CN104288756A - Glucagon-like peptide 1 (GLP-1) pharmaceutical formulations - Google Patents

Glucagon-like peptide 1 (GLP-1) pharmaceutical formulations Download PDF

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Publication number
CN104288756A
CN104288756A CN201410514033.8A CN201410514033A CN104288756A CN 104288756 A CN104288756 A CN 104288756A CN 201410514033 A CN201410514033 A CN 201410514033A CN 104288756 A CN104288756 A CN 104288756A
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glp
fdkp
diketopiperazine
molecule
solution
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CN201410514033.8A
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Inventor
斯蒂芬尼·戈恩
大卫·布兰特
柯哈瓦·盖尔伯
马克·金
韦曼·温蒂尔·师特汉姆
凯斯·奥伯格
安德里亚·勒龙-贝
马克·J·豪肯森
玛丽·法瑞斯
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Mannkind Corp
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Mannkind Corp
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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Abstract

The invention relates to glucagon-like peptide 1 (GLP-1) pharmaceutical formulations. Specifically, the invention discloses a composition which is table both in vivo and in vitro. The composition comprises glucagon-like peptide 1 (GLP-I) particles in combination with diketopiperazine (DKP). The composition can be used as a pharmaceutical formulation for treating diseases such as diabetes, cancers, and obesity but is not limited to such diseases or conditions. In particularly, the composition can be used as a pharmaceutical formulation for pulmonary delivery.

Description

Glucagon-like peptide 1 (GLP-1) pharmaceutical preparation
the cross reference of related application
The application to be the applying date be April 16 in 2007 day, are called the divisional application of the 200780013424.X Chinese invention patent application of " glucagon-like peptide 1 (GLP-1) pharmaceutical preparation ".
The application is the U. S. application No.10/632 submitted on July 22nd, 2003, the part continuation application (continuation-in-part) of 878, and require the U.S. Provisional Application No.60/744 that on April 14th, 2006 submits, the priority of 882 according to 35U.S.C. § 119 (e).During every part of above-mentioned priority application is incorporated herein by reference in their entirety.
Technical field
The present invention relates to the field of pharmaceutical preparation.The invention discloses the dry powder formulations comprising diketopiperazine (DKP) granule combined with glucagon-like peptide 1 (Glucagon-Like Peptide 1, GLP-1).The present invention can be used as the pharmaceutical preparation of disease therapy, and described disease is as diabetes, cancer and obesity but be not limited only to this kind of disease.The present invention more specifically can be used as the pharmaceutical preparation of pulmonary delivery.
Background technology
Glucagon-like peptide 1 disclosed in document (GLP-1) is 30 or 31 amino acid whose increasing secretins (incretin), and its response is released from enteroendocrine L cell from the fat of having meal, carbohydrate picked-up and protein.Find that the secretion of this peptide hormone is impaired in the individuality suffering from type 2 diabetes mellitus, becomes the possible candidate of this disease for the treatment of and other relevant diseases.
Without under the state of disease, the nutrient (especially sugar) that GLP-1 responds oral uptake is secreted from intestinal L cell, stimulate the insulin releasing of inducing from the dining of pancreas, suppress the glucagon from liver to discharge, and other effects to digestive tract and brain.GLP-1 effect in pancreas depends on glucose, and the risk of hypoglycemia making exogenous peptide use period minimizes.GLP-1 further promote biological insulin synthesis in institute in steps, and directly stimulate beta cell growth and survival and beta cell break up.The combination of these effects causes the beta cell amount improved.In addition, GLP-1 receptors signal transduction causes the minimizing of beta cell apoptosis, and this contributes to the beta cell amount improved further.
In the gastrointestinal tract, GLP-1 suppresses GI energy, improves the insulin secretion of response glucose, and reduces the secretion of glucagon, thus contributes to reducing glucose excursion (glucose excursion).In Rodents, the maincenter of GLP-1 is used and is shown suppression food intake, and the GLP-1 that prompting periphery reduces directly can affect brain.This is feasible, because the GLP-1 having shown circulation can reach the GLP-1 receptor in some brain district; I.e. Subfornical organ and area postrema.These regions of known brain relate to the conciliation of appetite and energy homeostasis.What is interesting is, gastric distension activates containing the neuron of GLP-1 in the oculomotor nucleus of solitary tract, and the GLP-1 that expresses of the maincenter that imply that is as the effect of appetite suppressant.These hypothesis, by use GLP-1 receptor antagonist---the research support of Exendin (9-39), observe adverse effect in described research.In people, the GLP-1 used has full effect (Verdich et al., 2001), when being provided by continuous print subcutaneous infusion during 6 weeks, diabetics display appetite reduces, and this causes significantly alleviating (Zander et al., 2002) of body weight.
GLP-1 has also been presented in the patient suffering from type 2 diabetes mellitus effective, and when providing as continuous subcutaneous infusion, it improves insulin secretion and also makes both fasting and post-prandial glycemia normalization (Nauck et al., 1993).In addition, GLP-1 inculcate the glucose level showing and reduce following patient: previously with the patient of non-insulin oral drug therapy and sulphonylurea therapy unsuccessfully after need the patient (Nauck et al., 1993) of insulinize.But, as this area neutralization below herein be recorded to, the effect of single subcutaneous injection GLP-1 provides disappointed result.Although reach the high blood plasma level of immunoreactivity GLP-1, insulin secretion returns value before treatment rapidly, and blood sugar concentration is not by normalization (Nauck et al., 1996).When only carrying out the subcutaneous administration of repetition, intravenous administration (Nauck et al., 1996) can be comparable to the effect of fasting serum glucose.Continuous subcutaneous administration 6 weeks display reduces fasting and postprandial glucose concentration and reduces HbA1c level (Zander et al., 2002).The of short duration effect of single subcutaneous injection GLP-1 is relevant to the unstability of its circulation.Evidence suggests that GLP-1 is by blood plasma In vitro metabolism, enzyme dipeptidyl peptidase-IV (DPP-IV) is responsible for this degraded (Mentlein et al., 1993).
Consider the fact that the physiology significance of GLP-1 in diabetics and external source GLP-1 are degraded by quick amino-terminal end in both health volunteer and type 2 diabetes mellitus experimenter, the probability (Deacon et al., 2004) of body internal stability as the novel way of the antidiabetic for the treatment of diabetes of operation GLP-1 has been devoted in much research.Have studied two kinds of independently approach: 1) exploitation is to the insensitive GLP-1 analog of enzymatic degradation, and 2) use selectivity enzyme inhibitor prevent vivo degradation and improve level that is complete, biologically active peptide.In clinical trial, have studied long-acting GLP-1 analog (the such as Liraglutide (Novo Nordisk, Copenhagen, Denmark) of " increasing secretin analogies " degradation-resistant, by name; Exenatide (exendin-4; ) (Amylin Inc., San Diego, CA) and exenatide-LAR, Eli Lilly, Indianapolis, IN).Suppress the inhibitors of dipeptidyl IV being responsible for the enzyme increasing secretin degraded (such as by Novartis, Basel, the Vildagliptin (Galvus) of Switzerland's exploitation) and by Merck, Whitehouse Station, the Januvia (sitagliptin) that New Jersey develops) also under study for action (Deacon et al., 2004).Therefore, the hypoglycemia tendency that the multiple binding mode (satiety of the insulin releasing such as improved, the gastric emptying of delay and raising) of GLP-1 is low with it seems to be it and brings the advantage exceeding current obtainable therapy.
But, although have these approach/progress in GLP-1 treatment, the obtainable medicine of current diabetics all can not achieve the goal (HbA1c, fasting serum glucose, glucose excursion) in all patients, and they all can not avoid side effect as toxicity, hypoglycemia, body weight increase, feel sick and from vomiting pressure.Therefore in the art, the stable GLP-1 preparation as having long-term effectiveness and optimal absorption during medicament administration is still needed.
Summary of the invention
Glucagon-like peptide 1 (GLP-1) preparation that is stable, that can suck as medicine is that this area lacks.In order to overcome the defect in this area, the invention provides combine with diketopiperazine (DKP) granule, as the GLP-1 preparation of medicine.
Therefore, in specific embodiment of the present invention, provide the dry powder composite comprising GLP-1 molecule and diketopiperazine or its officinal salt.In other embodiments; dry powder composite of the present invention comprises the GLP-1 molecule being selected from lower group, and described group of GLP-1, GLP-1 analogies, Exendin (exendin), GLP-1 peptide analogues or the biosynthetic GLP-1 analog protected by natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivant, dipeptides acyl-peptidase-IV (DPP-IV) forms.Also in another embodiment of the present invention, dry powder composite comprises the diketopiperazine with formula 2,5-diketone-3,6-bis-(4-X-ammonia butyl) piperazine, and wherein X is selected from by succinyl group, glutaryl, maleoyl and the fumaroyl group formed.In another embodiment, dry powder composite comprises diketopiperazine salt.Also provide dry powder composite in another embodiment of the present invention, wherein diketopiperazine is 2,5-diketone-3,6-bis-(4-fumaroyl-ammonia butyl) piperazine.
The present invention also comprises dry powder composite, and wherein GLP-1 molecule is natural GLP-1 or amidated GLP-1 molecule, and wherein amidated GLP-1 molecule is GLP-1 (7-36) amide.
Also in another specific embodiment of the present invention, provide the method for the preparation of the granule comprising GLP-1 molecule and diketopiperazine, described method comprises step: providing package is containing the GLP-1 solution of GLP-1 molecule; The suspension of granulopotent diketopiperazine solution or diketopiperazine granule is provided; Combine with by GLP-1 solution and diketopiperazine solution or suspension.In other specific embodiments of the present invention, the method for the preparation of the granule comprising GLP-1 molecule and diketopiperazine also comprises removes solvent by lyophilization, filtration or spraying dry from solution or suspension.Also in yet another embodiment, granule of the present invention is formed by removing solvent, or is formed before removal solvent.
In one embodiment of the invention; for the preparation of having in the method for granule of GLP-1 molecule and diketopiperazine; provide the GLP-1 molecule being selected from lower group, described group of GLP-1, GLP-1 analogies protected by natural GLP-1, GLP-1 analog, GLP-1 derivant, dipeptidyl peptidase-IV (DPP-IV), Exendin, GLP-1 peptide analogues or biosynthetic GLP-1 analog form.In another embodiment, the method for the preparation of the granule with GLP-1 molecule and diketopiperazine comprises the diketopiperazine provided with particle suspension liquid form.In yet another embodiment, diketopiperazine provides in the solution, and the method comprises the pH regulating solution, thus precipitates diketopiperazine and form granule.
In other specific embodiments of the present invention, GLP-1 solution is in the concentration of about 1 μ g/ml-50mg/ml, more preferably about 0.1mg/ml-10mg/ml.Also in another specific embodiment of the present invention, GLP-1 solution is in the concentration of about 0.25mg/ml.
For the preparation of containing in the other method of GLP-1 molecule and diketopiperazine granule, the method also comprises adds a kind of reagent in solution, and wherein said reagent is selected from salt, surfactant, ion, penetrant (osmolyte), chaotropic agent (chaotrope) and lyotrope (lyotrope), acid, alkali and organic solvent.This reagent promotes the association between GLP-1 and diketopiperazine granule, also improves stability and/or the pharmacokinetics of GLP-1 molecule.In some embodiments of the present invention, this reagent is salt, such as but not limited to sodium chloride.Also contemplating this reagent can be surfactant, such as but not limited to Tween, Triton, pluronic acid, CHAPS, cetrimide and Brij, H (CH 2) 7sO 4na.This reagent can be ion, such as cation or anion.This reagent can be penetrant (stabilizing agent), such as but not limited to hexanediol (Hexylene-Glycol, Hex-Gly), trehalose, glycine, Polyethylene Glycol (PEG), front three amine n-oxide (TMAO), mannitol and proline.This reagent can be chaotropic agent or lyotrope, such as but not limited to cesium chloride, sodium citrate and sodium sulfate.This reagent can be organic solvent, such as, be selected from the alcohol of methanol (MeOH), ethanol (EtOH), TFE (TFE) and hexafluoroisopropanol (HFIP).
In another specific embodiments of the present invention, consider the method for the preparation of the granule containing GLP-1 molecule and diketopiperazine, wherein the method comprises the pH regulator of particle suspension liquid to about 4 or larger.In other embodiments of the present invention, the method for the preparation of granule comprises GLP-1 molecule and diketopiperazine, and the GLP-1 molecule wherein in granule has larger stability.
Also contemplate the method experimenter needed being used to the GLP-1 molecule of effective dose in the present invention, comprise and provide GLP-1/ diketopiperazine granule to experimenter.This application process can be intravenous, subcutaneous, per os, per nasal, through cheek, per rectum or by pulmonary delivery, but be not limited only to this.In one embodiment, application process passes through pulmonary delivery.Also in another embodiment of the present invention, the method used comprises disease or the disease that treatment is selected from lower group, and described group is made up of diabetes, ischemia, Reperfu-sion tissue injury, dyslipidemia, diabetic cardiopathy, myocardial infarction, acute coronary syndrome, obesity, the change of postoperative catabolism, hyperglycemia, irritable bowel syndrome, apoplexy, neurodegenerative disorders, memory and learning disorder, islet cell transplantation and regenerative therapies.
The method using GLP-1/ diketopiperazine particulate composition in another embodiment of the present invention causes GLP-1 pharmacokinetic half-life and the bioavailability of improvement.
Also in another specific embodiments of the present invention, provide the method that preparation has the dry powder composite of the pharmacokinetic profile of improvement, the method comprising the steps of: the solution providing GLP-1 molecule; Granulopotent diketopiperazine is provided; Form granule; Combine with by GLP-1 and diketopiperazine; Remove solvent by dry method afterwards, obtain dry powder, wherein said dry powder has the pharmacokinetic profile of improvement.Pharmacokinetic profile through improving comprises the GLP-1 half-life of raising and/or the GLP-1 bioavailability through improving.The GLP-1 half-life of improving is more than or equal to 7.5 minutes.
In one embodiment of the invention, the dry powder composite comprising GLP-1 molecule and diketopiperazine compositions or its officinal salt is provided.In another embodiment, GLP-1 molecule is selected from by the following group formed: GLP-1, GLP-1 analogies that natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivant, dipeptides acyl-peptidase-IV (DPP-IV) protects, GLP-1 peptide analogues or biosynthetic GLP-1 analog.
In one embodiment of the invention, diketopiperazine is the diketopiperazine with formula 2,5-diketone-3,6-bis-(4-X-ammonia butyl) piperazine, and wherein X is selected from by succinyl group, glutaryl, maleoyl and the fumaroyl group formed.In another embodiment, diketopiperazine is diketopiperazine salt.In another embodiment, diketopiperazine is 2,5-diketone-3,6-bis-(4-fumaroyl-ammonia butyl) piperazine.
In one embodiment of the invention, GLP-1 molecule is natural GLP-1.In another embodiment, GLP-1 molecule is amidated GLP-1 molecule.In another embodiment, amidated GLP-1 molecule is GLP-1 (7-36) amide.
In one embodiment of the invention, provide the method being formed and comprise the granule of GLP-1 molecule and diketopiperazine, the method comprising the steps of: provide GLP-1 molecule; There is provided the diketopiperazine of following form, described form is selected from granulopotent diketopiperazine, diketopiperazine granule and combination thereof; Combine with the form of common solution with by GLP-1 molecule and diketopiperazine, wherein form the granule comprising GLP-1 molecule and diketopiperazine.
In one embodiment of the invention, the method also comprises and from described altogether solution, removes solvent by lyophilization, filtration or spraying dry.In another embodiment, form by removing solvent the granule comprising described GLP-1 molecule and diketopiperazine.In another embodiment, before removing solvent, form the granule comprising described GLP-1 molecule and diketopiperazine.
In another embodiment, GLP-1 molecule is selected from by the following group formed: GLP-1, GLP-1 analogies, GLP-1 peptide analogues or biosynthetic GLP-1 analog that natural GLP-1, GLP-1 analog, GLP-1 derivant, dipeptidyl peptidase-IV (DPP-IV) are protected.In another embodiment, GLP-1 molecule provides as a solution, and described solution comprises the GLP-1 concentration of about 1 μ g/ml-50mg/ml.In another embodiment, GLP-1 molecule provides as a solution, and described solution comprises the GLP-1 concentration of about 0.1mg/ml-10mg/ml.In another embodiment, GLP-1 molecule provides as a solution, and described solution comprises the GLP-1 concentration of about 0.25mg/ml.
In another embodiment of the present invention, diketopiperazine provides with the form of diketopiperazine particle suspension liquid.In another embodiment, diketopiperazine provides with the form of the solution comprising granulopotent diketopiperazine, and the method also comprises the pH of adjustment solution to form diketopiperazine granule.In another embodiment, the method also comprises adds following reagent in described solution or suspension, and wherein said reagent is selected from the group be made up of salt, surfactant, ion, penetrant, chaotropic agent and lyotrope, acid, alkali and organic solvent.In another embodiment, this reagent promotes the association between GLP-1 molecule and diketopiperazine granule or granulopotent diketopiperazine.In another embodiment, this reagent improves stability or the pharmacokinetics of GLP-1 molecule.In another embodiment, this reagent is sodium chloride.
In another embodiment of the present invention, the method also comprises the pH regulating suspension or solution.In another embodiment, pH is adjusted to about 4.0 or larger.Also in another embodiment, the GPL-1 molecule in granule has the stability larger than natural GPL-1.
In another embodiment, solution comprises the GLP-1 concentration of about 1 μ g/ml-50mg/ml altogether.In another embodiment, solution comprises the GLP-1 concentration of about 0.1mg/ml-10mg/ml altogether.In another embodiment, solution comprises the GLP-1 concentration of about 0.25mg/ml altogether.
Also in another embodiment of the present invention, the method also comprises adds following reagent in common solution, and wherein said reagent is selected from the group be made up of salt, surfactant, ion, penetrant, chaotropic agent and lyotrope, acid, alkali and organic solvent.In another embodiment, this reagent promotes the association between GLP-1 molecule and diketopiperazine granule or granulopotent diketopiperazine.In another embodiment, this reagent improves stability or the pharmacokinetics of GLP-1 molecule.In another embodiment, this reagent is sodium chloride.
In another embodiment, the method also comprises the pH regulating solution altogether.In another embodiment, pH is adjusted to about 4.0 or larger.
In one embodiment of the invention, provide the method experimenter needed being used to the GLP-1 molecule of effective dose, comprise granule experimenter's providing package being contained to GLP-1 and diketopiperazine.In another embodiment, by intravenous, subcutaneous, per os, per nasal, provide through cheek, per rectum or completed by pulmonary delivery.In other embodiments, completed by pulmonary delivery and provide.
In another embodiment, these needs comprise the treatment to the disease or disease being selected from lower group, and described group is made up of diabetes, ischemia, Reperfu-sion tissue injury, dyslipidemia, diabetic cardiopathy, myocardial infarction, acute coronary syndrome, obesity, the change of postoperative catabolism, hyperglycemia, irritable bowel syndrome, apoplexy, neurodegenerative disorders, memory and learning disorder, islet cell transplantation and regenerative therapies.
In another embodiment, granule the GLP-1 pharmacokinetic half-life and bioavailability that cause improvement compared with natural GLP-1 molecule are provided.
In one embodiment of the invention, provide the method being formed and have the powder composition of the GLP-1 pharmacokinetic profile of improvement, the method comprising the steps of: provide GLP-1 molecule; Granulopotent diketopiperazine in solution is provided; Form diketopiperazine granule; GLP-1 molecule and solution combination are formed common solution; From common solution, remove solvent with by spraying dry, form the powder with the GLP-1 pharmacokinetic profile of improvement.
In another embodiment, the GLP-1 pharmacokinetic profile of improvement comprises the GLP-1 half-life of raising.In another embodiment, the GLP-1 half-life of raising is more than or equal to 7.5 minutes.In another embodiment, the GLP-1 pharmacokinetic profile of improvement comprises the GLP-1 bioavailability of improvement compared with natural GLP-1.
Accompanying drawing explanation
The following drawings forms the part of this description, for further illustrating particular aspects of the present invention.By the following drawings, and in conjunction with the detailed description of detailed description of the invention, the present invention may be better understood.
Figure 1A-1D.GLP-1 structural analysis at various concentrations (pH 4,20 DEG C).-UV the circular dichroism (CD) far away of Figure 1A-GLP-1 has set forth the raising along with concentration, and the secondary structure of peptide is helical conformation from being mainly amorphous Conformation transition.Figure 1B-nearly-UV CD has set forth tertiary structure to be increased along with the peptide concentration improved, the self-association of prompting GLP-1.Fig. 1 C – is excited the fluorescent emission of GLP-1 (pH 4,20 DEG C) under the multiple concentration caused by 280nm tryptophan.The transmission FTIR of GLP-1 (pH 4,20 DEG C) under Fig. 1 D – many kinds of concentration.1656cm -1the amide I band at place shows that GLP-1 has α-helixstructure under the concentration of>=2mg/mL.
The structural analysis of Fig. 2 A-2D. (pH 4,20 DEG C) low concentration GLP-1 under different kinds of ions intensity.It is more orderly α-helixstructure by unordered GLP-1 thaumatropy that the-UV CD far away of Fig. 2 A-1.0mg/mL GLP-1 illustrates raising salinity.Nearly-UV the CD of Fig. 2 B – 1.0mg/mL peptide confirms and improves the tertiary structure that NaCl concentration also strengthens GLP-1.Fig. 2 C – interior fluorescent emission of (pH 4,20 DEG C) 1.0mg/mL GLP-1 under multiple NaCl concentration after 280nm place tryptophan excites.Under high peptide concentration, maximum intensity reduces and migrates to shorter wavelength, this indicates clearly defined tertiary structure.The tertiary structure analyses of Fig. 2 D – (pH 4,20 DEG C) 10mg/mL GLP-1 under different kinds of ions intensity.Closely-UV CD composes and confirms that the ionic strength improved enhances the tertiary structure of the GLP-1 of self-association.
The structural analysis of (pH 4) 10mg/mL GLP-1 under Fig. 3 A-3B. various temperature.Under the nearly-UV CD of Fig. 3 A – illustrates the temperature of raising, GLP-1 oligomer dissociates.The structural analysis of (pH 4) 10mg/mL GLP-1 under Fig. 3 B-various temperature.The structural analysis of (pH 4) 0.05mg/mL GLP-1 under Fig. 3 C-various temperature.Far-UV CD illustrates peptide to temperature is insensitive.
The structural analysis of (20 DEG C) GLP-1 under Fig. 4 A-4B. many kinds of pH.-UV the CD far away of (20 DEG C) 10mg/mL GLP-1 under Fig. 4 A-many kinds of pH.When pH is enhanced, the GLP-1 of self-association precipitates between pH 6.3 and 7.6, but retains helical structure pH 1.5 and 11.7 time.The secondary structure that Fig. 4 B – amplifies the pedigree announcement GLP-1 at pH 7.6 place is unordered, is reduced cause by concentration.
Fig. 5. the 1mg/mL GLP-1 confirmed by HPLC is to the resistance of both desamidation and Oxidation.Deacylated tRNA amine condition within 5 days, is reached by being hatched in 40 DEG C when pH 10.5 by GLP-1.By by GLP-1 at 0.1%H 2o 2in within 2 hours, reach oxidizing condition in incubated at room temperature.
Fig. 6 A-6B. stirs the impact on the tertiary structure of 1.5mg/mL GLP-1 and 9.4mg/mL GLP-1 (pH 4).The tertiary structure that closely fluorescent emission (Fig. 6 B) of-UV CD (Fig. 6 A) and GLP-1 all illustrates GLP-1 peptide does not significantly change due to stirring.Sample at room temperature stirs 30 and 90 minutes and collect fluorescence emission spectrum after 280nm place tryptophan excites.
Fig. 7 A-7C.10 freeze-thaw cycle is on 1.6,5.1 and the impact of 8.4mg/mL GLP-1 (pH 4) tertiary structure.The tertiary structure that closely fluorescent emission (Fig. 7 B) of-UV CD (Fig. 7 A) and GLP-1 all shows peptide does not significantly change because of multiple freeze-thaw cycle.Also at room temperature thaw at sample is frozen in-20 DEG C.Fluorescence emission spectrum is collected after 280nm place tryptophan excites.Carry out similar experiment by-UV CD far away, this experiment shows 11 freeze-thaw cycle to the impact (Fig. 7 C) of 10mg/mL GLP-1 (pH 4) secondary structure.
Fig. 8 A-8B. salt is studied.As the GLP-1/FDKP load curve (Fig. 8 A) of the function of pH and NaCl concentration.Load is carried out under 5mg/mL FDKP and 0.25mg/mL GLP-1.NaCl concentration represents with mM.Fig. 8 B – as the function of pH and NaCl concentration, describe rebuild without FDKP control sample in the GLP-1 that detects measure.
Fig. 9 A-9B. surfactant is studied.As the GLP-1/FDKP load curve (Fig. 9 A) of the function of pH and surfactant.Load is carried out under 5mg/mL FDKP and 0.25mg/mL GLP-1.Fig. 9 B – as the function of the surfactant of pH and interpolation, describe rebuild without FDKP control sample in the GLP-1 that detects measure.
Figure 10 A-10D. ion is studied.As the GLP-1/FDKP load curve of the function of pH and ion.Load (Figure 10 A and 11C) is carried out under 5mg/mL FDKP and 0.25mg/mL GLP-1.Ion concentration is (mM) shown in legend.Right side graph describes the result of 1M NaCl.Figure 10 B and 10D – as the function of pH, ion and 1M NaCl, describe rebuild without FDKP control sample in the GLP-1 that detects measure.
Figure 11 A-11B. penetrant is studied.As the function of pH and the GLP-1/FDKP load curve (Figure 11 A) when there is common stabilizing agent (penetrant).Load is carried out under 5mg/mL FDKP and 0.25mg/mL GLP-1.Figure 11 B – as the function of pH and penetrant, describe rebuild without FDKP control sample in the GLP-1 that detects measure." N/A " represents in sample to there is not penetrant.
Figure 12 A-12B. chaotropic agent/lyotrope research.As the GLP-1/FDKP load curve of the function of chaotropic agent or lyotrope concentration under pH 3.0 (Figure 12 A) and pH 4.0 (Figure 12 C).Load is carried out under 5mg/mL FDKP and 0.25mg/mL GLP-1.Figure 12 B and 12D – as the function of pH with when there is multiple chaotropic agent or lyotrope, describe rebuild without FDKP control sample in the GLP-1 that detects measure." N/A " represents in sample there is not chaotropic agent or lyotrope.
Figure 13 A-13B. alcohol is studied.As the GLP-1/FDKP load curve of the function of pH and alcohol.Load is carried out under 5mg/mL FDKP and 0.25mg/mL GLP-1.Four kinds of determining alcohols are evaluated to often kind of alcohol, 5%, 10%, 15% and 20%v/v (Figure 13 A).TFE=TFE; HFIP=hexafluoroisopropanol.Figure 13 B – as the function of pH and alcohol (20%), describe rebuild without FDKP control sample in the GLP-1 that detects measure.
Figure 14 A-14B. is from the load (Figure 14 A) of GLP-1/FDKP concentration studies.Load is carried out under 5mg/mL FDKP, and the GLP-1 concentration of analysis is listed in X-axis.Spherical and the bar-shaped GLP-1/FDKP granule composition of scanning electron microscopy (SEM) image display of Figure 14 B – many kinds of GLP-1/FDKP formula (amplifying 10000x doubly).(figure A) 0.5mg/mL GLP-1 and 2.5mg/mL FDKP; (figure B) 0.5mg/mL GLP-1 and 10mg/mL FDKP; (figure C) 20mM sodium chloride, 20mM potassium acetate and 20mM potassium phosphate, 0.5mg/mL GLP-1 and 10mg/mL FDKP in pH 4.0; (figure D) 20mM sodium chloride, 20mM potassium acetate and 20mM potassium phosphate, 10mg/mL GLP-1 and 50mg/mL FDKP in pH 4.0.
Figure 15. show the impact of coercing multiple GLP-1/FDKP preparation.Before legend points out lyophilizing, GLP-1 is to the quality-mass percent of other components existed in FDKP granule and solution.Sample hatches 10 days at 40 DEG C.
The structure of Figure 16 A-16C.GLP-1.Figure 16 A – describes form (SEQ ID NO.1) and the amidated form (SEQ ID NO.2) of the glycine-extension of GLP-1.FIG.16B – aprotinin is to the suppression of DPPIV activity.Figure 16 C – DPPIV inhibitor is to the suppression of DPPIV activity.
Figure 17. detect GLP-1 after hatching in lung-douching fluid.
Figure 18 A-18B. describes quantitative in blood plasma of GLP-1.It is quantitative that Figure 18 A shows in 1:2 plasma extender.It is quantitative that Figure 18 B shows in 1:10 plasma extender.
Figure 19 A-19B.GLP-1 and GLP-1 analog are on the impact of cell survival.GLP-1 is on the impact (Figure 19 A) of pancreas in rat epithelium (ARIP) cell death.When individualism and combination exist GLP-1 and staurosporine (Stau), anchorin V dyes and shows inhibited apoptosis (Figure 19 B).The concentration of GLP-1 is 15nM, and the concentration of staurosporine is 1 μM.
Figure 20 .GLP-1 analog exendin-4 is on the impact of cell viability.With 0,10,20 and 40nM Exendin by ARIP cell process 16,24 and 48 hours.
Figure 21. multiple GLP-1/FDKP preparation is on the impact of the cell death that staurosporine is induced.To be exposed in 5 μMs of staurosporines 4 hours with the ARIP cell of GLP-1 sample pretreatment, and to use Cell Titer-Glo tManalyze and measure cell viability.4 DEG C and 40 DEG C by sample Stress treatment 4 weeks.The control sample (culture medium, GLP-1, STAU, GLP+STAU) of right side display shows in culture medium (not containing GLP-1 or staurosporine), containing GLP-1, containing staurosporine and the viability (noting: legend is not suitable for control sample) containing cell when GLP-1 and staurosporine.All results of display are the triplicate meansigma methodss run.
The research of Figure 22 A-22B. pharmacokinetics describes the GLP-1/FDKP preparation azygos vein injection (IV in rats using multiple concentration; Figure 22 A) and lung insufflation (IS; Figure 22 B).Legend points out that in the preparation analyzed, GLP-1 is to the quality-mass percent of FDKP granule.
After Figure 23 A-23B. administration, 2 hours (Figure 23 A) and 6 hours (Figure 23 B) use the minimizing of accumulation food consumption in the rat of GLP-1/FDKP preparation administration.
Figure 24. the pharmacokinetic study of the GLP-1/FDKP used by lung insufflation in Male fatty Zucker rats.Data describe matched group (air; 1st group) and GLP-1/FDKP processed group (the 2nd group) the glucose measurements of the 0th, 15,30,45,60 and 90 minute.
Figure 25. the pharmacokinetic study of the GLP-1/FDKP used by lung insufflation in Male fatty Zucker rats.Data describe matched group (air; 1st group) and GLP-1/FDKP processed group (the 2nd group) the GLP-1 measurement result of the 0th, 15,30,45,60 and 90 minute.
Figure 26. the pharmacokinetic study of the GLP-1/FDKP used by lung insufflation in Male fatty Zucker rats.Data describe matched group (air; 1st group) and GLP-1/FDKP processed group (the 2nd group) the insulin measurement result of the 0th, 15,30,45,60 and 90 minute.
Figure 27. with the GLP-1/FDKP pharmacokinetic study that the multiple GLP-1 concentration used by lung insufflation is carried out in female rats.Data describe matched group (air; 1st group) and application of respectively 5%, 10% and the GLP-1/FDKP process the 2nd, 3 and 4 groups of 15%GLP-1 the GLP-1 measurement result of the 0th, 2,5,10,20,30,40 and 60 minute.
Figure 28. with the GLP-1/FDKP pharmacokinetic study that the multiple GLP-1 concentration used by lung insufflation is carried out in female rats.Data describe matched group (air; 1st group) and application of respectively 5%, 10% and the GLP-1/FDKP process the 2nd, 3 and 4 groups of 15%GLP-1 the FDKP measurement result of the 0th, 2,5,10,20,30,40 and 60 minute.
Figure 29. the GLP-1/FDKP pharmacokinetic study in female rats (n=10), continuous 4 days of described rat application of the GLP-1/FDKP containing 15%GLP-1 (0.3mg GLP-1) by lung insufflation single every day.The average food consumption that continuous 4 days of data display before administration, 1,2,4 and 6 hour measures after administration.
Figure 30. the GLP-1/FDKP pharmacokinetic study in female rats (n=10), continuous 4 days of described rat application of the GLP-1/FDKP containing 15%GLP-1 (0.3mg GLP-1) by lung insufflation single every day.The average weight that continuous 4 days of data display before administration, 1,2,4 and 6 hour measures after administration.
Figure 31. the GLP-1/FDKP toxicokinetics research in monkey, continuous 5 days of described monkey is GLP-1/FDKP by (30 minutes every days) mouth and nose administrations once a day.Data show peak plasma concentration (C that is male and female middle GLP-1 max).Animal accepts contrast (air; 1st group), 2mg/kg FDK (the 2nd group) or 0.3,1.0 or 2.0mg/kg GLP-1/FDKP (being respectively the 3rd, 4 and 5 group).
preferred embodiment describes in detail
Glucagon-like peptide 1 (GLP-1) preparation that is stable, that can suck as medicine is that this area lacks.This is owing to the unstability in GLP-1 peptide body.GLP-1 compound trends towards retaining in the solution under a large amount of condition, and has relatively short Half-life in vivo when being applied as pharmaceutical solutions.In addition, find that being present in various biological fluid such as the dipeptidyl-peptidase IV (DPP-IV) in lung and blood greatly reduces the biological half life of GLP-1 molecule.Such as, the biological half life of GLP-1 (7-37) is shown as 3 to 5 minutes; See U.S. Patent No. 5,118,666.GLP-1 be also presented at parenteral use after through going through body absorption fast.Similarly, amide GLP-1 (7-36) is had the half-life of about 50 minutes during subcutaneous administration; Also see U.S. Patent No. 5,118,666.
In state of the art, the quick removing of GLP-1 compositions and short-half-life represent the defect that the present invention overcomes.The defect of the present invention by providing optimized natural GLP-1/FDKP (FDKP) preparation being specially adapted to pulmonary delivery to overcome state of the art.In other are concrete, the invention provides the preparation of natural GLP-1 molecule, described preparation can cause GLP-1 to reply in vivo.Also contemplate the variant using natural GLP-1 in this kind of preparation.
In order to overcome the defect of state of the art, the invention provides the preparation of the GLP-1 combined with diketopiperazine (DKP) granule.In specific embodiment of the present invention, provide GLP-1/DKP preparation for being administered to experimenter.In other specific embodiments; GLP-1/DKP preparation comprises FDKP (FDKP) but is not limited only to this; and other KDP (asymmetric DKP, xDKP) can be comprised as 2; 5-diketone-3; 6-bis-(4-succinyl group-ammonia butyl) piperazine (SDKP); asymmetric diketopiperazine, comprises those (" single armed " analog of such as FDKP) that only on DKP ring, a position replaces, and DKP salt.In other specific embodiments of the present invention, use GLP-1/FDKP preparation by pulmonary delivery.
When developing the treatment preparation of GLP-1 molecule, by the architectural feature using multiple biophysics and analytical technology to evaluate GLP-1 in solution, described technology comprises far ultraviolet rays circular dichroism (-UV CD far away), nearultraviolet rays circular dichroism (nearly-UV CD), interior fluorescence, Fourier transform infrared spectroscopy (FTIR), high pressure liquid chromatography (HPLC) and mass spectrum (MS).Circular dichroism (CD) technology is the powerful for analyzing altering protein structure under kinds of experiments condition, and is well known in the art.Carry out these experiment conditions analyzed to comprise: concentration, ionic strength, temperature, pH, oxidative stress, stirring and multiple freeze-thaw cycle are on the impact of GLP-1 peptide.These are analyzed the condition of main path and the establishment operation GLP-1 peptide structure being designed to characterize degraded thus reach preferred GLP-1/DKP preparation, and described preparation has the pharmacokinetics (PK) and pharmacokinetics (PD) feature wanted.
Observe along with GLP-1 concentration improves, the secondary structure of peptide is the conformation of more spiral from mainly amorphous Conformation transition.The ionic strength improved in solution causes the structure of GLP-1 to increase, until it reversibly precipitates.The existence of NaCl improves the tertiary structure of GLP-1, and this intensity by the nearly CD band shown in Fig. 2 D improves display.This even occurs under the low peptide concentration not showing self-association.The ionic strength improved easily amorphous GLP-1 is converted into alpha-helix form (as in Fig. 2 A towards shown in the CD minima far away migration of 208nm and 222nm) and self-association conformation (as in Fig. 2 B and 2D of the salt and nearly CD pattern that use raising to shown in the tryptophan excitation transfer of more short wavelength).Temperature and pH differentially affect GLP-1 conformation because the disordered structure of GLP-1 not by these parameters any one change.On the other hand, find that the self-association conformation of GLP-1 is responsive to thermal denaturation and its its dissolubility is responsive to pH, as shown in Figure 4A and 4B, under this figure is presented at the peptide concentration of 10mg/ml, between pH 6.3-7.6, GLP-1 peptide reversibly precipitates.Find GLP-1 multiple conformation generally to stirring and multiple freeze-thaw cycle be stable.Deacylated tRNA amine is not observed to GLP-1, does not observe oxidation yet.
Also observe the absorption of GLP-1 to FDKP granule under numerous conditions, described condition comprises the change of pH, GLP-1 concentration, and kinds of surface activating agent, salt, ion, chaotropic agent and lyotrope, stabilizing agent and determining alcohol change.Find that GLP-1 is on the absorption of FDKP granule affecting by force by pH, particularly combines when about pH 4.0 or larger.Find that other excipient have limited impact to the absorption of GLP-1 on FDKP granule.
When developing GLP-1/DKP preparation of the present invention, have rated the quantity of parameters that may affect or impact sending property and absorption in its body.This kind of parameter comprise the structure of such as GLP-1 peptide, surface charge under certain preparation condition on molecule, as the dissolubility of preparation and stability, and to the susceptibility of serine stretch protein enzymatic degradation and body internal stability; These all play a key role when producing and easily can being absorbed and show the preparation of the biological half life extended.
In vitro and in vivo tests the stability of the GLP-1/FDKP preparation of acquisition under numerous conditions.Analyzed and analyze based on the algoscopy of cell the stability of GLP-1 by HPLC.In addition, in lung-douching fluid (it contains DPP-IV), checked the stability of GLP-1.Also find that the stability of natural GLP-1 is concentration dependent in the solution.
Also use external GLP-1 bioactivity research for the research of GLP-1/FDKP load, and determine effect in body.This strategy contributes to the GLP-1/FDKP compound method that qualification is leading further.In addition, show by inhibited apoptosis based on GLP-1, stimulated beta-cell proliferation and regenerating islets improving the fact worked in beta cell amount, by checking propagation and the anti-apoptotic potential of GLP-1/FDKP preparation of the present invention based on the algoscopy of cell.
Therefore, the invention provides the optimized preparation comprising the natural human GLP-1 combined with FDKP (FDKP), said preparation is stable and anti-degraded.
iI.GLP-1 molecule
In specific embodiment of the present invention, provide optimized preparation, it comprises the natural human glucagon-like peptide 1 (GLP-1) combined as FDKP (FDKP) with diketopiperazine.This kind of GLP-1/FDKP preparation of the present invention is stable and anti-degraded.
People GLP-1 is well known in the art, and derives from distal ileum (distal ileum), in pancreas and front proglucagon (preproglucagon) polypeptide of L-cell synthesis in brain.GLP-1 is 30-31 amino acid whose peptide, and it exists with two kinds of molecular forms: 7-36 and 7-37, and wherein 7-36 form is main.Front proglucagon becomes the processing that GLP-1 (7-36) amide and GLP-1 (7-37) extend form and mainly occurs in L-cell.This area has shown in the fasted state, and the blood plasma level of GLP-1 is about 40pg/ml.After dining, GLP-1 blood plasma level is increased to rapidly about 50-165pg/ml.
Term used herein " GLP-1 molecule " refers to GLP-1 protein, peptide, polypeptide, analog, analogies, derivant, isotype, fragment etc.This kind of GLP-1 molecule can comprise naturally occurring GLP-1 polypeptide (GLP-1 (7-37) OH, GLP-1 (7-36) NH 2) and GLP-1 metabolite as GLP-1 (9-37).Therefore; in specific embodiment of the present invention, GLP-1 molecule comprises: GLP-1, GLP-1 analogies, GLP-1 peptide analogues or biosynthetic GLP-1 analog that natural GLP-1, GLP-1 analog, GLP-1 derivant, dipeptidyl peptidase-IV (DPP-IV) are protected.
Term used herein " analog " comprises the compound similar to the structure of another compound.Such as, antiviral compound acyclovir (acyclovir) is a kind of nucleoside analog, its with derived from similar in the nucleoside guanosine structure of base guanine.Therefore, acyclovir mimics guanosine (biologically similar with guanosine), and disturb DNA to synthesize and stop and translate by the G residue (or competing with it) in displacement viral nucleic acid/transcribe.Therefore, the compound having structural similarity with another (parent compound) is analog, the biology of its simulation parent compound or chemism.Compound being defined as analog does not need maximum or minimum basic or functional group to replace quantity, as long as analog can with the biological characteristics of some relevant patterns (same, addedly or competition ground) simulation parent compound or chemical characteristic.Analog can be and the derivant (see below " derivant ") of normally parent compound.The analog of compound disclosed herein can have equaling, be less than or greater than the activity of their parent compound.
" derivant " used herein refers to natively or makes the compound of (or derivative) synthetically from parent compound.Derivant can be analog (see above " analog ") and thus can have similar chemistry or biologic activity.But different from analog, derivant must not simulate biology or the chemism of parent compound.Compound being defined as derivant does not need maximum or minimum basic or functional group to replace quantity.Such as, although antiviral compound Gan Keluowei (ganclovir) is the derivant of acyclovir, Gan Keluowei has the antiviral activity pedigree different from acyclovir and different toxicology characteristics.Equal, less, larger or even dissimilar activity can be had when the derivant of compound disclosed herein compares with its parent compound.
Term used herein " metabolite " refers to any intermediate product or the product of metabolism, and comprises macromole and micromolecule.When using herein and time suitable, this definition is applicable to primary and secondary metabolite.Primary metabolite is directly involved in that living body biological grows normally, development and fecundity.Secondary metabolite is not directly involved in these processes, but typically has important Ecological Functions (such as antibiotic).
Term used herein " biosynthetic " refers to any production of living body biological to compound.
Term used herein " granulopotent " refer to chemistry, biosynthetic or biological entities or compound, it can form solid particle, is formed in usual liquid medium within.The formation of granule typically occurs in granulopotent entity when being exposed under certain condition, and described condition is such as pH, temperature, humidity and/or Morie osmolarity/Osmolality.Can cause such as combining to the exposure of described condition, coalescent, solidify and/or dewater, thus form granule.Precipitation is an example that can form granule event.
Any medium that " altogether solution " used herein is made up of at least two kinds of chemistry, biology and/or biosynthetic entity.Such as, can by by comprise at least one chemistry, biology and/or biosynthetic entity liquid with comprise chemistry, the solid compositions of biology and/or biosynthetic entity forms common solution.In another example, can by by comprise at least one chemistry, biology and/or biosynthetic entity liquid with comprise chemistry, the another kind of liquid combination of biology and/or biosynthetic entity forms common solution.In another example, can form common solution by adding at least two kinds of solids in single solution, described often kind of solid comprises at least one chemistry, biology and/or biosynthetic entity.
The natural GLP-1 considered in the present invention is the polypeptide of the aminoacid sequence with SEQ ID NO.1 or SEQ ID NO.2.Natural GLP-1 peptide was cut fast and deactivation in vivo in several minutes.
GLP-1 analog of the present invention can comprise Exendin, and it is the peptide being found to be GLP-1 receptor stimulating agent; This kind of analog also can comprise Exendin 1 to 4.Exendin is found in the venom of Heloderma suspectum (Gila-monster), and shares the amino acid identity of about 53% with mammal GLP-1.Exendin also has the similar affinity to GLP-1 receptor.The cAMP that it is reported in Exendin-3 and exendin-4 stimulating pancreas acinous cell produce and amylase from release (Malhotra et al., 1992 pancreatic acinar cell; Raufman et al., 1992; Singh et al., 1994).Propose Exendin-3 is used for the treatment of diabetes and prevention hyperglycemia purposes (U.S. Patent No. 5,424,286) as pancreotropic hormone agent.
The carboxy terminal fragment of Exendin is as Exendin [9-39] (molecule of carboxy amidation) and to be reported as through the fragment 3-39 of 9-39 be the strong of GLP-1 and optionally antagonist (Goke et al., 1993; Raufman et al., 1991; Schepp et al., 1994; Montrose-Rafizadeh et al., 1996).Document have also demonstrated Exendin [9-39] and closes endogenous GLP-1 in vivo, causes insulin secretion (Wang et al., 1995 of reducing; D'Alessio et al., 1996).Exendin-4 is effectively in conjunction with the GLP-1 on the β-TC1 cell of excreting insulin, the acinous cell from the dispersion of pancreas and the parietal cell from stomach.Exendin-4 peptide is also in separated stomach moderate stimulation somatostatin release and work in suppressing gastrin to discharge (Goke et al., 1993; Schepp et al., 1994; Eissele et al., 1994).In the cell of the GLP-1 receptor transfection with clone, exendin-4 is reported as agonist (namely it promotes cAMP), and Exendin [9-39] is defined as antagonist (namely it blocks the stimulation of exendin-4 and GLP-1).Also find that Exendin is degradation-resistant.
Another embodiment of the present invention considers the purposes of peptide mimics.As is known to persons skilled in the art, peptide mimics is the peptide of biologically active determinants on mimic hormone, cytokine, zymolyte, virus or other biological molecule, and can antagonism, stimulation or otherwise regulate the physiologically active of native ligand.Consult such as BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, the Johnson et al. in New York (1993), " Peptide Turn Mimetics ".The potential principle of peptide mimics is used to be that the peptide main chain of protein is mainly to determine that the mode that amino acid side chain direction contributes to interaction of molecules exists.Estimate that peptide mimics allows the interaction of molecules similar to natural molecule.
In other embodiments, GLP-1 molecule of the present invention can have at least one biological activity of natural GLP-1, such as, also cause the ability of the signal transduction pathway causing insulinotropic activity with GLP-1 receptors bind.In other embodiments of the present invention, GLP-1 molecule can be peptide, polypeptide, protein, analog, analogies, derivant, isotype, fragment etc., and it maintains at least one biological activity of naturally occurring GLP-1.GLP-1 molecule also can comprise officinal salt and prodrug, with the salt of prodrug, the polymorph (polymorph) of naturally occurring people GLP-1, hydrate, solvate, bioactive fragment, bioactive variants and stereoisomer, and the agonist of naturally occurring people GLP-1, analogies and antagonist variants.Comprise the Exendin family of Exendin 1 to 4, and Polypeptide fusions.GLP-1 molecule of the present invention also can comprise the GLP-1 that dipeptidyl peptidase-IV (DPP-IV) is protected, and it stops or suppresses the degraded of GLP-1.
GLP-1 molecule of the present invention comprises peptide, polypeptide, protein and derivant thereof, and it contains aminoacid replacement, improves dissolubility, gives the resistance be oxidized, improves biopotency, or improves the half-life in circulation.Therefore, the GLP-1 molecule considered in the present invention comprises aminoacid replacement, deletion or interpolation, wherein said aminoacid be selected from well known in the art those.N end or the C end of molecule also can be modified, such as but not limited to being acylated, acetylation, amidatioon.Therefore, in the present invention, term " aminoacid " refers to the aminoacid that naturally occurring and non-natural exists, and amino acid analogue and amino acid analog thing, and it is to play function with the mode of naturally occurring amino acid similarity.The aminoacid of natural coding is 20 kinds of common aminoacid (alanine, arginine, N, aspartic acid, cysteine, glutaminase, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and burnt lysine (pyrolysine) and selenocysteine (selenocysteine).Amino acid analogue refers to the compound with the basic chemical structure identical with naturally occurring aminoacid, the α carbon that namely described basic chemical structure is combined with hydrogen, carboxyl, amino and R group, such as homoserine, nor-leucine, norvaline, methionine sulfoxide, methionine methyl sulfonium, citrulline, hydroxygultamic acid, hydroxyproline and praline.This kind of analog has modified R group (such as nor-leucine), but retains the basic chemical structure identical with naturally occurring aminoacid.The aminoacid considered in the present invention also comprises the beta-amino acids similar to a-amino acid, because they contain aminoterminal and c-terminus.But in beta-amino acids, two carbon atoms separate these functional end.Beta-amino acids with specific side chain can exist as R or the S isomer on α (C2) carbon or β (C3) carbon.This causes any given side chain four kinds of possible diastereomers altogether.
GLP-1 molecule of the present invention also can comprise hybrid GLP-1 albumen, its fusion rotein, oligomer and polymer, homologue, glycosylation pattern variants and mutein, wherein GLP-1 molecule keeps at least one biological activity of natural molecule, and have nothing to do with its synthesis or manufacture method, described method includes but not limited to recombinate (no matter being produce from the DNA of cDNA, genomic DNA, synthesis or the nucleic acid of other form), synthesis and gene activation method.Recombinant DNA technology is (see Russell, D.W., et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, N.Y., 2001) known to a person of ordinary skill in the art.
iII. diketopiperazine
Diketopiperazine is because it forms the ability of microgranule but well known in the art, and described ability is applicable to drug delivery and stable.In the present invention, use diketopiperazine to contribute to the absorption of GLP-1 molecule, thus degradation-resistant stabilization formulations is provided.
Can use multiple method, wherein diketopiperazine can form whole and granule that is GLP-1 molecule, or GLP-1 molecule can be adsorbed on granule above.This can relate to the solution of diketopiperazine solution and GLP-1 molecule or suspension to mix and then precipitates, and forms the granule comprising diketopiperazine and GLP-1 subsequently.Or, diketopiperazine can be precipitated and form granule, and mix with the solution of GLP-1 molecule subsequently.Association between diketopiperazine granule and GLP-1 molecule can be undertaken by removal of solvents, or can comprise specific step (as pH regulator) before the drying, thus promotes to associate.
In a preferred embodiment, diketopiperazine of the present invention includes but not limited to 3,6-bis-(4-fumaroyl-ammonia butyl)-2,5-diketopiperazine, also (E)-3 is known as, 6-bis-[4-(N-carboxyl-2-acrylic) ammonia butyl]-2,5-diketopiperazines (it is also referred to as FDKP or FDKP).
Other diketopiperazines considered in the present invention comprise, but be not limited to 3,6-bis-(4-ammonia butyl)-2, the derivant of 5-diketopiperazine is such as: 3,6-bis-(succinyl-4-ammonia butyl)-2,5-diketopiperazine is (in this article also referred to as 3,6-bis-(4-carboxylic propyl group) ammonia butyl-2,5-diketopiperazine; SDKP or SDKP); 3,6-bis-(maleoyl-4-ammonia butyl)-2,5-diketopiperazines; 3,6-bis-(citraconoyl-4-ammonia butyl)-2-5-diketopiperazine; 3,6-bis-(glutaryl-4-ammonia butyl)-2,5-diketopiperazines; 3,6-bis-(malonyl-4-ammonia butyl)-2,5-diketopiperazines; 3,6-bis-(oxalyl-4-ammonia butyl)-2,5-diketopiperazines and derivant thereof.In other embodiments, contemplated by the invention the purposes of diketopiperazine salt.This kind of salt can comprise such as any pharmaceutically useful salt, as Na, the K of diketopiperazine, Li, Mg, Ca, ammonium or single, two or trialkyl ammonium (as derived from triethylamine, butylamine, diethanolamine, triethanolamine or pyridine etc.) salt.Salt can be the salt of single salt, disalt or mixing.Have also contemplated that the more senior salt of diketopiperazine, wherein R group contains more than one acidic group.In other side of the present invention, the primitive form of reagent can be mixed to form the drug salts of diketopiperazine with diketopiperazine, thus this medicine is the balance cation (counter cation) of diketopiperazine.An example of the salt considered herein comprises FDKP disodium in a non limiting manner.U.S. Patent application No:11/210,710 teach the drug delivery using DKP, and its all the elements relating to DKP salt are by reference to being incorporated to herein.
As herein disclosed in other places, invention also uses the novel asymmetric analog xDKP of FDKP, such as (E)-3-(4-(3,6-dioxopiperazine-2-base) Butylcarbamoyl)-acrylic acid; (E)-3-(3-(3,6-dioxopiperazine-2-base) propyl-amino formoxyl) acrylic acid; (E)-3-(4-(5-isopropyl-3; 6-dioxopiperazine-2-base)-Butylcarbamoyl) acrylic acid; and open in the U.S. Provisional Patent Application being entitled as " Asymmetrical FDKP Analogs for Use as Drug Delivery Agents ", this application is submitted on the date consistent therewith and is incorporated to herein (attorney docket No.51300-00041) with its entirety.
Diketopiperazine can be formed: as Katchalski, et al., (J.Amer.Chem.Soc. 68:879-80; 1946) the described cyclodimerization body effect by amino acid ester derivative, as Kopple, et al., (J.Org.Chem.33:862-64; 1968) dewater by the cyclisation of dipeptide ester derivatives or by the heat of amino acid derivativges in high boiling solvent described in, the instruction of described file is introduced into herein.
For the synthesis of being known to a person of ordinary skill in the art with the method preparing diketopiperazine, and be disclosed in United States Patent (USP) 5,352,461; 5,503,852; 6,071,497; 6,331,318; 6,428,771 and U.S. Patent application No.20060040953 in.U.S. Patent No. 6,444,226 and 6,652,885 to describe in waterborne suspension preparation and provide Diketopiperazine microparticle, be added the solution of active agent, thus active agent is combined with granule in described waterborne suspension.These patents also describe and produce by lyophilization removal liquid medium the method comprising the microgranule of activating agent, change the solvent condition of this kind of suspension to promote that the combination of active agent and granule is then at US Pat Appl Ser No:60/717, 524 and 11/532, 063 (the two is all entitled as " Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces ") and 11/532, instruction in 065 (being entitled as " Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces. ").Also See U. S. Patent No.6, the US Pat Appl Ser No:11/210 that on August 23rd, 440,463 and 2005 submits to, 709 and U.S. Patent application No.11/208,087.In some cases, consider the US Pat Appl Ser No.11/678 being entitled as " A Method For Improving the Pharmaceutic Properties of Microparticles Comprising Diketopiperazine and an Active Agent. " by such as submitting on February 22nd, 2006, spray drying process disclosed in 046 is dry of the present invention by the diketopiperazine granule of load.Every part of patent and patent application relate to the content of diketopiperazine by reference to being incorporated to herein for them.
the treatment preparation of IV.GLP-1/DKP granule
Present invention also offers the GLP-1/FDKP preparation for being administered to the experimenter needing treatment.The experimenter considered in the present invention can be household house pet or people.In certain embodiments, treatment is for type ii diabetes, obesity, cancer or its any relevant disease and/or disease.People is especially preferred experimenter.
The Other diseases considered in the present invention or disease include, but are not limited to irritable bowel syndrome, myocardial infarction, ischemia, Reperfu-sion tissue injury, dyslipidemia, diabetic cardiomyopathy, acute coronary syndrome, metabolism syndrome, the change of Post operation catabolism, neurodegenerative disorders, memory and learning disorder, islet cell transplantation and regenerative therapy or apoplexy.The Other diseases that the present invention considers and/or disease comprise and relevant any disease listed above and/or disease, and it can by using GLP-1/FDKP dry powder formulations to treat to the experimenter needed.GLP-1/FDKP dry powder formulations of the present invention also may be used for the induction of β cell differentiation in people's cell for the treatment of type ii diabetes and hyperglycemia.
Also in yet another embodiment of the present invention, consider that experimenter can be household house pet or animal, comprise rat, rabbit, hamster, guinea pig, pallasiomy (gerbil), marmot, cat, dog, sheep, goat, pig, cattle, horse, monkey and ape (comprising orangutan, Gibbon and baboon).
Also consider that GLP-1/FDKP granular preparation of the present invention can by known to a person of ordinary skill in the art and use for multiple route of administration that is clinical or non-clinical object.GLP-1/FDKP compositions of the present invention can be administered to the biomembrane of any targeting, the mucosa of preferred experimenter.Use and can be undertaken by any approach, described approach is included but not limited to per os, per nasal, uses through cheek, general intravenous injection, subregion that is subcutaneous, that provided by blood or lymph, be applied directly to affected site or even undertaken by local means.In a preferred embodiment of the present invention, using of GLP-1/FDKP compositions passes through pulmonary delivery.
In the present invention, other alternative route of administration operable can comprise: intradermal, intra-arterial, intraperitoneal, intralesional, intracranial, intraarticular, in prostate, in pleura, tracheal strips, in vitreous body, intravaginal, rectum, in tumor, intramuscular, Ink vessel transfusing (intravesicular), mucosa, in pericardium, bronchus local application, use aerosol, injection, inculcate, inculcate continuously, concentrated perfusion is directly taken a shower target cell, pass through conduit, pass through lavation, in cream, in fluid composition (such as liposome), or other method that to be manufactured by the conventional counting personnel in this area or aforesaid any combination (consult such as Remington's Pharmaceutical Sciences, 1990, its full content relating to medication is by reference to being incorporated to herein).
As dry powder formulations, GLP-1/DKP granule of the present invention can by sucking the specific region being delivered to respiratory system, and this depends on the size of granule.In addition, GLP-1/DKP granule can be manufactured into enough little and can mix in intravenous injection liquid suspension dosage form.For oral delivery, can be impregnated in suspension, tablet or capsule.GLP-1/DKP compositions can be delivered from suction apparatus, and described suction apparatus is nebulizer, metered dose inhaler, Diskus and aerosol apparatus such as.
In other embodiments, the GLP-1/DKP preparation using " effective dose " to the patient needed is considered.The GLP-1/DKP dry powder formulations of " effective dose " considered in the present invention refers to the amount of GLP-1 compound, analog or peptide mimics etc., and this consumption can alleviate one or more symptoms of disease, disease or the obstacle be treated in some degree.In one embodiment, the GLP-1/DKP dry powder formulations of " effective dose " should be by plasma insulin level is improved, fasting blood sugar level reduces or reduces and pancreas beta cell amount improves the amount for the treatment of the GLP-1 molecule of diabetes at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more (but being not limited thereto).In another preferred embodiment, the present invention considers by carrying out treatment of obesity to the GLP-1 molecule of the experimenter's drug administration effective dose needing this kind for the treatment of.In such cases, the GLP-1/DKP dry powder formulations of " effective dose " should be the amount by losing weight or reduce the GLP-1 molecule at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more (but being not limited thereto).Invention also contemplates that the GLP-1/DKP dry powder formulations using " effective dose " is used for by controlling satiety to the GLP-1 molecule of the experimenter's drug administration effective dose needing this kind for the treatment of.With regard to nonrestrictive mode, GLP-1 molecule can be that exendin peptide molecule is as Exendin-1 or-4.In such cases, the GLP-1/DKP dry powder formulations of " effective dose " should be by hunger sensation by and food intake (such as being measured by quality or calorie content) be reduced by least about 5%, the amount of the GLP-1 molecule of 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50% or more (but being not limited thereto).The GLP-1/DKP dry powder formulations of " effective dose " can also be defined as enough can detect and repeatedly can improve, reduce, minimize or limit the amount of degree of disease or disease or its symptom.Also the invention formulation of " effective dose " may be used to eliminate, remove or cure diseases or disease.
When using GLP-1/FDKP compositions of the present invention to the experimenter needed, the actual dose of compositions can be determined based on physics and physiologic factor, and described factor is as the seriousness of body weight, disease, the disease type be treated, previously or the treatment interference coexisted, the idopathy (idiopathy) of patient and route of administration.Technical staff should determine actual dosage based on these factors one or more.
GLP-1/DKP preparation of the present invention can be applied once or more than once, depend on disease to be treated or disease.Using of GLP-1/DKP preparation can be provided to experimenter with following interval, be interposed between described several minutes, a few hours, a couple of days, several weeks or several months scope in change.In some cases, the timing of therapeutic scheme can relate to the half-life of using rear GLP-1 molecule.In other embodiments, such as, when treating the disease of concrete or complexity or disease as cancer, such as, the GLP-1/DKP preparation of the present invention using drug containing excipient or reagent can be expected.In such cases, application program can be instructed by drug excipient or reagent.
v. embodiment
Following embodiment is included in herein for setting forth certain embodiments of the present invention.It will be appreciated by those skilled in the art that, technology disclosed in embodiment illustrates the representative art suitably playing function in the practice of the invention.But those skilled in the art it should be understood that and can carry out many changes in the specific embodiments which are disclosed under inspiration of the present invention, and still obtains similar or similar result, and does not depart from the purpose and scope of the invention.
embodiment 1
the biophysics Epidemiological Analysis of GLP-1 structure and analytic process analysis
In order to analyze structure and the behavior of GLP-1, employ a large amount of Biophysical techniques and analytical technology.These technology comprise far ultraviolet rays circular dichroism (-UV CD far away), nearultraviolet rays circular dichroism (nearly-UV CD), interior fluorescence, Fourier transform infrared spectroscopy (FTIR), high pressure liquid chromatography (HPLC) and mass spectrum (MS); It is all known to a person of ordinary skill in the art.Use large-scale condition research concentration, ionic strength, temperature, pH, oxidative stress, stirring and multiple freeze-thaw cycle on the impact of GLP-1 peptide; It is all hereafter describing in more detail.Also use the main path that these analysis and characterizations are degraded, and establish the peptide structure thus the condition reaching certain GLP-1/DKP preparation that operate GLP-1.
experimental procedure
GLP-1 is purchased from American Peptide (Sunnyvale, CA) or AnaSpec (San Jose, CA), or inner manufacture (MannKind Corporation, Valencia, CA).At pH 4.0 and the aqueous GLP-1 sample analyzing multiple concentration under 20 DEG C (except as otherwise noted).Sample is generally fresh preparation and before each experiment and suitable additive (such as salt, pH buffer, H 2o 2deng, mix if any).The secondary structure measurement result of GLP-1 under multiple condition is collected with-UV CD far away and transmission fourier transform infrared spectrometry (FTIR).In addition, using nearly-UV CD and interior fluorescence, analyzing the tertiary structure of GLP-1 by monitoring its aromatic residue (i.e. tryptophan) environment around.
the concentration dependent structure of GLP-1
Use alpha-helix, random coils, beta sheet lamella, β-corner and random coils that circular dichroism (CD) analysis of spectrum molecule (as protein or peptide) may show.Particularly ,-UV CD far away is used to measure the type of secondary structure in protein and peptide, such as pure alpha-helix, β-lamella etc.On the other hand, the tertiary structure of nearly-UV CD analyzing molecules is used.Therefore, in order to check the impact of concentration on GLP-1, use-UV CD far away and nearly both-UV CD technology.
UV-CD far away in Figure 1A confirms that GLP-1 forms two kinds of different structures, and described structure comprises alpha-helix and random coils under large-scale concentration (such as 1.8,4.2,5.1,6.1,7.2 and 8.6mg/ml).Determined by the huge single minima at 205nm place, under low concentration (≤2mg/mL), GLP-1 is mainly amorphous.Determined by two minima at 208nm and 224nm place, when concentration improves, peptide takes α-helixstructure (Figure 1A).
The GLP-1 structure of tertiary structure analyses prompting high concentration is the conformation (i.e. oligomer) of self-association.Closely-UV CD and fluorescent emission data all support this hypothesis.Positive band closely in-UV CD (Figure 1B) between 250-300nm discloses GLP-1 and has the tertiary structure determined, this structure increases under higher concentration.More particularly, these bands show that the aromatic residue of peptide is fixed in large quantities, and are present in clearly defined environment.
Similarly, under multiple concentration, the fluorescent emission display aromatic residue tryptophan (it also shows strong band in nearly-UV CD composes) of (pH 4.0,20 DEG C) GLP-1 is present in clearly defined tertiary structure; The tryptophan that the data shown derive from 280nm place excites (Fig. 1 C).The fluorescence maximum at low concentration GLP-1 355nm place is shown that tryptophan is exposed in solvent, and there is not significant tertiary structure.Under high peptide concentration, maximum intensity reduces and migrates to shorter wavelength, shows the tertiary structure determined more.
In order to measure the potential secondary structure of GLP-1 self-association conformation further, (pH 4.0,20 DEG C) carries out FTIR analysis at various concentrations.1656cm -1the amide I band at place clearly illustrates that GLP-1 has α-helixstructure (Fig. 1 D) under the concentration of>=2mg/mL.Therefore, GLP-1 does not form β-lamellar structure; Replace, peptide more may produce helical bundle in higher concentrations.
In addition, these different GLP-1 structures of experiment display are not produced by sample treatment.With peptide is directly dissolved in compared with the GLP-1 for preparing in buffer, the diluent from concentrated storage solutions produces similar-UV CD far away, nearly-UV CD and fluorescence emission spectrum.
ionic strength is on the impact of GLP-1
Also carry out some researchs and measure the impact of ionic strength on GLP-1 peptide.It is alpha-helix conformation by unordered GLP-1 thaumatropy that Fig. 2 A (far away-UV-CD) has set forth the concentration (from 100mM to 1000mM) improving salt, as 208nm and 224nm place minima disclose.After NaCl concentration is increased to 1M, a lot of peptide (when 1.0mg/mL) is precipitated out (Fig. 2 A) from solution.But, dissolve after such precipitation display dilute with water, thus determine GLP-1 under high ionic strength and can reversibly be precipitated.
Salt also shows and produces and promote the tertiary structure of GLP-1.This is illustration in Fig. 2 B (near-UV-CD), wherein 1.0mg/mL GLP-1 not shows signal when salt-free, but shows clearly tertiary structure, and this tertiary structure is strengthened along with the ionic strength improved.After 280nm place tryptophan excites, under multiple NaCl concentration, the fluorescent emission (Fig. 2 C) of (pH 4.0,20 DEG C) 1.0mg/mL GLP-1 confirms these results.The ionic strength improved causes fluorescence maximum to migrate to shorter wavelength, shows that the tertiary structure of 1.0mg/mL GLP-1 had not only been produced but also had been enhanced.
In addition, use nearly-UV CD to compose under different kinds of ions intensity (pH 4.0,20 DEG C) and the tertiary structure analyses of 10mg/mL GLP-1 is confirmed that the ionic strength improved also enhances the self-association conformation (Fig. 2 D) of GLP-1.
Therefore, the structure of Notes of Key Data ionic strength to GLP-1 has tremendous influence, causes protein not only to take alpha-helix conformation but also associated to become oligomer.In addition, the ionic strength improved in solution causes the oligomerization of GLP-1 to improve, until it reversibly precipitates.This event is obvious under low peptide concentration (wherein there is not tertiary structure at first) and high peptide concentration (it has shown a large amount of secondary and tertiary structure).Therefore, amorphous GLP-1 is easily converted into the conformation of alpha-helix and self-association by the ionic strength of raising.In addition, the spectroscope result observed is comparable with the impact of the peptide concentration of the raising previously shown.
temperature and pH are on the impact of GLP-1
Whether the conformation being also studied the self-association measuring GLP-1 is responsive to the change of temperature or pH.Fig. 3 A (nearly-UV-CD) confirms along with temperature improves, the tertiary structure dissociation significantly of 10mg/mL GLP-1.On the other hand, at various temperature and pH 4.0 times, the GLP-1 of temperature on low concentration (0.05mg/mL) does not have impact; See Fig. 3 B and 3C (-UV-CD far away).Far-UV CD illustrates peptide to temperature is insensitive.Therefore, the molecular motion of raising hampers the self-association of GLP-1 significantly.
On the contrary, Fig. 4 A (-UV-CD far away) confirms that the dissolubility of alpha-helix GLP-1 conformation is pH sensitivity.Although pH 4.4 and following time, the structure of 10mg/mL GLP-1 is relatively homogeneous (namely GLP-1 keeps spiral), but when pH is increased to close to or is in neutrality (between pH 6.3 and 7.6), some precipitations occur and produce unordered pedigree.The sample that wherein there occurs precipitation has lower intensity, because there is less soluble g LP-1 in solution.The single minima that this unordered structure is observed by 208nm place in Fig. 4 A (far away-UV-CD) is determined, this further describe in Fig. 4 B (nearly-UV-CD) and likely the minimizing of GLP-1 in solution after autoprecipitation.When pH be increased to higher than GLP-1 5.5 pI time, this precipitation can be there is.But along with pH is increased to 11.7 from close to neutrality, major part precipitation is dissolved again, shows that this precipitation is reversible.Peptide amount in solution can be caused to reduce in the remaining undissolved GLP-1 precipitation of pH11.7, thus reduce the intensity of-UVCD pedigree far away, as viewed in Fig. 4 A.Also observe when the GLP-1 powder of lyophilizing and pH 9 buffer are mixed into high concentration GLP-1, GLP-1 is extremely insoluble.
the stability of GLP-1
By measuring GLP-1 peptide on the resistance of desamidation and Oxidation and on to stir and the resistance of freeze-thaw cycle impact measures its stability.
The GLP-1 (1mg/mL) being in pH 10.5 is hatched 5 days at 40 DEG C, carries out reversed-phase HPLC and electrospray mass spectrometry (MS) afterwards for desamidation and Oxidation analysis.Also using HPLC and MS to carry out oxidation research to GLP-1 sample (1mg/mL), there is 0.1%H in described sample 2o 2time hatch 2 hours.
Fig. 5 describes the stability of GLP-1 under deacylated tRNA amine and oxidizing condition.HPLC chromatogram illustrates GLP-1 eluting under the identical holdup time, and the instability condition analyzed does not produce degradation peak.In addition, MS analyzes and creates similar quality 3297g/mol to all samples, shows that this quality is not changed.Data also illustrate peptide when hatching under numerous conditions and keep pure and complete.Therefore, the deacylated tRNA amine of GLP-1 is not observed.It is stable that GLP-1 also shows oxidative stress, as there is 0.1%H 2o 2time observed, wherein the purity of GLP-1 and quality keep complete, as measured respectively by HPLC and MS.In a word, there is not the change of holdup time or mass value, and do not produce degradation peak, thus confirm that GLP-1 peptide is all resistances to deacylated tRNA amine and oxidation.
Stirring and continuous print freeze-thaw cycle are on the impact of the GLP-1 of multiple concentration nearly-UV CD and interior fluorescence analysis.9.4 and the stirring of 1.5mg/mL GLP-1 do not produce the remarkable change of peptide, as by nearly-UV CD (Fig. 6 A) and fluorescent emission (Fig. 6 B) viewed.Sample is at room temperature stirred 30 and 90 minutes, and collect fluorescence emission spectrum after 280nm place tryptophan excites.In independently freeze thawing research, by freezing and at room temperature melt at-20 DEG C for the solution containing 1.6,5.1 and 8.4mg/mL GLP-1 (pH 4.0).Instructed by nearly-UV CD (Fig. 7 A) and fluorescent emission (Fig. 7 B) and analyze 10 freeze-thaw cycle to the impact of GLP-1.Fluorescence emission spectrum is collected after 280nm place tryptophan excites.Analyze the tertiary structure all showing peptide for two kinds appreciably not change due to multiple freeze-thaw cycle.In similar experiment, analyze 11 freeze-thaw cycle to the impact (Fig. 7 C) of 10mg/mL GLP-1 (pH 4.0) secondary structure.The secondary structure that far-UV CD illustrates peptide does not significantly change because of multiple freeze-thaw cycle.
In a word, the structure deriving from the biophysics Epidemiological Analysis display GLP-1 peptide of above experiment is by its strongly the affecting of concentration in the solution.When the concentration of GLP-1 is enhanced, alpha-helix is more outstanding.In addition, improve ionic strength and strengthen (producing in some cases) three grades of GLP-1 structures.
embodiment 2
gLP-1/FDKP adsorbs research
By carrying out the interaction of adsorbing GLP-1 and diketopiperazine (DKP) granule in research evaluation suspension.Variable in absorption research has been probed into electrostatic, hydrogen bond, water-bound, protein flexibility and special salt-pairing and has been interacted on the interactional impact of GLP-1/DKP.In addition, several common protein stabilizing agent is tested on the impact of GLP-1 and DKP surface adsorption.
Use preformed DKP suspension particle (i.e. FDKP), have studied the condition that GLP-1 is adsorbed to preformed DKP particle surface.The solution combination of FDKP particle suspension liquid (wherein FDKP granule is preformed) and 3X pH buffer and 3X additive or excipient.Final solution has the FDKP concentration of 5, mg/ml and the GLP-1 concentration of 0.25, mg/ml (5%w/w).Unconjugated GLP-1 filtering from suspension in supernatant.With the GLP-1 protein of 100, mM ammonium bicarbonate solubility FDKP granule and combination, and filter with the GLP-1 protein being separated any gathering.By the GLP-1 amount in the fraction of the quantitative supernatant of HPLC and reconstruction.Carry out series of experiments, the condition wherein used comprises the GLP-1 using additive (such as salt, surfactant, ion, penetrant, chaotropic agent, lyotrope) and multiple concentration.The result deriving from these researchs is hereafter describing.
salt is studied.The impact that – is combined with FDKP granule by HPLC analysis and observation salt pair GLP-1.Under 5mg/mL FDKP and 0.25mg/mL GLP-1, the load (Fig. 8 A) of GLP-1/FDKP granule is carried out when existence 0,25,50,100,250,500,1000 and 1500mM NaCl.Also as the function of pH and NaCl concentration, have rated rebuild without FDKP control sample in the GLP-1 that detects measure (Fig. 8 B).PH in two groups of data sets all controls with 20mM phosphate/20mM acetate mixture.
As viewed in fig. 8 a, the best combination (absorption) of GLP-1 on FDKP is subject to the strong impact of pH of suspension.Under 4 and above pH, when GLP-1/FDKP ratio is 5%w/w in solution, observe the GLP-1 of about 3.2% to about 4% to the combination of FDKP.When existence 0 and 25mM NaCl, there is no the absorption of obvious GLP-1 to FDKP granule for 2.0 times at pH, but observe some obvious loads by the ionic strength improved.Containing >=1M NaCl without FDKP contrast in observe GLP-1 and precipitate, Fig. 8 B.Under >=1M NaCl, this obvious load is the result of GLP-1 peptide reversible precipitation (saltouing) under high ionic strength.High salt GLP-1 contrast not containing FDKP granule also shows high GLP-1 level in the sample rebuild, and when showing to collect supernatant, GLP-1 is caught by filter.During lower than 1M NaCl, the sign not having GLP-1 to precipitate when there is not FDKP granule.
surfactant is studied. the impact that – is combined GLP-1 and FDKP granule by HPLC analysis and research surfactant.Load is carried out (Fig. 9 A) under 5mg/ml FDKP and 0.25mg/mL GLP-1 when there is surfactant.Also as pH and surfactant function evaluates rebuild without FDKP control sample in the GLP-1 that detects measure (Fig. 9 B).PH is identical with the above-described condition for ionic strength research with control sample condition.The surfactant used in this research comprises: the H (CH of Pluronic F68,0.9mM of Triton X, 0.12mM of the Brij 78 of 0.09mM, the Tween 80 of 0.01mM, 0.2mM 2) 7sO 4the CHAPS of Na, 0.9mM, the Cetrimide of 0.9 mM.The load curve that there is GLP-1 during often kind of surfactant shows for GLP-1/FDKP as the function of pH.
Data show needle does not affect by the existence close to the surfactant of its critical micelle concentration (CMC) the pH adsorption curve of GLP-1/FDKP granule, the described critical micelle concentration i.e. concentration among a small circle of separately following boundary: the boundary that in fact can't detect aggregation/micelle below it, and more than it in fact all extra surfactant molecules all form the boundary of aggregation.Therefore, these surfactants any have also been pointed out can be used to optimization stability as described below and/or pharmacokinetics (PK).As verifiable in the research of salt above, the interaction of GLP-1 and FDKP granule affects by the pH of suspension.
ion is studied.For this experiment, carry out two kinds of different ion researchs and measure the impact that ion pair GLP-1 is combined with FDKP granule.In two kinds of research, Cl -for cationic equilibrium ion, Na +for the equilibrium ion of anion.The load of GLP-1/FDKP granule is carried out as described in earlier experiments.Control pH as described above.When existing and there is not NaCl (it is used to evaluate better the situation of high ionic strength) with pH3.0,3.5, the pH buffer of 4.0 or 5.0 prepares sample.Other ions are comprised as follows: 20 or the LiCl, 20 or the NH of 250mM of 250mM in individual sample 4cl, 20 or the NaF and 20 of 250mM or the NaCH of 250mM 3cOO.
As shown in Figure 10 A, from the data display of first ion research as the GLP-1/FDKP load curve of the function of pH and ion.When without NaCl, 20 or the fluoride of 250mM concentration affect absorption under (enhancing) low pH consumingly, the wherein maximum combination that has nothing to do with pH of the NaF concentration display of 250mM.Observing this pattern is due to the fluorine in solution instead of sodium, because sodium bicarbonate does not have identical effect in 20 and 250nM.In addition, these effects are not the results of sodium in sample, because as shown in Figure 8, the salt of similar concentration does not show this effect.When there is 1M NaCl, all ions give high " apparent " load.To 1M NaCl, " apparent " load should be saltoutd from solution by GLP-1 peptide when there is high ionic strength and cause.This illustrates in fig. 1 ob further, described Figure 10 B show GLP-1 be present in containing 1M NaCl reconstruction without in FDKP control sample.The amount of the GLP-1 detected for these control samples improves, because they add the total ionic strength in sample under larger ion concentration.
In second ion experiment (Figure 10 C), at existence 20 or 250mM KCl, 20 or 250mM imidazoles, 20 or 250mM NaI or 20 or 250mM NaPO 4time prepare GLP-1/FDKP sample.Data display 250mM imidazoles reduces load when there is 1M NaCl, and 250mM phosphate and 250mM all provide high " apparent " load (Figure 10 C).According to rebuild under 0M and 1M NaCl concentration without FDKP control sample in detect GLP-1 amount (Figure 10 D), these impacts cause by the impact of ion pair GLP-1 peptide self instead of on the impact of peptide and FDKP Interaction between particles.Sodium phosphate and sodium iodide cause saltouing of some GLP-1 when there is not NaCl.In addition, imidazoles helps GLP-1 to be dissolved in 1M NaCl sample, thus provides lower " apparent " load.Precipitation is also been observed in the 0M NaCl of 250mM phosphate and iodide contrasts.
penetrant is studied.Also HPLC analysis and observation GLP-1 is passed through to the combination of FDKP granule.Figure 11 A shows the GLP-1/FDKP load curve as pH function when there is common stabilizing agent (penetrant).The load of GLP-1/FDKP granule is carried out as described in earlier experiments.Similarly, control pH as described above.Sample is prepared at pH 3.0 with when there is 20,50,100,150,200 or penetrant (stabilizing agent) of 300mM.Penetrant is hexanediol (Hex-Gly), trehalose, glycine, PEG, TMAO, mannitol or proline; N/A indicates without penetrant.In similar experiment, in sample, the concentration of penetrant (stabilizing agent) is kept constant at 100mM, and pH is from 2.0 to 4.0 changes.
The penetrant (stabilizing agent) of research does not all have considerable impact to the absorption on GLP-1 to FDKP surface, and no matter pH is maintained at concentration change (Figure 11 A of pH 3.0 and penetrant; Leftmost curve) or permeate concentration is maintained and constantly changes pH (Figure 11 A at 100mM; Right side graph).Rebuild without FDKP control sample in do not detect GLP-1 precipitate (Figure 11 B).These penetrants can be used to optimization stability and/or pharmacokinetics.
chaotropic agent and lyotrope research.Research affects the ionic species (chaotropic agent and lyotrope) of water and protein structure, determines these factors at GLP-1 to the effect in FDKP absorption.The load of GLP-1/FDKP granule is carried out as described in previous experiment.Similarly, control pH as described above.PH 3.0 and exist 0,20,50,100,150,200 the following chaotropic agent of 300mM or lyotrope time prepare sample, described chaotropic agent or lyotrope are: NaSCN, CsCl, Na 2sO 4, (CH 3) 3n-HCl, Na 2nO 3, sodium citrate and NaClO 4.In similar experiment, in sample, chaotropic agent or lyotropic concentration are kept constant at 100mM, and pH is from 2.0 to 4.0 changes.
Figure 12 A shows the GLP-1/FDKP load curve as pH and chaotropic agent and/or lyotropic function.When low pH (≤3), the different chaotropic agents analyzed be there occurs to the significant change in load, particularly under higher chaotrope concentration.But when pH 4, do not observe change (Figure 12 C).Therefore, these reagent promote under being presented at disadvantageous lower pH that GLP-1 is to the combination of FDKP granule, but have considerably less impact under in conjunction with favourable higher pH condition.The load changing section observed for 3.0 times at pH from the Notes of Key Data without FDKP control sample of rebuilding affects saltout (precipitation) (Figure 12 B and 12D) of GLP-1 peptide in multiple degree owing to specific chaotropic agent.This to strong chaotropic agent as NaSCN and NaClO 4significant.
organic substance is studied.Evaluate following alcohol to determine helical conformation role in the absorption of GLP-1 to FDKP, the known bond strength inducing cycloidic conformation in amorphous peptide by improving hydrogen bond of described alcohol.The load of GLP-1/FDKP granule is carried out as described in earlier experiments.Similarly, control pH as described above.The effect of often kind of alcohol is observed for 2.0,3.0,4.0 and 5.0 times at pH.The alcohol used is: methanol (MeOH), ethanol (EtOH), TFE (TFE) or hexafluoroisopropanol (HFIP).5%, 10%, 15% and 20%v/v concentration under evaluate often kind of alcohol.
Figure 13 A display is for the GLP-1/FDKP load curve of often kind of alcohol of often kind of concentration as pH function.PH 3.0 times, the HFIP (5%) of low concentration causes high absorption, as GLP-1 to the mass ratio of FDKP granule confirm.Only have the strongest H-key to strengthen (one-tenth spiral) alcohol HFIP, on the absorption in the suspension of buffering, there is impact.Under the HFIP (20%) of higher concentration, GLP-1/FDKP absorption is suppressed.Under Figure 13 B is presented at the determining alcohol of 20%, rebuild without FDKP control sample in be not recorded to significant GLP-1 and precipitate.
The Conformational flexibility (entropy of the FDKP-contactant that namely can be formed and quantity) of this prompting medicine can work in absorption.Can work in the interaction on Notes of Key Data H-bonding GLP-1 and FDKP surface under these conditions.Based on these data, if also infer that H-bonding is main with general power effect in interacting as FDKP-GLP-1, then more and stronger effect should be expected.
concentration studies.– have studied the absorption of GLP-1 to FDKP particle surface under multiple GLP-1 concentration.Figure 14 A shows the load curve from GLP-1/FDKP as GLP-1 concentration function under multiple pH.GLP-1 concentration is 0.15,0.25,0.4,0.5,0.75,1.0,1.5,2.0,5.0 or 10mg/mL.The pH of sample is 2.5,3.0,3.5,4.0,4.5 or 5.0.
When GLP-1 concentration improves when FDKP concentration is kept constant at 5mg/mL, observe the raising of GLP-1 load on FDKP granule.When pH 4 times concentration as GLP-1 are 10mg/mL, observe the GLP-1 absorption close to 20% on FDKP granule.Surprisingly, the GLP-1 absorption not observing load on FDKP granule under high concentration GLP-1 is saturated.This observed result may become polylayer forest owing to GLP-1 self-association.
Existed as crystal or dull and stereotyped spline structure by the analysis display GLP-1/FDKP granule of scanning electron microscope (SEM) to GLP-1/FDKP dosage form, it can form the aggregation (Figure 14 B) comprising more than one GLP-1/FDKP granule.Prepare these preparations by Solutions in Freeze-drying, described solution contains: (figure A) 0.5mg/mL GLP-1 and 2.5mg/mL FDKP; (figure B) 0.5mg/mL GLP-1 and 10mg/mL FDKP; (figure C) 20mM sodium chloride, 20mM potassium acetate and 20mM potassium phosphate, 0.5mg/mL GLP-1 and 10mg/mL FDKP in pH 4.0; (figure D) 20mM sodium chloride, 20mM potassium acetate and 20mM potassium phosphate, 10mg/mL GLP-1 and 50mg/mL FDKP in pH 4.0
result is summarized
In a word, the absorption of GLP-1 and FDKP Interaction between particles research display GLP-1 is combined with DKP particle surface in the dependent mode of pH-, pH 4 or on there is high absorption.Find that the absorption of DKP particle surface on GLP-1 is subject to pH and affects the most by force, substantially do not adsorb when pH 2.0, have great interaction when pH >=4.0.According to the observation, sodium and fluorion strengthen absorption at a low ph.Other additives such as surfactant and conventional stabilizing agent only have minimal effect to the absorption of GLP-1 and FDKP particle surface.
In addition, the result of these experiments of properties influence of GLP-1 self.Find that the behavior of GLP-1 is atypia and surprising, because do not observe saturated adsorption, this is owing to the self-association of GLP-1 under high concentration.Under high concentration, the self-association of GLP-1 allows possible multilamellar GLP-1 peptide to the parcel of DKP granule, thus promotes the GLP-1 peptide load of higher percentage ratio.This surprising self-association character confirms that be useful in the GLP-1 administration form that preparation is stable.In addition, the self-association conformation of GLP-1 can reduce or postpone its degraded in blood.But noticing must be careful when operating the GLP-1 associated, because it is responsive to temperature and high pH.
embodiment 3
the integrity analysis of GLP-1/FDKP preparation
Based on the result from experiment in embodiment 1 and 2, have selected a series of GLP-1 preparations with feature described in table 1 and measure for the cell viability discussed herein.Major part preparation contains GRAS (" being generally considered to safe ") excipient, but some are by the relation selecting to allow to study between stability and absorption.
The GLP-1/FDKP that table 1. is selected for integrity facies analysis (Integrity Phase Analysis).
In addition, based on the result obtained in embodiment 1 and 2, also have selected a series of preparation and study for the phase II integrity of GLP-1/FDKP.Following table 2 shows the six kinds of GLP-1 preparations selecting to be used for phase II integrity.After preparing powder, by them and blank FDKP fusion, in often kind of preparation, produce similar a large amount of GLP-1 peptide and FDKP.
The GLP-1/FDKP preparation that table 2. is selected for II phase integrity.The preparation that 10mg/ml GLP-1 makes from 20mM NaCl and pH 4.0 buffer is described to salt-association preparation.
The impact (Figure 15) of coercing GLP-1/FDKP preparation in his-and-hers watches 2 is analyzed by HPLC.Containing load at H 2in O 5%, 10% or the sample of 20%GLP-1/FDKP; Load in NaCl+pH4.0 buffer 5% or the sample of 10%GLP-1/FDKP hatch 10 days at 40 DEG C.HPLC chromatogram to confirm that GLP-1 peptide does not exist degradation peak with identical holdup time eluting.In addition, MS analyzes and produces the quality 3297g/mol similar for all samples, shows that the sample of this quality for all analyses is homogeneous.Before data display lyophilizing, GLP-1 is to the quality-mass ratio that there are other compositions in FDKP granule and solution.In a word, the display of GLP-1/FDKP preparation is stable to coercing.
embodiment 4
the stability of the GLP-1 of hatching in lung-douching fluid
Consider and in biofluid, to find dipeptidyl-peptidase IV (DPP-IV) cutting and deactivation GLP-1, the stability of GLP-1 in analyzing biologic fluids (as lung fluid and blood).
Dipeptidyl-peptidase IV (DPP-IV) is the membrane-bound serine protease in a kind of extracellular, and it is at several cell type (especially CD4 +t cell) surface on express.Also in blood and lung fluid, find DPP-IV.DPP-IV has related to the control of glucose metabolism, because its substrate comprises insulinotropic hormone GLP-1, described GLP-1 is inactivated by the removal of its n terminal amino acid; See Figure 16 A.DPP-IV cuts the Ala-Glu key of the main circulation form of people GLP-1 (GLP-1 (7-36)), two residues of release N-end.DPP-IV display is by degraded GLP-1 thus reduce incretin and be used for the process of negative regulator glucose to pancreas beta cell.
Carry out some researchs and measure GLP-1 degraded when there is aprotinin or DPP-IV inhibitor in rat serum and lung fluid.In the sample after collection, add naturally occurring serpin aprotinin with 1,2,3,4 and 5TIU/ml, it is degraded in Profilin matter known in the art.Then it is active that DPP-IV is measured in the cutting by detecting luminous substrate, and described substrate contains the Gly-Pro sequence that DPP-IV identifies.Broncho-pulmonary eluate and front-fluorogenic substrate are hatched 30 minutes, and by luminous detection cleaved products.
As the suppression of being degraded by peptide in the multiple biofluid (as described herein) with the aprotinin concentration increased progressively detect, the raising (Figure 16 B) that data display DPP-IV activity suppresses.After collection, add DPP-IV inhibitor with 1.25,2.5,5,10 and 20 μ l/ml in sample observe similar result (Figure 16 C).After sample collection, the interpolation of inhibitor allows more accurate sample evaluation.
Also use and catch the stability that ELISA mAb (it identifies GLP-1 aminoacid 7-9) checked GLP-1 in lung eluate.GLP-1 hatches 2,5,20 and 30 minutes in lung eluate (LLF).As shown in figure 17, incubation conditions is: 1 or 10 μ g (w/w) LLF and 1 or 10 μ g (w/w) GLP-1.GLP-1 is not detected separately in LLF.Use the combination of LLF and GLP-1 of multiple concentration, exist and can detect by the high GLP-1 compared with independent GLP-1, show that GLP-1 is in time stable (Figure 17) in lung eluate.The stability of GLP-1 in undiluted lung eluate is confirmed in similar research; The GLP-1 integrity (data are not shown) of 70-72% when having marked 20 minutes.
In addition, checked the stability of GLP-1 in rat plasma.Blood plasma derives from different rats (as shown in blood plasma in legend 1 and blood plasma 2) and is diluted by 1:2 or 1:10 (v/v).1 milligram of GLP-1 is added in 10 μ l blood plasma or PBS.Sample is hatched 5,10,30 or 40 minutes at 37 DEG C.The cessation reaction on ice also adds the aprotinin of 0.1U.Data are presented at the GLP-1 (Figure 18 A and 18B) of the plasma extender middle and high concentration of all time point 1:2 and 1:10 of test.In a word, data show that GLP-1 is all surprising stable in the lung eluate having found serine stretch protein enzyme DPP-IV and blood plasma.
embodiment 5
gLP-1 molecule is on the impact of apoptosis and cell proliferation
In order to check GLP-1 whether inhibited apoptosis, carry out Screening test to determine the impact that GLP-1 suppresses beta cell death.With 0,2,5,10,15 or the GLP-1 of 20nM concentration by pancreas in rat epithelium (ARIP) cell (as pancreas beta cell model; Purchased from ATCC, Manassas, VA) pretreatment 10 minutes.Then make cell untreated or process 4.5 hours with 5 μMs of staurosporines (apoptosis inducers).Use Cell Titer-Glo tM(Promega, Madison, WI) evaluates cell viability.In the cell of staurosporine process, improve by the GLP-1 concentration up to 10nM the reduction (Figure 19 A) being recorded to cell death percentage ratio
Anchorin V dyeing is used to determine the further inspection of GLP-1 on apoptotic impact by facs analysis.Anchorin V dyeing is the useful tool detecting apoptotic cell, and is well known to a person skilled in the art.Anchorin and the combination of cell membrane analyze phospholipid (PS) asymmetric change before allowing to lose with film integrality before the morphological change relevant with apoptosis occurs.Therefore, GLP-1 is being measured on apoptotic impact with in 15nm GLP-1,1 μM of staurosporine, 4 hours, 1 μM staurosporine+15nmGLP-1 or the cell that not only processed without staurosporine but also without GLP-1 (experiment contrast).The apoptosis that staurosporine is induced by data display GLP-1 suppresses about 40% (Figure 19 B).
Use GLP-1 analog exendin-4 to observe similar apoptosis and suppress result, described exendin-4 is with the mode of similar GLP-1 and GLP-1 receptors bind.With 5 μMs of staurosporines, ARIP cell is processed 16,24 or 48 hours respectively when existence 0,10,20 or 40nM Exendin.When data (Figure 20) are presented at 10nM, Exendin is completely invalid to inhibited apoptosis, because 100% cell death.Under 20 and 40nM, Exendin same degree inhibited apoptosis, suppression in 48 hours about 50% when there is 40nM exendin-4.
embodiment 6
the impact of material standed for GLP-1/FDKP preparation on cells death
Carry out the ability evaluating GLP-1/FDKP preparation (disclosed in embodiment 3, upper table 1) T suppression cell death based on the determination experiment of cell.These GLP-1/FDKP granular preparations are in suspension or are lyophilized.Analyze the ability of the cell death that preparation suppresses staurosporine to be induced in ARIP cell.To be exposed in 5 μMs of staurosporines 4 hours with the ARIP cell of GLP-1 sample pretreatment, and to use Cell Titer-Glo tM(Promega, Madison, WI) analyzes to determine cell viability.
The sample of multiple GLP-1/FDKP preparation is not coerced or coerce 4 weeks at 4 DEG C or 40 DEG C.In based on the algoscopy of cell, use often kind of GLP-1/FDKP sample with 45nM, determine that they suppress the ability of the cell death of staurosporine induction.Shown in right side, control sample is illustrated in independent culture medium, separately containing GLP-1, separately containing staurosporine or there is both GLP-1 and staurosporine culture medium in the viability of cell (note: legend is not suitable for control sample.Every bar representative is independently triplicate).Result shown in all is the meansigma methods of carrying out in triplicate.
The all GLP-1/FDKP lyophilized formulations of coercing of data display suppress the cell death (Figure 21) of staurosporine induction.But many GLP-1/FDKP suspension preparations not T suppression cell are dead.
embodiment 7
the lung insufflation of GLP-1/DKP granule
In order to check the pharmacokinetics of GLP-1/FDKP, evaluate the plasma concentration of GLP-1 in female Sprague Dawley rat, described female rats application of multiple GLP-1/FDKP preparation by vein (IV) injection or lung insufflation.In preliminary research, use the GLP-1 accounting for GLP-1/FDKP granular preparation about 4% and 16% (w/w).It is 12 groups that rat is randomized, and wherein the 1st, 4,7 and 10 group of acceptance injects by lung fluid drip or IV the GLP-1 solution used.2nd, 5,8 and 11 groups accept the preparation (disclosed in chart 2) being injected the GLP-1/FDKP salt binding used by lung insufflation or IV.3rd, 6,9,12 groups accept the mix preparation being injected the GLP-1/FDKP salt binding used by lung insufflation or IV.GLP-1/FDKP preparation is the preparation of the salt binding of about 16% load.In order to reach the load of about 4%, the fusion in the mixture of 3:1 by the preparation of 16% and DKP granule.For total GLP-1 dosage of 0.08mg, lung inhalation or intravenous injection are the granule (be respectively 16% or 4%GLP-1 load) of 0.5 or 2.0mg.
In independently animal groups (7-12 group), the 2nd day repetitive administration.80 μ g GLP-1 solution are used to the 1st, 4,7 and 10 group.2nd, 5,8 and 11 group is used to the preparation (~ 16%GLP-1 load) of GLP-1/DKP salt binding.3rd, 6,9,12 groups accept the blended formulations (~ 4%GLP-1 load) of GLP-1/DKP salt binding.
Use identical preparation that experiment is carried out twice, continuous print in two days administration and collect blood.For every group on administration same day, 2,5,10,20,30,60 and 120 minutes blood samplings after (time 0), administration before administration.At each time point, by the about 150 μ L whole blood collections from trailing edge vein in the cyro-vial pipe containing about 3U/mL aprotinin and 0.3%EDTA, be inverted and be stored on ice.Blood sample is centrifugal and move in the flat board of 96-hole by 40 μ l blood plasma pipets at 4000rpm, described flat board is stored in-80 DEG C, afterwards according to the recommendation (Linco Research, St Charles, MO) of manufacturer by elisa assay GLP-1 level.Determine optimum condition be only exist serum (5%FBS) and without when substrate when measure buffer be GLP-1 time.
intravenous administration: 5th, 6,10,11 and 12 groups of veins (IV) receive multiple GLP-1/FDKP preparation and GLP-1 solution (Figure 22 A).5th group and the 6th group is applied to 15.8%GLP-1/FDKP, and the 11st was applied to the another kind of dosage of 15.8%GLP-1/FDKP with 12 groups at one day that is connected; 10th group is applied to GLP-1 solution in contrast.The concentration of GLP-1/FDKP is detected at the time point of the 0th, 2,5,10,20,40,60,80,100 and 120 minute.All groups show the detected raising of GLP-1 blood plasma level after intravenous administration, within 2 minutes, observe maximum concentration after treatment.Background level is returned for the blood plasma level of all groups of process latter 20 minutes active GLP-1.When being used by intravenous injection, the kinetics of these multiple GLP-1/FDKP preparations and GLP-1 solution does not observe significant difference.Notice that, in the rat by intravenous injection process, after administration, the blood plasma level of 10-20 minute GLP-1 returns back to baseline values, prompting physiological dynamics (namely the GLP-1 of about 95% was eliminated in 10 minutes).
single insufflation is used: 1st, 2,3,7,8 and 9 groups receive multiple GLP-1/FDKP formula or GLP-1 solution (Figure 22 B) by lung insufflation.1st group application of the GLP-1 contrast of 80 μ g by lung liquid insufflation (LIS); 2nd group application of 15.8%GLP-1/FDKP by lung insufflation (IS); 3rd group application of 3.8%GLP-1/FDKP by lung insufflation (IS); 7th group application of the GLP-1 contrast of 80 μ g by lung liquid insufflation (LIS); 8th group application of 15.8%GLP-1/FDKP by lung insufflation (IS); 3.8%GLP-1/FDKP is application of by lung insufflation (IS) with the 9th group.In the concentration of the point in time measurement GLP-1/FDKP of the 0th, 2,5,10,20,40,60,80,100 and 120 minute.
All groups show the detected raising of blood plasma GLP-1 concentration after lung is used.GLP-1 maximal plasma concentration changes with the preparation/compositions used.Indicated by AUC, 2nd group and the 8th group shows the maximum GLP-1 blood plasma level of 10-20 minute after treatment, and the 3rd group and the 9th group is presented at the remarkable activity GLP-1 level of 5-10 minute, the 1st group and the 7th group quicker and instantaneous raising showing active GLP-1 blood plasma level.In the 2nd, 3,7 and 8 group, the blood plasma level processing latter 60 minutes active GLP-1 is returned to background level, and the 1st and 7 groups within 20 minutes, reach background level after treatment.
In diabetes rat model, the GLP-1 of 8 nanomoles is seemingly effective; GLP-1 dosage is 80 μ g (being greater than the effective dose 3000 times of report); Within 30 minutes, using the lung blood plasma GLP-1 level of sending upon administration and within 3 hours, inculcating (Chelikani et al., 2005) compares high 10 times; The bioavailability of the GLP-1/FDKP sent by lung insufflation is 71%.These results are reported further in following table 4.In the most of rat passing through lung delivery process, after administration, the blood plasma level of 30-60 minute GLP-1 returns back to baseline values.Except No. 1 rat in the 2nd group, all rats show the raising of GLP-1 plasma concentration after intravenous injection or lung are blown into multiple GLP-1/FDKP preparation.
conclusion: in the pharmacokinetic profile of GLP-1/FDKP preparation, observed the difference compared with GLP-1 solution.For the rat by GLP-1 solution-treated, more lasting by the plasma concentration of GLP-1 in the rat of GLP-1/FDKP preparation lung insufflation process.Upon administration between 20 to 60 minutes, the reduction of all animal display GLP-1 plasma concentration.The relative uniformity of continuous 2 experiments carried out for 2 days of these results display.
The bioavailability of table 4.GLP-1/FDKP preparation
* with FDKP granule 3:1 fusion
embodiment 8
gLP-1/FDKP reduces the food intake of rat
This area also known GLP-1 acts in the brain, to trigger full sensation and to reduce food intake.According to GLP-1 this effect in satiety and food intake, carry out testing and determine GLP-1/FDKP preparation of the present invention whether as to reduce the reagent of raising be effective and thus have the potential of obesity controlling disease.
The 15.8%GLP-1/FDKP preparation (0.32mg GLP-1/ agent) of (air) or 2mg/ days dosage is contrasted to the administration of two groups of female Sprague Dawley rats by lung insufflation.Matched group is made up of five rats, and test group is made up of ten rats.Each rat is provided to single dose in continuous 5 days, and 2 and 6 hours after each administration measure food intake.Collect the body weight of every rat every day.
Preliminary data display 2 and 6 hours upon administration, the entirety that there is accumulation food consumption in the rat with the administration of GLP-1/FDKP preparation reduces (Figure 23 A and 23B).After administration in the 4th day, this minimizing in 2 hours significantly (p=0.01).1st day and 6 little these minimizings constantly in the 2nd day more remarkable (p<0.02).After administration 24 hours not on the impact of food consumption.
embodiment 9
toxicity research
Carry out the Dose Toxicity research of repetition, to evaluate the possible toxic action and toxicokinetics pattern of repeatedly using rear GLP-1/DKP.Carry out the research of in the research of in rat 14 days and monkey 28 days.GLP-1/DKP administration is carried out every day by inhalation route.To in the animals administer research of 28 days, a part of animal will be put to death immediately after dosage regimen, and other animal allows the restore cycle having one month at the most before execution.Evaluate the clinical sign of all animals, multiple physiological parameter, comprise the histopathology of GLP-1, glucose, insulin, organ weight and clinical pathology and multiple organ.
Carry out a series of GLP mutagenicity research, to evaluate the mutation potential of diketopiperazine granule.These researchs comprise external Ames and chromosomal aberration algoscopy, and it all well known to a person skilled in the art.In addition, micronuclei in mice algoscopy in body known to the skilled has also been carried out.The display of genetoxic data does not have evidence to represent, and diketopiperazine granule has mutagenicity or genotoxic potential.
Also carry out some research evaluation diketopiperazine granules to the impact of breeding toxicity.These study the fertility be included in rat and rabbit, EF is grown and postnatal development research.The diketopiperazine granule used by subcutaneous injection does not damage fertility or implantation in rats, and does not have the evidence of teratogenecity in rat or rabbit.Diketopiperazine granule does not adversely affect fertility and early embryonic development, fetal development or in utero or postnatal development.
Consider that high amount of drug is removed because they cause the tendency of LQT syndrome (acquired LQTS or long-term Q-T syndrome are a kind of hereditary disorders of the rare cardiac electric rhythm and pace of moving things occurred in medication crowd) from clinical market, use hERG algoscopy to check the pharmacology of diketopiperazine granule.Consider and cause most of medicine of acquired LQT to cause acquired LQT's by closing people's ether-à-go-go related gene (hERG) potassium channel, therefore use hERG algoscopy, described potassium channel is responsible for the repolarization of ventricle cardia action potential (ventricular cardiac action potential).Result from hERG algoscopy shows the IC of diketopiperazine granule 50>100 μM.In addition, the result display from diketopiperazine non-clinical study does not affect, because do not observe prolongation (9 months or safety pharmacology cardiovascular research) in dog QTc interval (the QT interval that heart rate corrects).When intravenous is used, diketopiperazine granule does not affect the CND evaluated in safety pharmacology core set of cells or cardiovascular system.
embodiment 10
gLP-1 is on the impact of beta cell quality
Institute during known GLP-1 promotes biological insulin to synthesize also directly stimulates beta cell growth in steps and survives and beta cell differentiation.The combination of these effects causes the beta cell quality increased.In addition, GLP-1 receptors signal transduction causes the minimizing of beta cell apoptosis, and this contributes to increasing beta cell quality further.Known GLP-1 passes through three kinds of possible approach adjustment beta cell quality: strengthen beta-cell proliferation; Suppress beta cell apoptosis; With the stem cell estimated in differentiation ductal epithelium.
In order to confirm the impact of GLP-1 on beta cell, compared with untreated cell with GLP-1/FDKP process cell at the 1st, 3 and 5 day.As described in document, beta cell quality improves 2 times (Sturis et al., 2003) by active GLP-1 use at the most.In addition, multiple GLP-1 receptor (GLP-1R) agonist confirms the prevention of GLP-1R agonist to the inspection of the impact of diabetes or is delayed generation or the development of diabetes.
GLP-1/FDKP is evaluated on the impact of beta-cell proliferation, insulin and glucose in male Zucker diabetic obese/obesity (ZDF) rat (n=8/ group).Animal accepts to contrast (air) or the 2mg GLP-1/FDKP containing 15% (0.3mg) GLP-1 for three days on end.Carry out the test of intraperitoneal (IP) glucose-tolerant, and before administration, within after administration 15,30,45,60 and 90 minutes, collect blood sample and be used for blood plasma GLP-1 and glucose analysis.Collect pancreatic tissue to be used for by immunohistochemical analysis insulin secretion, beta cell quality and apoptosis.
Within 4th day, IP glucose-tolerant test (IPGTT, Figure 24) is carried out in administration.At the 3rd day after overnight fast, animal accepts glucose bolus by peritoneal injection, uses immediately afterward through lung insufflation acceptance contrast (air) or GLP-1/FDKP.Before glucose excites and to administration, multiple time points of 90 minutes collect blood.30 minutes upon administration, the 1st group of display compared with before administration 47% glucose level improve, and the 2nd group (GLP-1/FDKP) display to be worth with before administration compared with 17% glucose level improve.In time points all after glucose-tolerant test, process compared with control animal, glucose level significantly lower (p<0.05).
Also measured GLP-1 level (Figure 25) at the 3rd day of administration.In 2nd group, the Cmax of blood plasma GLP-1 level is after administration 15 minutes 10,643pM.
In addition, together with the glucose measurement after testing with IP glucose-tolerant, at multiple point in time measurement insulin levels of the 3rd day.Contrast (air) the 1st group and the 2nd group (GLP-/DKP) all confirm administration after 15 minutes for level before administration respectively the insulin concentration of 46% and 30% initially reduce (Figure 26).But 30 minutes upon administration, the insulin level in the 2nd group was returned to baseline, and the insulin level in the 1st group continues to be reduced to 64% of the front value of administration.In processed animal, the insulin level of 45 minutes, 60 minutes and 90 minutes is worth close to before administration, and deviation is less than 1.5%.
Prepare the microscope slide for insulin immunostaining and microscopic evaluation insulin expression.According to by the quantitative assessment of optical microscope to insulin expression, there is the relevant insulin expression of process in the pancreas of male ZDF rats and improve (its be correlated with for dosage), although do not reach significance,statistical (p=0.067); This determined by the percentage ratio of the β islet cells of expression of insulin.
Also Apoptosis assay is carried out to the pancreatic tissue of ZDF rat.External secretion and endocrine pancreatic cell (Tornusciolo D.R.et al., 1995) is evaluated by TUNEL algoscopy.To in pancreas about 10,000 cell (exocrine and endocrine) scoring.Major part TUNEL-positive cell is exocrine.The difference of apoptotic marker index is not had in process and matched group.
In addition, in the pancreas of Zucker diabetic fatty rat, have rated Beta cell proliferation, described rat contrasts (air) or GLP-1/FDKP administration once a day 3 days by lung insufflation.Prepare the microscope slide that use immunohistochemistry is located altogether to insulin and Ki67 (proliferation marker).The microscopic evaluation of cell proliferation is carried out in the islets of langerhans and exocrine pancreas of the insulin positive of total 17 ZDF rats.According to the quantitative assessment of cell proliferation, in the pancreaticβ-cell of male ZDF rats or Exocrine Pancreas In Rats, there is no the impact that the process of on cell proliferation is relevant.
In a word, the GLP-1/FDKP that this research display is used with 2mg or 0.3mg GLP-1 by lung insufflation reduces the blood sugar level in diabetic obese mice (model of type ii diabetes) after glucose-tolerant test, and improves the insulin secretory cell quantity of each islets of langerhans.
embodiment 11
the preparation of GLP-1/FDKP granular preparation
Also use the alternative approach of preparation GLP-1/FDKP granular preparation.Preparation is prepared as follows: prepare 10wt%GLP-1 storage solutions by 1 part of GLP-1 (by weight) being added in 9 parts of deionized waters the glacial acetic acid formation settled solution also added in a small amount.The storage suspension (granule of about 10wt%) of FDKP granule is divided into three parts.In every partial suspended liquid, add the GLP-1 storage solutions of appropriate amount, the target compositions by dried powder 5 and 15wt%GLP-1 is provided.After adding protein solution, the pH of suspension is about 3.5.Then suspension is adjusted to about pH 4.4-4.5, afterwards suspension is made bead in liquid nitrogen and lyophilizing except deicing.
The aerodynamics loading of powder can suck a grade proportion by subtraction (respirable fraction on fill) (RF Based on Fill) and characterize, namely can powder percentage ratio (%) within the scope of suction with the gauge of powder in cartridge case, it measures as follows: fill five cartridge cases with the powder of 5mg is manual and passes through MannKind ' s inhaler (being described in U.S. Patent application No.10/655, in 153) discharges.
The method produces the preparation with good loading RF.The powder with 5wt%GLP-1 is measured as 48.8%RF/ loading, and is 32.2%RF/ loading containing the powder of about 15wt%GLP-1.
embodiment 12
containing the pharmacokinetics of the GLP-1/FDKP of multiple GLP-1 concentration
In order to evaluate the pharmacokinetics of the GLP-1/FDKP with multiple GLP-1 concentration, 18 that weigh between 192.3 grams to 211.5 grams male Sprague Dawley rats are divided into four processed group: contrast GLP-1 (the 1st group, n=3); GLP-1/FDKP preparation (2-4 group, n=5/ group).Animal accepts one of following test article: by the contrast (air) of lung insufflation; Containing the 2.42mg GLP-1/FDKP (0.12mg GLP-1) of 5%GLP-1; By the 1.85mg GLP-1/FDKP (0.19mg GLP-1) containing 10%GLP-1 of lung insufflation, or contain the 2.46mg GLP-1/FDKP (0.37mg GLP-1) of 15%GLP-1.Collect blood sample and measure before administration and multiple time point (2,5,10,20,30,40 and 60 minutes) serum FDKP and blood plasma GLP-1 level after administration.
Use the maximum blood plasma GLP-1 concentration (C after GLP-1/FDKP (5% preparation) max) be: 5 minutes T after administration maxthe 2321pM at place; 10 minutes T after administration max4,887pM (10% preparations) at place; With 10 minutes T after administration max10,207pM (15% preparations) at place.As shown in figure 27, until after administration 30 minutes still observe significant GLP-1 level.The area under curve (AUC) of the GLP-1 level of 1-4 group is respectively 10622,57101,92606,227873pM* minute.To 10% or 15%GLP-1 load GLP-1/FDKP estimate the GLP-1 half-life be 10 minutes.
As shown in figure 28,5%, 10% and the maximum FDKP concentration of GLP-1/FDKP preparation of 15%GLP-1 be measured as 8.5 μ g/mL (the 2nd group), 4.8 μ g/mL (the 3rd group) and 7.1 μ g/mL (the 4th group) respectively.To the time (T of Cmax max) be 10 minutes.These data display FDKP and GLP-1 shows similar adsorption dynamics adsorption kinetics, and the FDKP of similar quantity is had nothing to do with the GLP-1 load on granule by adsorbing.
In a word, after research is presented at and uses GLP-1/FDKP by lung insufflation single dose in Sprague Dawley rat, blood plasma GLP-1 level is detected as significant level.The relevant raising of the dosage observing blood plasma GLP-1 level reaches Cmax in 10 minutes about upon administration, within 40 minutes, has observable GLP-1 level upon administration.All animals survived, until at the end of research.
embodiment 13
the pharmacokinetic properties of the GLP-1/FDKP used by lung insufflation
In order to evaluate the pharmacokinetic properties of GLP-1/FDKP, female Sprague Dawley rat is divided into 2 processed group.Animal (n=10) accepts contrast (air by single lung insufflation every day in continuous 4 days; N=5) or containing the 2mg GLP-1/FDKP of 15%GLP-1 (0.3mg GLP-1).
Within continuous 4 days 1,2,4 and 6 hour before administration, after administration in dark circulation, measure food consumption (Figure 29).Compared with contrast (air) group, decreasing food consumption (p<0.05) after using GLP-1/FDKP at the 1st, 2 and 3 day by lung insufflation single dose every day in the animal be subject to processing.Before the administration of on 1 hour of the 1st day and 6 hours point and 4 hours, 6 hours of the 2nd day and the 3rd day, there is the decreasing food consumption of statistically significant in the animal be subject to processing in group relative to contrast (air).
Within continuous 4 days, measure body weight (Figure 30) before administration every day.Body weight when administration is initial changing in the scope of about 180 to 209 grams.Although do not reach significance,statistical between process and contrast (air) animal, in the animal of process, body weight is lower.All animals survived are until predetermined execution.
embodiment 14-16
toxicokinetics (TK) is studied
Examples below 14 to 16 discloses the repeated dose toxicity research carried out in rat and monkey to evaluate the possible toxic action of GLP-1/FDKP inhalation of dust and toxicokinetics pattern.Data show that, under the dosage of the clinical application dosage several times higher than plan, GLP-1/FDKP inhalation of dust does not have obvious toxicity.In addition, show in each species between male and jenny and there is no difference.
embodiment 14
the toxicokinetics of the GLP-1/FDKP of 5 days is used by lung insufflation in monkey
Carry out studying and measure the toxicity of GLP-1/FDKP and toxicokinetics pattern, continuous 5 days of described GLP-1/FDKP once a day (30 minutes every days) use (the human therapy route of administration of expection) to stump-tailed macaque (Macaca fascicularis) by mouth and nose.Mouth and nose are used to relate to and on the mouth and nose of monkey, are worn mask and breath test preparation 30 minutes.
Animal is made to adapt to restriction (restraint) and dosing step starting to process front fortnight.When processing beginning (the 1st day), buck is between 30 and 56 monthly ages, and weight range is from 2.3 to 4.0kg; Female between 31 and 64 monthly ages, weight range is from 1.6 to 3.4kg.As shown in hereafter table 5 and table 6, ten (5 male and 5 female) non-stump-tailed macaques of testing first are appointed as 5 groups (often organizing 2 animals).Non-monkey of testing first is the compoundanimal previously having accepted preparation to be tested.But these preparations have short-half-life, and expection can not show or have any impact on monkey at administration experimental session disclosed herein.Animal accepts contrast (air), 2mg/kg FDKP or 0.3 (0.04mg GLP-1), 1.0 (0.13mg GLP-1) or 2.0 (0.26mg GLP-1) mg/kg GLP-1/FDKP.
Table 5: target with assessment actual dose level (pass through gravimetric analysis *measure):
*by before administration, during administration and the filter paper of weighing after administration in suction chamber carry out gravimetric analysis, calculate aerocolloidal concentration in chamber and measure persistent period of administration.
1based on the 2.5kg body weight of supposition.
2based on the body weight measured (to male and female average).
3in the gas that the target listed and the supposition of actual dosage level produce, the ratio of GLP-1 is 13%.Valuation supposition 100% deposition in respiratory tract of total inhalation dose.
Table 6: target (pass through gravimetric analysis with the Mean aerosol concentrations of reality *measure):
*by before administration, during administration and the filter paper of weighing after administration in suction chamber carry out gravimetric analysis, calculate aerocolloidal concentration in chamber and measure persistent period of administration.
1in the gas that the target listed and the supposition of actual aerosol concentration produce, the ratio of GLP-1 is 13%.Valuation supposition 100% deposition in respiratory tract of total inhalation dose.
Whole blood sample (1.4mL/ blood sample) is obtained: before administration, after administration 10,30,45,60,90,120 minutes at the following time point of the 5th day.From femoral vein, blood is collected by venipuncture.Blood sample is divided into 2 deciles; The a blood plasma GLP-1 of being used for analyzes (0.8mL), and another part (0.6mL) is analyzed for serum FDKP.For blood plasma GLP-1 analyzes, at each time point, whole blood (0.8mL) is collected in 1.3mL EDTA pipe (0.1%EDTA).After blood is collected, about 5-10 adds (10 μ L/mL blood) DPP-IV inhibitor (Millipore-Billerica, MA) second in pipe, obtains the DPP-IV concentration of 100 μMs.Pipe reversion being existed side by side for several times, it is on ice moistening to be namely placed in.Whole blood sample remains on ice moistening, until within about 10 minutes, produce blood plasma at 4000rpm centrifugal (2 °-8 DEG C).Plasma sample is transferred in suitable pipe, and remains on dry ice before being stored in-70 (± 10) DEG C refrigerator.Mensuration GLP-1's must by concentration (C max), T max, AUC and T 1/2.
Within continuous 4 days, suck after using GLP-1/FDKP, within the 5th day, before all administrations, find detectable GLP-1 level in sample.At the 5th day, the peak plasma concentration (C reaching GLP-1 in latter about 10 minutes was used in administration max) (Figure 31).
Within 5th day, in male and female monkey, all observe the GLP-1C as dose function maxand AUC lastthe dosage of (area from the zero-time to the concentration-time curve of last measurable concentration-time) is relevant to be increased.In the dosage range of research, in male and female monkey, observe with the dosage increased progressively the GLP-1AUC be less than with dose proportional lastincrease, 1mg/kg/ days dosage levels male except.Male middle AUC is only caused from the 6.7 multiple dose increases of 0.3 to 2.0mg/kg/ day last2.9 times increase and female middle AUC last1.1 times of increases.
When using GLP-1/FDKP with 0.3,1.0 and the dosage level of 2.0mg/kg/ days respectively, average GLP-1 peak concentration is 17.2,93.1 and 214pg/mL in male, is 19.3,67.9 and 82.8pg/mL in female.The blood plasma level of GLP-1 declines fast, and its apparent removing half-life scope was at 4 minutes to 24 minutes.
When using GLP-1/FDKP with 0.3,1.0 and the dosage level of 2.0mg/kg/ days respectively, the AUC value of GLP-1 is 21.6,105 and 62.3pg*h/mL in male, is 33.4,23.7 and 35.4pg*h/mL in female.
Obvious sex difference is there is not in the GLP-1TK parameter that lowest dosage levels is observed.But in research and under high dose level, male monkey shows the AUC higher than female monkey all the time lastvalue.Some samples from vehicle control and contrast (air) monkey show measurable GLP-1 level.This air pollution that can be sucked by animal causes, and can be maybe the tolerance of endogenous GLP-1 in these concrete monkeys.It should be noted that control animal is exposed on the room different from the animal of GLP-1/FDKP process.
Because the biological half-life of GLP-1 is less than 15 minutes, so the GLP-1 used from GLP-1/FDKP should be completely removed in 24 hours.Therefore, gageable GLP-1 level all the time in the zero-time sample collected in the animal of all GLP-1/FDKP process for the 5th day of the endogenous levels possible explanation of GLP-1.After the administration observed, GLP-1 concentration, deduct zero-time value can reflect because GLP-1/FDKP uses the GLP-1 change caused.
For serum FDKP analyzes, whole blood (0.6mL) collected into not containing in the pipe of anticoagulant at each time point, allow at room temperature minimal condensation 30 minutes, and obtain serum by centrifugalize.FDKP analyzes and measures serum-concentration (C max), T max, AUC and T 1/2.After GLP-1/FDKP is used in absorption in continuous four days, within the 5th day, after all administrations, find detectable FDKP level in sample.5th day, after administration is used, within about 10 to 30 minutes, reach the peak plasma concentration (C of FDKP max).
In male and female monkey, within 5th day, all observe the relevant increase of dosage of the FDKP AUC ∞ (being pushed into the area the concentration-time curve of Infinite Time from the zero-time) as dose function.But in male, between 0.3 and 1.0mg/kg/ days, there is no the difference of FDKP AUC ∞, but between 1 and 2mg/kg/ days, be recorded to the relevant raising of dosage.Under all situations observing increase, it is less than and dose proportional.Male middle AUC is only caused from the 6.7 multiple dose increases of 0.3 to 2.0mg/kg/ day last2.7 times increase and 3.0 times of increases of female middle AUC ∞.When using GLP-1/DKP with 0.3,1.0 and the dosage level of 2.0mg/kg/ days respectively, average FDKP peak concentration (C max) be 200,451 and 339ng/mL in male, be 134,161 and 485ng/mL in female.When using GLP-1/FDKP with 0.3,1.0 and the dosage level of 2.0mg/kg/ days respectively, average FDKP AUC ∞ value is 307,578 and 817ng.h/mL in male, is 268,235 and 810ng.h/mL in female.Only with middle AUC ∞ and C of the animal (the 2nd group) of using FDKP for 2.1mg/kg/ days maxlevel is in the identical order of magnitude with the animal accepting 2.13mg/kg/ days GLP-1/FDKP, and exception is the T of after administration is used 30 to 45 minutes maxslight longer.
In a word, GLP-1/FDKP is well tolerable, does not have clinical sign or affects body weight, food consumption, Clinicopathological Parameters, naked eyes or microscopical evaluation.Also be recorded to the GLP-1/FDKP used 30 minutes every days 5 days stump-tailed macaque to suck and use the toxicity without any dose limitation, described in the actual dose of assessment used up to 2.13mg/kg/ days (the GLP-1 dosage corresponding to 0.26mg/kg/ days).
embodiment 15
the toxicokinetics of the GLP-1/FDKP of 14 days is used in rats by lung insufflation
This research evaluation to use the possible toxicity of rear GLP-1/FDKP for continuous 14 days every day by lung insufflation.Continuous 14 days of rat (n=24/ sex/group) accepts the FDKP granule of contrast (air), 10mg/kg, or 1 (0.15mg GLP-1), 3 (0.45mg GLP-1) or 10 (1.5mg GLP-1) mg/kg GLP-1/FDKP are blown into as lung every day.Observe the toxicity clinical sign of animal every day; Also body weight and food consumption is recorded.
At the 1st day and the 14th day, in all dosage groups, after administration is used, reach GLP-1C in about 10 to 15 minutes max.Male and female in, the peak concentration of GLP-1 average under 10mg/kg/ days GLP-1/FDKP is respectively the 1st day 6714 and 6270pg/mL and the 14th days 2979 and 5834pg/mL.The blood plasma level of GLP-1 declines, and apparent removing half-life scope was from 0.7 hour to 4.4 hours.Under the maximum dose level of 10mg/kg/ days GLP-1/FDKP, the average A UC level of GLP-1 is 2187pM*h in male, is 2703pM*h in female.Observe minimum or do not have GLP-1 to accumulate, and C max, half-life and T maxthere is no sex difference.Under all dosage, in female rats, the AUC value of GLP-1 is slight higher than in male rat.Within continuous 14 days, using sightless ill effect level (NOAEL) in the rat of GLP-1/FDKP by lung insufflation is 10mg/kg/ days GLP-1/FDKP (1.5mg/kg/ days GLP-1).
Within after final administration about 24 hours, put to death animal (12/ sex/group); Carry out clinical pathology, naked eyes and microscopic evaluation.Within the 14th day of administration, after final blood is collected, put to death animal metabolism kinetics (TK) satellite animal (12/ sex/group).There is not the death relevant to GLP-1/DKP or clinical observation result.The difference of body weight or food consumption is not had between contrast and the animal be subject to processing.Only 10mg/kg GLP-1/FDKP female in, liver weight regulating liver-QI is obviously lower compared with contrast (air) group to the ratio of body weight.
From hematology, solidify, chemistry, urinalysis or urochemistry result in, not being recorded to application of between the rat of carrier and air controls has obvious difference.There are not following naked eyes in tissue to find or histopathology finds, described discovery is confirmed as owing to application of GLP-1/FDKP and the possible toxicity of tool.
embodiment 16
used the toxicokinetics of the GLP-1/FDKP of 28 days by lung insufflation in monkey
At least 4 weeks of this research evaluation are by sucking toxicity and the toxicokinetics of daily GLP-1/FDKP.In order to evaluate reversibility, the persistency of any effect or send out evening, there is the restore cycle of 4 weeks.
Animal accepts one of following process: the 1st group: contrast (air); 2nd group: 3.67mg/kg/ days FDKP granules; 3rd group: 0.3mg/kg/ days GLP-1/FDKP (0.045mg/kg/ days GLP-1); 4th group: 1mg/kg/ days GLP-1/FDKP (0.15mg/kg/ days GLP-1) or the 5th group: 2.6mg/kg/ days GLP-1/FDKP (0.39mg/kg/ days GLP-1).
42 stump-tailed macaques are divided into the recovery (n=2/ sex/group) in the 2 groups: 1st, 2 and 5 group and main research (n=3/ sex/group).1st group: air controls, the 2nd group: FDKP (~ 4mg/kg/ days); 3rd group: 0.3mg/kg/ days GLP-1/FDKP (low dosage); 4th group: .0mg/kg/ days GLP-1/FDKP (middle dosage); 5th group: 2.6mg/kg/ days GLP-1/FDKP (high dose).Typically, in monkey research, only have high dose and evaluate when impinging upon recovery.
Observe mortality rate or the sickness rate of animal twice daily, within 30 minutes, observe exception and signs of toxicity upon administration at least once a day.Collect weight data weekly, evaluate food consumption qualitatively every day.Collect blood at the 1st, 28 and 56 day and be used for toxicokinetics.At the 29th day, three animal/sex/groups anaesthetized, weigh, blood-letting and necropsy.At the 57th day, the residue animal (n=2/ sex/group) in the 1st, 2 and 5 group anaesthetized, weigh, blood-letting and necropsy.During necropsy, weighed by selected organ, the tissue that collection is selected is also anticorrosion.Microscopy from each animal institute in a organized way.
There is accidental fluctuation in the body weight of all groups; But there is not the impact on body weight relevant to process.Usually, all animals maintain body weight or increase little weighing sb. in research process.Observe the lax or liquid manure of more high incidence and frequency at high doses.Be not recorded to the remarkable change of any clinical chemistry parameters being considered to relevant to process, exception is that the moderate of the 29th day female middle lactic acid dehydrogenase (LDH) of (at the end of process) high dose and aspartate aminotransferase (AST) increases; In table 7.The level of male middle LDH also slightly improves.These changes are eliminated at the end of the restore cycle, and find uncorrelated with any microscope in liver.In male group of high dose, the change of AST level is mainly owing to one of five animals.
The average of table 7:ALT, AST and LDH changes %
Under the dosage level up to 2.6mg/kg/ days GLP-1/FDKP, there is not the sign of any macroscopic change relevant to process or Histological change.Up to 2.6mg/kg/ days GLP-1/FDKP (0.39mg/kg/ days GLP-1) dosage under, GLP-1/FDKP is well tolerable, there is no significant clinical sign, do not affect body weight, food consumption, hematology, urinalysis, insulin analysis, ophthalmoscopy, ECG, the change under unobserved macroscopic or microscope.Not relevant to any toxicity with using FDKP up to suction 30 minutes every days with the actual dose of the assessment up to 3.67mg/kg/ days in 28 days yet.
Within 1st day, in male and female monkey, all observe GLP-1 and FDKPC as dose function maxand AUC lastdosage relevant to increase.In the dosage range of research, within the 28th day, in male and female monkey, all observe with the dosage increased progressively the GLP-1C be less than with dose proportional maxincrease, and do not find AUC lastincrease.GLP-1 peak concentration average under 2.6mg/kg/ days GLP-1/FDKP is 259pg/mL in male, is 164pg/mL in female.The blood plasma level of GLP-1 reduces, and removes half-life scope from change in 0.6 to 2.5 hours.Under high dose, the average AUC value of GLP-1 is 103pg*hr/mL in male, is 104pg*hr/mL in female.FDKP peak concentration average under 2.6mg/kg/ days GLP-1/FDKP is 1800ng/mL in male, is 1900pg/mL in female.
Generally speaking, suck to use to the GLP-1/FDKP of stump-tailed macaque and tolerated well by clinical, described in use with the actual dose of the assessment up to 2.6mg/kg/ days GLP-1/FDKP or 0.39mg/kg/ days GLP-1, to use GLP-1/FDKP up to sucking 30 minutes every days.NOAEL is 2.6 mg/kg/ days GLP-1/FDKP (0.39mg/kg/ days GLP-1).As described in Examples below 19, the I phase study in most adult's dosage should be 1.5mg GLP-1/FDKP or ~ 0.021mg/kg GLP-1 (being assumed to the people of 70kg) every day.Other research dosage should be 3.0mg GLP-1/FDKP or ~ 0.042mg/kg GLP-1 every day.
embodiment 17
prepare Exendin/FDKP preparation
By acid exendin-4 peptide (SEQ ID No.3) solution and FDKP particle suspension liquid are combined to prepare exendin-4/FDKP.Acidity peptide solution is be dissolved in 10% in 2% acetic acid (w/w) peptide.FDKP suspension is containing 10% (w/w) FDKP granule of having an appointment.Acid exendin-4 peptide solution is added in FDKP particle suspension liquid, mildly mixes simultaneously.With 25% ammonia solution, exendin-4/FDKP mixture is progressively titrated to pH 4.50.Then mixture is made bead and lyophilizing in liquid nitrogen.
It is 36% that the loading of 15% exendin-4/FDKP powder can suck fraction % (%RF on Fill) content, and the emptying percentage ratio of cartridge case (Percent Cartridge Emptying) is 99%.With the %RF on Fill of the 15%GLP-1/FDKP powder of similar large-scale production display 34%, the emptying percentage ratio of cartridge case is 100%.
embodiment 18
the pharmacokinetics of the Exendin/FDKP used by lung insufflation
Carry out the initial toxicity research of dosage of repetition, with under checking multiple concentration and repeatedly used pharmacokinetics and the pharmacokinetic profile of rear exendin-4 (GLP-1 analog) by pulmonary route.
The research of 28 days has been carried out in rat and monkey.Exendin/FDKP administration is carried out every day by inhalation route.To in the animals administer research of 28 days, a part of animal will be put to death immediately after dosage regimen, and other animal allows the restore cycle having nearly one month before execution.Evaluate the clinical sign of all animals, multiple physiological parameter, comprise the blood level of exendin-4, glucose and insulin; The clinical pathology of organ weight and multiple organ and histopathology.
Initial seminar forms by often organizing five animals and has two matched groups: air and intravenous administration Exendin.There are six lungs and be blown into group, it accepts about 2.0mg dosage, the Exendin/FDKP of 5%, 10%, 15%, 20% and 25% and 30% Exendin load (w/w).Collect whole blood and be used for blood glucose and exendin peptide concentration, until the time point of 8 hours.
Collect data (C max, T 1/2and T max), it confirms that Exendin/FDKP preparation has or better pharmacokinetics comparable relative to GLP-1/FDKP.
embodiment 19
the pharmacokinetics of the GLP-1/xDKP used by lung insufflation in rat
In order to determine whether different DKP can affect the pharmacokinetic profile of GLP-1/FDKP preparation, as be entitled as " Asymmetrical FDKP Analogs for Use as Drug Delivery Agents " U.S. Provisional Patent Application disclosed in, manufacture multiple GLP-1/xDKP preparation, described application is being submitted to the date consistent herein and is being incorporated to herein (attorney docket No.51300-00041) with its entirety.
Study in the rat being divided into 6 processed group, described group forms by often organizing 5 animals.Matched group (n=3) accepts GLP-1 by fluid drip.Also use and be blown into the GLP-1/FDKP (0.3mg GLP-1) that uses as the second contrast by lung.The group of each GLP-1/xDKP process by lung be blown into load 10% and 15%GLP-1 ~ the xDKP dosage of 2.0mg accepts GLP-1/xDKP preparation.The xDKP used is (E)-3-(4-(3; 6-dioxopiperazine-2-base) Butylcarbamoyl)-acrylic acid), (3; 6-bis-(4-carboxylic propyl group) aminobutyl-2; 5-diketopiperazine) and ((E)-3; 6-bis-(4-(carboxyl-2-acrylic) aminobutyl)-2,5-diketopiperazine disodium salts) load.5,10,20,30,45,60 until collect whole blood for 90 minutes for evaluating GLP-1 concentration upon administration.
embodiment 20
1a phase of GLP-1/FDKP inhalation of dust, single dose, open label, ascending-dose, controlled safety and tolerance test in healthy adult masculine subjects
When being inculcated by vein (iv) or subcutaneous (sc) or given by multiple subcutaneous injection, GLP-1 has been presented in people the blood glucose controlling to improve.Because the half-life of hormone is very short, may need continuous subcutaneous infusion or repeatedly every day subcutaneous injection.These approach are not all suitable for Long-term clinical purposes.Treatment level can be reached when zoopery display is by sucking and using GLP-1.
The several effect of GLP-1 (comprise reduce gastric emptying, improve satiety and suppress unsuitable glucagon and secrete) seem to discharge with the GLP-1 had meal when starting break out relevant.Fluctuated in early days by this supplementing GLP-1 with GLP-1/FDKP inhalation of dust, can in diabetic animal, cause pharmacokinetics to reply.In addition, can be fluctuated by the late period of using the GLP-1/FDKP inhalation of dust simulation natural GLP-1 relevant to the insulin secretion improved after the meal.
The 1a clinical trial phase of GLP-1/FDKP inhalation of dust is designed to safety and the tolerability of the novel suction-type glycemic control treatment product of the selected dosage of first time test in people experimenter.Administration make use of previously to have been tested inhaler apparatus.The main purpose of this clinical trial is the dosage range identifying the GLP-1/FDKP inhalation of dust sucked by lung, and described dosage range is the evidence that also can be further used for setting up effectiveness and safety in clinical trial that is safe, that can tolerate.For 1a clinical trial phase select dosage based on following animal safety result, described animal safety result is from the nonclinical test of GLP-1/FDKP inhalation of dust in rat and primates described in above-described embodiment.
26 (26) individual experimenters are registered in 5 teams, reach every team in the 1st and 2 teams and reach 4 appreciable experimenters, in 3 to 5 team, every team reaches 6 appreciable experimenters, and described appreciable experimenter meets criterion of acceptability and completes clinical trial.The glucagon-like peptide-1 (GLP-1) that each experimenter is used as GLP-1/FDKP inhalation of dust is administered once, and dosage level is as follows: the 1st team: 0.05mg; 2nd team: 0.45mg; 3rd team: 0.75mg; The GLP-1 of the 4th team: 1.05mg and the 5th team: 1.5mg.Do not replace the person of dropping by the wayside.The body weight of these dosage supposition 70kg.Those skilled in the art can determine extra dosage level according to above-disclosed.
The object of this test is safety and the toleration of the GLP-1/FDKP inhalation of dust of ascending-dose in the male subject determining healthy adult.The toleration of the GLP-1/FDKP inhalation of dust of ascending-dose will be evaluated, it is by monitoring pharmacology or determine the detrimental effect of variable, comprises the adverse events (AE) of report, vital sign, physical examination, clinical laboratory's test and electrocardiogram (ECG).
Second object evaluates other safety and pharmacokinetic parameter.This comprises other security parameters, is expressed as the change of pulmonary function between the sickness rate of lung and other AE and investigation 1 (screening) and investigation 3 (following up a case by regular visits to); By pharmacokinetics (PK) parameter of blood plasma GLP-1 and serum FDKP (FDKP) after the administration of GLP-1/FDKP inhalation of dust, pass through AUC 0-120 (minute)blood plasma GLP-1 and AUC 0-480minute serum FDKP measures; Blood plasma GLP-1PK parameter with other, comprising: t maxblood plasma GLP-1; C maxblood plasma GLP-1; And T 1/2blood plasma GLP-1.Other serum FDKP PK parameter comprises: T maxserum FDKP; C maxserum FDKP; And T 1/2serum FDKP.
Test end of the final point (endpoint) is based on testing the comparison of following pharmacology and the security parameters measured in population of subjects.Elementary end of the final point should comprise: safety end of the final point by AE (comprise cough and dyspnea, feel sick and/or vomiting) sickness rate and the seriousness according to report, from vital sign screening change, clinical laboratory tests and physical examination is evaluated.Secondary ending point should comprise: the PK distribution (AUC of blood plasma GLP-1 and serum FDKP 0-120 minuteblood plasma GLP-1 and AUC 0-480 minuteserum FDKP); Other blood plasma GLP-1PK parameter (T maxblood plasma GLP-1, C maxblood plasma GLP-1, T 1/2blood plasma GLP-1); Other serum FDKP PK parameter (T maxserum FDKP, C maxserum FDKP); With other security parameters (pulmonary function test (pft) (PFTs)) and ECG.
1a phase, single dose test incorporate open label, ascending-dose structure and meet 21CFR312, the layout strategy of Good Clinical Practice:Consolidated Guidance (ICH-E6) and Guidance on General Considerations for Clinical Trials (ICH-E8), for measuring drug products (IMP) safety and the toleration of research.
Clinical trial should be made up of 3 clinical investigations: a 1) screening investigation (investigation 1); 2) a treatment investigation (investigation 2); With 3) follow-up investigation (investigation 3) of 8-14 days after investigation 2.The using of GLP-1/FDKP inhalation of dust of single dose will occur during investigation 2.
This clinical trial can evaluate the security parameters in every team.Before all safeties of looking back first time administration or previously administration main investigator (PI) and tolerance data, administration is not carried out to the squad that plan accepts subsequent dose concentration.Halfhour administration lag time should be performed, to guarantee experimenter's safety between the experimenter of every team.If the experimenter of 3 or more stands serious feeling sick and/or vomiting in squad, maybe when reaching maximal dose, or under the judgement of PI, administration can be stopped.
Evaluate the GLP-1/FDKP inhalation of dust GLP-1 of 1.5mg (0.05,0.45,0.75,1.05 and) of 5 kinds of dosage.In order to adapt to all dosage, the GLP-1/FDKP prepared will mix with FDKP inhalation of dust.Former state is used or single dose cartridge case containing 10mg dry powder used in combination with the FDKP inhalation of dust of appropriate amount, described dry powder is made up of (GLP-1/FDKP of 15% weight by weight) GLP-1/FDKP inhalation of dust, to obtain GLP-1 (0.05mg, 0.45 mg, 0.75mg, 1.05mg and 1.5mg) dosage wanted: 1. 2 minimum dosage levels are evaluated in 2 teams of every team 4 experimenters, 3 higher dose food are evaluated in 3 teams of every team 6 experimenters.Each experimenter only accepts 1 dosage in 5 dosage levels to be evaluated.Except getting blood and carrying out GLP-1 (active with overall) and FDKP and measure, sampling is used for glucagon, glucose, insulin and C-peptide mensuration.
The publication of a large amount of patent and printing has been refer in this description full text.The publication of each above-mentioned list of references and printing is individually incorporated to herein with its entirety by reference.
Unless otherwise, what use in the specification and claims is expressed as dosis refracta, and all numerals of the character such as molecular weight, reaction condition are all appreciated that: in all cases, with term " about " in addition modification.Therefore, unless there are the explanation of contrary, the number parameter shown in this specification and the appended claims is all approximate number, the character that they can go for according to the present invention and changing.At least, and not limited the application of Claims scope doctrine of equivalents, each number parameter at least according to the number of the significant digits of report, and should be applied common rounding techniques to explain.Although illustrate that the digital scope of broad range of the present invention and parameter are approximate numbers, special numerical value shown in embodiment is but accurately reported as much as possible.But any numerical value, must contain certain error, this is that the standard deviation found in their respective checking measurements methods must cause.
One of ordinary skill in the art will readily recognize that without departing from the scope and spirit in the present invention, various improvement can be carried out to the present invention.
In this article, " one " that uses together with " comprising " in claims and/or description generally represents " one ", but its meaning represented also is consistent with " one or more ", " at least one " and " one or more than one ".
Any method as herein described or compositions realize by any other method as herein described or compositions.
Mutually exclusive between certain optional manner or optional manner unless explicitly stated otherwise, in claim, term "or" used refers to "and/or".
In this article, the value represented by term " about " comprises the standard deviation determining this value device therefor or method.
According to the previous description that embodiment and claims provide, can clearly be seen that other objects, features and advantages of the present invention.But should be appreciated that detailed description and specific embodiment are only exemplary, and those skilled in the art can make various changes and improvements within the spirit and scope of the present invention according to these detailed descriptions.
list of references
Thering is provided in the supplementary exemplary steps of disclosure herein or the degree of other details, following list of references is hereby expressly incorporated into this detailed description herein by reference.
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Claims (38)

1. a dry powder composite; it comprises the microgranule of the diketopiperazine containing the polypeptide having adsorbed GLP-1 molecule, and wherein said GLP-1 molecule is selected from the group be made up of following material: GLP-1, GLP-1 analogies, GLP-1 peptide analogues or biosynthetic GLP-1 analog that natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivant, dipeptidyl peptidase-IV (DPP-IV) are protected.
2. the dry powder composite of claim 1, wherein said diketopiperazine is the diketopiperazine with formula 2,5-diketone-3,6-bis-(4-X-ammonia butyl) piperazine, and wherein X is selected from by succinyl group, glutaryl, maleoyl and the fumaroyl group formed.
3. the dry powder composite of claim 2, wherein said diketopiperazine is 2,5-diketone-3,6-bis-(4-fumaroyl-ammonia butyl) piperazine.
4. the dry powder composite of claim 1, wherein said GLP-1 molecule is natural GLP-1.
5. the dry powder composite of claim 1, wherein said GLP-1 molecule is amidated GLP-1 molecule.
6. the dry powder composite of claim 5, wherein said amidated GLP-1 molecule is GLP-1 (7-36) amide.
7., for the formation of the method for granule comprising the diketopiperazine having adsorbed GLP-1 molecule, described method comprises step:
GLP-1 molecule is provided;
There is provided the diketopiperazine of following form, described form is selected from granulopotent diketopiperazine, diketopiperazine granule and combination thereof; With
Described GLP-1 molecule and described diketopiperazine are combined with the form of common solution; wherein formed and comprise the described described granule having adsorbed the diketopiperazine of GLP-1 molecule, and wherein said GLP-1 molecule is selected from the group be made up of following material: GLP-1, GLP-1 analogies, GLP-1 peptide analogues or biosynthetic GLP-1 analog that natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivant, dipeptidyl peptidase-IV (DPP-IV) are protected.
8. the method for claim 7, it also comprises removes solvent by lyophilization, filtration or spraying dry from described solution altogether.
9. the method for claim 8, wherein comprises the described described granule having adsorbed the diketopiperazine of GLP-1 molecule by removing the formation of described solvent.
10. the method for claim 8, was wherein formed and comprises the described described granule having adsorbed the diketopiperazine of GLP-1 molecule before the described solvent of removal.
The method of 11. claim 7, wherein said GLP-1 molecule provides as a solution, and described solution comprises the GLP-1 concentration of about 1 μ g/ml-50mg/ml.
The method of 12. claim 7, wherein said GLP-1 molecule provides as a solution, and described solution comprises the GLP-1 concentration of about 0.1mg/ml-10mg/ml.
The method of 13. claim 7, wherein said GLP-1 molecule provides as a solution, and described solution comprises the GLP-1 concentration of about 0.25mg/ml.
The method of 14. claim 7, wherein said diketopiperazine provides with the form of diketopiperazine particle suspension liquid.
The method of 15. claim 7, wherein said diketopiperazine provides with the form of the solution comprising granulopotent diketopiperazine, and described method also comprises and regulates the pH of described solution to form diketopiperazine granule.
The method of 16. claims 14 or 15, it also comprises add reagent in described solution or suspension, and wherein said reagent is selected from the group be made up of salt, surfactant, ion, penetrant, chaotropic agent and lyotrope, acid, alkali and organic solvent.
The method of 17. claim 16, wherein said reagent promotes the association between described GLP-1 molecule and described diketopiperazine granule or described granulopotent diketopiperazine.
The method of 18. claim 16, wherein said reagent improves stability or the pharmacokinetics of described GLP-1 molecule.
The method of 19. claim 16, wherein said reagent is sodium chloride.
The method of 20. claims 14 or 15, it also comprises the pH regulating described suspension or solution.
The method of 21. claim 20, wherein said pH is adjusted to about 4 or larger.
The method of 22. claim 7, the described GLP-1 molecule in wherein said granule has higher stability.
The method of 23. claim 7, wherein said solution altogether comprises the GLP-1 concentration of about 1 μ g/ml-50mg/ml.
The method of 24. claim 7, wherein said solution altogether comprises the GLP-1 concentration of about 0.1mg/ml-10mg/ml.
The method of 25. claim 7, wherein said solution altogether comprises the GLP-1 concentration of about 0.25mg/ml.
The method of 26. claim 7, it also comprises add reagent in described altogether solution, and wherein said reagent is selected from the group be made up of salt, surfactant, ion, penetrant, chaotropic agent and lyotrope, acid, alkali and organic solvent.
The method of 27. claim 26, wherein said reagent promotes the association between described GLP-1 molecule and described diketopiperazine granule or described granulopotent diketopiperazine.
The method of 28. claim 26, wherein said reagent improves stability or the pharmacokinetics of described GLP-1 molecule.
The method of 29. claim 26, wherein said reagent is sodium chloride.
The method of 30. claim 7, it also comprises the pH regulating described solution altogether.
The method of 31. claim 30, wherein said pH is adjusted to about 4 or larger.
32. Diketopiperazine microparticle having adsorbed GLP-1 molecule are manufacturing the purposes be used for the treatment of in the medicine of disease or disease, wherein said disease or disease are selected from by diabetes, ischemia, Reperfu-sion tissue injury, dyslipidemia, diabetic cardiopathy, myocardial infarction, acute coronary syndrome, obesity, postoperative catabolism changes, hyperglycemia, irritable bowel syndrome, apoplexy, neurodegenerative disorders, memory and learning disorder, the group of islet cell transplantation and regenerative therapies composition, wherein said GLP-1 molecule is selected from the group be made up of following material: natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivant, the GLP-1 that dipeptidyl peptidase-IV (DPP-IV) is protected, GLP-1 analogies, GLP-1 peptide analogues or biosynthetic GLP-1 analog.
The purposes of 33. claim 32, wherein said medicine by intravenous, subcutaneous, per os, per nasal, through cheek, per rectum or carry out administration by pulmonary delivery.
The purposes of 34. claim 32, wherein said medicine carries out administration by pulmonary delivery.
35. form the method with the powder composition of the GLP-1 pharmacokinetic profile of improvement, and described method comprises step:
There is provided GLP-1 molecule, wherein said GLP-1 molecule is selected from the group be made up of following material: GLP-1, GLP-1 analogies, GLP-1 peptide analogues or biosynthetic GLP-1 analog that natural GLP-1, GLP-1 metabolite, GLP-1 analog, GLP-1 derivant, dipeptidyl peptidase-IV (DPP-IV) are protected;
Granulopotent diketopiperazine in solution is provided;
Form diketopiperazine granule;
Described GLP-1 molecule and described solution combination are formed common solution; With
From described solution altogether, remove solvent by spraying dry, form the GLP-1 pharmacokinetic profile and the powder comprising following microgranule with improvement, described microgranule contains the diketopiperazine having adsorbed GLP-1 molecule.
The method of 36. claim 35, the GLP-1 pharmacokinetic profile of wherein said improvement comprises the GLP-1 half-life of raising.
The method of 37. claim 36, the GLP-1 half-life of wherein said raising is more than or equal to 7.5 minutes.
The method of 38. claim 35, the GLP-1 pharmacokinetic profile of wherein said improvement comprises the GLP-1 bioavailability of improvement compared with natural GLP-1.
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