Method of preventing adverse effects by glp-1

A technology of GLP-1 and preparations, which can be used in pharmaceutical formulas, medical preparations containing active ingredients, metabolic diseases, etc., and can solve problems such as toxicity to patients

Inactive Publication Date: 2011-02-09
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AI-Extracted Technical Summary

Problems solved by technology

In some cases, metabolites can be toxic to p...
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Method used

Figure 16 shows the representative data from following experiment, described experiment is measured by IV administration, by pulmonary administration with various formulations comprising fumaryl-diketopiperazine at various times after administration. Plasma concentrations of PYY in female ZDF rats and air control rats administered PYY formulations. The results of these measurements are also shown in Table 6. The data showed that Group 1 rats administered PYY IV maintained higher plasma PYY concentrations (30.7 μg/mL) compared to rats treated by pulmonary insufflation. The peak plasma concentration (Tmax) of PYY was approximately 5 minutes for rats in groups 1, 6 and 7 and 10 minutes for rats in groups 2, 4 and 5. The data showed that all rats treated with PYY/FDKP by pulmonary insufflation had measurable amounts of PYY in their plasma samples, however, group 7 rats had the highest plasma PYY concentration (4.9 μg/mL), and the value Remain higher than the other groups until about 35 minutes after dosing. The data also indicated that plasma concentrations of PYY administered by pulmonary insufflation were dose dependent. Although administration by IV injection at the doses used resulted in higher venous plasma concentrations of PYY than pulmonary administration of PYY/FDKP, greater inhibition of food consumption was achieved with pulmonary administration of PYY/FDKP.
In another embodiment, the pulmonary administration of the respirable dry powder formulation comprising GLP-1 and diketopiperazine can be used as a diagnostic tool for diagnosing the level of development of type 2 diabetes in patients afflicted with diabetes, A specific treatment regimen suitable for the patient to be treated is thereby identified. In this embodiment, there is provided a method of diagnosing the level of diabetes progression in a patient identified as having diabetes, the method comprising administering to the patient a predetermined amount of an inhalable dry powder formulation comprising GLP-1 and diketopiperazine , and measure endogenous insulin production or response. Administration of the inhalable dry powder formulation comprising GLP-1 can be repeated with a predetermined amount of GLP-1 until an appropriate level of insulin response is achieved for the patient, thereby determining the desired treatment regimen required by the patient. In this embodiment, if the patient's insulin response is insufficient, the patient may require alternative therapy. Patients who are sensitive or responsive to insulin can be treated with GLP-1 formulations comprising diketopiperazines as therapy. In this way, specific amounts of GLP-1 can be administered to patients so that an appropriate insulin response is achieved to avoid hypoglycemia. In this and other embodiments, GLP-1 is capable of inducing rapid endogenous insulin release, which mimics the no...
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A method for preventing or reducing adverse effects such as profuse sweating, nausea and vomiting, which normally are associated with subcutaneous and intravenous administration of glucagon-like peptide 1 (GLP-1 ) therapy is provided. In particular, the method comprises the rapid administration of a GLP-1 formulation into the pulmonary circulation such as by inhalation, directly into pulmonary alveolar capillaries using a dry powder drug delivery system.

Application Domain

Powder deliveryOrganic active ingredients +4

Technology Topic

VomitingAdverse effect +12


  • Method of preventing adverse effects by glp-1
  • Method of preventing adverse effects by glp-1
  • Method of preventing adverse effects by glp-1


  • Experimental program(8)
  • Effect test(1)

Example Embodiment

[0115] Example 1
[0116] Administration of GLP-1 to healthy adult males in the form of an inhalable dry powder
[0117] GLP-1 has been shown to control elevated blood glucose in humans when given by intravenous (iv) or subcutaneous (sc) infusion or by multiple subcutaneous injections. Due to the extremely short half-life of the hormone, continuous subcutaneous infusions or multiple daily subcutaneous injections are required to achieve clinical efficacy. None of these routes is practical for prolonged clinical use. The applicant has found in animal experiments that therapeutic levels can be achieved when GLP-1 is administered by inhalation. The results of these studies can be found, for example, in US Patent Application No. 11/735,957, the disclosure of which is incorporated herein by reference.
[0118] In healthy individuals, several effects of GLP-1, including decreased gastric emptying, increased satiety and suppression of inappropriate glucagon secretion, appear to be related to the burst of GLP-1 released upon initiation of a meal. By supplementing this early surge of GLP-1 with a formulation of GLP-1 and 2,5-diketo-3,6-bis(4-fumaryl-aminobutyl)piperazine (FDKP) as an inhalation powder, one can Pharmacodynamic responses were elicited in diabetic animals, including reductions in endogenous insulin production, glucagon, and glucose levels. Additionally, the late surge of native GLP-1 associated with increased insulin secretion can be mimicked by postprandial administration of GLP-1/FDKP inhalation powder.
[0119] The Phase 1a clinical trial of GLP-1/FDKP Inhalation Powder was designed to test for the first time the safety and tolerability of selected doses of a novel inhaled glycemic control therapeutic product in human subjects. Use pre-tested device, administering GLP-1/FDKP inhalation powder. This experiment was designed to characterize the safety and tolerability of various doses of GLP-1/FDKP inhalation powder via pulmonary inhalation. Animal safety studies from nonclinical studies in rats and primates using GLP-1/FDKP administered by inhalation as described in U.S. Application Serial No. 11/735,957 (which is incorporated herein by reference) Based on the results, dosages for human use are selected.
[0120]Enroll 26 subjects into 5 groups, providing up to 4 worthy subjects each in Groups 1 and 2 and up to 6 valuable subjects each in Groups 3 to 5 Or, the subject of interest met the eligibility criteria and completed the study. Each subject was dosed once with GLP-1 as GLP-1/FDKP powder at the following dose levels: Group 1: 0.05 mg GLP-1; Group 2: 0.45 mg GLP-1; Group 3 : 0.75 mg GLP-1; Group 4: 1.05 mg GLP-1 and Group 5: 1.5 mg GLP-1. Dropouts are not replaced. These doses assume a body weight of 70 kg. Other dosage levels can be determined by one of ordinary skill in the art based on the studies disclosed herein.
[0121] In these experiments, the safety and tolerability of increasing doses of GLP-1/FDKP inhalation powder was determined in healthy adult male subjects. Tolerability of escalating doses of GLP-1/FDKP inhalation powder was determined by monitoring pharmacological or adverse effects on variables including reported adverse events (AEs), fatal signs, physical examination, clinical laboratory Tests and electrocardiogram (ECG).
[0122] Additional pulmonary safety and pharmacokinetic parameters were also evaluated. Pulmonary safety, expressed as the incidence of pulmonary and other adverse events and the change in lung function between Visit 1 (screening) and Visit 3 (follow-up), was studied. Pharmacokinetic (PK) parameters of plasma GLP-1 and serum fumaryl diketopiperazine (FDKP) following administration of GLP-1/FDKP inhalation powder were measured as AUC 0-120分钟 Plasma GLP-1 and AUC 0-480分钟 Serum FDKP. Other PK parameters of plasma GLP-1 include the time to maximum plasma GLP-1 concentration, T max Plasma GLP-1; maximum GLP-1 concentration in plasma, C max Plasma GLP-1, and half of the total time to reach maximum GLP-1 concentration in plasma, T 1/2 Plasma GLP-1. Other PK parameters of serum FDKP include T max Serum FDKP, C max Serum FDKP and T 1/2 Serum FDKP. The clinical trial endpoints are based on the comparison of the following pharmacological and safety parameters determined in the trial subject population. Primary endpoints included the incidence and severity of reported AEs (including cough and dyspnea, nausea and/or vomiting), and changes from fatal sign screens, clinical laboratory tests, and physical examinations. Secondary endpoints included plasma GLP-1 and serum FDKP (AUC 0-120分钟 Plasma GLP-1 and AUC 0-480分钟 Serum FDKP), plasma GLP-1 (T max Plasma GLP-1, C max Plasma GLP-1, T 1/2 plasma GLP-1); serum FDKP (T max Serum FDKP, C max Pharmacokinetic profile of serum FDKP); pulmonary function tests (PFTs) and ECG.
[0123] The clinical trial consisted of 3 clinical visits: 1) a screening visit (Visit 1); 2) a treatment visit (Visit 2); and 3) a follow-up visit 8-14 days after Visit 2 ( Visit 3). A single dose of GLP-1/FDKP inhalation powder was administered at Visit 2.
[0124] Five doses of GLP-1/FDKP inhalation powder (0.05, 0.45, 0.75, 1.05 and 1.5 mg of GLP-1) were evaluated. To accommodate all doses, the reconstituted GLP-1/FDKP is mixed with FDKP inhalation powder containing granules without active agent. A single-dose cartridge containing 10 mg of dry powder consisting of GLP-1/FDKP inhalation powder (15% by weight of GLP-1/FDKP) is used as such or mixed with an appropriate amount of FDKP inhalation powder for obtaining the desired GLP-1 Doses (0.05 mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg). The first 2 lower dose levels were evaluated in 2 groups of 4 subjects each and the last 3 higher dose levels were evaluated in 3 groups of 6 subjects each. Each subject received only 1 dose at one of the 5 dose levels evaluated. In addition to blood draws for GLP-1 (activity and total) and FDKP measurements, samples were also taken for glucagon, glucose, insulin and C-peptide assays. The results from these experiments are described with reference to the figures and tables below.
[0125] figure 1 Depicted are active GLP-1 plasma concentrations in Group 5 following pulmonary administration of a GLP-1 dose of 1.5 mg. The data showed that peak GLP-1 concentrations occurred before the first sampling point at 3 minutes, very similar to intravenous (IV) bolus administration. GLP-1 plasma concentrations were greater than the assay limit of 500 pmol/L in some subjects. The peak concentration of active GLP-1 ranged from about 150 pmol/L to about 500 pmol/L. Intravenous bolus administration of GLP-1 as reported in the literature (Vilsboll et al. 2000) resulted in a total GLP-1:active GLP-1 ratio of 3.0-5.0 compared to a ratio of 1.5 in group 5 of this study . At comparable active concentrations, metabolite peaks were 8-9 times higher after intravenous administration compared to pulmonary administration, suggesting that pulmonary delivery resulted in faster delivery and less GLP-1 degradation.
[0126] Table 1.
[0128] a divided by t max is the median (range), all other parameters are mean (SD)
[0129] AULQ - two or more subjects in the dose group had an analyte plasma concentration of AULQ; NA = due to short sampling time (20 minutes), the pharmacokinetic model did not meet the specification of the model; ND = due to Insufficient data were available in some subjects to calculate parameters.
[0130] In healthy individuals, the physiological postprandial venous plasma concentration of GLP-1 typically ranges from 10-20 pmol/L (Vilsboll et al. J. Clin. Endocr. & Metabolism. 88(6):2706-13, June 2003). Some subjects in Group 2 who received 0.45 mg of GLP-1 achieved these levels. Higher doses of GLP-1 produced significantly higher peak plasma GLP-1 concentrations than physiological peak venous concentrations. However, because of the short half-life of GLP-1 (approximately 1-2 minutes), the plasma concentration of active GLP-1 at 9 minutes after administration falls within the physiological range. Although peak concentrations are much higher than those observed physiologically in the venous circulation, there is evidence that local concentrations of GLP-1 can be much higher than those observed systemically.
[0131] Table 1 presents the pharmacokinetic profile of GLP-1 in formulations containing FDKP from this study.
[0132] The FDKP pharmacokinetic parameters of groups 4 and 5 are also shown in Table 1. Other groups were not analyzed. The data also showed that subjects treated with 1.05 mg and 1.5 mg GLP-1 had mean plasma FDKP concentrations of 184 and 211 pmol/L, respectively. For the respective doses with a half-life of approximately 2 hours (127 and 123 minutes), maximum plasma FDKP concentrations were maintained at approximately 4.5 and 6 minutes post-administration, respectively.
[0133] Figure 2A Depicted are mean insulin concentrations in subjects treated with an inhalable dry powder formulation of GLP-1 at a dose of 1.5 mg. Data show that 1.5 mg GLP-1 induces endogenous insulin release from β-cells, as insulin concentrations were detected in all subjects, and a mean peak insulin of approximately 380 pmol/L occurred at 6 minutes or earlier after dosing concentration. Insulin release is rapid but sustained because plasma insulin concentrations drop rapidly after the initial response to GLP-1. Figure 2B Depicted are GLP-1 plasma concentrations in GLP-treated subjects administered a 1.5 mg dose by pulmonary inhalation compared to subcutaneously administered GLP-1 doses. The data demonstrate that pulmonary administration of GLP-1 occurs relatively quickly and peak plasma concentrations of GLP-1 occur more rapidly compared to subcutaneous administration. Additionally, pulmonary inhalation of GLP-1 resulted in a much faster return of GLP-1 plasma concentrations to basal levels than subcutaneous administration. Thus, the patient's exposure to GLP-1 provided by pulmonary inhalation using the drug delivery system of the present invention is shorter in time than by subcutaneous administration, and the total exposure to GLP-1 measured by AUC is higher for inhaled insulin. few. Figure 2C It was stated that pulmonary administration of a dry powder formulation of GLP-1 induced a similar insulin response compared to the response obtained after intravenous administration of GLP-1, but the peak time and the amount of endogenous insulin produced were different from subcutaneous GLP-1 administration, which It was shown that pulmonary administration of GLP-1 using formulations of the invention is more effective in inducing insulin response.
[0134] image 3 Depicted are plasma C-peptide concentrations in subjects treated with inhalable dry powder formulations containing doses of 1.5 mg GLP-1, measured at various times post-inhalation. The data demonstrate that C-peptide is released following GLP-1 inhalation, confirming endogenous insulin release.
[0135] Figure 4 Fasting plasma glucose concentrations in subjects treated with GLP-1-containing GLP-1 formulations are depicted. For 1.5 mg GLP-1 treated subjects, the mean fasting plasma glucose (FPG) concentration was approximately 4.7 mmol/L. GLP-1 mediated insulin release is glucose dependent. Hypoglycemia was not historically observed in euglycemic subjects. In this experiment, the data clearly showed that the glucose concentration in these normoglycemic healthy subjects was reduced after pulmonary administration of GLP-1. At the 1.5 mg GLP-1 dose, two of the six subjects had concentrations reduced by GLP-1 to below 3.5 mmol/L, the laboratory value that defines hypoglycemia. Plasma glucose was lowered by more than 1.5 mol/L in two of the six subjects who received the 1.5 mg GLP-1 dose. Additionally, the decrease in plasma glucose was dose-related to GLP-1. The smallest reduction in glucose concentration was observed with the 0.05 mg dose and the largest reduction was observed with the 1.5 mg dose. The three intermediate GLP-1 doses produced approximately equal reductions in plasma glucose. The data indicate that GLP-1 glucose-dependence is overcome based on GLP-1 concentrations above the physiological range. The physiological range of GLP-1(7-36)amide in normal individuals has been reported to be in the range of 5-10 pmol/L during fasting and rapidly increases to 15 to 50 pmol/L after feeding (Drucker, D. and Nauck, M. The Lancet 368:1696-1705, 2006).
[0136] Figure 5 It was further delineated that insulin concentrations in plasma following GLP-1 pulmonary administration are dose-dependent. In most subjects, insulin release was not sustained because plasma insulin concentrations fell rapidly after the initial response to GLP-1 administration. In most subjects, peak plasma insulin responses ranged from 200-400 pmol/L, with one subject showing peak plasma insulin levels in excess of 700 pmol/L. Thus, the data suggest that the insulin response is GLP-1 dose dependent.
[0137] Image 6Depicted are glucagon concentrations in plasma following GLP-1 pulmonary administration in various dosing groups. Across the multiple dose groups, baseline glucagon levels ranged from 13.2 pmol/L to 18.2 pmol/L. The greatest change in plasma glucagon was observed at 12 minutes post-dose. The maximum decrease in plasma glucagon was approximately 2.5 pmol/L and was observed in the 1.5 mg dose group. The maximal suppression of glucagon secretion may have been underestimated because the minima did not always occur at 12 minutes.
[0138] Tables 2 and 3 report adverse events or side effects symptoms recorded for the patient population in the study. The list of adverse events reported in the literature for GLP-1 administered by injection is not exhaustive; and those reported were described as mild or moderate, and tolerable. The main adverse events reported were profuse sweating, nausea and vomiting at GLP-1 concentrations above 100 pmol/L. as in Tables 1 and 3 and figure 1 As shown in , pulmonary administration at doses of 1.05 mg and 1.5 mg resulted in active GLP-1 concentrations well over 100 pmol/L without the use of parenteral (subcutaneous, intravenous [bolus or infusion either]) GLP-1 1 commonly observed side effects. None of the subjects in the study reported nausea, profuse sweating, or vomiting. Subjects in Group 5 achieved a C comparable to that observed with the 50 μg/kg IV bolus (reported by Vilsboll et al. 2000). max , the majority of subjects in the data reported significant adverse events.
[0139] Table 2. Adverse Events
[0141] Table 3. GLP-1: Comparative Adverse Events for IV Versus Pulmonary Administration
[0143] Vilsboll et al Diabetes Care, June 2000; * Comparable C max
[0144] Tables 2 and 3 show that no serious or severe adverse events were reported in any of the subjects receiving GLP-1 by pulmonary inhalation in this study. The most commonly reported adverse events were those related to dry powder inhalation, cough and throat irritation. Surprisingly, among patients treated by pulmonary inhalation, no subjects reported nausea or pathological dysphoria, and there was no vomiting associated with any of these subjects. The inventors also found that pulmonary administration of GLP-1 in a dry powder formulation lacked inhibition of gastric emptying in the above subjects (data not shown). Inhibition of gastric emptying is a commonly encountered unwanted side effect associated with injected standard GLP-1 formulations.
[0145] In conclusion, clinical GLP-1/FDKP powders contained up to 15 wt% GLP-1, which provided a maximum dose of 1.5 mg GLP-1 in 10 mg powder. Andersen cascade measurements showed that 35-70% of the particles had an aerodynamic diameter of <5.8 μm. A dose of 1.5 mg GLP-1 produced a mean peak concentration of >300 pmol/L active GLP-1 at the first sampling time (3 minutes); resulted in a mean insulin of 375 pmol/L at the first measurement time point (6 minutes) peak concentration; reduced mean fasting plasma glucose from 85 to 70 mg/dL 20 minutes after dosing; and was well tolerated and did not cause nausea or vomiting.

Example Embodiment

[0146] Example 2
[0147] Comparison of pulmonary administration of GLP-1 and Exented to male Zucker diabetic obese rats for and subcutaneous administration of Exented
[0148] Considerable effort has been expended in developing GLP-1 analogs with longer circulating half-lives that would lead to clinically useful treatments. As demonstrated herein, pulmonary administration of GLP-1 also provided clinically meaningful activity. It is therefore of interest to compare these two pathways.
[0149] Preparation of FDKP particles
[0150] Fumaryl diketopiperazine (FDKP) and polysorbate 80 were dissolved in dilute ammonia water to obtain a solution containing 2.5 wt% FDKP and 0.05 wt% polysorbate 80. The FDKP solution was then mixed with an acetic acid solution containing polysorbate 80 to form granules. The particles were washed and concentrated by tangential flow filtration to achieve about 11% solids by weight.
[0151] Preparation of GLP-1 stock solution.
[0152] A 10 wt% GLP-1 stock solution in deionized water was prepared by combining 60 mg GLP-1 solids (86.6% peptide) with 451 mg deionized water. Add approximately 8 µL of glacial acetic acid to dissolve the peptide.
[0153] Preparation of GLP-1/FDKP particles.
[0154] A 1 g portion (108 mg of particles) of the stock FDKP suspension was transferred to a 2 mL polypropylene tube. Add the appropriate amount of GLP-1 stock solution (Table 1) to the suspension and mix gently. The pH of the suspension was adjusted from pH~3.5 to pH~4.5 by adding 1 [mu]L aliquots of 50% (v/v) ammonium hydroxide. The GLP-1/FDKP particle suspension was then pelleted in liquid nitrogen and lyophilized. The dry powder was analyzed by high performance liquid chromatography (HPLC) and found to be comparable to theoretical values.
[0155] Prepare Exented stock solution.
[0156] A 10 wt% exendin stock solution was prepared in 2% wt acetic acid by mixing 281 mg exendin solid (88.9% peptide) with 2219 mg 2% wt acetic acid.
[0157] Preparation of Exented/FDKP particles.
[0158] A 1533 mg portion of the stock FDKP particle suspension (171 mg particles) was transferred to a 4 mL glass tube. A 304 mg portion of exendin stock solution was added to the suspension and mixed gently. The pH of the suspension was adjusted from pH~3.7 to pH~4.5 by adding 3-5 [mu]L aliquots of 25% (v/v) ammonium hydroxide. The Exented/FDKP particle suspension was then pelleted in liquid nitrogen and lyophilized. The dry powder was analyzed by high performance liquid chromatography (HPLC) and found to be comparable to theoretical values.
[0159] Pharmacokinetic and pharmacodynamic evaluations were performed in rats.
[0160] Male Zucker diabetic fat (ZDF) rats (5/group) were assigned to one of four test groups. Animals were fasted overnight and then administered glucose (1 g/kg) by intraperitoneal injection just prior to test item administration. Animals in the control group received air via lung insufflation. Animals in Group 1 received Exented (0.3 mg) in saline (0.1 mL) by subcutaneous (SC) injection. Animals in Group 2 received 15% by weight Exented/FDKP (2 mg) by pulmonary insufflation. Animals in Group 3 received 15% by weight of GLP-1/FDKP (2 mg) by pulmonary insufflation. Blood samples were collected from the tail before dosing and at 15, 30, 45, 60, 90, 120, 240 and 480 minutes after dosing. Harvest plasma. Determination of blood glucose and plasma GLP-1 or plasma Exentedide concentration.
[0161] Figure 7 Exented pharmacokinetics are reported in A. These data show that Exented is rapidly absorbed after insufflation of Exented/FDKP powder. The bioavailability of inhaled Exented was 94% compared to subcutaneous injection. This may indicate that pulmonary administration is particularly beneficial for Exented. In rats receiving subcutaneous Exentedide, time to maximum peak circulating Exentedide concentration (T max ) was 30 minutes compared to <15 minutes in rats receiving inhaled Exentedide. this T max Similar to insufflated GLP-1/FDKP (data not shown).
[0162] Figure 8 Comparative pharmacokinetics are reported in . Data show changes in blood glucose for all four test groups. Glucose excursions after the glucose tolerance test were lower in animals receiving inhaled Exented/FDKP compared to animals receiving SC Exented. Exsented exposures were therefore comparable in the two groups ( Figure 7 ), so these data suggest that the shorter time to peak ixenated concentration provides better glucose control in the Exented/FDKP group. Additionally, glucose excursions were comparable in animals receiving either GLP-1/FDKP or Exented/FDKP. These data are surprising because the circulating half-life of Exented (89 minutes) is considerably longer than that of GLP-1 (15 minutes). In fact, Exented was developed to maximize the circulating half-life for the purpose of enhancing potency. These data suggest that the longer circulating half-life of Exentide offers no advantage in controlling hyperglycemia when using pulmonary administration. Additionally, pulmonary administration of either molecule provided superior blood glucose control over SC Exentide.
[0163] Figure 7 Depicted are mean plasma exendin concentrations in male ZDF rats receiving exendin-4/FDKP administered by pulmonary insufflation relative to exendin-4 subcutaneously. Solid squares represent lung responses after exendin-4/FDKP powder insufflation. Open squares represent the response following administration of exendin-4 administered subcutaneously. Data are plotted as mean ± standard deviation. The data showed that rats insufflated with powder providing doses of 0.12, 0.17 and 0.36 mg GLP-1 produced maximum plasma GLP-1 concentrations of 2.3, 4.9 and 10.2 nM, respectively (C max ) and exposures (AUC) (t max = 10 minutes, t 1/2 = 10 minutes). In the intraperitoneal glucose tolerance test performed after daily administration of 0.3 mg GLP-1 for 4 consecutive days, treated animals showed significantly lower glucose concentrations than controls (p<0.05). At 30 minutes after challenge, glucose increased by 47% in control animals and only by 17% in treated animals.
[0164] Figure 8 Depicted is the exposure to air control, exendin-4/FDKP powder, or GLP-1 by lung insufflation compared to subcutaneous exendin-4 and exendin-4 administered by lung insufflation. Changes in blood glucose from baseline in male ZDF rats/FDKP powder. Closed diamonds represent lung responses to exendin-4/FDKP powder insufflation. Closed circles represent responses following subcutaneous exendin-4 administration. Closed triangles represent responses after administration of GLP-1/FDKP powder. Closed squares represent individual lung responses to air insufflation. Open squares represent the response to rats administered 2 mg GLP-1/FDKP by insufflation followed by 2 mg exendin-4/FDKP powder by insufflation.

Example Embodiment

[0165] Example 3
[0166] Oxyntomodulin/FDKP Powder Preparation
[0167] Oxyntomodulin (also known as glucagon-37) is a peptide consisting of 37 amino acid residues. This peptide is manufactured by and obtained from American Peptide Company, Inc. of Sunnyvale, CA. FDKP particles in suspension were mixed with oxyntomodulin solution, then snap-frozen into pellets in liquid nitrogen, and lyophilized to produce sample powders.
[0168] Six powders were prepared with target peptide contents between 5% and 30%. The actual peptide content determined by HPLC was between 4.4% and 28.5%. The aerodynamic properties of powders containing 10% peptide were analyzed using cascade impact.
[0169] The FDKP solution was then mixed with a polysorbate 80-containing acetic acid solution to form granules. The particles were washed and concentrated by tangential flow filtration to achieve about 11% solids by weight.
[0170] The FDKP particle suspension (1885 mg x 11.14% solids = 210 mg FDKP particles) was weighed into a 4 mL clean glass tube. Cap the tube and mix using a magnetic stirrer to prevent settling. Oxntomodulin solution (909 mg of 10% peptide in 2 wt% acetic acid) was added to the tube and allowed to mix. The ratio of the final composition was approximately 30:70 oxyntomodulin:FDKP particles. The oxyntomodulin/FDKP suspension had an initial pH of 4.00, which was adjusted to pH 4.48 by adding 2-10 μL aliquots of 1:4 (v/v) ammonium hydroxide/water. The suspension was pelleted in a small crystallization dish containing liquid nitrogen. Plates were placed in a lyophilizer and lyophilized at 200 mTorr. The shelf temperature was slowly raised from -45°C to 25°C at a rate of 0.2°C/min, and then held at 25°C for about 10 hours. The resulting powder was transferred to a 4 mL clean glass tube. The total yield of powder after transfer to the tube was 309 mg (103%). The samples were tested for oxyntomodulin content as follows: the oxyntomodulin preparation was diluted in sodium bicarbonate and determined by high pressure liquid chromatography in a Waters 2695 separation system using Fluoroacetic acid (TFA) in deionized water and acetonitrile containing 0.1% TFA were used as the mobile phase, and the detection wavelength was set at 220 nm and 280 nm. Using Waters Empower TM A software program analyzes the data.
[0171] Pharmacokinetic and pharmacodynamic evaluation in rats.
[0172] Male ZDF rats (10/group) were assigned to one of four groups. Animals in one group received oxyntomodulin by intravenous injection. Animals in the other three groups received 5% oxyntomodulin/FDKP powder (containing 0.15 mg oxyntomodulin), 15% oxyntomodulin/FDKP powder (containing 0.45 mg oxyntomodulin) by pulmonary insufflation. oxyntomodulin) or 30% oxyntomodulin/FDKP powder (containing 0.9 mg oxyntomodulin). Blood samples were collected from the tail prior to dosing and at various times after dosing for the measurement of plasma oxyntomodulin concentrations ( Figure 9A ). Food consumption was also monitored at various times after administration with oxyntomodulin ( Figure 9B ).
[0173] Figure 9A Plasma concentrations of oxyntomodulin were compared in control rats receiving oxyntomodulin by intravenous injection and in male ZDF rats after administration of different amounts of the inhalable dry powder formulation. These data show that oxyntomodulin is rapidly absorbed following insufflation of oxyntomodulin/FDKP powder. Time to maximum circulating oxyntomodulin concentration in rats receiving inhaled oxyntomodulin (T max ) less than 15 minutes. This study showed that the half-life of oxyntomodulin ranged from about 22 to about 25 minutes after pulmonary administration.
[0174] Figure 9B is a bar graph showing cumulative food consumption in male ZDF rats treated with intravenous oxyntomodulin or oxyntomodulin/FDKP powder administered by pulmonary insufflation compared to control animals receiving air flow . The data show that pulmonary administration of oxyntomodulin/FDKP reduces food consumption to a greater extent than a single dose of intravenous oxyntomodulin or air controls.
[0175] In a similar set of experiments, rats received airflow as a control (group 1) or 30% oxyntomodulin/FDKP powder by lung insufflation. Rats administered oxyntomodulin/FDKP inhalation powder received 0.15 mg oxyntomodulin (as 0.5 mg oxyntomodulin/FDKP powder; group 2), 0.45 mg oxyntomodulin prepared as described above. Oxyntomodulin (as 1.5 mg oxyntomodulin/FDKP powder, group 3) or 0.9 mg oxyntomodulin (as 3 mg oxyntomodulin/FDKP powder, group 4) was either dosed. The studies were performed in ZDF rats fasted for 24 hours before starting the experiment. Rats were allowed to eat after receiving the experimental dose. Rats were given a predetermined amount of food, and the amount of food consumed by the rats was measured at various times after initiation of the experiment. Rats were administered the oxyntomodulin/FDKP dry powder formulation by pulmonary insufflation, and food measurements and blood samples were taken at various points after dosing.
[0176] Figure 10A and 10B Circulating oxyntomodulin concentrations, and changes in food consumption relative to controls are shown, respectively, for all animals tested. Up to 6 hours after dosing, rats administered oxyntomodulin consumed significantly less food than control rats. Higher doses of oxyntomodulin were shown to suppress appetite more significantly than lower doses, suggesting that appetite suppression is dose-dependent, as rats given higher doses consumed minimal amount of food.
[0177] The maximum concentration of oxyntomodulin was detected in the blood at 10 to 30 minutes, and the maximum concentration of oxyntomodulin was dose-dependent, because rats receiving 1.5 mg oxyntomodulin had 311 μg/ mL, while rats receiving 3 mg oxyntomodulin had a maximum plasma concentration of 660 μg/mL. Half-life of oxyntomodulin (t 1/2 ) ranges from about 25 minutes to 51 minutes.


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