxylic acid, 1, 1,3-ciclohexanosicarboxílico acid, 1,4-cyclohexanedicarboxylic acid, 1 -adamantanocarboxílico, phenylpropanoic acid, adipic acid, ciclohexanopentanoico acid, ciclohexanobutanoico acid, ácidopentilciclohexanoico, 2-ciclopentanohexanoico, ciciohexano pentanioci acid, hexanedioic acid, ciclohexanobutanoico acid and acetic ciclohexno (4-methylphenyl) acid.

Other Examples of Perturban Complexing Agents Although all interfering agents forming complexes that may form the 'supramolecular complexes described in this section are within the scope of the present invention, other examples of the interfering agents complexing include but not limited to, 2 -carboxymethyl-phenylalanine-leucine; 2-benzyl succinic acid, and actinonin, phenylsulfonyl aminophenyl butyric acid.

LXIII LXtV Mimetic Agents The mimetics within the scope of the present invention are structural and / or functional equivalents of an original entity. The structurally and chemically functional mimetics of the supramolecular complexes and the transportable and reversible intermediate states of the active agents are not necessarily peptidic, since the non-peptidic mimetics can be prepared having the appropriate structural chemical properties. However, the preferred mimetics are peptides which have a different primary structure than the supramolecular complex or the intermediate state, but retain the same secondary and tertiary structure of the supramolecular complex or intermediate state complex. Although the mimetics have less bioactivity than in the natural state or in an intermediate state of active agent or supramolecular complex, the mimetics may possess other important properties that they do not possess in the natural state, such as, for example, greater increase in the ability to administer orally.

Methods of preparation of these mimetic agents are described, for example, in Yamazaki et al., Chirality 3: 268-276 (1991); Willey et al., Peptidomimetics Derived From Natural Products, Medicinal Research Teviews, Vol. 13, No. 3, 327-384 (1993); Gurrath et al., Eur. J. Biochem 210: 991-921 (1992); Yamazaki et al., Int. J. Peptide Protein Res. 37: 314-323 (1991); Clark et al., J. Med. Chem. 32: 2026-2038 (1989); Portoghese, J. Med. Chem. 34: (6) 1715-1720 (1991); Zhou et al., J. Immunol. 149 (5) 1763-1769 (September 1, 1992); Holzman et al., J. Protein Chem. 10: (5) 553-563 (1991); Masier et al., Biotechnology 10 (July 1992); Olmsteel et al., J. Med. Chem. 36: (1) 179-180 (1993); Mallin et al., Peptides 14: 47-51 (1993); and Kouns et al., Blood 80 (10) 2539-2537 (1992); Tanaka et al., Biophys. Chem. 50 (1994) 47-61; DeGrado et al., Sciuence 243 (February 3, 1989); Regan et al., Science 241: 976-978 (August 19, 1988); Matouschek et al., Nature 340: 122-126 (July 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); Ptitsyn et al., Biopolymers 22: 15-25 (1983); Ptitsyn et al., Protein Engineering 2 (6): 443-447 (1989).

For example, protein structures are determined by the collective intra- and intermolecular interactions of the constituent amino acids. In alpha helices, the first and fourth amino acids in the helix interact non-covalently with one another. This pattern is repeated through the complete helix except for the last 4 and the first 4 amino acids. In addition, the side chains of amino acids can interact with each other. For example, the phenyl-side chain of phenylalanine will probably not be exposed to a solvent if this phenylalanine is found in the helix. If the interactions of this phenylalanine contribute to the stability of the helix, then replacing an alanine by a phenylalanine interrupts the helix and changes the conformation of the protein.

Therefore, a mimetic agent is manufactured by first determining which side chains of amino acids become exposed by a solvent and at least they are removed from contributing to the stabilization of the natural state such as the exploratory mutagenesis technique. Mutants containing amino acid substitutions in these same locations can be created so that the substituted amino acids provide the protein conformation in a more intermediate way than in a more natural way. Confirmation that the proper structure is sintered can come from spectroscopy and other analytical methods.

Distribution Compositions The distribution compositions including the supramolecular complex described above, are typically formulated by mixing an effective amount of a disrupting agent appropriate for the chosen route of distribution, i.e., subcutaneous, nasal, or sublingual, with the active agent. The components can be prepared long before administration or can be mixed just prior to administration.

The distribution compositions of the present invention may also include one or more enzyme inhibitors. Such enzyme inhibitors include, but are not limited to compounds such as actinonin or epiactinonin and derivatives thereof. These compounds have the following formulas: Actinonin Epiactinonin LXV LXVI Derivatives of these compounds are disclosed in U.S. Patent No. 5,206,384. The actinonin derivatives have the following formula: LXVII wherein R 31 is sulfoxymethyl or carboxyl or a substituted carboxyl group selected from the carboxamide, hydroxyaminocarbonyl and alkoxycarbonyl groups; and R32 is a hydroxyl, alkoxy, hydroxyamino or sulfoxyamino group. Other enzyme inhibitors include, but are not limited to, aprotinin (Trasylol) and the Bo man-Birk inhibitor.

The stabilizing additives can be incorporated into a carrier solution of the supramolecular complex. With some drugs, the presence of such additives promotes the stability and dispersibility of the agent in the solution.

The stabilizing additives can be used at a concentration with a range between about 0.1 and 5% (W / V), preferably about 0.5% (W / V). suitable but not limiting examples of the stabilizing additives include acacia gum, gelatin, methyl cellulose, polyethylene glycol, carboxylic acids and salts thereof, and polylysine. The preferred stabilizing additives are acacia, gelatin and methyl cellulose.

The amount of the active agent is an amount effective to achieve the purpose of the particular active agent. The amount of the composition is typically a pharmacologically or biologically effective amount. However, the amount may be less than a pharmacologically or biologically effective amount when the composition is used in unit dosage form, such as a capsule, tablet or liquid, because the unit dosage form may contain a multiplicity of carrier compositions. / or of biologically or chemically active agents or may contain a pharmacologically or biologically effective divided amount. The total effective amounts can then be administered in cumulative units containing, in total, pharmacologically or biologically or chemically active amounts of a biologically or pharmacologically active agent.

The total amount of the active agent, and in particular of a biologically or chemically active agent to be used, can be determined by those skilled in the art. However, it has been surprisingly found by some biologically or chemically active agents, the use of the disclosed carriers provides an extremely efficient distribution, particularly in the intranasal, sublingual, or subcutaneous systems. Therefore, smaller amounts of biologically or chemically active agents than those used in the above forms of unit doses or delivery systems can be administered to the subject, while still achieving the same blood levels and therapeutic effects.

The amount of the disturbing agent used is an effective distribution amount.

The compositions of the present invention may be formulated in unit doses by the addition of one or more excipients, diluents, disintegrants, lubricants, plasticizers, colorants, or dosing vehicles. Preferred unit dosage forms are oral unit dose forms. The most preferred unit dosage forms include, but are not limited to, tablets, capsules, or liquids. The unit dosage forms may include biologically, pharmacologically, or therapeutically effective amounts of the active agent or may include less than such amount if the multiple forms of unit doses are used to administer a total dose of the active agent. Unit dosage forms are prepared by conventional methods in the art.

The invention of the subject is useful for administering biologically active agents to any animal such as birds, mammals, such as primates and particularly humans, and insects. The systems are particularly advantageous for distributing chemically or biologically active agents that are otherwise destroyed or less effective by the conditions encountered before the active agent in its natural state reaches its target zone (i.e. which the active agent is distributed) and by the. conditions inside the body of the animal to which they are administered. Particularly, the present invention is useful in administering the active agents subcutaneously, intranasally or sublingually.

DESCRIPTION OF THE PREFERRED MODALITIES The following examples illustrate the invention without limitation. All parts and percentages are by weight unless otherwise indicated.

Example 1 - Natural gels from a-Interferon Natural gradient gels were run (Pharmacia) with 647 μg / ml of α-Interferon, (Intron-A-Schering Plow) and increased amounts (10-500 mg / ml) of the disrupting agent (mixture of L-Valine, L-Leucine, L-phenylalanine, L-lysine and L-arginine modified with benzenesulfonylchloride) (valine-7.4%, leucuna-16.5%, phenylalanine-40.3%, lysine-16.2%, and arginine-19.6%). 4μl of the material was loaded into the gel using a 6/4 comb for loading.

The results are illustrated in Figure 1 Path 1 = High molecular weight marker (Bio-Rad) - in 1:20 dilution w / d H20 - (5μl -> 100μl). Path 2 = α-interferon A (67μg / mL) control 5μl + 5μl bromophenol blue (BPB) - (1.29μg loaded). Path 3 = a-interferon + disturbing agent (lOmg / mL) - 50μl of a-interferon + 50μl of BPB = lOOμl (1.29μg loaded). Path 4 = a-interferon + disturbing agent (50mg / mL) 50μl of a-interferon + 50μl BPB = lOOμl (1.29μg loaded). Path 5 = a-interferon + disrupting agent (lOOmg / mL) 50μl of a-interferon + 50μl BPB = lOOμl (1.29μg loaded). Path 6 = a-interferon + disturbing agent (500mg / mL) 5μl of a-interferon + 50μl BPB = lOμl (1.29μg loaded).

Example 1 A - Natural Gradient α-Interferon Gel The method of Example 1 was followed by substituting the thermal condensation product of glutamic acid, aspartic acid, tyrosine and phenylalanine (Glu-Asp-Tir-Phe) which was fractionated through a molecular weight truncation filter. 3000 for the disturbing agent. The results are illustrated in Figure 2.

Samples Path 1 = High molecular weight marker (Bio-Rad).

Path 2 = a-interferon (647μg / mL) - 5μl + 5μl BPB control. Path 3 = a-interferon + disturbing agent (lOmg / mL) - 50μl + 50μl BPB = lOOμl. Path 4 = a-interferon + disturbing agent - 50μl + 50μl BPB = lOOμl. Path 5 = a-interferon + disturbing agent (lOOmg / mL) - 50μl Intron A + 50μl BPB = lOOμl. Path 6 = a-interferon + disturbing agent (500mg / mL) - 5μl Intron A + 50μl BPB = lOOμl.

Examples 1 and IA illustrate that the individual α-interferon (Lane 2 in Figures 1 and 2) shows bands in appropriate molecular weight (approximately 19,000 daltons). As the amount of aggregate disturbing agent is increased in each subsequent path relative to a fixed concentration of α-Interferon, α-Interferon migrates to a lower molecular weight, rather than a high molecular weight. The change observed with the disrupting agent of Example 1 is more pronounced than that observed with the disrupting agent of Example Ia. This indicates that the structure of the α-Interferon is changing due to two different disturbing agents, because if the structure does not change, there would be a shift towards a higher molecular weight while the disrupting agent is formed complete with the active agent.

Example 2 - Isothermal Titration Calorimetry A dosing composition of the disrupting agent of Example 1 at 2.4mM and sCt at 0.3mM was prepared, and an isothermal titration calorimetry at a pH of 6.5 and 4.5 was carried out. the buffer at a pH of 6.5 was 30mM Hepes-30mM NaCl, and the buffer at pH 4.5, was 30mM sodium acetate-NaCl.

All experiments were carried out at 30 ° C using 8. OmM of disturbing agent in a drip syringe and 1. OmM of calcitonin in the calorimeter cell. In all experiments, increments of 15.10μl of disturbing agent were added in additions of 10 seconds duration with 2 minutes of equilibration between the additions.

The results were validated in experiments where the disrupting agent (8mM) was placed in the drip syringe, and equivalent increments were added to a pH buffer of 4.5 (without sCt) and where the disturbing agent was put into the syringe of drip and lOμl increments were added to a buffer of pH 6.5 (without sCt). The titration curves were not obtained in these experiments, and the results showed that the heat of the mixture with the dilution of the disturbing agent was negligible. Therefore, the experimental isotherms were not corrected by a background subtraction. The results are illustrated in Table 1 below. Example 2A The method of Example 2 was followed by replacing the disrupting agent of Example 1 A. The results were validated in the experiments where the disrupting agent was placed in the drop syringe. and equivalent increments were added to a pH 4.5 buffer (without sCt). The results are shown in Table 1 below.

Example 3 - Denaturation with a-Interferon GuHCl A solution is prepared in the presence of 9.1 mg / mL of α-Interferon (Schering Plow Corp.) in 20 mM of a sodium phosphate buffer at a pH of 7.2.

The examples were prepared by diluting the α-Interferon with the sodium phosphate buffer and 10 M guanidine hydrochloride (GuHCl) (Sigma Chmeical Co. - St. Louis, MO) at 200 ug / mL of a concentration of α-Interferon a various concentrations of GuHCl. The diluted samples were allowed to reach equilibrium by incubation for approximately 30 minutes at room temperature before quantification. 1 The calorimetry experiments were performed essentially as detailed by You, J.L., Scarsdale, J.N., and Harris, R.B., J. Prot. Chem. 10: 301-311, 1991; You, Jng, Page, Jimmy D., Scarsdale, J. Neel., Colman, Robert W., and Harris, R. B., Peptides 14: 867-876, 1993; Tyler-Cross, R., Sobel, M., D. F. and Harris, R. B., Arch. Biochem biophys. 306: 528-533, 1993; Tyler-Cross, R., Sobel, M. Marques, D., and Harris, R. B., Protein Science 3: 620-627, 1994.

The fluorescence quantifications were made at 25 ° C using a Hitachi F-4500. Protein fluorescence of tryptophan was observed at a wavelength of 298 nm and at a wavelength of 343 nm. Fluorescence ANS (l-anilinonaphthalene-8-sulfonate) was observed at an excitation wavelength of 355 nm and at an emission wavelength of 530 nm. For all fluorescence quantifications, a spectral collision of 5 nm was chosen for both excitation and emission. The results are illustrated in Figure 3.

Example 4 - Effect of Concentration of GuHCl in the Configuration of α-Interferon A stock solution of 5 M GuHCl was prepared using 20 mM sodium phosphate, and buffer at pH 7.2. After dilution, the pH of the stock solution was verified and adjusted by concentrated HCl. To determine the concentration of the final solution of the refractive index referenced in Methods in Enzymology, Vol. 6, page 43 by Yasuhiko Nozaki was used.

A stock solution of α-Interferon (9.1 mg / mL) was mixed with sufficient amounts of GuHCl to produce the concentrations of Table 1 A below: Table 1 A - α-Interferon / GuHCl solutions An exploratory differential calorimetry (DSC) was run, and the results are illustrated in Figure 4.

Example 5 - pH Titration of Intron A as Quantified by Intrinsic Fluorescence of Tryptophan A solution is prepared in the presence of 9.1 mg / mL of α-Inferred in 20mm of a sodium phosphate buffer at a pH of 7.2 (Schering Plow, Co.).

The samples were prepared by diluting the a-Interfer- ponte to a concentration of 200 μg / mL in a buffer solution at various pH values using the following buffers: glycine at a pH of 2 and 12, sodium phosphate at a pH of 3, 4, 5, 7 and boric acid at a pH of 8. These buffers are prepared as described in Practical Handbook of Biochemistry and Molecular Biology, Edited by Gerald D. Fashman, 1990. Diluted samples were allowed to reach equilibrium by incubation for approximately 30 minutes at room temperature before quantification.

The fluorescence was quantified according to the procedure of Example 3- the results are illustrated in Figure 5.

Example 6 - Titration of the pH of Insulin as Quantified by ANS Fluorescence A stock solution is prepared by dissolving 2 mg of insulin in 1 mL of deionized water. A solution in the presence of l-anilinonaphthalene-8-sulfonate (ANS) was prepared by dissolving 10 mg in 10 mL of deionized water. The samples are prepared by diluting the insulin to a concentration of 200 ug / mL where a buffered solution at various pH values using the following buffers: glycine at a pH of 2 and 12, sodium phosphate at a pH of 3.4 , 5, 7, and boric acid at a pH of 8. These buffers are prepared as described in Practical Handbook of Biochemistry and Molecular Biology, Edited by Gerald D. Fashman, 1990. The final concentration of ANS was 90ug / mL. The diluted samples were allowed to reach equilibrium by incubation for approximately 30 minutes at room temperature before quantification.

Fluorescence was quantified according to the procedure of Example 3. The results are illustrated in Figure 6.

Example 7 - Reversibility of the Circular Spectrum of a-Interferon Dichroism at a pH of 2 and 7.2 The circular spectrum of α-Interferon dichroism is generated at a pH of 7.2. the pH of the solution was adjusted again to a pH of 2, and the sample was re-scanned. The sample solution was re-adjusted to a pH of 7.2 and then re-scanned.

The concentration of α-Interferon is 9.2μM or 0.17848mg / mL, [(IFN) solution in existence = 9.1mg / mL)].

The buffers that are used with 2OmM of Na phosphate at a pH of 1.2; and 2OmM of glycine at pH 2.0.

The inversion of the pH to 7.2 results in a complete restoration of the natural structure, demonstrating the reversibility of the intermediate state. It is believed that the difference in free energy between the natural state and the intermediate state is small.

The results are illustrated in Figures 7 A and 7B.

Example 8 - Circular Spectrum of a-Interferon Dichroism to a pH Unit at 7.2 The extension of the ordered secondary structure of the α-Interferon at different pH is determined by the circular dichroism quantification (CD) in the far UV range. The greater factor of dilution of the solution in the presence of Interferon (~ 50 times) results in the sample being at the appropriate pH. The concentration of α-Interferon is 9.2μM or 0.17848mg / mL [(IFN) solution in stock = 9.1mg / mL)]. The buffers used are 2OmM of sodium phosphate at a pH of 6.0 and 7.2; 2OmM NaAc at pH 3.0, 4.0, 4.5, 5.0 and 5.5: and 20mM glycine at a pH of 2.0.

The content of the secondary structure is estimated with various adjustment programs, each of which decomposes the CD curve into 4 major structural components: a-helix, ß-leaf, turns and random winding. All these programs are provided with the CD instrument as an analysis facility. The first program uses 7 reference proteins: myoglobin, lysozyme, papain, cytochrome C, hemoglobin, ribonuclease A and chymotrypsin. The second uses Yang. REF as a reference file.

A third program, CCAFAST, uses the Convex Constraint Algorithm and is described in "Analysis of the Circular Dichroism Spectrum of Proteins Using the Convex Constraint Algorithm: A Practical Guide". (A. Perczel, K. park and G. D. Fas an (1992) Anal. Biochem, 203: 83-93).

The descircunvolución of the distant explorations of UV on a range of volumes of pH (2.0-7.2) indicates a significant compactación of the secondary structure to a pH of 3.5. The near UV scan indicates an interruption of the tertiary structure packing, and the far UV scanning indicates that there is still a significant secondary structure at this pH. The results are illustrated in Figure 8.

Example 9 - DSC of Insulin and Growing Concentrations of GuHCl The DSC was carried out with 6mg / mL of insulin (0.83mM assuming a molecular weight of 6,000) in 50mM of phosphate buffer, at a pH of 7.5. each subsequent term is adjusted by a historical subtraction of a 0.6M guanidine-phosphate buffer solution.

Insulin was prepared fresh as a concentrated solution in stock in 50 mM phosphate buffer at a pH of 7.5, and an appropriate aliquot is diluted in the buffer, filtered through a 2 micron PTFE filter, and degassed for minus 20 minutes The reference cell contains the degassed damper.

Exploratory calorimetry was carried out using 5mg 0.83mM porcine insulin (MW 6,000) per mL in 50mM phosphate buffer at a pH of 7.5. all thermograms were run on a Microcal MC-2 scanning calorimeter equipped with the DA2 data acquisition system operating in over-grade mode at ° C / min (up to 90 ° C), and the data points were collected at 20 second intervals. All scans start at least 20 ° C lower than the transitions observed for the active agent. All the thermograms are corrected for the subtraction of the baseline and normalized for the concentration of the macromolecule. According to the methods of Johns Hopkins Biocalori etry Center, see, for example Ramsay et al., Biochemistry (1990) 29: 8677-8693; Schon et al., Biochemistry, (1989) 28: 5019-5024 (1990) 29: 781-788. The DSC data analysis programs are based on the mechanical statistical decoupling of a thermally induced macromolecular fusion profile.

The effect of GuHCl on the structure is evaluated in the DSC experiments where the individual solutions are prepared in the phosphate amrotiguator, pH 7.5, containing the diluted denaturant of a 5M solution in stock at concentrations with a range of 0.5- 2M. The results are illustrated in Table 2 below.

Example 10 - Effect of Ionic Force on the DSC Spectrum of Insulin A sample containing 6mg / mL of insulin (0.83mM in 50mM of a phosphate buffer, at pH 7.5, containing 0.25, 0.5 or 1.0 M NaCl) was used. The thermograms were run according to the procedures of Example 9 and corrected by subtracting a vacuum of 0.5M NaCl phosphate buffer as described above.

The effect of increasing the ionic strength in the structure was evaluated in the DSC experiments where the individual solutions are prepared to contain NaCl at concentrations ranging from 0.25-3M.

The results are illustrated in Table 3 below.

Example 10 A - Effect of the Ionic Force on the DSC Spectrum of the rhGh In the method of Example 9, 5mg / mL of recombinant human growth hormone (rhGH) (225μM based on M, 22,128 HGH) is still substituted in 50mM of phosphate buffer at a pH of 7.5 containing either 0.5 or 1.0 M NaCl, for insulin. The thermograms are corrected by subtracting a vacuum of 0.5M NaCl-phosphate buffer.

The results are illustrated in the Table below.

Example 11 - Effect of pH on the DSC Spectrum of rhGH 5mg / mL of rhGh is dissolved in the buffer (0.17mM in 50mM of phosphate buffer, assuming a molecular weight of 20,000). The pH of the solution was adjusted to the desired value, and all the curves are corrected by a baseline subtraction.

The effect of the pH on the structure was evaluated by DSC according to the procedure of Example 9 wherein the individual solutions are prepared in the phosphate buffer with a pH of 2.0 to 6.0. The results are illustrated in Table 5 below. the rhGH An initial scan of rhGH was carried out at 10mg / mL in the absence of GuHCl (0.33mM assuming a molecular weight of 20,000). Therefore, the concentration of rhGH is decreased to 5mg / mL (0.17mM) in 50mM of phosphate buffer, at a pH of 7.5 containing various concentrations of GuHCl. Each subsequent thermogram is corrected by a background subtraction of a 0.5M solution of guanidine-dosate buffer. The thermograms are corrected by a subtraction of a vacuum of 0.5M NaCl phosphate buffer. The scans are executed according to the procedure of Example 9.

The results are illustrated in Table 6 below.

Example 13 - Dependence of the pH in the Conformation of α-Interferon A solution in the presence of α-Interferon (9.1mg / mL) was diluted with a buffer at a concentration of 0.6mg / mL. The sample was dialysed overnight in the buffer (a-Interferon volume ratio for the buffer is 1: 4,000). Since an extinction coefficient is not provided, the concentration of the sample used is determined by comparing the absorption spectrum of the sample before and after dialysis. For each particular pH, the absorbance of the non-dialyzed α-interferon of the known concentration was quantified at 280 nm. Then after the dialysis, the aobsorbancy was read again to take into account the protein loss, the dilution, etc. The conditions of the buffer and the concentrations of α-Interferon are: pH 3.0: Shock absorber - 20mM NaAc [IFN] 0.50 mg / mL pH 4.1: Shock absorber - 20mM NaAc [IFN] 0.53 mg / mL pH 5.0: Shock absorber - 2OmM NaAc [IFN] 0.37 mg / mL pH 6.0: Shock absorber - 2OmM Na [ IFN] 0.37 mg / mL Phosphate pH 7.2: Shock absorber 20mM Na [IFN] = 0.48 mg / mL Phosphate The DSC scans were carried out according to the procedure of Example 9. Although clear, the clear solutions of α-Interferon are obtained for each pH at room temperature, there were no noticeable signs of precipitation at pH 5.0 and 6.0 after the scans. Of temperature.

The results are illustrated in Table 7 below.

Example 14 - Effect of Concentration of GuHCl in the Conformation of α-Interferon The GuHCl / α-Interferon samples were prepared according to the method of Example 4. The DSC scans were carried out according to the procedure of Example 9.

The results are illustrated in Table 8 below: Examples 3-14 illustrate that the ionic strength, the concentration of the guanidine hydrochloride and the pH result in changes in the Tm of the active agents, indicating a change in conformation. This is confirmed by fluorescence spectroscopy. Reversible intermediate conformational states can be used as templates to prepare mimetics.

Example 15 - Preparation of Intermediate State Mimetic Agents of α-Interferon An intermediate conformational state of α-Interferon is determined. A peptide mimetic with a secondary and tertiary structure of the intermediate state is prepared.

Example 16 - Preparation of Intermediate State Mimetic Agents of Insulin The method of Example 15 is followed by replacing an insulin with α-Interferon.

Example 17 - Preparation of Intermediate State Mimetic Agents of rhGH The method of Example 15 is still substituted by recombinant human growth hormone by α-Interferon.

Example 18 - Titration of α-Interferon as Quantified by Intrinsic Fluorescence of Tryptophan A solution is prepared in the presence of 9.1mg / mL of α-Interferon in 20mM of a sodium phosphate buffer at a pH of 7.2. a solution is prepared in the presence of a disturbing agent by dissolving 800mg of the disturbing agent (L-arginine acylated with cyclohexanoyl chloride) in 2mL of 2OmM of a Sodium Phosphate buffer (pH 7).

The samples are prepared by diluting the α-Interferon with the sodium phosphate buffer and the disturbing agent stock solution at various concentrations of the disturbing agent. The diluted samples were allowed to reach equilibrium by incubation for approximately 30 minutes at room temperature before quantification.

The fluorescence from the endogenous tryptophan resident of α-Interferon was quantified according to the procedure of Example 3. The disrupting agent does not contain a fluorophore. The results are illustrated in Figure 9.

Example 19 - Differential Exploratory Calorimetry of α-Interferon and Perturban Agent The DSC of the agglutination of the disturbing agent is carried out using 20 mM of a Na Phosphate buffer at a pH of 7.2. the dry disturbing agent is weighed to make solutions in the presence of the disturbing agent. A solution in the presence of a-Interferon is diluted in the buffer. The α-Interferon solution was not dialyzed prior to the experiments for the purpose of having the same active concentration for the full set.

DSC thermograms were generated with α-Interferon at a concentration of 0.64 mg / mL and a disturbing agent (Purified fenulsulfonyl-para-aminobenzoic acid a> 98% (as determined by reversible phase chromatography before generation of the spectrum)) at concentrations of the disrupting agent of 5, 10, 25 and 100 mg / ml. The DSC was carried out in an exploratory differential calorimetry DASM-4 interconnected to a PC IBM pair an automatic data collection. The scanning speed was 60 ° C / h.

The results are illustrated in Table 9 below and in Figure 10.

Comparative Example 19 - Differential Exploratory Calorimetry of α-Interferon The method of Example 19 is followed by substituting the α- Interferon without another disturbing agent. The results are illustrated in Table 9 below and in Figure 10.

The DSC scans were the aggregate concentration of the perturbing agent with a range from 0-100 mg / mL and show conformational changes of induced a-interferon that occur in a concentration dependent of the form. At 100 mg / mL of the perturbing agent, the thermogram indicates that the α-Interferon Cp against the Tm curve was a flat line. The flat Cp against the Tm curve obtained at 100 mg / mL of the disturbing agent indicates that the hydrophobic residues within the α-Interferon molecule become exposed to the solvent. It is clear that the disturbing agent was able to change the structure of the α-Interferon in a concentration dependent on the form.

Example 20 - Dialysis Experiments - Reversibility of Complex Formation with the Perturban Agent A solution in the presence of α-Interferon is diluted to a concentration of 9.1 mg / mL with an α-Interferon buffer of 0.6 mg / mL. The DSC was executed according to the procedure of Example 19.

The result is illustrated in Figure 11 A.

The α-Interferon (0.6 mg / mL) and the disrupting agent of Example 19 (100 mg / mL) are mixed without apparent changes in the Cp of the solution. This solution was then dialyzed overnight to a phosphate buffer and the thermogram was rerun. The results are illustrated in Figure 11B: The dialyzed example has essentially the same Tm and the same area under the Cp against the Tm curve as it was before the addition of the disturbing agent. This indicates that not only the disturbing agent is capable of inducing conformational changes in the protein but also that this process is reversible. The dilution was sufficient to induce a release of the disturbing agent from the active agent.

Example 21 - DSC of the Perturbante Agent and a- Interferon The method of Example 4 was followed, replacing the disrupting agent of Example 19 with GuHCl. The results are illustrated in Figure 12.

The DSC experiments of the equilibrium denaturation of α-Interferon indicate the existence of intermediate conformations of the molecule. The graphs? H against Tm indicate the energetics of the intermediate conformations occupied by the? -Interferon in each group of experimental conditions.

Example 22 - Dependent Change by Concentration of Perturbate Agent in a-Interferon The method of Example 19 is followed by replacing the cyclohexanoyl phenylglycine of the disrupting agent.

The results are illustrated in Table 10 below and in Figure 13.

Cyclohexanoyl phenylglycine induces conformational changes in a-Interferon where the concentration is dependent.

Example 23 - DSC of the Disturbing Agent and a- Interferon The method of Example 4 is followed by replacing the disrupting agent of Example 30 with GuHCl. The results are illustrated in Figure 14.

The graphs of? H against Tm indicate the existence of an intermediate conformation of equilibrium for α- Interferon which is stable below 5 and 25 mg / ml of the disturbing agent cyclohexanoyl phenylglycine added.

Example 24 - Isothermal Calorimetry of a-Interferon Titration with the Perturban Agent The isothermal calorimetry titration of the disrupting agent forming complexes with the α-Interferon was carried out at 25 ° C at two different pH. The buffers used are 2OmM NaFosphate with a pH of 7.2 and 2OmM of NaAc for a pH of 3.0. A solution of α-Interferon was dialyzed before the experiment reached the appropriate pH. The dry disrupting agents are weighed and diluted in a dialysate.

The ITC was carried out in an OMEGA McroCal titration calorimeter (MicroCal Inc. - Northampton, MA). The data points were collected every 2 to 30 seconds, without subsequent filtering. A solution of α-Interferon placed in a 1.3625 mL cell was titrated using one. 250 μL syringe filled with a concentrated solution of disturbing agent solution. A certain amount of the titration compound was tried every 3-5 minutes up to 55 injections.

A reference experiment to correct the heat of the mixture of two solutions was carried out so that the intercept of the reaction cell was filled with the buffer without the active agent.

The analysis of the data was carried out using the programs developed at the Johns Hopkins University Biocalorimetry Center.

Titration at a pH of 7.2 includes 53 injections of 2μl of the disrupting agent of Example 30 (50 mg / ml = 191.6mM (MW 261)) and α-Interferon (1.3 mg / mL = 0.067mM (MW 19400)).

The results are illustrated in Figure 15.

The curve adjustment indicates multiple independent sites: n (l) = 121.0354 where n = # of complete molecules of disturbing agent.

? H (1) = 58.5932 cal / Moles per disturbing agent log 10 Ka (1) = 2.524834 where KA = association constant the units of the X-axis are the concentration of the carrier in mM.

Y-axis units represent heat / injection expressed in calories.

At a pH of 3, the formation of the complex results in a negative enthalpy.

Comparative Example 24 * - Isothermal Calorimetry of Disturbing Agent Titration The method of Example 24 was followed, substituting 53 injections of 2μl of the disrupting agent (50 mg / mL = 191.6mM) [IFN] = Omg of Example 22 without the active agent.

Example 24 and Comparative Example 24 * illustrate that α-Interferon has a positive enthalpy and an agglutination constant of (Kd = 10_3M).

Example 25 - Isothermal Calorimetry of a-Interferon Titration and Complex Formation of the Perturban Agent The method of Example 24 was followed by substituting the disrupting agent of Example 19 for the disrupting agent of Example 22.

Titration at a pH of 7.2 includes 2 sets of 55 injections each of 5μl of the disrupting agent (50mg / mL = 181mM (FW 277)) and a_interferon (2.31mg / mL = 0.119mM, (MW 19400)).

The results are illustrated in Figure 16.

The adjustment of the curve indicates an independent multiple site: n (l) = 55.11848 where n = # of complete molecules of disturbing agent.

? H (1) = -114.587 cal / Moles per disturbing agent log 10 Ka (1) = 2.819748 where KA = association constant the units of the X-axis are the concentration of the carrier in mM.

Y-axis units represent heat / injection expressed in calories.

The formation of interfering agent complexes at α-Interferon at a pH of 3.0 results in precipitation of the complex out of the solution. Due to the effect of heat produced by this process, it is impossible to quantify the parameters of complex formation.

Comparative Example 25 * - Isothermal Calorimetry of Disturbing Agent Titration The method of Example 25 was followed, substituting 55 injections of 5μl of the disrupting agent of Example 26 (50mg / mL = 181mM) in 20mM sodium phosphate at a pH of 7.2 without the active agent.

The disrupting agent of Example 19 formed complexes with α-Inteferon and resulted in a negative enthalpy and an agglutination constant comparable to those with the disrupting agent of Example 22 and α-Inferred.

The samples 24 and 25 indicate that the stronger the disturbing agent is, the more complex it is with the active substance and the more thermodynamically stable the intermediate state of the active agent, the greater the bioavailability of the active agent.

Therefore, by schematizing the curve? H against Tm of an active agent and disturbing agent, these disturbing agents induce little or no enthalpy change over the wider range of Tm are the preferred disruptive agents. It is believed that disturbing agents that stabilize the intermediate states to a greater extent result in a more efficient distribution of the active agent.

Example 26 - Comparison of the Effects of Three Perturban Agents in Charts of? H against Tm with a- Interferon DSC experiments were carried out according to the procedure of Example 19, with 0.5 mg / mL of a-interferon mixed with (1) benzoyl para-amino pheutyric acid, (2) the disrupting agent of Example 22, or (3) the disturbing agent of Example 19.

The benzoyl para-amino pheutyric acid is poorly soluble under the conditions of the buffer. The maximum concentration at which the solution is still transparent at room temperature is 8mg / mL. Therefore, the presentations used for the concentrations are 2, 4 and 6 mg / mL. The results are illustrated in Figures 17 and 18. The dotted line in Figure 17 represents the smaller linear boxes, and the regression equation is at the top of Figure 17.

Y = -1.424e5 + 3148.8x R = 0.9912 Figures 17 and 18 illustrate the conformational changes in α-interferon is more readily produced by benzoyl para-amino pheutyric acid than by the disrupting agents of Examples 22 and 19, and that these changes are more easily produced by the disruptive agent of Example 22 than by the disruptive agent of Example 19.

Example 27 - Isothermal Calorimetry of a-Interferon Titration and Complex Formation ITC was carried out according to the method of Example 24 with 40 injections of 5μL of the disturbing agent benzoyl para-amino pheutyric acid (7.5 mg / mL = 24.59mM, (FW 305)) and α-Interferon (2.5 mg / mL = 0.129mM, (MW 19400)).

The results are illustrated in Figure 19.

The adjustment of the curve indicates an independent multiple site: n (l) = 23.69578 where n = # of complete molecules of disturbing agent.

? H (1) = 791.5726 cal / Moles per disturbing agent log 10 Ka (1) = 3.343261 where KA = association constant The units of the X-axis are the concentration of the carrier in mM.

Y-axis units represent heat / injection expressed in calories.

Comparative Example 27 * An ITC was carried out according to the method of Example 27 with 40 injections of 5μl of the disturbing agent benzoyl para-amino pheutyric acid (7.5 mg / mL = 24.59mM) in 20mM NaFosphate buffer at a pH of 7.2, without active agent. .

The apparent dissociation constant for the disrupting agent of Example 26 is greater than that of the disrupting agent of Example 30 (10"4M) at pH 7.

Thus, benzoyl para-amino pheutyric acid forms complexes more strongly with α-Interferon and induces the natural intermediate conformational state in a reversible state at lower concentrations of the disturbing agent.

Examples 28-30 - Isothermal Titration Calorimetry of rhGH at pH 7.5 and 4.0 with Different Perturban Agents The ability of rhGH to form complexes with different disturbing agents was evaluated by Itc using a Micrical Omega titrant, normally equilibrated at 30 ° C . The sample cell of the calorimeter was filled with unggregated rhGH (usually 0.25mM) prepared in 50mM of a phosphate buffer, at a pH of 7.5 or 4.0. the disturbing agent (L-tyrosine modified with cyclohexanoyl chloride (a), L-phenylalanine modified with salicyloyl, or phenylsulfonyl para-amino benzoic acid (c)) in the drip jersey at lmM (for a pH of 7.5) and 2.5 mM. (for a pH of 4.0). Twenty to 25 injections of lOμl were made into a rapidly mixed solution (400 rpm) at 2 minute intervals between the injections.

The initial concentration of the disturbing agent placed in the sample cell in the calorimeter assumed a formula weight of 200 for each disturbing agent. The pH of each solution was verified after dissolution, but no pH adjustments were required. All the experiments were carried out at 30 ° C. The initial concentration of the rhGh placed in the drip syringe assumed a weight of 20,000 for the rhGH. The pH of each solution was verified after dissolution but no pH adjustment was required.

The heat of the reaction was determined by determining the peaks observed. To correct the heat of the mixture and dilution, a control experiment was also carried out under identical conditions where the aliquots of the test disrupting agent or the rhGH were only due to the buffer solution. The sum total of the developed heat was plotted against the total reaction of the disturbing agent to produce the isotherm of which the association constant (KA, M), enthalpy change (? H, kcal / mol), entropy change (? S (eu), and N, and stoichiometry of the perturbing agent molecules formed in complexes or an equivalent of the supramolecular complex forming complexes were determined by adjusting curves of the binder isotherm against the agglutination equation described for the formation of disturbing agent complexes in a supramolecular complex that has a set of complex formation sites independent of the disturbing agent. circumvolve using a nonlinear least-squares algorithm provided in the manufacturer's program.The results are illustrated in Table 11 below.

A = L-tyrosine modified with cyclohexanoyl chloride B = L-phenylalanine modified with salicyloyl C = phenylsulfonyl para-aminobenzoic acid Positive values of? S at a pH of 7.5 indicate that the formation of complexes at this pH results in a change structural.

Examples 31 and 32 - Assessment of Inhibition of Pancreatin with α-Interferon and Perturban Agents The pancreatin activity titration is prepared as follows: O.lmL of a solution in the presence of α-interferon (9.1 mg / mL, 2OmM NaH2P04, pH 7.2) (Schering Plow, Corp.) at 2.5 mL was added. either of a disturbing agent of phenylsufonyl-para-aminobenzoic acid (46) or a disturbing agent of cyclohexanoyl phenylglycine (47) (200mg / mL) in 5mM KH2P04, pH 7.0. Incubation was carried out at 37 ° C for 30 and 60 minutes followed by the addition of O.lmL of pancreatin USP (20mg / mL) (Sigma Chemical Co.) aliquots of 0.1 mL were separated at these time intervals. Enzymatic reactions were stopped by the addition of protease inhibitors (Aprotinin and Bowman-Birk Inhibitor (BBI), each at 2 mg / mL) and were diluted 5 times to confirm that the α-Interferon was left intact. A reversible phase HPLC method using a C-4 butyl cartridge (3.0 x 0.46 cm, Tainin) and using a gradient elution between 0.1% TF a / water and 90% ACN in 0.1% TFA pooled with UV detection 220 nm was used to separate and quantify α-Interferon. The α-Interferon at 0 minutes was quantified from an aliquot before the addition of pancreatin and was taken as 100%. The results are illustrated in Figure 20.

Examples 31 and 32 illustrate that both the supramolecular complexes withstood enzymatic degradation. However, in additional analyzes there was no correlation between the potency of the enzyme inhibitors and the ability to distribute the drug.

Example 33 - DSC of Heparin at a pH of 5.0 DSC thermograms of heparin at a pH of 5.0 were carried out according to the method of Example 9 using a pH, GuHCl, and ionic strength as disturbing agents.

The thermograms were corrected by subtracting the 0.05M NaCl-phosphate heparin in target, but an individual target was not used for each NaCl concentration.

The results are illustrated in Tables 12-14 below and in Figure 21. (a) domain a (b) domain b TABLE - 13 Effects of 10M Guanidine Hydrochloride in 50mM of a Phosphate Buffer in the DSC Spectrum of Heparin 0. 50M NaCl 41.6 0.094 Not present These data indicate that non-proteinaceous active agents are capable of changing the response conformation of the disturbing agent.

Example 34 - Columnar Chromatography of Heparin and Disturbing Agents The following materials were used: Column: Pharmacia glass column of 10mm x 30cm, low pressure, with adjustable bed volume. The bed volume used was 22 cm at a pressure of 0.8 mPa.

Packing: Heparin was covalently bound to Sepharose CL-6B without a binding molecule.

Fractionation speed of the Sepharose: 10,000 - 4,000,000. The density of the heparin was 2mg / cc as per the Department Q. C. Pharmacy.

Conditions: The mobile phase was 67 mM phosphate buffer, at a pH of 7.4. The flow velocity was 1.5mL / min. The shift time was 45 minutes. The detection of samples was carried out with a Perkin Elmer refractive index detector.

The columnar integrity was confirmed by injecting protamine and observing a retention time of more than one hour. The empty volume was determined by injecting water and measuring the elution time.

Each of the disrupting agents of Table 15 below (5mg) was dissolved independently in lmL of mobile phase and injected (100μl) into the column. The elution time was measured. The K 'value was determined by using the following equation (as it is in USP): K '= (carrier retention time / water retention time) - 1 The results were compared between each disturbing agent as well as with their respective in vivo performance in Figure 29. The values of K '(degree of delay) in the figure are corrected by subtracting the K' value determined from the column of Sepharose of the K 'value determined from the heparin-sepharose column.

TABLE - 15 PERTURBATING AGENTS Cyclohexyldenebutyric acid (2) -sal Na # 1 Cyclohexanebutyroyl (2-) aminobutyric acid (4) # 2 Phenylacetyl-para-aminobutyric acid # 3 Ortho-methyl-cyclohexanoyl-aminobutyric acid (4) # 4 Phenylacetylaminohexanoic acid (6-) # 5 Cinamoyl-para-aminophenylbutyric acid # 6 Cyclohexanebutyroyl (2-) -para-aminophenylbutyric acid # 7 Hydrocinyl para-aminophenylbutyric acid # 8 Cyclohexanebutyroyl (2-) -leu-leu # 9 Cyclohexanebutyroyl (2 -) - gli # 10 Example 35 - Comparison of the Effects of Six Disturbing Agents with DPPC in Charts of? H vs. Tm The DSC experiments were carried out according to the procedure of Example 19, with 1.0 mg / ml dipalmitolfosphatidylcholine (DPPC) mixed with disrupting agents XI, L, LII, Lili and LIV. The concentrations of the disturbing agents varied from 0 to 20 mg / mL. The results are illustrated in Figure 22.

Example 36 - Exploratory Differential Colorimetry of DPPC and Compound of Perturbate Agent L The DSC of the agglutination of the disturbing agent was carried out using 2OmM of a Na buffer.

Phosphate at a pH of 7.2. the dry disturbing agent L is weighed to make solutions in the presence of the disturbing agent. The solution in the presence of DPPC was prepared in the buffer.

The DSC thermograms were generated with DPPC at a concentration of 1.0 mg / mL. The concentrations of the disturbing agent that were used were 0, 5, 10 and 20 mg / mL. The DSC was carried out as described in Example 19. The results are illustrated in Figure 23.

Example 37 - Exploratory Differential Colorimetry of DPPC Compound of Disturbing Agent L and rhGH The distressing agent agglutination DSC was carried out using 20 mM Na Phosphate buffer at a pH of 7.2. the dry disturbing agent L is weighed to make solutions in the presence of the disturbing agent. The solution in the presence of DPPC is prepared in the buffer. The rhGh solution is prepared as described in Example 10 A.

DSC thermograms are generated with DPPC at a concentration of 1.0 mg / mL. Samples that have DPPC alone; DPPC with 10 mg / mL of disturbing agent; DPPC with 0.3 mg / mL of rhGH; and DPPC, 10 mg / mL of disrupting agent and 0.3 mg / mL of rhGh were prepared and analyzed. The DSC is carried out as described in Example 19. The results are illustrated in Figure 24.

Example 38 - Exploratory Differential Colorimetry of the DPPC Compound of Disturbing Agent LII and rhGH The agglutination DSC of the disturbing agent is carried out using 20 mM of a Na buffer.

Phosphate at a pH of 7.2. The dry disturbing agent LII is weighed to make solutions in the presence of the disturbing agent. The solution in the presence of DPPC is prepared in the buffer. The rhGH solution is prepared as described in Example 10 A.

DSC thermograms are generated with DPPC at a concentration of 1.0 mg / mL. The samples have only DPPC; DPPC with 5 mg / mL of the disturbing agent; DPPC with 0.3 mg / mL of rhGH; and DPPC, 5 mg / ml of the disrupting agent and 0.3 mg / ml of rhGH are prepared and analyzed. The DSC is carried out as described in Example 19.

Example 39 - Exploratory Differential Colorimetry of the DPPC Compound of the Disturbing Agent XI and rhGH The agglutination DSC of the disturbing agent is carried out using 20 mM of a Na Phosphate buffer at a pH of 7.2. The dry disturbing agent XI is weighed to make solutions in the presence of the disturbing agent. The solution in the presence of DPPC is prepared in the buffer. The rhGH solution is prepared as described in Example 10 A.

DSC thermograms are generated with DPPC at a concentration of 1.0 mg / mL. The samples have only DPPC; DPPC with 2 mg / mL of the disturbing agent; DPPC with 0.3 mg / mL of rhGH; and DPPC, 2 mg / ml of the disrupting agent and 0.3 mg / ml of rhGH are prepared and analyzed. The DSC is carried out as described in Example 19.

The results are illustrated in Figure 26, Example 40 - Dynamic Light scattering of Compound L The solutions of the compound L are prepared in lOmM phosphate buffer at a pH of 7.0. The concentrations analyzed were 10, 15, and 20 mg / ml. The solutions were analyzed using normal techniques of microscopic scattering of light.

The results are illustrated in Figure 27 Examples 41 and 42 - In vivo evaluation of the Growth Hormone Recombinant in Rats The dosage compositions are prepared by mixing the modified amino acids and human growth hormone (rhGH) as listed in Table 16 below in a phosphate buffer at a pH of about 7-8.

The rats were administered the dosage composition by sublingual or nasal administration. The distribution is evaluated by using an ELISA assay for rhGH from Medix Biotech, Inc. For intracolonic administration, a sample is prepared and dosed in fasted rats at a dose of 25mg / kg carrier in a buffered solution containing propylene glycol. (0-50%) and 1 mg / kg of rhGH.

The results are illustrated in Table 16 below.

Comparative Example 41 A rhGH (6 mg / ml) was administered by oral priming to the rat, and the distribution was evaluated according to the procedure of Example 41 A.

The results are illustrated in Table 16 below.

Examples 43-54 - Live Evaluation of Recombinant Growth Hormone in Rats Preparation of Dosing Solutions The distribution agents are reconstituted with distilled water and adjusted to a pH of 7.2-8.0 with either aqueous hydrochloric acid or aqueous sodium hydroxide. A solution in the presence of rhGh is prepared by mixing rhGH, D-mannitol and glycine and dissolving this mixture in 2% glycerol / water. The solution in existence is then added to the solution of the distribution agent. Various proportions of the distribution agent and active agent were studied.

In vivo experiments Male Sprague-Dawley rats weighing 200-250g were fasted for 24 hours and were administered ketamine (44 mg / kg) and chlorpromazine (1.5 mg / kg) 15 minutes before dosing. The rats were administered one of the dosage solutions described above by subcutaneous injection, intranasal instillation, or sublingual instillation. Blood samples were collected from the stem serially from the tail artery for determination of serum calcium concentration or serum rhGH concentrations. The dose of rhGh administered in these experiments was 0.1 mg / kg.

The concentrations of serum rhGH were quantified by an enzyme immunoassay analysis kit. The results are given in Table 17 and Figures 28 and 29.

In Figure 29 the circles represent the response after the SL dosage of an aqueous solution of the compound CXXII-H and rhGH. The tables represent the response after the IN dosing of an aqueous solution of the compound CXXIII-H and rhGH. The triangles represent the response after the IC dosage of an aqueous solution of the compound CXXIII-H and rhGh. The dose of compound CXXIII-H was 25 mg / kg and the dose for rhGH was 1 mg / kg.

Comparative Example 43 A A rhGh (1 mg / kg) was orally administered by priming to a rat, and the distribution was evaluated according to the procedure of Example 43. The results are illustrated in Table 17 below.

TABLE - 17 Examples 55-60 - In vivo Evaluation of Calcitonin from Salmon in Rats Preparation of the Dosing Solution The distribution agents are made with distilled water and adjusted to a pH of 7.2-8.0 with either aqueous hydrochloric acid or sodium hydroxide. A solution in existence of sCT is prepared by dissolving sCT in citric acid (0.085N). The solution in existence is then added to the solution of the distribution agent. Different proportions of distribution agent and active agent were studied.

In vivo experiments Male Sprague-Dawlry rats weighing 200-250 g were fasted for 24 hours and were administered ketamine (44 mg / kg) and chlorpromazine (1.5 mg / kg) 15 minutes before dosing. The rats were administered one of the dosage solutions described above by subcutaneous injection. Blood samples were collected serially from the tail artery for serum calcium concentration.

Serum calcium concentrations were quantified by the o-cresophthalein complexone method (Sigma) using a UV / VIS spectrophotometer (Perkin Elmer). The results are given in Table 5.

Example 55 A Salmon calcitonin was administered by oral priming to the rats, and the distribution was evaluated according to the procedure of Example 55. The results are given in Table 18 below.

All patents, applications, methods of analysis and publications mentioned in this section are hereby incorporated for reference.

Many variations of the present invention are also suggested for those skilled in the art in light of the above detailed relationship. All these modifications are within the full scope extended in the appended claims.

Claims (57)

1. CLAIMS A method for distributing, via the subcutaneous route, a biologically active agent to a subject in need of this biologically active agent, characterized in that this method comprises: (a) providing a biologically active agent that can exist in a natural conformational state, a denatured conformational state, and an intermediate conformational state, which is reversible in this natural state and is conformationally between these natural and denatured states. (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent to this intermediate state to form subcutaneously distributable supramolecular complexes this disrupting agent has a molecular weight between about 150 and about 600 daltons, and has at least a hydrophilic portion and at least a hydrophobic portion, this supramolecular complex comprises this disturbing agent by complexing non-covalently with this biologically active agent, this biologically active agent does not form a microsphere with this disturbing agent, and this disturbing agent is present in an effective amount for the subcutaneous distribution of this biologically active agent; and (c) subcutaneously administering this supramolecular complex to the subject.
2. 2. A method as defined in accordance with claim 1, characterized in that it also comprises (d) after 1 step of administration, remove the disturbing agent from this supramolecular complex to transform this biologically active agent into this natural state.
3. 3. A method as defined in accordance with claim 2, characterized in that step (d) comprises this supramolecular complex diluted.
4. 4. A method as defined in accordance with any of claims 1 to 3, characterized in that the intermediate state has a? G from about -20 kcal / moles to about 20 kcal / moles relative in this natural state.
5. 5. A method as defined according to any one of claims 1 to 4, characterized in that this biologically active agent is selected from the group consisting of a peptide, a small peptide, a hormone, a polysaccharide, a mucopolysaccharide, a carbohydrate , a lipid, a pesticide, or any combination of the above.
6. A method as defined according to any one of claims 1 to 5, characterized in that this biologically active agent is selected from a group consisting of human growth hormone, bovine growth hormone, the hormone releasing the growth hormone, interferon, interleukin-I, interleukin-II, insulin, heparin, low-molecular-weight heparin, calcitonin, erythropoietin, atrioventricular factor, antigen, monoclonal antibody, somatostatin, adenocortinotropin, gonadotropin-releasing hormone, oxytocin , vasopressin, cromolyn sodium, vancomycin, desferrixamine, an antimicrobial, an antifungal agent and any combination of any of the above.
7. A method as defined according to any one of claims 1 to 6, characterized in that the disrupting agent is selected from the group consisting of a proteinoid, an acylated amino acid, an acylated polyamino acid, a sulfonated amino acid, a sulfonated polyamino acid, an acylated aldehyde of an amino acid, an acylated aldehyde of a polyamino acid, an acylated ketone of an amino acid, and an acylated ketone of a polyamino acid.
8. 8. A method as defined according to any one of claims 1 to 7, characterized in that this disrupting agent comprises a carboxylic acid having the formula: R-C02H wherein R is Ci to C24 alkyl, C2 to C2 alkenyl, C3 cycloalkyl a Cio, C3 to Cio cycloalkenyl, phenyl, naphthyl, (Ci to Cio alkyl) phenyl, (C2 to Cio alkenyl) phenyl, (Ci to Cio alkyl) naphthyl, (C2 to Cio alkenyl) naphthyl, phenyl (Ci to Cio alkyl) ), phenyl (C2 to C2 alkenyl), naphthyl (C1 to C14 alkyl), or naphthyl (C2 to C2 alkenyl); R is optionally substituted with Ci to Cι alkyl, C 2 to Cι alkenyl, C x to C 4 alkoxy, -OH, -SH, -CO Í R 1, C 3 to C cycloalkyl. C3 to Cio cycloalkenyl, heterocycle having from 3 to 10 ring atoms wherein the heteroatom is one or more N, O, S atoms, or any combination thereof; aryl, (C 1 -Cy alky) aryl, aryl (Ci a Cio) alkyl or any combination thereof; R is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; Y R1 is hydrogen, C1 to C4 alkyl, or C2 to C4 alkenyl; and a salt of these.
9. 9. A method for preparing an active subcutaneously distributable agent, characterized in that this method comprises: (a) providing a biologically active agent that can exist in a natural conformational state, a denatured conformational state, and an intermediate conformational state, which is reversible in this natural state and is conformationally between these natural and denatured states. (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent to this intermediate state to form subcutaneously distributable supramolecular complexes, this disturbing agent has a molecular weight between about 150 and about 600 daltons, and has at least one hydrophilic portion and at least one hydrophobic portion, this supramolecular complex comprises this disturbing agent by complexing non-covalently with this biologically active agent, this biologically active agent does not form a microsphere with this disturbing agent, and this disturbing agent is present in an amount effective for the subcutaneous distribution of this biologically active agent; Y
10. 0. A method as defined in accordance with claim 9, characterized in that it also comprises the step of: (c) preparing a mimetic agent of the supramolecular complex.
11. 1. A method as defined in accordance with claim 10, characterized in that said biologically active agent comprises a peptide and this mimetic agent comprises a mimetic agent of a peptide.
12. 2. A subcutaneous distribution composition comprising a subcutaneously distributed mimetic prepared by the method according to claim 9.
13. 3. A composition of subcutaneous distribution comprising a supramolecular complex characterized in that it comprises: (a) a biologically active agent that can exist in an intermediate conformational state by complexing non-covalently with (b) a disturbing complexing agent having a molecular weight range from about 150 to about 600 and having at least a hydrophilic portion and at least one hydrophobic portion; wherein this intermediate state is reversible to this natural state and is conformationally between a natural conformational state and a denatured conformational state of this biologically active agent; this composition is not a microsphere; and this disrupting agent is present in an effective amount for the subcutaneous distribution of this biologically active agent.
14. 14. A composition of subcutaneous distribution as defined in accordance with claim 13, characterized in that this biologically active agent is selected from the group consisting of human growth hormone, bovine growth hormone, growth hormone releasing hormone. , an interferon, interleukin-I, interleukin-II, insulin, heparin, low-molecular-weight heparin, calcitonin, erythropoietin, atrioventricular factor, an antigen, a monoclonal antibody, somatostatin, adenocortinotropin, gonadotropin-releasing hormone, oxytocin, vasopressin, cromolyn sodium, vancomycin, desferrixamine, an antimicrobial, an antifungal agent and any combination of any of the foregoing.
15. 5. A composition of subcutaneous distribution as defined in claim 13, characterized in that this disrupting agent is selected from the group consisting of a proteinoid, an acylated amino acid, an acylated polyamino acid, a sulfonated amino acid, a sulfonated polyamino acid, an aldehyde acylated of an amino acid, an acylated aldehyde of a polyamino acid, an acylated ketone of an amino acid, and an acylated ketone of a polyamino acid.
16. 6. A subcutaneous distribution composition as defined in any of claims 13 to 15, characterized in that this disrupting agent comprises a carboxylic acid having the formula: R-C02H wherein R is Ci to C2 alkyl, C2 to C2 alkenyl, cycloalkyl C3 to Cío. C3 to Cio cycloalkenyl, phenyl, naphthyl, (Ci to Cio alkyl) phenyl, (C2 to Cio alkenyl) phenyl, (Ci to Cio alkyl) naphthyl, (C2 to Cio alkenyl) naphthyl, phenyl (Ci to Cio alkyl), phenyl (C2 to C2 alkenyl), naphthyl (C1 to C14 alkyl), or naphthyl (C2 to C2 alkenyl) R is optionally substituted with Ci to Cio alkyl, C2 to Cio alkenyl, Ci to C4 alkoxy, -OH, -SH, -C02R1, C3 to Cio cycloalkyl. C3 to Cio cycloalkenyl, heterocycle having from 3 to 10 ring atoms wherein the heteroatom is one or more N, O, S atoms, or any combination thereof; aryl, (C 1 -Cy alky) aryl, aryl (Ci a Cio) alkyl or any combination thereof; R is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; Y R1 is hydrogen, C1 to C4 alkyl, or C2 to C4 alkenyl; or any salt of these.
17. 17. A unit dose form characterized in that it comprises: (A) a composition as defined in accordance with claim 13; Y (B) (a) an excipient, (b) a diluent, (c) a disintegrant, (d) a lubricant, (e) a plasticizer, (f) a dye, (g) a dosing vehicle, or (h) ) any combination of these.
18. 18. A method for preparing an agent that is capable of being distributed via the subcutaneous route to a subject in need of this agent, characterized in that this method comprises: (a) provide a biologically active agent that can exist in a natural conformational state, a denatured conformational state and an intermediate conformational state, which is reversible in this natural state and is conformationally between this natural state and this denatured state (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent to this intermediate state, where this disrupting agent is present in an effective amount for the subcutaneous distribution of this agent biologically active; Y (c) preparing a mimetic agent of this intermediate state.
19. 9. A method as defined in accordance with claim 18, characterized in that this disturbing agent comprises a pH-changing agent, an ion-strength-altering agent, or guanidine hydrochloride.
20. 0. A method for distributing, via the sublingual route, a biologically active agent to a subject in need of this biologically active agent, characterized in that this method comprises: (a) provide a biologically active agent that can exist in a natural conformational state, a denatured conformational state and an intermediate conformational state, which is reversible in this natural state and is conformationally between this natural state and this denatured state (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent to this intermediate state, and to form sublingually distributable supramolecular complexes, this disrupting agent has a range of molecular weight from about 150 to about 600 daltons, and having at least one hydrophilic portion and at least one hydrophobic portion, this supramolecular complex comprises this disturbing agent forming non-covalent complexes with this biologically active agent, this biologically active agent does not form microspheres with this disturbing agent, and this disturbing agent is present in an effective amount for the sublingual distribution of this biologically active agent, and (c) sublingually administer this supramolecular complex to this subject.
21. 21. A method as defined in accordance with claim 20, characterized in that it further comprises: (d) after this administration step, remove this disturbing agent from this supramolecular complex to transform this biologically active agent into this natural state.
22. 2. A method as defined in accordance with claim 21, characterized in that step (d) comprises diluting this supramolecular complex.
23. 3. A method as defined in accordance with any of claims 20 to 22, characterized in that this intermediate state has a? G spanning from about -20 kcal / moles to about 20 kcal / moles relative in this natural state.
24. 4. A method as defined according to any of claims 20 to 23, characterized in that this biologically active agent is selected from the group consisting of a peptide, a small peptide, a hormone, a polysaccharide, a mucopolysaccharide, a carbohydrate , a lipid, a pesticide, and any combination of precedents.
25. 5. A method as defined in accordance with any of claims 20 to 24, characterized in that this biologically active agent is selected from a group consisting of human growth hormone, bovine growth hormone, the hormone releasing the growth hormone, interferon, interleukin-I, interleukin-II, insulin, heparin, low-molecular-weight heparin, calcitonin, erythropoietin, atrioventricular factor, antigen, monoclonal antibody, somatostatin, adenocortinotropin, gonadotropin-releasing hormone, oxytocin , vasopressin, cromolyn sodium, vancomycin, desferrixamine, an antimicrobial, an antifungal agent and any combination of any of the preceding.
26. 6. A method as defined in any of claims 20 to 25, characterized in that this disrupting agent is selected from the group consisting of a proteinoid, an acylated amino acid, an acylated polyamino acid, a sulfonated amino acid, a sulfonated polyamino acid, an acylated aldehyde of an amino acid, an acylated aldehyde of a polyamino acid, an acylated ketone of an amino acid, and an acylated ketone of a polyamino acid.
27. 27. A method as defined in any of claims 20 to 26, characterized in that this disrupting agent comprises a carboxylic acid having the formula: R-C02H wherein R is Ci to C24 alkyl, C2 to C24 alkenyl, C3 cycloalkyl to Cío. C3 to Cio cycloalkenyl, phenyl, naphthyl, (Ci to Cio alkyl) phenyl, (C2 to Cio alkenyl) phenyl, (Ci to Cio alkyl) naphthyl, (C2 to Cio alkenyl) naphthyl, phenyl (Ci to Cio alkyl), phenyl (C2 to C2 alkenyl), naphthyl (C1 to C14 alkyl), or naphthyl (C2 to C2 alkenyl); R is optionally substituted with Ci to Cio alkyl, C2 to Cio alkenyl, Ci to C4 alkoxy, -OH, -SH, -CO? R1, C3 cycloalkyl to C3 cycloalkenyl to Cio, heterocycle having from 3 to 10 atoms rings wherein the hetero atom is one or more atoms of N, O, S, or any combination thereof; aryl, (C 1 -Cy alky) aryl, aryl (Ci a Cio) alkyl or any combination thereof; R is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; and R1 is hydrogen, C1 to C4 alkyl / or C2 to C4 alkenyl; or any salt of these.
28. 28. A method for preparing a biologically active sublingually distributable agent, characterized in that this method comprises: (a) provide a biologically active agent that can exist in a natural conformational state, a denatured conformational state and an intermediate conformational state, which is reversible in this natural state and is conformationally between this natural state and this denatured state (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent into this intermediate state, and to form a supramolecular sublingually distributable complex, this disturbing agent has a molecular weight range from about 150 to about 600 daltons, and has at least one hydrophilic portion and at least one hydrophobic portion, this supramolecular complex comprises this disturbing agent forming non-covalent complexes with this biologically active agent, this biologically active agent does not form microspheres with this disturbing agent, and this disturbing agent is present in an effective amount for the sublingual distribution of this biologically active agent.
29. 9. A method as defined in claim 28, characterized in that it further comprises the step of: (c) preparing a mimetic agent for a supramolecular complex.
30. 0. A method as defined in claim 29, characterized in that said biologically active agent comprises a peptide and this mimetic agent comprises mimetic agent of a peptide.
31. . A sublingual distribution composition comprising a sublingual distribution mimetic prepared in accordance with claim 28.
32. A sublingual distribution composition comprising a supramolecular complex characterized in that it comprises: (a) a biologically active agent in an intermediate conformational state complexing non-covalently with (b) a disturbing complexing agent having a molecular weight ranging from about 150 to about 600 and having at least one hydrophobic portion and at least one hydrophilic portion wherein this intermediate state is reversible in this natural state and is conformational between the natural conformational state and this denatured conformational state of this biologically active agent; this composition is not a microsphere; this disturbing agent is in an effective amount for the sublingual distribution of this biologically active agent.
33. 3. A sublingual distribution composition as defined in claim 32, characterized in that this biologically active agent is selected from a group consisting of human growth hormone, bovine growth hormone, hormone releasing hormone, growth, an interferon, interleukin-I, interleukin-II, insulin, heparin, low molecular weight heparin, calcitonin, erythropoietin, atrial naturétic factor, an antigen, a monoclonal antibody, somatostatin, adenocortinotropin, gonadotropin-releasing hormone, oxytocin, vasopressin , sodium cromolyn, vancomycin, desferrixamine, an antimicrobial, an antifungal agent and any combination of any of these precedents.
34. 34. A method as defined according to any one of claims 32 to 33, characterized in that the disrupting agent is selected from the group consisting of a proteinoid, an acylated amino acid, an acylated polyamino acid, a sulfonated amino acid, a sulfonated polyamino acid, an acylated aldehyde of an amino acid, an acylated aldehyde of a polyamino acid, an acylated ketone of an amino acid, and an acylated ketone of a polyamino acid.
35. 35. A sublingual distribution composition as defined in any of claims 32 to 34, characterized in that this disrupting agent comprises a carboxylic acid having the formula: R-C02H wherein R is Ci to C24 alkyl, C2 to C24 alkenyl , C3 cycloalkyl to Cio. C3 to Cio cycloalkenyl, phenyl, naphthyl, (Ci to Cio alkyl) phenyl, (C2 to Cio alkenyl) phenyl, (Ci to Cio alkyl) naphthyl, (C2 to Cio alkenyl) naphthyl, phenyl (Ci to Cio alkyl), phenyl (C2 to C2 alkenyl), naphthyl (C12 alkyl), or naphthyl (C2 to C2 alkenyl); R is optionally substituted with Ci to C, C2 to Cio alkenyl, Ci to C4 alkoxy, -OH, -SH, -CO ^ 1, C3 cycloalkyl to C3 cycloalkenyl to Cio. heterocycle having from 3 to 10 ring atoms wherein the hetero atom is one or more atoms of N, O, S, or any combination thereof; aryl, (C 1 -Cy alky) aryl, aryl (Ci a Cio) alkyl or any combination thereof; R is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; Y R1 is hydrogen, C1 to C4 alkyl, or C2 to C4 alkenyl; or any salt of these.
36. 36. A unit dose form characterized in that it comprises: (A) a composition as defined in accordance with claim 32; Y (B) (a) an excipient, (b) a diluent, (c) a disintegrant, (d) a lubricant, (e) a plasticizer, (f) a dye, (g) a dosing vehicle, or (h) any combination of these.
37. 7. A method for preparing an agent that is capable of being distributed via the sublingual route to a subject in need of this agent, characterized in that this method comprises: (a) provide a biologically active agent that can exist in a natural conformational state, a denatured conformational state and an intermediate conformational state, which is reversible in this natural state and is conformationally between this natural state and this denatured state (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent to this intermediate state, where this disrupting agent is present in an effective amount for the sublingual distribution of this agent biologically active; Y (c) preparing a mimetic agent of this intermediate state.
38. 8. A method as defined in accordance with claim 37, characterized in that this disrupting agent comprises a pH-changing agent, an ion-strength-altering agent, or guanidine hydrochloride.
39. 9. A method for distributing, via the intranasal route, a biologically active agent to a subject in need of this biologically active agent, characterized in that this method comprises: (a) providing a biologically active agent that can exist in a natural conformational state, a denatured conformational state and an intermediate conformational state, which is reversible in this natural state and is conformationally between this natural state and this denatured state (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent to this intermediate state, and to form an intranasally distributable supramolecular complex, this disrupting agent has a molecular weight range from about 150. up to about 600 daltons, and has at least one hydrophilic portion and at least one hydrophobic portion, this supramolecular complex comprises this disturbing agent forming non-covalent complexes with this biologically active agent, this biologically active agent does not form microspheres with this disturbing agent, and this disturbing agent is present in an amount effective for the intranasal distribution of this biologically active agent, and (c) intranasally administering this supramolecular complex to this subject.
40. 0. A method as defined in accordance with claim 39, characterized in that it further comprises: (d) after this administration step, remove this disturbing agent from this supramolecular complex to transform this biologically active agent into this natural state.
41. 1. A method as defined in accordance with claim 40 characterized in that step (d) comprises diluting this supramolecular complex.
42. 2. A method as defined in accordance with any of claims 39 to 41, characterized in that this intermediate state has a? G ranging from about -20 kcal / moles to about 20 kcal / moles relative to this natural state.
43. 43. A method as defined in accordance with any of claims 39 to 42, characterized in that this biologically active agent is selected from the group consisting of a peptide, a small peptide, a hormone, a polysaccharide, a mucopolysaccharide, a carbohydrate , a lipid, a pesticide, and any combination of the above.
44. 44. A method as defined according to any one of claims 39 to 43, characterized in that this biologically active agent is selected from a group consisting of human growth hormone, bovine growth hormone, hormone-releasing hormone, growth hormone, interferon, interleukin-I, interleukin-II, insulin, heparin, low-molecular-weight heparin, calcitonin, erythropoietin, atrioventricular factor, antigen, monoclonal antibody, somatostatin, adenocortinotropin, gonadotropin-releasing hormone, oxytocin , vasopressin, cromolyn sodium, vancomycin, desferrixamine, an antimicrobial, an antifungal agent and any combination of any of the above.
45. 45. A method as defined according to any one of claims 39 to 44, characterized in that this disrupting agent is selected from the group consisting of a proteinoid, an acylated amino acid, an acylated polyamino acid, a sulfonated amino acid, a sulfonated polyamino acid, an acylated aldehyde of an amino acid, an acylated aldehyde of a polyamino acid, an acylated ketone of an amino acid, and an acylated ketone of a polyamino acid.
46. 46. A method as defined in accordance with any of claims 39 to 45, characterized in that this distng agent comprises a carboxylic acid having the formula: R-C02H wherein R is Ci to C24 alkyl, C2 to C24 alkenyl, C3 to Cio cycloalkyl. C 3 to Cι cycloalkenyl, phenyl, naphthyl, (C 1 to C 1 alky) phenyl, (C 2 to C 1 alkenyl) phenyl, (C 1 to C 1 alky) naphthyl, (C 2 to C 1 alkenyl) naphthyl, phenyl (C 1 to C 1 alkyl) phenyl ( C2 to Cio alkenyl) naphthyl (Ci to Cio alkyl) / or naphthyl (C2 to Cio alkenyl); R is optionally substituted with Ci to Cι alkyl, C 2 to Cι alkenyl, Ci to C 4 alkoxy, -OH, -SH, -C02R x, C 3 cycloalkyl to Cι / cycloalkenyl C 3 to Cιo heterocycle having from 3 to 10 ring atoms in where the heteroatom is one or more atoms of N, O, S, or any combination thereof; aryl, (C 1 -Cy alky) aryl, aryl (Ci a Cio) alkyl or any combination thereof; R is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; Y R1 is hydrogen, C1 to C4 alkyl, or C2 to C4 alkenyl; or any salt of these.
47. 47. A method for preparing an intranasally distributable biologically active agent, characterized in that this method comprises: (a) providing a biologically active agent that can exist in a natural conformational state, a denatured conformational state and an intermediate conformational state, which is reversible in this state natural and is in a conformational way between this natural state and this denatured state (b) exposing this biologically active agent to a distng complexing agent to reversibly transform this biologically active agent to this intermediate state, and to form an intranasally distributable supramolecular complex, this distng agent has a molecular weight range from about 150 to about 600 daltons, and having at least one hydrophilic portion and at least one hydrophobic portion, this supramolecular complex comprises this distng agent forming complexes in a non-covalent way with this biologically active agent, this biologically active agent does not form microspheres with this distng agent, and this distng agent is present in an amount effective for its intranasal distribution of this biologically active agent.
48. 48. A method as defined in accordance with claim 47, characterized in that it further comprises the step of: (c) preparing a mimetic agent for a supramolecular complex.
49. 49. A method as defined in accordance with claim 48, characterized in that said biologically active agent comprises a peptide and this mimetic agent comprises a mimetic agent of a peptide.
50. 50. An intranasal distribution composition comprising a mimic agent of the intranasal distribution composition prepared in accordance with the method of claim 47.
51. . An intranasal distribution composition comprising a supramolecular complex characterized in that it comprises: (a) a biologically active agent in an intermediate conformational state complexing non-covalently with (b) a disturbing complexing agent having a molecular weight ranging from about 150 to about 600 and having at least one hydrophobic portion and at least one hydrophilic portion wherein this intermediate state is reversible in this natural state and is conformational between the natural conformational state and this denatured conformational state of this biologically active agent; this composition is not a microsphere; this disturbing agent is present in an amount effective for the intranasal distribution of this biologically active agent.
52. 2. An intranasal distribution composition as defined in claim 51, characterized in that said biologically active agent is selected from a group consisting of human growth hormone, bovine growth hormone, hormone releasing hormone, growth, an interferon, interleukin-I, interleukin-II, insulin, heparin, low molecular weight heparin, calcitonin, erythropoietin, atrial naturétic factor, an antigen, a monoclonal antibody, somatostatin, adenocortinotropin, gonadotropin-releasing hormone, oxytocin, vasopressin , sodium cromolyn, vancomycin, desferrixamine, an antimicrobial, an antifungal agent and any combination of any of these precedents.
53. 3. An intranasal distribution composition as defined according to any one of claims 51 to 52, characterized in that this disrupting agent is selected from the group consisting of a proteinoid, an acylated amino acid, an acylated polyamino acid, a sulfonated amino acid, a sulfonated polyamino acid, an acylated aldehyde of an amino acid, an acylated aldehyde of a polyamino acid, an acylated ketone of an amino acid, and an acylated ketone of a polyamino acid.
54. 54. An intranasal distribution composition as defined in any of claims 51 to 53, characterized in that this disrupting agent comprises a carboxylic acid having the formula: R-C02H wherein R is Ci to C24 alkyl, C2 to C2 alkenyl, C3 to Cio cycloalkyl / C3 to Cio cycloalkenyl, phenyl, naphthyl, (Ci to Cio alkyl) phenyl, (C2 to C2 alkenyl) phenyl, (Ci to Cio alkyl) naphthyl, (C2 to C2 alkenyl) naphthyl, phenyl (C1 to C14 alkyl) / phenyl (C2 to C2 alkenyl) / naphthyl (C1 to C14 alkyl) or naphthyl (C2 to C2 alkenyl); R is optionally substituted with C 1 to C 1 alkyl. C2 to Cio alkenyl, Ci to C4 alkoxy, -OH, -SH, -CO..R1, C3 cycloalkyl to Cι / cycloalkenyl C3 to Cι / heterocycle having from 3 to 10 ring atoms wherein the hetero atom is one or more atoms of N, O, S, or any combination thereof; aryl, (C 1 -Cy alky) aryl, aryl (Ci a Cio) alkyl or any combination thereof; R is optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; Y R1 is hydrogen, C1 to C4 alkyl, or C2 to C4 alkenyl; or any salt of these.
55. 55. A unit dose form characterized in that it comprises: (A) a composition as defined in accordance with claim 51; Y (B) (a) an excipient, (b) a diluent, (c) a disintegrant, (d) a lubricant, (e) a plasticizer, (f) a dye, (g) a dosing vehicle, or (h) ) any combination of these
56. A method for preparing an agent that is capable of being distributed by the intranasal route to a subject in need of this agent, characterized in that this method comprises: (a) provide a biologically active agent that can exist in a natural conformational state, a denatured conformational state and an intermediate conformational state, which is reversible in this natural state and is conformationally between this natural state and this denatured state (b) exposing this biologically active agent to a disturbing complexing agent to reversibly transform this biologically active agent to this intermediate state, wherein this disrupting agent is present in an amount effective for intranasal distribution of this agent biologically active; and (c) preparing a mimetic agent of this intermediate state.
57. 57. A method as defined in accordance with claim 56, characterized in that this disrupting agent comprises a pH-changing agent, an ion-strength-altering agent, or guanidine hydrochloride. SYSTEM. OF TRANSPORTATION OF ACTIVE AGENTS SUMMARY The present invention relates to methods and compositions for transporting active agents, in particular to biologically active agents, through cell membranes or lipid bilayers. These methods and compositions facilitate the distribution of an active agent to an objective, such as the distribution of a pharmaceutical agent through an adverse environment to a particular location of the body.