Pharmaceutical formulations comprising insulin complexed with a diketopiperazine

The present invention is generally in the field of pharmaceutical formulations, and more specifically, relating to methods and compositions for purifying and stabilizing peptides and proteins (eg, insulin) used in pharmaceutical applications.

In normal individuals, the insulin produced by the β-cells of the islets of Langerhans in the pancreas produces the insulin needed by the body for glucose metabolism in response to increased blood glucose levels. Insulin metabolizes incoming glucose and temporarily discontinues the conversion of liver glycogen and lipids to glucose, thereby allowing the body to support the metabolic activity between meals. However, type I diabetes has a reduced ability or absolute inability to produce insulin due to β-cell destruction and requires daily injections or insulin replacement via an insulin pump. However, type II diabetes is more common than type I diabetes, which is characterized by insulin resistance and progressively impaired pancreatic β-cell function. Type II diabetes can still produce insulin, but these may also require insulin replacement therapy.

Type II diabetes typically exhibits a delayed response to increased blood glucose levels. Normal people usually release insulin within a few minutes of consuming food, but type II diabetes cannot secrete endogenous insulin even hours after consumption. As a result, endogenous glucose production persists after consumption (Pfeiffer, Am.J.Med., 70: 579-88 (1981)), and patients experience hyperglycemia due to elevated blood glucose levels. To do.

Deletion of glucose-induced insulin secretion is one of the early disorders of β-cell function (Cerasi et al., Diabetes, 21: 224-34 (1972); Polonsky et al., N. Engl. J. Med., 318: 1231-39 (1988)), the cause and extent of β-cell dysfunction is unknown in most cases. While genetic function plays an important role (Leahy, Curr. Opin. Endocrinol. Diabetes, 2: 300-06 (1995)), some insulin secretory disorders appear to be acquired and optimal. It can be at least partially reversible through a diabetic glucose control. Optimal glucose regulation via postprandial insulin treatment requires both normal tissue responsive to a sharp increase in administered insulin and serum insulin levels, thereby in natural glucose-induced insulin release. Significant improvements can occur. Therefore, the challenge presented in early-stage treatment of patients without excessive deletion of β-cell function in type II diabetes is to restore postprandial insulin release.

The earliest stages of type II diabetes are currently treated with oral preparations with little success. Subcutaneous injections of insulin are also rarely effective in delivering insulin to patients with type II diabetes, and this is due to delayed, variable and superficial onset of insulin. The action can actually be exacerbated. However, when insulin is administered intravenously with a meal, early-stage type II diabetes experiences a dysfunction of hepatic glucose production and exhibits increased physiological glucose regulation. In addition, these free fatty acid levels fall at a faster rate than without insulin treatment. Insulin intravenous administration is probably effective in treating type II diabetes, but it is not a suitable solution. This is because intravenous administration with each meal is neither safe nor feasible for the patient.

Insulin is a polypeptide with a nominal molecular weight of 6,000 daltons and has traditionally been produced by treating porcine and bovine pancreas to isolate natural products. However, more recently, recombinant techniques have been used to produce human insulin in vitro. Natural and recombinant human insulin in aqueous solution, when dissolved in water in the presence of zinc ions, has a hexamer configuration, i.e., six molecules of recombinant insulin are non-covalently bound to the hexamer complex. Meet. Hexamer insulin is not absorbed rapidly. In order for recombinant human insulin to be absorbed into the patient's circulation, this hexamer is first in the form of a dimer and / or monomer before the material can be transferred into the blood stream. Must meet. Delayed absorption requires that recombinant human insulin be administered approximately half an hour before meal time to produce therapeutic insulin blood levels, which accurately predicts meal time. Can be annoying for patients who need it. To overcome this delay, recombinant human insulin analogs (eg, HUMALOG)^TM) Was developed. It rapidly dissociates into monomeric form substantially completely after subcutaneous administration. Clinical research is HUMALOG^TMHowever, it was shown that after subcutaneous administration, it was absorbed quantitatively faster than recombinant human insulin. See, for example, US Pat. No. 5,547,929 to Anderson Jr. et al.

Administration of monomeric analogs of insulin via the pulmonary pathway has been developed in an effort to avoid the disadvantages associated with delivery by injection and accelerate absorption. For example, US Pat. No. 5,888,477 to Gonda et al. Discloses that a patient is forced to inhale an aerosol formulation of monomeric insulin to accumulate insulin particles on the patient's lung tissue. However, monomeric formulations are unstable and rapidly lose their activity, while the rate of uptake remains unchanged.

Although it is desired to produce rapidly absorbable insulin from natural sources, the conversion from hexameric to monomeric form, for example by removing zinc from the complex, is unstable. Produces insulin with a short shelf life that is present and undesired. Therefore, it is desired to provide the monomeric form of insulin while maintaining its stability in the absence of zinc. It is advantageous to provide diabetics with a monomeric insulin composition that is suitable for pulmonary administration, provides rapid absorption and can be produced into off-the-shelf formulations with a commercially useful shelf life.

These problems with impurities (metal ions that affect stability or bioavailability) arise with many other proteins and peptides.

US Pat. No. 6,071,497 to Steiner et al. Is stable at pH below 6.4 and unstable at pH above 6.4, diketopiperazine microparticles, or stable at acidic and basic pH, but about 6.4 and 8 Disclosed is a particulate drug delivery system in which a drug is encapsulated in diketopiperazine particulates, which is unstable at a pH between This patent does not disclose a monomeric insulin composition that is stable for pulmonary administration, provides rapid absorption, and can be produced into off-the-shelf formulations with a commercially useful shelf life.

Therefore, it is advantageous to develop an alternative insulin delivery composition for type II diabetes that provides a faster rise in blood levels of insulin and is easily administered to ensure patient compliance. Is. It is also desirable to apply this delivery composition and delivery method to other active agents.

Therefore, it is an object of the present invention to provide improved methods for purifying peptides and proteins, especially in the preparation of compositions suitable for pulmonary administration.

It is another object of the present invention to provide a stable monomeric peptide composition suitable for pulmonary delivery.

It is a further object of the present invention to provide methods and compositions for facilitating the transport of insulin and other biologically active factors across biological membranes.

It is another object of the invention to provide methods and compositions for improved absorption of insulin or other biologically active factors in the bloodstream.

It is a further object of the present invention to provide methods and compositions for improved absorption of insulin or other biologically active factors in the bloodstream, characterized by ease of administration.

Purify the peptide or protein by incorporating the peptide or protein into diketopiperazine or a competitive complexing agent to facilitate the removal of one or more impurities (ie, undesired components) from the peptide or protein. A method for is provided. In a preferred embodiment, a peptide such as insulin containing one or more impurities (eg, zinc ions) is trapped in diketopiperazine to form a peptide / diketopiperazine / impurity precipitate, which is then , Diketopiperazine is non-solvent and peptide is non-solvent, washed with a solvent for the impurities to be removed. Alternatively, the impurities can be removed by using a complexing agent that selectively forms a complex with the impurities and, for example, removes by dialysis.

Formulations and methods are also provided for improved transport of active factors across biological membranes, which results in, for example, a rapid increase in factor concentration in the blood. Formulations include (i) an active factor that may be charged or neutral, and (ii) a transport enhancer and / or target biomembrane that masks the charge of the factor to facilitate transport. Examples include fine particles formed from transport enhancers that form hydrogen bonds. In a preferred embodiment, insulin is administered via pulmonary delivery of microparticles containing fumaryldiketopiperazine and a biologically active form of insulin. The charge on the insulin molecule is masked by the hydrogen that binds the insulin molecule to diketopiperazine, which allows insulin to cross the target membrane. This method of delivering insulin results in a rapid increase in blood insulin levels comparable to the increase resulting from intravenous delivery.

<figref num="1">FIG. 1a is a graph of average blood glucose level over time (minutes). FIG. 1b is a graph of mean C-peptide concentration during experiments comparing insulin levels (ng / ml) over time (minutes) when insulin was administered intravenously, subcutaneously and by inhalation. Is.</figref><figref num="2">FIG. 2a is a graph of glucose infusion rate (mg / kg / min) over time comparing insulin administered by intravenous administration, subcutaneous administration and inhalation. FIG. 2b is a graph of mean insulin concentration (μU / ml) over time comparing insulin administered intravenously, subcutaneously and by inhalation.</figref>

Encapsulation or trapping of large polymers (eg, proteins and peptides) in diketopiperazine can be used to remove impurities or contamination (eg, metal ions or other small molecules). Diketopiperazine also acts in both stabilizing and enhancing the delivery of trapped material. Formulations have also been developed for enhanced transport of active factors across biological membranes. These formulations are formed from (i) an active factor that may be charged or neutral, and (ii) a transport enhancer that masks the charge of the factor and / or a transport enhancer that hydrogen bonds to the membrane. Fine particles can be mentioned. This formulation may provide a rapid increase in the concentration of active factor in the blood after administration of the formulation.

For example, it was discovered that hexamer insulin is delivered to the lungs in a fumalyl diketopiperazine formulation and can reach peak blood levels within 3-10 minutes. In contrast, insulin administered by the pulmonary route without fumaryldiketopiperazine typically takes between 25-60 minutes to reach peak blood levels, while hexamer. Insulin, when administered by subcutaneous injection, takes 30-90 minutes to reach peak blood levels. This behavior has been successfully reproduced several times and in several species, including humans.

Removal of zinc from insulin typically results in unstable insulin with an undesired short shelf life. Purification, stabilization and enhanced delivery for the removal of zinc in insulin are illustrated by way of example. Insulin formulations trapped in fumaryldiketopiperazine have been found to be stable and have an acceptable shelf life. Measurements of zinc levels showed that zinc was largely removed during the trapping process, resulting in monomeric insulin in a stable delivery formulation.

Rapid absorption of many other peptides, including salmon calcitonin, parathyroid hormone 1-34, octreotide, leuprolide and RSV peptides, means that the peptide is concentrated within 3-10 minutes after lung delivery. It is observed when delivered pulmonary in fumalyl diketopiperazine, which provides a peak of.

(I. Material) (A. Delivered factor) A factor delivered herein refers to a molecule as an active factor, or encapsulated or trapped. It may or may not be a charged species. Examples of classes of active factors suitable for use in the compositions and methods described herein include therapeutic agents, prophylactic and diagnostic agents, and dietary supplements (eg, vitamins).

Although the exact mechanism by which diketopiperazine forms a complex with the material to which it is delivered is unknown, diketopiperazine is thought to form a complex with the material to be purified. This process is referred to herein as interchangeable as trap or encapsulation.

These materials can be any polymer or large organic molecule, most preferably peptides and proteins. Generally speaking, any form of drug is trapped. Examples include synthetic inorganic and organic compounds, proteins and peptides, polysaccharides and other sugars, lipids and nucleic acid sequences that have therapeutic, prophylactic or diagnostic activity. A protein is defined as consisting of 100 or more amino acid residues; a peptide is less than 100 amino acid residues. Unless otherwise stated, the term protein refers to both proteins and peptides. Factors incorporated can have a variety of biological activities (eg, vasoactive factors, neuroactive factors, hormones, anticoagulants, immunomodulatory factors, cytotoxic factors, antibiotics, antiviral agents). , Antisense, antigens, and antibodies). In some examples, the protein can be an antibody or antigen, otherwise it must be administered by injection to elicit an appropriate response. Representative polymers include proteins, peptides, polysaccharides, nucleic acid molecules, and combinations thereof.

Preferred peptides and proteins include hormones, cytokines and other immunomodulatory peptides, as well as antigens / vaccines. In a preferred embodiment, the active factor is monomeric insulin or a stabilized form of insulin purified to remove zinc. In another preferred embodiment, the active factor is glucagon.

The active factor (ie, drug) can be an antigen, where this molecule preferentially infects the lung with a protective immune response (eg, mycoplasma, bacteria that cause pneumonia, and RS (respiratory synticial) virus. ) Is intended to provoke. In these cases, it may also be useful to administer the drug in combination with an adjuvant to increase the immune response to the antigen.

Any gene that is useful in replacing or supplementing the desired function or in achieving the desired effect, such as inhibiting tumor growth, is introduced using the matrix described herein. Can be done. As used herein, a "gene" is an isolated nucleic acid molecule with a nucleotide length greater than 30, preferably 100 or greater. Examples of genes that replace or replace function are missing enzymes such as adenosine deaminase (ADA), which have been used in clinical trials to treat ADA deficiency, as well as insulin and coagulation factor VIII. Examples include genes encoding cofactors. The gene that results in regulation can also be administered alone or in combination with a gene that supplements or replaces a particular function. For example, a protein that suppresses the expression of a particular protein-encoding gene and, conversely, a gene that encodes a protein that induces the expression of a protein-encoding gene can be administered in the matrix. Examples of genes that are useful in stimulating an immune response include viral and tumor antigens, as well as cytokines (tumor necrosis factor) and cytokine inducers (endotoxins), as well as various pharmacological factors.

Other nucleic acid sequences that may be utilized include antisense molecules that bind to complementary DNA and inhibit transcription, ribozyme molecules, and externally induced sequences that are used to target cleavage by RNAase P.

As used herein, a vector is a factor that transports the gene into a target cell and contains a promoter that results in the expression of the gene in the cell to which the vector is delivered. Promoters are common promoters that produce expression in a variety of mammalian cells, are cell-specific, or are even nucleus-to-cytoplasmic-specific. These are known to those of skill in the art and can be constructed using standard molecular biology protocols. Vectors that increase infiltration (eg, lipids, liposomes, lipid conjugate-forming molecules, detergents, and other membrane permeability increasing factors) are commercially available and can be delivered with nucleic acids.

Imaging agents, including metals, radioisotopes, radiopaque agents, fluorescent dyes, and radiopaque agents, can also be incorporated. Examples of radioisotopes and radiopaque agents include gallium, technetium, indium, strontium, iodine, barium, and phosphorus.

Impurities that can be removed from the active factor composition include metal ions (eg, zinc) and other divalent or polyvalent ions, as well as small inorganic molecules and solvent residues.

(B. Diketopiperazine) Diketopiperazines useful in the compositions and methods of the invention are described, for example, in US Pat. No. 6,071,497, which is hereby incorporated by reference in its entirety.

((i) General formula) Diketopiperazine or its substituted analog is a rigid planar ring with at least 6 ring atoms, including heteroatoms and unbonded electron pairs. One or both of the nitrogens are replaced with oxygen to produce the substituted analogs diketomorpholine and dioxanedioxane, respectively. It is possible to replace nitrogen with sulfur atoms, but this does not give a stable structure.

The general formula of diketopiperazine and its analog is shown below.

<chemistry num="1"><img id="000002" he="45" wi="52" file="JP5774828B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Where n is between 0 and 7, and Q is independently C_1~20Linear alkyl, branched alkyl or cyclic alkyl, aralkyl, alkaline, alkenyl, alkynyl, heteroalkyl, heterocyclic, alkyl-heterocyclic or heterocyclic-alkyl; T is -C (O) O, -OC (O), -C (O) NH, -NH, -NQ, -OQO, -O, -NHC (O), -OP (O), -P (O) O, -OP (O)₂, -P (O)₂O, -OS (O)₂, Or -S (O)₃U is an acid group (eg, carboxylic acid, phosphoric acid, phosphonic acid, and sulfonic acid) or a basic group (eg, primary amine, secondary amine and tertiary amine, quaternary ammonium salt, guanidine, aniline). ), Heterocyclic derivatives (eg, pyridine and morpholin), or diionic C containing at least one acidic group and at least one basic group (eg, the groups described above)._1~20A chain, where the side chain can be further functionalized with an alkene or alkyne group at any position, and one or more carbons on the side chain can be replaced with oxygen, eg, a short polyethylene glycol chain. It can occur, one or more carbons can be functionalized with an acidic or basic group as described above, where the ring atoms X at positions 1 and 4 are either O or N.

As used herein, "side chain" is defined as QTQU or QU, where Q, T, and U are as defined above.

Examples of acidic side chains include, but are not limited to: cis and trans, -CH = CH-CO.₂H, -CH (CH₃) = CH (CH)₃)-CO₂H,-(CH₂)₃-CO₂H, -CH₂CH (CH)₃)-CO₂H, -CH (CH₂CO₂H) = CH₂,-(Tetrafluoro) Benzoic Acid,-Benzoic Acid and-CH (NHC (O) CF₃)-CH₂-CO₂H.

Examples of basic side chains include, but are not limited to: -aniline, -phenyl-C (NH) NH.₂, -Phenyl-C (NH) NH (alkyl), -Phenyl-C (NH) N (alkyl)₂And-(CH₂)₄NHC (O) CH (NH₂) CH (NH₂) CO₂H.

Examples of zwitterionic side chains include, but are not limited to: -CH (NH).₂)-CH₂-CO₂H and -NH (CH)₂)_1~20CO₂H.

The term aralkyl refers to an aryl group having an alkyl substituent.

Terminology Heterocyclic-alkyl refers to a heterocyclic group having an alkyl substituent.

The term alkaline refers to an alkyl group having an aryl substituent.

The term alkyl-heterocyclic refers to an alkyl group having a heterocyclic substituent.

The term alkene, as referred to herein, and unless otherwise specified, is C.₂~ C₁₀Alkene group, especially containing vinyl and allyl.

The term alkyne, as referred to herein, and unless otherwise specified, C.₂~ C₁₀Alkyne group.

As used herein, "diketopiperazine" includes diketopiperazine and its derivatives and variants that fall within the scope of the general formula above.

Fumalyl diketopiperazine is most preferred for pulmonary application.

((ii) Synthesis) Diketopiperazine can be formed by cyclic dimerization of amino acid ester derivatives, as described in Kathalski et al., J.Amer.Chem.Soc.68: 879-80 (1946), or of dipeptide ester derivatives. It can be formed by cyclization, or by thermal dehydration of amino acid derivatives in a high boiling solvent, as described by Kopple et al., J. Org. Chem. 32 (2): 862-64 (1968). Can be done. 2,5-Diketo-3,6-di (aminobutyl) piperazine (Katchalski et al. Called it lysine anhydride) in molten phenol, similar to J. Org. Chem.'S Kopple method. It was prepared by cyclic dimerization of N-ε-PL-lysine in (1) and then removal of the butyl (P) -group using HBr of 4.3 M in acetic acid. Because this pathway uses commercially available starting materials, includes reaction conditions that have been reported to preserve the stereochemistry of the starting materials in the product, and all steps can be easily scaled up for production. ,preferable.

Diketomorpholin and diketooxetane derivatives can be prepared by stepwise cyclization in a manner similar to that disclosed in Kattalski et al., J. Amer. Chem. Soc. 68: 879-80 (1946).

Diketopiperazine can be radiolabeled. Means for binding the radiolabel are known to those of skill in the art. Radiolabeled diketopiperazine can be prepared, for example, by reacting tritium gas with a compound listed above that contains a double or triple bond. C-14 radiolabeled carbon is readily available¹⁴It can be incorporated into the side chain by using a C-labeled precursor. These radiolabeled diketopiperazines can be detected in vivo after administering the resulting microparticles to the subject.

((a) Synthesis of symmetric diketopiperazine derivatives) Diketopiperazine derivatives are symmetric if both side chains are identical. Side chains can contain acidic groups, basic groups, or combinations thereof.

An example of a symmetric diketopiperazine derivative is 2,5-diketo-3,6-di (4-succinylaminobutyl) piperazine. 2,5-Diketo-3,6-di (aminobutyl) piperazine is thoroughly succinylated with succinic anhydride in a slightly alkaline aqueous solution and is readily soluble in a weakly alkaline aqueous solution, The acidic aqueous solution gives a product that is completely insoluble. When a concentrated solution of a compound in a weakly alkaline aqueous solution is rapidly acidified under appropriate conditions, the substance separates from the solution as fine particles.

Other preferred compounds can be obtained by replacing the succinyl group in the above compounds with a glutaryl group, a maleyl group, or a fumaryl group.

((B) Synthesis of asymmetric diketopiperazine derivatives) One way to prepare an asymmetric diketopiperazine derivative is to protect the functional groups on the side chains, selectively deprotect one of the side chains, and react the deprotected functional groups. Forming the first side chain, deprotecting the second functional group, and reacting the deprotected functional group to form the second side chain.

Diketopiperazine derivatives with protected acidic side chains (eg, cyclo-Lys (P) Lys (P), where P is a benzyloxycarbonyl group, or a protecting group known to those of skill in the art) are selective. Can be deprotected. These protecting groups can be obtained by using limiting reagents (eg, HBr for benzyloxycarbonyl groups, or fluoride ions for silicon protecting groups) and by using controlled time intervals. Can be selectively disconnected. In this way, reaction mixtures containing unprotected, mono-protected, and di-protected diketopiperazine derivatives can be obtained. These compounds have different solubilities in different solvents and pH ranges and can be separated by selective precipitation and removal. A suitable solvent (eg, ether) can then be added to such a reaction mixture to precipitate all of these substances together. This allows the deprotection reaction to be stopped before completion by removing diketopiperazine from the reactants used to deprotect the protecting group. By stirring the mixed precipitate with water, both partially reacted and fully reacted species can be dissolved in the aqueous medium as salts. Unreacted starting material can be removed by centrifugation or filtration. By adjusting the pH of the aqueous solution to weakly alkaline conditions, the asymmetric monoprotective product containing a single protecting group precipitates from the solution, leaving a completely deprotected substance in the solution.

In the case of diketopiperazine derivatives with basic side chains, the basic groups can also be selectively deprotected. As mentioned above, the deprotection step can be stopped prior to completion, for example by adding a suitable solvent to the reaction system. By carefully adjusting the pH of the solution, the deprotected derivatives can be removed by filtration, and the partially deprotected and fully deprotected derivatives remain in the solution. By adjusting the pH of the solution to slightly acidic conditions, the monoprotected derivative can precipitate from the solution and be isolated.

Zwitterionic diketopiperazine derivatives can also be selectively deprotected as described above. In the final step, adjusting the pH to slightly acidic conditions causes the monodeprotecting compound with free acidic groups to precipitate. By adjusting the pH to slightly basic conditions, monodeprotective compounds with free basic groups precipitate.

Limited removal of protecting groups by other mechanisms includes, but is not limited to, cleavage of protecting groups that are cleaved by hydrogenation using a limited amount of hydrogen gas in the presence of a palladium catalyst. The resulting product is also an asymmetric, partially deprotected diketopiperazine derivative. These derivatives can be isolated essentially as described above.

Monoprotected diketopiperazine is reacted to produce diketopiperazine with one side chain and a protecting group. Removal of the protecting group and coupling with other side chains results in diketopiperazine asymmetrically substituted with a mixture of acidic, basic, and zwitterionic side chains.

Other materials that exhibit this reaction to pH can be obtained by functionalizing the amide ring nitrogen of the diketopiperazine ring.

(C. Transport Enhancer) In a preferred embodiment, the active factor is complexed with a transport enhancer that is degradable and capable of forming hydrogen bonds with the target biological membrane to facilitate transport of the factor across the membrane. Transport enhancers can also form hydrogen bonds with active factors to mask the charge when charged and to facilitate the transport of factors across the membrane. A preferred transport enhancer is diketopiperazine.

The transport enhancer is preferably biodegradable and may provide linear, pulsed or bulk release of the activator. Transport enhancers can be natural or synthetic polymers and via substitution or addition of chemical groups, including alkyly, alkylene, hydroxylation, oxidation, and other modifications customarily made by one of ordinary skill in the art. Can be modified.

A preferred transport enhancer is fumalyl diketopiperazine. Other diketopiperazines that may be useful as transport enhancers are described above.

Like most proteins and peptides, insulin is a charged molecule, which impedes its ability to cross charged biological membranes. It has been found that when insulin hydrogen-bonds to fumaryldiketopiperazine, the charge of the peptide is masked, thereby facilitating or enhancing the passage of insulin across the membrane (eg, mucosa) and into the blood. It has been issued.

(II. Method) (A. Encapsulation) In one embodiment, the active factor dissolves diketopiperazine having an acidic side chain in bicarbonate or other basic solution, the active factor in the solution or suspension is added, and then an acid (eg, eg). By precipitating the fine particles by adding 1M citric acid), they are encapsulated in the fine particles.

In another embodiment, the active factor dissolves diketopiperazine having a basic side chain in an acidic solution (eg, 1M citric acid), adds the active factor in solution or suspension, and then bicarbonate. Alternatively, the fine particles are encapsulated in the fine particles by precipitating the fine particles by adding another basic solution.

In yet another embodiment, the active factor dissolves diketopiperazine having both acidic and basic side chains in an acidic or basic solution, and the encapsulated active factor is added to the solution or suspension. It is encapsulated within the microparticles by adding and then precipitating the microparticles by neutralizing the solution.

The microparticles can be stored dry and suspended for administration to a patient. In the first embodiment, the reconstructed microparticles maintain their stability in an acidic medium and dissociate as the medium approaches a physiological pH in the range between 6 and 14. In a second embodiment, the suspended microparticles remain stable in a basic medium and dissociate at a pH between 0 and 6. In a third embodiment, the reconstructed microparticles maintain their stability in an acidic or basic medium and dissociate as the medium approaches a physiological pH in the range between 6 and 8.

The impurities are typically removed when the fine particles precipitate. However, impurities can also be removed by washing the particles to dissolve the impurities. A preferred wash solution is water or an aqueous buffer. Solvents other than water can also be used to wash the microspheres or to precipitate diketopiperazine to remove impurities that are not soluble in water. Any solvent in which neither cargo nor fumaryl diketopiperazine is soluble is suitable. Examples include acetic acid, ethanol, and toluene.

In an alternative embodiment, the diketopiperazine microparticles are prepared and provided in a suspension (typically an aqueous suspension), to which a solution of the active factor is then added. The suspension is then lyophilized or lyophilized to produce diketopiperazine microparticles with an active factor coating. In a preferred embodiment, the active factor is insulin in hexamer form. Zinc ions can then be removed by washing the particles with a suitable solvent.

As used herein, with respect to an active factor containing / diketopiperazine in diketopiperazine, the term "trapped" includes coating the active factor on fine particles of diketopiperazine.

Diketopiperazine microparticles were found to have a higher affinity for insulin than zinc. Insulin was found to be stabilized within a designated grid array of fumaryldiketopiperazine. In this case, in a sufficient deficiency of zinc ions, insulin is predominantly a dimer or monomer, as opposed to its hexameric state. Therefore, insulin dissociates more easily into its monomeric state, which is the state in which insulin exerts its biological activity.

Other complexing agents can be used in place of diketopiperazine. Other representative complexing agents include serum albumin and other proteins, arginine acid, antibodies, cyclodextrins, phospholipids, and lecithin. For example, zinc-contaminated insulin can be complexed with bovine serum albumin. This complex can be dialyzed off in a tube with a molecular weight cutoff of less than 1,000 daltons to separate and remove zinc. Once a sufficient amount of zinc has been dialyzed, the dispersion is transferred to a dialysis tube with a molecular weight cutoff of 10,000 daltons or less, as evidenced by its presence in the dialysate. Only the monomeric insulin passes through the tube into the dialysate, leaving any remaining hexameric zinc complexed insulin. Purified insulin is trapped from the dialysate.

However, these materials may not provide sufficient stability for unstable or labile drugs.

(B. administration) The composition of the active factor described herein can be administered to a patient in need of the active factor. The composition is preferably administered in the form of particulates, which can be in the form of a dry powder for pulmonary administration or can be suspended in a suitable pharmaceutical carrier (eg, saline).

The microparticles are preferably stored in a dry or lyophilized form until just prior to administration. The microparticles can then be directly administered as a dry powder, for example, by an inhalation method (eg, using a dry powder inhaler known in the art). Alternatively, the microparticles can be suspended in a sufficient amount of pharmaceutical carrier, for example as an aqueous solution for administration as an aerosol.

The microparticles can also be administered via the oral, subcutaneous, and intravenous routes.

The composition can be administered to any targeted biological membrane (preferably the patient's mucosa). In a preferred embodiment, the patient is a human suffering from type II diabetes. In a preferred embodiment, the composition delivers a biologically active form of insulin to the patient, which provides a spike in serum insulin concentration that stimulates a normal response to eating.

In a preferred embodiment, hexameric insulin is trapped in fumaryldiketopiperazine to form a solid precipitate of monomeric insulin in fumaryldiketopiperazine, which then removes free zinc. Therefore, it is washed with an aqueous solution. This formulation shows blood uptake after pulmonary administration at 2.5 times the rate of insulin uptake after subcutaneous injection, where blood level peaks are between 7.5 and 10 minutes after dosing. occured.

The range of load of the delivered drug is typically between about 0.01% and 90%, depending on the form and size of the delivered drug and the target tissue. In a preferred embodiment using diketopiperazine, the preferred range is a loading of 0.1% to 50% by weight of the drug. Appropriate dosing can be determined, for example, by the amount of incorporated / encapsulated factor, the rate of release of the factor from the microparticles, and, in a preferred embodiment, the blood glucose level of the patient.

One preferred application is in the treatment of hyperinsulinemia. In a preferred embodiment, microparticles of the composition in which the active factor is glucagon can be administered by continuous subcutaneous injection. Glucagon is a very unstable peptide, but can be stabilized in particles of diketopiperazine, for example. Stabilized glucagon / diketopiperazine microparticles can be made by adding glucagon to a solution of diketopiperazine, which hydrogen bonds to glucagon, and this solution can be made, for example, from food acid (food). When acidified by the addition of acid), both diketopiperazine and glucagon self-assemble to form uniform microspheres with an average particle size of, for example, about 2 μm. In this process, about 95% glucagon is withdrawn from the solution and evenly distributed within the diketopiperazine microparticles. These particles can be easily suspended and injected subcutaneously using a standard infusion pump. The glucagon / diketopiperazine particles are then contacted with a nearly neutral pH environment of the subcutaneous fluid, where these particles dissolve, thereby releasing glucagon in its pharmacologically active state.

The compositions and methods described herein are further described by the following non-limiting examples.

(Example 1: Removal of zinc from USP injectable insulin) Insulin trapped in fumaryldiketopiperazine was analyzed to assess whether zinc was removed during the trapping process. The insulin used as a starting material met the USP standard for injectable insulin, and according to the certificate of analysis, this insulin contained a significant amount of zinc (ie 0.41%). The insulin was then trapped in fumaryldiketopiperazine to form a solid fumaryldiketopiperazine / insulin mixture as described above.

Following the trapping of insulin on fumaryldiketopiperazine, this amount of zinc should theoretically be present in the same proportion as it would be in neat insulin. Using the certificate of analytical values, it was calculated that it would be expected to find 697 ppm zinc per gram in solid yield of fumaryldiketopiperazine / insulin. Surprisingly, the amount of zinc present in solid fumalyl diketopiperazine / insulin was measured to be only 6 ppm. The "lost" zinc was probably eliminated with the water used to wash the insulin / fumaryldiketopiperazine precipitate.

(Example 2: Bioavailability of insulin in diketopiperazine lung formulation) (Subjects and methods) This study was reviewed and certified by the Heinrich-Heine-University, Dusseldorf Ethical Review Board and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice rules.

This study was conducted on 5 healthy male volunteers. Comprehensive criteria are good health as judged by physical examination, age: 18-40 years, body mass index: 18-26 kg / m², Ability to reach peak expiratory flow 4 I / sec, as measured by computer-assisted vital capacity measurement, and FEV predicted to be normal above 80%₁(FEV₁(= Forced exhalation volume for 1 second). Exclusion criteria are true diabetes type 1 or 2, prevalence of human insulin antibody, history of hypersensitivity to study medication or drugs with similar chemical structure, history or severity or multiple of allergies, before study initiation Treatment with some other investigative factor in the last 3 months of the disease, progressive lethal disease, history of drug or alcohol abuse, current drug treatment with other drugs, prominent cardiovascular disease, respiratory disease, gastrointestinal illness A smoker with a history of illness, liver illness, renal illness, neurological illness, psychiatric illness, and / or hematological illness, ongoing airway infection, or evidence or history of tobacco or nicotine use. It was a patient who was defined as.

(Implementation of research) On the morning of the test day (7:30 a.m.), the subject (fasting except water after midnight) came to the hospital. These subjects were restricted from excessive exercise and alcohol intake for 24 hours prior to each treatment day. These subjects were randomly assigned to one of the three treated upper arms. These subjects were given a constant intravenous infusion of regular human insulin. This injection is 0.15 mU min so that serum insulin levels are established at 10-15 U / ml over the 2 hours prior to time point 0.^-1kg^-1Maintained in. This low dose injection was continued throughout the study to suppress endogenous insulin secretion. Glucose-controlled infusion system (BIOSTATOR) for blood glucose^TM) Maintained a constant level of 90 mg / dl during the glucose clamp. This glucose clamp algorithm was based on actually measured blood glucose levels and a grade of variability for a few minutes before calculating the glucose infusion rate to keep the blood glucose levels constant. Insulin application (5U iv or 10U sc injection, or 3 deep breathing inhalations per capsule applied with a commercial inhalation device (Boehringer Ingelheim) (2 capsules of 50U each)) was completed shortly before time point 0. I had to do it. The duration of the clamp experiment was 0 to 6 hours at time points. Glucose infusion rate, blood glucose, serum insulin, and C-peptide were measured.

(Biopotency and bioavailability) To determine bioefficiency, the area below the glucose infusion rate curve, the first 3 hours after administration (AUC)_0-180) And a full observation period of 6 hours after administration (AUC)_0-360) And correlated with the amount of insulin applied. To determine bioavailability, the area below the insulin concentration curve, the first 3 hours after administration (AUC)_0-180) And a full observation period of 6 hours after administration (AUC)_0-360) And correlated with the amount of insulin applied.

In this clamp study, 100U TECHNOSPHERE^TM/ Insulin inhalation is well tolerated and can have a significant blood glucose-lowering effect with a relative bioavailability of 25.8% for the first 3 hours, calculated from the serum insulin levels achieved. Demonstrated. TECHNOSPHERE^TMAre fine particles (also referred to herein as microspheres) formed from diketopiperazine that self-assemble into regular lattice arrays at a particular pH (typically low pH). These are typically produced to have an average diameter between about 1 μm and about 5 μm.

(result) The drug kinetics results are shown in Figures 1 and 2 and Table 1.

(Result of effect) 100U TECHNOSPHERE^TM/ Insulin inhalation (100U inhalation) peaks insulin concentration after 13 minutes (intravenous (iv) (5U): 5 minutes, subcutaneous (sc) (10U): 121 minutes), and after 180 minutes (iv) : 60 minutes, sc 360 minutes) revealed the return of insulin levels to baseline. Biological effects, as measured by glucose infusion rate, peaked after 39 minutes (iv 14 minutes, sc: 163 minutes) and lasted longer than 360 minutes (iv: 240 minutes, sc:&gt; 360 minutes). Absolute bioavailability (compared to iv application) was 14.6 ± 5.1% for the first 3 hours and 15.5 ± 5.6% for the first 6 hours. Relative bioavailability (compared to sc application) was 25.8 ± 11.7% for the first 3 hours and 16.4 ± 7.9% for the first 6 hours.

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(Safety result) TECHNOSPHERE^TM/ Insulin has been shown to be safe in all patients. One patient was coughing during inhalation without any further symptoms or signs of respiratory alteration.

(Conclusion) 100U TECHNOSPHERE^TM/ Insulin inhalation is well tolerated and can have a significant blood glucose-lowering effect with a relative bioavailability of 25.8% for the first 3 hours, calculated from the serum insulin levels achieved. Demonstrated.

(wrap up) In this study, TECHNOSPHERE^TM/ Inhalation of insulin (formulation of Example 1) has a time-action in healthy human subjects with a rapid peak of insulin concentration (Tmax: 13 minutes) and a rapid onset of action (Tmax: 39 minutes). It was demonstrated to have a profile, as well as a long-lasting effect over 6 hours. 100U TECHNOSPHERE^TM/ The total metabolic effect measured after inhalation of insulin was greater than after subcutaneous injection of 10 U insulin. TECHNOSPHERE^TMThe relative bioefficiency of / insulin was calculated to be 19.0%, while the relative bioavailability was determined to be 25.8% in the first 3 hours.

These data are also TECHNOSPHERE^TM/ Insulin inhalation results in a much faster onset of action than the onset of action of sc insulin injection (which was close to the onset of action of iv insulin injection), while TECHNOSPHERE^TM/ Indicates that the duration of action of insulin was comparable to the duration of action of sc insulin injection.

The drug was well tolerated, and no serious adverse events were reported during the entire trial.

(Example 3: Removal of impurities from proprietary peptide) The proprietary peptide containing impurities was trapped in fumaryldiketopiperazine to form a peptide / fumaryldiketopiperazine precipitate. The precipitate was washed with water to remove impurities. The peptide is fairly unstable, and trapping this peptide on fumaryldiketopiperazine significantly improves its stability, both as a dry powder and in an aqueous suspension for injection.

(Example 4: Stabilized glucagon formulation) (Prescription) Precipitating glucagon with fumalyl diketopiperazine (3,6-bis [N-fumaryl-N- (n-butyl) amino] -2,5-diketopiperazine) in an acidic solution under sterile conditions. Was prescribed to the stabilizing complex. The complex was washed and lyophilized to give a sterile dry powder formulation of diketopiperazine / glucagon (referred to herein as "TG"). It contains 1.2-8.2 wt% glucagon, depending on the desired formulation parameters (allowing the surgeon to increase the dose while keeping the volume constant). The TG powder was suspended in a suitable medium suitable for subcutaneous delivery with a MiniMed 507C infusion pump.

(Stability protocol) Glucagon and TG were suspended in the infusion medium and incubated at 40 ° C. in a water bath for various hours up to 150 hours.

(HPLC analysis of glucagon) An adaptation of the USP method for glucagon analysis was used. Waters Symmetry Shield RP8 columns (5 μm, 3.9 × 150 mm) and guard RP8 columns (5 μm, 3.9 × 20 mm) were used at a flow rate of 1 mL / min and a detection wavelength of 214 nm. Inclination method consisting of the following mobile phases: A: 9.8 g NaH per liter of HPLC grade water₂PO₄Adjust pH to 2.6 with (0.0816M) and 170mg L-Cysteine (1.4mM), phosphoric acid; and B: acetonitrile. The glucagon solution was diluted with water as needed and injected. TG samples were prepared by adding 1/10 volume of 1M Tris (pH 10.0) to the samples to solubilize fumalyl diketopiperazine.

(Rat research protocol) Sprague Dawley rats 200-250 g are fasted overnight, and glucagon or TG (0.75 mg / kg) in a suitable medium is injected subcutaneously and these rats are injected at 25 ° C for 0 hours, 24 hours, or 48 hours. Retained. Blood samples were taken at -10 minutes, -5 minutes, 0 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes after dosing and analyzed for blood glucose ( HemCue B-Glucose Analyzer, Hemocue AB, Angelholm Sweden). Mean baselines (pre-medication measurements) were determined and subtracted from the subsequent data and plotted against time. By doing this, it was ensured that the TG formulation, which did not appear to be significantly degraded, exhibited adequate pharmacological activity.

(result) Following incubation at 40 ° C, HPLC analysis showed an increase in cleavage products in the glucagon preparation. In contrast, TG has only one small degradation peak (RT = 6). This degradation peak correlates with the slightly less active form of oxidation of glucagon. No diketopiperazine (ie TECHNOSPHERE)^TMGlucagon (without) had many degradation peaks, some of which contributed to the enhanced effect, and others reduced the potency of glucagon.

The sterile lyophilized powder of TG was transferred to the hospital in a frozen state, where it was resuspended in a sterile medium. The material was well resuspended and each vial was continuously injected over 72 hours.

(Conclusion) Standard preparations for glucagon are not suitable for the regulation of blood glucose by continuous subcutaneous injection. Administration of preparations such as containing variable amounts of deamidated and hydrolyzed forms resulted in highly variable blood glucose levels. Stable TECHNOSPHERES^TM/ Glucagon suspensions do not aggregate and contain clinically inappropriate amounts of cleavage products. TG itself can and has been used as a treatment for hyperinsulinemia. It then provides consistently elevated glucose levels when administered subcutaneously over time. 1. A method for purifying an active factor, which is as follows. A step of providing an active factor containing impurities to be removed; a step of trapping the active factor in diketopiperazine to form a mixture; and a step of essentially removing all the impurities from the mixture. Including, method; 2. The method according to 1, wherein the active factor is a peptide or protein; 3. The method according to 2, wherein the peptide or protein is selected from the group consisting of insulin, salmon calcitonin, parathyroid hormone 1-34, octreotide, leuprolide, and RSV peptide; 4. The method according to 3, wherein the active factor is insulin; 5. The method according to 1, wherein the impurity is a multivalent ion; 6. The method according to 4, wherein the active factor in step (a) is an insulin complex and the impurity is a zinc ion; 7. The method according to 6, wherein the complex is hexamer insulin; 8. The method according to any one of 1 to 7, wherein the diketopiperazine is fumaryl diketopiperazine; The method according to any one of 9.1 to 8, wherein the active factor is described below. The step of preparing the diketopiperazine solution, the step of preparing the solution or suspension of the active factor, the step of combining the diketopiperazine solution with the solution or suspension of the active factor, and the step of combining the active factor. Precipitates the fine particles of the diketopiperazine in which the diketopiperazine is dispersed, thereby A method trapped by a process, including the step of forming the mixture; 10. The method according to 9, further comprising the step of washing the fine particles with an aqueous solution of the impurities; 11. Compositions for administration of peptides to patients, including peptides stabilized in diketopiperazine; 12. The composition according to 11, wherein the peptide is dimeric or monomeric insulin; 13. The composition according to 11, wherein the peptide is glucagon; 14. The composition according to any one of 11 to 13, wherein the diketopiperazine is fumaryl diketopiperazine; 15. The composition according to 12, which is substantially free of zinc ions; 16. The composition according to any one of 11 to 15, wherein the peptide is dispersed in the diketopiperazine or coated on fine particles of the diketopiperazine; 17. The composition according to 16, wherein the microparticles are provided in the form of a dry powder; 18. The composition according to 16, wherein the microparticles are provided as an aqueous suspension in a pharmaceutically acceptable carrier; 19. The composition according to any one of 11, 15, or 17, which is a suitable form for pulmonary administration; 2. The composition according to 11 which is prepared by any of the methods 20.1 to 10. 21. A method of administering an active factor to human mucosa that requires the active factor, the method of which is as follows: A step of administering the composition to the mucosa, wherein the composition is: By (i) containing fine particles formed from the active factor and (ii) an effective amount of transport enhancer, and by hydrogen bonding the transport enhancer to the mucosa, if any, by masking the charge of the factor. , Or a combination thereof, which facilitates the transport of the active factor across the mucosa, comprising a method; 22. The method according to 21, wherein the active factor is a charged molecule; 23. The method according to 22, wherein the active factor is insulin; 24. The method of 21; wherein the transport enhancer forms a hydrogen bond with the active factor to mask the charge of the active factor; 25. The method according to 21, wherein the transport enhancer is fumalyl diketopiperazine; 26. The method of any one of 21-25, wherein the composition is delivered to the lungs via inhalation; 27. A method for delivering insulin to patients in need of insulin: A method comprising administering to the patient an effective amount of insulin in a composition comprising microparticles of diketopiperazine in which the monomeric insulin is encapsulated; 28. The method according to 27, wherein the diketopiperazine is fumaryl diketopiperazine; 29. The method of 28, wherein the composition is in the form of a dry powder administered to the lungs via inhalation; 30. The method according to any one of 27-29, wherein the patient has type II diabetes; 31. The method of 30, wherein the composition is administered at the same time as or less than about 20 minutes prior to the patient eating a meal; 32. The method of any one of 27-31, wherein the composition is provided in one or more unit doses of insulin, each dose equal to about 6 IU of insulin.