Pharmaceutical formulations for the oral delivery of peptide drugs

Abstract

The present invention relates to improved pharmaceutical formulations, uses and methods for the oral delivery of peptide drugs with advantageously high bioavailability, safety and costeffectiveness. In particular, the invention provides a peptide drug having a molecular weight of equal to or less than 5 kDa for use as a medicament, wherein said peptide drug is to be administered orally in combination with a pharmaceutically acceptable copper salt/complex and/or a pharmaceutically acceptable zinc salt/complex and/or a pharmaceutically acceptable iron salt/complex, and with a pharmaceutically acceptable complexing agent. The invention also provides a pharmaceutical composition comprising: a peptide drug having a molecular weight of equal to or less than 5 kDa; a pharmaceutically acceptable copper salt/complex and/or a pharmaceutically acceptable zinc salt/complex and/or a pharmaceutically acceptable iron salt/complex; and a pharmaceutically acceptable complexing agent.

Claims

1. A method of orally delivering a peptide drug, the method comprising orally administering a pharmaceutical composition to a subject in need thereof, wherein the pharmaceutical composition comprises: a peptide drug having a molecular weight of equal to or less than 5 kDa and having at least one serine protease cleavage site; a pharmaceutically acceptable copper complex and/or a pharmaceutically acceptable zinc complex and/or a pharmaceutically acceptable iron complex, wherein said copper complex is selected from the group consisting of a copper(II) amino acid complex, copper(II) lysine complex, copper(II) glycinate, copper(II) EDTA complex, copper(II) chitosan complex, copper(II) citrate, copper(II) gluconate, copper(II) lactate, copper(II) lactate gluconate, and copper(II) orotate, wherein said zinc complex is a zinc(II) complex which is selected from the group consisting of zinc ascorbate, zinc caprylate, zinc gluconate, zinc stearate, zinc orotate, a zinc amino acid complex, zinc glycinate, zinc arginate, zinc picolinate, zinc pidolate, zinc carnosine, zinc undecanoate, zinc undecylenate, zinc methionine, zinc lactate, and zinc lactate gluconate, wherein the iron complex is selected from the group consisting of iron(II) gluconate, iron(II) orotate, iron(II) tartrate, iron(II) fumarate, iron(II) lactate, iron(II) lactate gluconate, iron(II) citrate, iron(II) ascorbate, an iron(II) amino acid complex, ferrous bis-glycinate, iron(III) tartrate, iron(III) lactate, iron(III) glycinate, iron(III) EDTA, iron(III) ascorbate, and ammonium iron(III) citrate; a pharmaceutically acceptable complexing agent; and an adsorption enhancer, wherein the absorption enhancer selected from the group consisting on N-[8-(2-hydroxybenzoyl)amino]caprylic acid, sodium N-[8-(2-hydroxybenzoyl)amino]caprylate, a sodium N-[8-(2-hydroxybenzoyl)amino]caprylate derivative, a C.sub.8-20 alkanoyl carnitine, sucrose laurate, and pharmaceutically acceptable salts thereof.

2. The method of claim 1, wherein the peptide drug has a molecular weight of about 500 Da to about 4 kDa.

3. The method of claim 1, wherein the peptide drug is selected from the group consisting of GLP-1, a GLP-1 analog, an acylated GLP-1 analog, a diacylated GLP-1 analog, a long-acting albumin-binding fatty acid-derivatized GLP-1 analog, a GLP-1 agonist, semaglutide, liraglutide, exenatide, exendin-4, lixisenatide, taspoglutide, langlenatide, GLP-1(7-37), GLP-1(7-36)NH.sub.2, a dual agonist of the GLP-1 receptor and the glucagon receptor, oxyntomodulin, GLP-2, a GLP-2 agonist or analog, teduglutide, elsiglutide, amylin, an amylin analog, pramlintide, a somatostatin analog, octreotide, lanreotide, pasireotide, goserelin, buserelin, peptide YY, a peptide YY analog, glatiramer, leuprolide, desmopressin, a glycopeptide antibiotic, teicoplanin, telavancin, bleomycin, ramoplanin, decaplanin, bortezomib, cosyntropin, sermorelin, luteinizing-hormone-releasing hormone, calcitonin, calcitonin-salmon, pentagastrin, oxytocin, neseritide, enfuvirtide, eptifibatide, glucagon, viomycin, thyrotropin-releasing hormone, leucine-enkephalin, methionine-enkephalin, substance P, a parathyroid hormone fragment, teriparatide, PTH(1-31), PTH(2-34), linaclotide, carfilzomib, icatibant, cilengitide, a prostaglandin F2 receptor modulator, PDC31, and pharmaceutically acceptable salts thereof.

4. The method of claim 1, wherein said complexing agent is selected from the group consisting of mannitol, sorbitol, saccharose, sucrose, trehalose, calcium phosphate, basic calcium phosphate, calcium hydrogen phosphate, dicalcium phosphate hydrate, disodium phosphate dihydrate, an amino acid, EDTA, EGTA, citrate, a complexing peptide, glycyl-histidyl-lysine peptide, polyacrylic acid, a polyacrylic acid derivative, a carbomer, a carbomer derivative, sodium alginate, a silicate, kaolin, hydroxypropyl methylcellulose, methylcellulose, glycerol, sodium dodecyl sulfate, calcium sulfate, calcium carbonate, and pharmaceutically acceptable salts thereof.

5. The method of claim 1, wherein said absorption enhancer is sodium N-[8-(2-hydroxybenzoyl)amino]caprylate.

Description

(1) The invention is also described by the following illustrative figures. The appended figures show:

(2) FIG. 1: Pharmacokinetics of the peptide drug liraglutide after oral administration of different liraglutide formulations to Sprague Dawley rats (see Example 2).

(3) FIG. 2: Concentration-dependent inhibition of chymotrypsin by copper(II)sulfate (see Example 9). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(4) FIG. 3: Concentration-dependent inhibition of trypsin by copper(II)gluconate (see Example 10). The X-axis shows copper gluconate concentrations in the final solutions (mg/ml), the y-axis shows percentage of trypsin inhibition.

(5) FIG. 4: Concentration-dependent inhibition of trypsin by zinc(II)bisglycinate (see Example 11). The X-axis shows zinc bisglycinate concentrations in the final solutions (mg/ml), the y-axis shows percentage of trypsin inhibition.

(6) FIG. 5: Concentration-dependent inhibition of trypsin by iron(II)gluconate (see Example 12). The X-axis shows concentrations of iron gluconate in the final solutions (mg/ml), the y-axis shows percentage of trypsin inhibition.

(7) FIG. 6: Inhibition of chymotrypsin by 3 different copper salts (see Example 13). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(8) FIG. 7: Influence of copper pre-incubation on chymotrypsin activity (see Example 14). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(9) FIG. 8: Chymotrypsin inhibition by copper gluconate, EDTA, sucrose laurate and combinations thereof (see Example 17). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(10) FIG. 9: Chymotrypsin inhibition by copper gluconate, EDTA, sodium caprylate and combinations thereof (see Example 18). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(11) FIG. 10: Chymotrypsin inhibition by copper gluconate, sodium caprylate and Tween 20+/EDTA (see Example 19). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(12) FIG. 11: Trypsin inhibition by copper gluconate, copper lysinate and iron gluconate (see Example 20). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(13) FIG. 12: Trypsin inhibition by copper gluconate, lauryl-glutamate and a combination thereof (see Example 21). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(14) FIG. 13: Trypsin inhibition by iron gluconate, lauryl-glutamate and a combination thereof (see Example 22). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(15) FIG. 14: Chymotrypsin inhibition by copper gluconate, lauryl-glutamate and a combination thereof (see Example 23). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(16) FIG. 15: Chymotrypsin inhibition by iron gluconate, lauryl-glutamate and a combination thereof (see Example 24). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(17) FIG. 16: Trypsin inhibition by iron gluconate, capryl-glucoside and a combination thereof (see Example 25). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(18) FIG. 17: Trypsin inhibition by copper sulfate, copper tartrate and zinc orotate (see Example 26). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(19) FIG. 18: Chymotrypsin inhibition by copper gluconate, beta-cyclodextrin and a combination thereof (see Example 27). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(20) FIG. 19: Trypsin inhibition by copper gluconate and a combination of copper gluconate+ manganese sulfate (see Example 28). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(21) FIG. 20: Trypsin inhibition by combinations of copper gluconate+SiO.sub.2 and iron gluconate+ SiO.sub.2 (see Example 29). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(22) FIG. 21: Chymotrypsin inhibition by combinations of copper gluconate+trisodium phosphate and iron gluconate+trisodium phosphate (see Example 30). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(23) FIG. 22: Chymotrypsin inhibition by copper gluconate, EDTA and a combination thereof (see Example 31). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(24) FIG. 23: Trypsin inhibition by copper gluconate, iron gluconate and combinations thereof (see Example 33). The X-axis shows time (minutes), the y-axis shows absorbance at 405 nm.

(25) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

Example 1

In Vitro Test of the Compatibility of Different Absorption Enhancers with Different Trace Elements

(26) Solid dry powder mixtures of desmopressin acetate, zinc sulfate or iron(III) chloride, and different absorption enhancers were prepared and dissolved in 2 ml aqua purificata. Visual examination was performed to observe either a clear solution or visible precipitation. The results of these experiments are summarized in the following table:

(27) TABLE-US-00001 Trace Dissolution in element Absorption enhancer aqueous medium Peptide (5 mg/ml) (10 mg/ml) (2 ml) Desmopressin ZnSO.sub.4 Sodium caprate Precipitation Desmopressin ZnSO.sub.4 Sodium caprylate Precipitation Desmopressin ZnSO.sub.4 Lauroyl sarcosinate Precipitation Desmopressin ZnSO.sub.4 Cholic acid Precipitation Desmopressin ZnSO.sub.4 Sodium cholate Precipitation Desmopressin ZnSO.sub.4 Sodium dodecyl sulfate Clear solution Desmopressin ZnSO.sub.4 Lauroyl carnitine HCl Clear solution Desmopressin ZnSO.sub.4 Sucrose laurate Clear solution Desmopressin ZnSO.sub.4 n-Dodecyl-b-D-maltoside Clear solution Desmopressin ZnSO.sub.4 n-Octyl-b-D- Clear solution glucopyranoside Desmopressin ZnSO.sub.4 Chitosan Precipitation Desmopressin ZnSO.sub.4 Labrasol Clear solution Desmopressin ZnSO.sub.4 Citric acid Clear solution Desmopressin FeCl.sub.3 Sodium caprate Precipitation Desmopressin FeCl.sub.3 Sodium caprylate Precipitation Desmopressin FeCl.sub.3 Lauroyl sarcosinate Precipitation Desmopressin FeCl.sub.3 Cholic acid Precipitation Desmopressin FeCl.sub.3 Sodium cholate Precipitation Desmopressin FeCl.sub.3 Sodium dodecyl sulfate Clear solution Desmopressin FeCl.sub.3 Lauroyl carnitine HCl Clear solution Desmopressin FeCl.sub.3 Sucrose laurate Clear solution Desmopressin FeCl.sub.3 n-Dodecyl-b-D-maltoside Clear solution Desmopressin FeCl.sub.3 n-Octyl-b-D- Clear solution glucopyranoside Desmopressin FeCl.sub.3 Chitosan Precipitation Desmopressin FeCl.sub.3 Labrasol Clear solution

(28) The term clear solution as used in this table refers to that no clear visible precipitation or flocculation has been observed. The term clear solution also includes slightly colored clear solutions such as yellowish or orange solutions.

(29) These results show that non-ionic and zwitter-ionic absorption enhancers are compatible with di- and trivalent trace elements.

Example 2

Pharmacokinetic Profiles of Liraglutide Formulations after Intestinal Administration to Sprague Dawley Rats

(30) Liraglutide formulations comprising a trace element, a complexing agent and an absorption enhancer were dissolved in distilled water and dosed into ileum in volume of 0.4 ml/kg (final concentration 6 mg/ml) to anaesthetized rats. Blood was taken from tail vessels at the time points 0, 30, 60, 90, 120, 180 and 240 min after dosing. The liraglutide plasma concentrations were determined using commercial liraglutide kit (AB Biolabs, USA, cat. number CEK 0130-03). A formulation comprising liraglutide and sodium dodecyl sulfate (SDS) without trace element served as control (LIRA-SDS).

(31) Control:

(32) LIRA-SDS

(33) 6 mg/ml Liraglutide

(34) 20 mg/ml SDS

(35) Composition:

(36) LIRA001

(37) 6 mg/ml Liraglutide

(38) 10 mg/ml TRIS

(39) 10 mg/ml ZnSO.sub.4

(40) 20 mg/ml SDS

(41) Composition:

(42) LIRA002

(43) 6 mg/ml Liraglutide

(44) 5 mg/ml TRIS

(45) 40 mg/ml Sodium ascorbate

(46) 5 mg/ml FeCl.sub.3

(47) 20 mg/ml SDS

(48) Composition:

(49) LIRA003

(50) 6 mg/ml Liraglutide

(51) 1 mg/ml CuSO.sub.4

(52) 20 mg/ml SDS

(53) Composition:

(54) LIRA004

(55) 6 mg/ml Liraglutide

(56) 40 mg/ml Mannitol (pharma grade with <0.1% reducing sugar impurities)

(57) 5 mg/ml ZnSO.sub.4

(58) 20 mg/ml SDS

(59) 5 mg/ml TRIS

(60) The observed pharmacokinetic properties of these compositions are summarized in the following table:

(61) TABLE-US-00002 AUG.sub.(0-t) Cmax Tmax (ng/ml min) (ng/ml) (min) LIRA-SDS 1304 298 12 1 40-90 LIRA001 30720 15848 232.8 149.5 60-120 LIRA002 11796 3192 66.5 21.5 60-240 LIRA003 16575 11291 116.4 85.4 30-120 LIRA004 17430 9124 111.9 65.4 60-240

(62) Moreover, the pharmacokinetic profiles obtained for LIRA001 and LIRA002 as well as LIRA-SDS (control) are illustrated in FIG. 1.

(63) These results show that the addition of a copper salt/complex, a zinc salt/complex or an iron salt/complex to an oral GLP-1 peptide formulation according to the invention improves oral bioavailability up to 23 fold compared to control. The presence of zinc in oral GLP-1 peptide formulations leads surprisingly to high bioavailability.

Example 3

Pharmacokinetic Profiles of PTH(1-34) Formulations after Intestinal Administration to Sprague Dawley Rats

(64) Teriparatide (PTH1-34) was dosed subcutaneously in volume of 1 ml/kg (final concentration 0.024 mg/ml teriparatide) to anaesthetized rats. TER001 and TER002 were dosed into ileum in volume of 0.4 mi/kg (final concentration 0.24 mg/ml teriparatide) to anaesthetized rats. Blood was taken from tail vessels at the time points 0, 10, 20, 40, 60, 90, 120 and 180 min after dosing. The teriparatide plasma concentrations were determined using commercial pTH (1-34) human ELISA kit (Biovendor, EU, cat. number RS-1163.0001).

(65) Composition:

(66) TER001

(67) 0.38 mg/ml PTH(1-34)

(68) 30 mg/ml Lauroylcarnitine HCl

(69) 7.5 mg/ml TRIS

(70) 5 mg/ml ZnSO.sub.4

(71) 5 mg/ml Mannitol (pharma grade with <0.1% reducing sugar impurities)

(72) (Final pH=5.3)

(73) Composition:

(74) TER002

(75) 0.38 mg/ml PTH(1-34)

(76) 30 mg/ml Lauroylcarnitine HCl

(77) 30 mg/ml TRIS

(78) 5 mg/ml ZnSO.sub.4

(79) 5 mg/ml Mannitol (pharma grade with <0.1% reducing sugar impurities)

(80) (Final pH=8.3)

(81) The observed pharmacokinetic properties of these compositions are summarized in the following table:

(82) TABLE-US-00003 AUC.sub.(0-t) Cmax Tmax F Half-life (ng/ml min) (ng/ml) (min) (%) (min) PTH 136 32 2.8 0.8 10 100 23 44 8 (1-34) s.c. TER001 94 38 1.1 0.4 10-90 11 4 86 13 TER002 82 29 1.1 0.4 10-40 10 3 66 1

(83) Compositions according to the invention comprising PTH(1-34), an absorption enhancer, the trace element zinc and a complexing agent thus resulted in significant oral bioavailability and sustained pharmacokinetic profile as shown by increasing half life.

Example 4

GLP-1 Peptide Formulations with SNAC and Trace Elements

(84) Compositions comprising a GLP-1 peptide, a complexing agent (disodium phosphate dihydrate), the absorption enhancer N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC) and various trace elements or metal salts were prepared and dissolved in 2 ml of aqua purificata and examined visually. The results of these experiments are summarized in the following table:

(85) TABLE-US-00004 GLP-1 Absorption peptide enhancer Trace element Dissolution 1.8 mg/ml (20 mg) (2 mg) in aqua purificata Liraglutide SNAC ZnSO.sub.4 precipitation Liraglutide SNAC Zinc acetate precipitation Liraglutide SNAC ZnCl.sub.2 precipitation Liraglutide SNAC Zinc orotate Clear solution Liraglutide SNAC Zinc picolinate Clear solution Liraglutide SNAC Zinc carnosine Clear solution Liraglutide SNAC CuSO.sub.4 precipitation Liraglutide SNAC Copper gluconate Clear yellowish solution Liraglutide SNAC Copper orotate Clear solution Liraglutide SNAC Fe(III)chlorid precipitation Liraglutide SNAC Ferrous gluconate Clear orange solution Liraglutide SNAC Iron bisglycinate Clear brownish solution

(86) These results show a good compatibility with regard to solubility of SNAC with trace elements comprising an organic salt whereas inorganic salts result in precipitations.

Example 5

Pharmacokinetic Profiles of Liraglutide after Intestinal Administration to Sprague Dawley Rats

(87) The liraglutide formulations were dosed into ileum in volume of 0.4 ml/kg (final concentration of 6 mg/ml) to anaesthetized rats. The anaesthesia was induced with Hypnorm/Dormicum mixture. After checking of the depth of anaesthesia the animal was placed on its back and a 3-5 cm long midline incision was made in the skin of abdomen.

(88) The caecum was exposed and the distal segment of small intestine was pulled out of the abdominal cavity and the position of the spot convenient for introduction of catheter was measured using a PE tubing with mark at a distance of 5 cm. The intestine was penetrated by the catheter tip and the catheter was inserted downstream into the ileum lumen at a distance of 5 cm from caecum in a spot without feces, outside the area with accumulated lymphatic tissue and outside the blood vessels and fixed with ligature.

(89) The pulled segment of small intestine was replaced into the abdominal cavity, 2 ml of sterile saline were flushed over the intestine and the abdominal cavity was closed with metal wound clips in two layers. Blood was taken from tail vessels at the time points 0, 30, 60, 120, 180 and 240 min after dosing. The liraglutide plasma concentrations were determined using commercial liraglutide kit (AB Biolabs, USA, cat.number CEK 0130-03). The results are summarized in the table further below.

(90) Reference Formulation

(91) 6 mg/ml Liraglutide

(92) 50 mg/ml SNAC

(93) LIRA026

(94) 6 mg/ml Liraglutide

(95) 50 mg/ml SNAC

(96) 35 mg Polysorbate 20

(97) 1.9 mg/ml Copper(II)orotate

(98) 1.9 mg/ml Mannitol

(99) LIRA027

(100) 6 mg/ml Liraglutide

(101) 50 mg/ml SNAC

(102) 35 mg Polysorbate 20

(103) 2.0 mg/ml Copper(II)glycinate

(104) 2.0 mg/ml Mannitol

(105) LIRA029

(106) 6 mg/ml Liraglutide

(107) 50 mg/ml SNAG

(108) 35 mg Polysorbate 20

(109) 4.0 mg/ml Zinc(II)orotate

(110) 4.0 mg/ml Mannitol

(111) LIRA033

(112) 6 mg/ml Liraglutide

(113) 50 mg/ml SNAC

(114) 35 mg Polysorbate 20

(115) 3.5 mg/ml Zinc(II)picolinate

(116) 3.5 mg/ml Mannitol

(117) Results:

(118) TABLE-US-00005 AUC.sub.(0-180) Cmax Tmax Improvement Formulation (ng/ml min) (ng/ml) (min) ratio Reference 4907 2921 40.5 14.4 60-120 LIRA026 14276 8207 85.3 41.2 60-180 2.9-fold LIRA027 19627 15401 123.5 87.8 30-120 4.0-fold LIRA029 5717 3207 43.0 18.7 60-240 1.2-fold LIRA033 10511 4980 71.3 27.5 30-120 2.1-fold

(119) These results show that the compositions according to the present invention, containing a peptide drug such as liraglutide in combination with a copper or zinc salt/complex and a complexing agent, exhibit an advantageously increased oral bioavailablity.

Example 6

Pharmacokinetic Profiles of Liraglutide after Oral Administration to Beagle Dogs

(120) Hard capsules comprising liraglutide (10 mg/dog) were dosed orally directly on the root of the tongue. Administered capsule was washed down by 3 ml of water via a syringe to ensure that the drug is correctly swallowed and to ensure complete oesophageal clearance. Blood was taken by venepuncture from v. cephalics antebrachii at the time points 0, 15, 30, 60, 90, 120 and 180 min before and after oral dosing.

(121) 2 ml of blood were sampled into Greiner Bio-one tubes containing K3EDTA (Greiner, Austria). Blood samples were centrifuged (10 min, 3500 rpm, 4 C.) and approximately 600 l of plasma were collected. The liraglutide plasma concentrations are determined using commercial liraglutide EIA kit (Peninsula Laboratories International, USA, cat. number S-1502.0001). The formulation LIRA042 exhibited the best pharmacokinetic profile. The liraglutide plasma concentrations reaching 10-15 ng/ml appeared 60 min after dosing and persisted up to the end of the study. The PK data is summarized in the table further below.

(122) Reference Formulation

(123) HPMC capsule

(124) 10 mg Liraglutide

(125) 200 mg SNAG

(126) LIRA042

(127) HPMC capsule

(128) 10 mg Liraglutide

(129) 200 mg SNAC

(130) 200 mg Sorbitol

(131) 1 mg Copper(II)glycinate

(132) 19 mg Mannitol

(133) LIRA043

(134) HPMC capsule

(135) 10 mg Liraglutide

(136) 200 mg SNAC

(137) 200 mg Sodium citrate

(138) 1 mg Copper(II)glycinate

(139) 19 mg Mannitol

(140) LIRA045

(141) HPMC capsule

(142) 10 mg Liraglutide

(143) 200 mg SNAC

(144) 100 mg TRIS

(145) 100 mg Polysorbate 20

(146) 2 mg Copper(II)glycinate

(147) 38 mg Mannitol

(148) LIRA046

(149) HPMC capsule

(150) 10 mg Liraglutide

(151) 200 mg SNAC

(152) 100 mg TRIS

(153) 100 mg Polysorbate 20

(154) 5 mg Copper(II)glycinate

(155) 95 mg Mannitol

(156) LIRA047

(157) HPMC capsule

(158) 10 mg Liraglutide

(159) 200 mg SNAC

(160) 100 mg TRIS

(161) 100 mg Polysorbate 20

(162) 10 mg Copper(II)glycinate

(163) 190 mg Mannitol

(164) LIRA048

(165) HPMC capsule

(166) 10 mg Liraglutide

(167) 200 mg SNAC

(168) 100 mg TRIS

(169) 100 mg Polysorbate 20

(170) 10 mg Zinc(II)picolinate

(171) PK-Profile of Liraglutide after Oral Administration to Beagle Dogs:

(172) TABLE-US-00006 AUC.sub.(0-180) Cmax Tmax Improvement Formulation (ng/ml min) (ng/ml) (min) ratio Reference 46.5 1.1 60 (n = 2) LIRA042 1866 16.7 90 40-fold LIRA043 167 2.3 90 3.6-fold LIRA045 167 3.0 60 3.6-fold LIRA046 90 2.8 180 1.9-fold LIRA047 341 4.1 120 7.3-fold LIRA048 147 4.9 180 3.2-fold

(173) These results demonstrate that compositions according to the invention comprising a copper or zinc salt/complex, a complexing agent and SNAC show several fold improved absorption of a GLP-1 peptide (liraglutide) after oral administration in comparison with SNAG alone.

Example 7

In Vivo Study in Non-Human Primates with Oral PTH(1-34) Formulations

(174) Capsule formulations comprising PTH(1-34) were dosed orally to female Cynomolgus macaques (Macaca fascicularis) with a body weight of 4 to 5 kg. Blood collection for PTH(1-34) analysis was performed at the time points: 1 time pre-dose (0 min), 15 min, 30 min, 60 min, 90 min, 120 min, 180 min and 240 min post-dose with heparinized syringe. Each sample is collected from a peripheral vein. After each tube of blood is drawn, it is inverted gently several times to ensure the mixing of anticoagulant. The sample is centrifuged at between 3-5 C. for 10 minutes at 3,000 g. The teriparatide plasma concentrations are determined using commercial high sensitivity teriparatide ELISA kit (Immutopics Inc., USA, cat.number 60-3900).

(175) Reference Formulation

(176) HPMC capsule

(177) 2.5 mg PTH(1-34)

(178) 100 mg SNAC

(179) TER071

(180) HPMC capsule

(181) 2.5 mg PTH(1-34)

(182) 0.5 mg Copper(II)orotate

(183) 9.5 mg Mannitol

(184) 100 mg TRIS

(185) 100 mg SNAC

(186) TER073

(187) HPMC capsule

(188) 2.5 mg PTH(1-34)

(189) 1 mg Copper(II)orotate

(190) 19 mg Mannitol

(191) 100 mg TRIS

(192) 100 mg SNAC

(193) TER075

(194) HPMC capsule

(195) 2.5 mg PTH(1-34)

(196) 1 mg Copper(II)orotate

(197) 19 mg Mannitol

(198) 100 mg Choline chloride

(199) 100 mg SNAC

(200) TER077

(201) HPMC capsule

(202) 2.5 mg PTH(1-34)

(203) 1 mg Copper(II)glycinate

(204) 19 mg Mannitol

(205) 100 mg Choline chloride

(206) 100 mg SNAC

(207) TER084

(208) HPMC capsule

(209) 2.5 mg PTH(1-34)

(210) 3 mg Copper(II)glycinate

(211) 27 mg Mannitol

(212) 200 mg Choline chloride

(213) 200 mg SNAC

(214) TABLE-US-00007 Formulation AUC Improvement ratio Reference formulation (n = 4) 18725 TER071 (n = 3) 43256 2.3-fold TER073 (n = 1) 70650 3.8-fold TER075 (n = 1) 45975 2.5-fold TER077 (n = 1) 42547 2.3-fold TER084 (n = 1) 65250 3.5-fold

(215) These results show that the compositions according to the present invention, particularly compositions containing a peptide drug such as teriparatide (PTH(1-34)) in combination with a copper salt/complex and a complexing agent, exhibit a considerably increased absorption and, thus, an improved oral bioavailablity.

Example 8

Pharmacokinetic Profile of Octreotide Formulations after Administration into Proximal Jejunum of Sprague Dawley Rats

(216) The formulations OCT002, OCT003 and OCT004 were dissolved in an octreotide stock solution 5-10 min prior to dosing into proximal jejunum in volume of 0.4 ml/kg to anaesthetized rats. The final octreotide concentration of each formulation was 0.36 mg/kg. Blood was taken from tail vessels at the time points 0, 10, 20, 40, 60, 90 and 120 min after dosing. The octreotide plasma concentration was determined using commercial octreotide kit (Peninsula Laboratories International, Inc., USA, cat.number S-1342.0001).

(217) OCT002

(218) 0.89 mg/ml Octreotide

(219) 50 mg/ml SNAC

(220) 50 mg/ml Polysorbate 20

(221) 2.5 mg/ml Copper(II)bisglycinate

(222) 2.5 mg/ml Sorbitol

(223) OCT003

(224) 0.89 mg/ml Octreotide

(225) 100 mg/ml Sucrose laurate

(226) 2.5 mg/ml Copper(II)bisglycinate

(227) 2.5 mg/ml CuSO.sub.4 penta hydrate

(228) 5 mg/ml Sorbitol

(229) OCT004

(230) 0.89 mg/ml Octreotide

(231) 100 mg/ml SMEDDS (50 mg/ml Labrasol, 40 mg/ml Polysorbate 20 and 10 mg/ml Glycerol)

(232) 2.5 mg/ml Copper(II)bisglycinate

(233) 2.5 mg/ml CuSO.sub.4 penta hydrate

(234) Results:

(235) The formulation OCT004 had the best pharmacokinetic profile with mean Cmax 6.1 ng/ml and relative biovailability of 9.3%, while the formulation OCT003 reached relative bioavailability of 7.7% and Cmax of 4.5 ng/ml. The effect of the formulation OCT002 was lower with the relative bioavailability of 2.2%. All formulations showed low variability of pharmacokinetic parameters. The octreotide data are summarized in the following table:

(236) PK Profile of Octreotide Formulations:

(237) TABLE-US-00008 AUC.sub.(0-120) Cmax Tmax F (ng/ml min) (ng/ml) (min) (%) Reference 14126 2727 148 36 90 100 19 (s.c.) OCT002 106 65 1.2 0.6 20-40 2.2 1.3 OCT003 389 63 4.5 0.7 10-20 7.7 1.2 OCT004 472 77 6.1 1.2 10-40 9.3 1.5

(238) It has thus been demonstrated that compositions comprising a peptide drug, the trace element copper, at least one polyol as complexing agent and further comprising a permeation enhancer such as a SMEDDS formulation or a classical permeation enhancer resulted in robust oral bioavailability with low variability relative to subcutanous administration.

Example 9

Concentration Dependent Inhibition of Chymotrypsin by Copper(II)Sulfate

(239) Stock Solutions:

(240) CuSO.sub.4.5H.sub.2O dilutions in 10 mM TRIS buffer pH 7 to 10 mM, 5 mM, 2.5 mM 1.25 mM Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL BTPNA in Acetone

(241) Study: (1) 100 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (2) 80 l buffer (10 mM TRIS, pH=7)+20 l CuSO.sub.4 stock (10 mM)+50 l Chymotrypsin Stock+50 l BTPNA (3) 80 l buffer (10 mM TRIS, pH=7)+20 l CuSO.sub.4 dilut. (5 mM)+50 l Chymotrypsin Stock+50 l BTPNA (4) 80 l buffer (10 mM TRIS, pH=7)+20 l CuSO.sub.4 stock (2.5 mM)+50 l Chymotrypsin Stock+50 l BTPNA (5) 80 l buffer (10 mM TRIS, pH=7)+20 l CuSO.sub.4 dilut. (1.25 mM)+50 l Chymotrypsin Stock+50 l BTPNA

(242) Final copper concentrations: 1 mM, 0.5 mM, 0.25 mM, 0.125 mM.

(243) Absorption was measured directly at 405 nm.

(244) Values were corrected (subtraction of blank).

(245) Results:

(246) Copper sulfate inhibits the proteolytic enzyme chymotrypsin in a dose dependent manner (see also FIG. 2).

Example 10

Concentration Dependent Inhibition of Trypsin by Copper(II)Gluconate

(247) Stock Solutions:

(248) 100 mg/ml copper gluconate in 50 mM TRIS pH 7; pH was adjusted to pH 7; stock solution was diluted to the following concentrations: 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, 2.5 mg/ml, 1.25 mg/ml, 0.625 mg/ml, 0.313 mg/ml, 0.156 mg/ml and 0.078 mg/ml

(249) 0.1 mg/ml Trypsin

(250) 0.5 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA)

(251) Study:

(252) 100 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(253) 100 l Copper solution (according to the above described dilutions)+50 L Trypsin Stock+50 L BAPNA Stock

(254) Absorption was measured after 15 minutes at 405 nm.

(255) Values were corrected (subtraction of blank).

(256) Results:

(257) Copper gluconate inhibits the proteolytic enzyme trypsin in a dose dependent manner (see FIG. 3).

Example 11

Concentration Dependent Inhibition of Trypsin by Zinc(II)Bisglycinate

(258) Stock Solutions:

(259) 100 mg/ml Zinc bisglycinate in 50 mM TRIS pH 7; pH was adjusted to pH 7; stock solution was diluted to the following concentrations: 50 mg/ml and 25 mg/ml

(260) 0.1 mg/ml Trypsin

(261) 0.5 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA)

(262) Study:

(263) 100 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(264) 100 l Zinc solution (according to the above described dilutions)+50 L Trypsin Stock+50 L BAPNA Stock

(265) Absorption was measured after 15 minutes at 405 nm.

(266) Values were corrected (subtraction of blank).

(267) Results:

(268) In high concentrations, zinc bisglycinate inhibits the proteolytic enzyme trypsin in a dose dependent manner (see also FIG. 4).

Example 12

Concentration Dependent Inhibition of Trypsin by Iron(II)Gluconate

(269) Stock Solutions:

(270) 10 mg/ml iron gluconate in 50 mM TRIS pH 7; pH was adjusted to pH 7; stock solution was diluted to the following concentrations: 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml and 0.625 mg/ml

(271) 0.1 mg/ml Trypsin

(272) 0.5 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA)

(273) Study:

(274) 100 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(275) 100 l Iron solution (according to the above described dilutions)+50 L Trypsin Stock+50 L BAPNA Stock

(276) Absorption was measured directly at 405 nm.

(277) Values were corrected (subtraction of blank).

(278) Results:

(279) Iron gluconate inhibits the proteolytic enzyme trypsin in a dose dependent manner (see FIG. 5).

Example 13

Inhibition of Chymotrypsin by 3 Different Copper Salts

(280) Stock Solutions:

(281) 10 mM CuSO.sub.4 and Copper gluconate stock solution were diluted 1:1 with 10 mM TRIS buffer pH 7

(282) Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL BTPNA in Acetone

(283) Study: (1) 100 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (2) 80 l buffer (10 mM TRIS, pH=7)+20 l CuSO.sub.4 dil. (5 mM)+50 l Chymotrypsin Stock+50 l BTPNA (3) 80 l buffer (10 mM TRIS, pH=7)+20 l Copper gluconate dilut. (5 mM)+50 l Chymotrypsin Stock+50 l BTPNA (4) 100 l Cu-bisglycinate stock (0.5 mM)+50 l Chymotrypsin Stock+50 l BTPNA

(284) Final copper concentrations: 0.5 mM (CuSO.sub.4 and copper gluconate), 0.25 mM copper bisglycinate

(285) Values were corrected (subtraction of blank)

(286) Results:

(287) Three different copper salts were tested and all showed similar inhibition profiles of chymotrypsin (see FIG. 6).

Example 14

Influence of Copper Pre-Incubation on Chymotrypsin Activity

(288) Stock Solutions:

(289) 10 mM CuSO.sub.4 stock solution was diluted 1:1 with 10 mM TRIS Puffer pH 7

(290) Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL in Acetone

(291) Study: (1) 30 l buffer (10 mM TRIS, pH=7)+20 l CuSO.sub.4 dil. (5 mM)+100 l Chymotrypsin Stock: 30 min. incubation, then addition of 50 l BTPNA (2) 30 l buffer (10 mM TRIS, pH=7)+100 l Chymotrypsin Stock: 30 min. incubation, then addition of 20 l CuSO.sub.4 dil. (5 mM)+50 l BTPNA

(292) Three independent experiments with the above setup were carried out.

(293) Final copper concentration: 0.5 mM

(294) Values were corrected (subtraction of blank)

(295) Results:

(296) Pre-incubation of copper and chymotrypsin increases the chymotrypsin inhibitory activity (see FIG. 7).

Example 15

Chymotrypsin Inhibition by Iron(II)-Bisglycinate and Copper Gluconate

(297) Stock Solutions:

(298) Iron(II)-bisglycinate solution containing 1.86 mM of iron in 10 mM TRIS buffer pH 7 was prepared

(299) Cu(II)-gluconate solution containing 2.0 mM of copper in 10 mM TRIS buffer pH 7 was prepared

(300) Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL BTPNA in Acetone

(301) Study: (1) 100 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (2) 100 l iron(II)-bisglycinate+50 l Chymotrypsin Stock+50 l BTPNA (3) 100 l copper(II)-gluconate+50 l Chymotrypsin Stock+50 l BTPNA

(302) Absorption was measured directly at 405 nm.

(303) Values were corrected (subtraction of blank). Inhibition was calculated by setting the absorption value of (1) at each time-point as 100%.

(304) Results:

(305) Iron bisglycinate and copper gluconate can inhibit chymotrypsin, as reflected by the inhibition data shown in the following table.

(306) TABLE-US-00009 Metal ion Inhibition % concentration mM 0 15 30 45 60 Control 0 mg/ml 0.92 0 0 0 0 0 Fe-bisglycinate 0.06 mg/ml 0.92 0 69 62 56 52 Cu-gluconate 0.065 mg/ml 1.0 0 82 84 82 79
Minutes

Example 16

Chymotrypsin Inhibition by Iron(II)-Bisglycinate and Copper(II)-Bisglycinate

(307) Stock Solutions:

(308) Iron(II)-bisglycinate solution containing 0.46 mM of iron in 10 mM TRIS buffer pH 7 was prepared

(309) Cu(II)- bisglycinate solution containing 0.46 mM of copper in 10 mM TRIS buffer pH 7 was prepared

(310) Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL BTPNA in Acetone

(311) Study: (1) 100 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (2) 100 l iron(II)-bisglycinate+50 l Chymotrypsin Stock+50 l BTPNA (3) 100 l copper(II)-bisglycinate+50 l Chymotrypsin Stock+50 l BTPNA

(312) Absorption was measured directly at 405 nm.

(313) Values were corrected (subtraction of blank). Inhibition (%) was calculated by setting the absorption value of (1) at each time-point as 100% degradation.

(314) Results:

(315) Iron-bisglycinate and copper-bisglycinate can inhibit chymotrypsin, as reflected by the inhibition data shown in the following table.

(316) TABLE-US-00010 Metal ion Inhibition % concentration mM 0 10 20 30 Copper-bisglycinate 0.015 mg/ml 0.23 0 36 36 35 Iron-bisglycinate 0.015 mg/ml 0.23 0 35 31 24 Control 0 mg/ml 0.23 0 0 0 0

(317) Minutes

Example 17

Chymotrypsin Inhibition by Copper Gluconate, EDTA, Sucrose Laurate and Combinations thereof

(318) Stock Solutions:

(319) Sucrose laurate 4 mg/ml in 10 mM TRIS pH 7 was prepared

(320) Copper gluconate 10 mM in 10 mM TRIS buffer pH 7 was prepared

(321) EDTA 5 mM in 10 mM TRIS buffer pH 7 was prepared

(322) Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL BTPNA in Acetone

(323) 0.1 mg/ml Chymotrypsin

(324) Study: (1) 100 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (2) 50 l Sucrose laurate+50 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (3) 25 l EDTA+75 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (4) 25 l copper gluconate+75 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (5) 25 l EDTA+50 l Sucrose laurate+25 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (6) 25 l EDTA+25 l copper gluconate+50 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (7) 50 l sucrose laurate+25 l copper gluconate+25 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (8) 25 l EDTA+25 l copper gluconate+50 l sucrose laurate (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA

(325) Absorption was measured directly at 405 nm.

(326) Values were corrected (subtraction of blank). pH was confirmed to be pH 7 after the experiment.

(327) Results:

(328) Copper gluconate and EDTA as well as combinations of copper gluconate+EDTA, EDTA+sucrose laurate and combinations of copper gluconate+EDTA+sucrose laurate can inhibit chymotrypsin (see FIG. 8).

Example 18

Chymotrypsin Inhibition by Copper Gluconate, EDTA, Sodium Caprylate and Combinations thereof

(329) Stock Solutions:

(330) Sodium caprylate 4 mg/ml in 10 mM TRIS pH 7 was prepared

(331) Copper gluconate 10 mM in 10 mM TRIS buffer pH 7 was prepared

(332) EDTA 5 mM in 10 mM TRIS buffer pH 7 was prepared

(333) Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL BTPNA in Acetone

(334) 0.1 mg/ml Chymotrypsin

(335) Study: (1) 100 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (2) 50 l sodium caprylate+50 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (3) 25 l EDTA+75 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (4) 25 l copper gluconate+75 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (5) 25 l EDTA+50 l sodium caprylate+25 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (6) 25 l EDTA+25 l copper gluconate+50 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (7) 50 l sodium caprylate+25 l copper gluconate+25 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (turbid, could not be measured) (8) 25 l EDTA+25 l copper gluconate+50 l sodium caprylate (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA

(336) Absorption was measured directly at 405 nm.

(337) Values were corrected (subtraction of blank). pH was confirmed to be pH 7 after the experiment.

(338) Results:

(339) Copper gluconate and EDTA as well as combinations of copper gluconate+EDTA, EDTA+sodium caprylate (C8) and combinations of copper gluconate+EDTA+sodium caprylate (C8) can inhibit chymotrypsin (see FIG. 9).

Example 19

Chymotrypsin Inhibition by Copper Gluconate, Sodium Caprylate and Tween 20+/ EDTA

(340) Stock Solutions:

(341) 1 mg/mL Cu(II)gluconate in 10 mM TRIS pH 7 (copper content=14%), 0.14 mg/ml copper

(342) EDTA 5 mM in 10 mM TRIS buffer pH 7 was prepared

(343) Sodium caprylate (C8): 1 mg/mL in 10 mM TRIS pH 7

(344) Tween 20 (1): 1 mg/mL in 10 mM TRIS pH 7

(345) Tween 20 (2): 2 mg/mL in 10 mM TRIS pH 7

(346) 0.1 mg/ml Chymotrypsin in 10 mM TRIS pH 7

(347) Benzoyl-Tyrosine p-nitroanilide (BTPNA): 0.5 mg/mL BTPNA in Acetone

(348) Study: (1) 50 l copper stock+20 l C8+20 L Tween 20 (1)+20 L EDTA+50 L Chymotrypsin Stock+50 L BTPNA Stock (2) 50 l copper stock+20 l C8+20 L Tween 20 (2)+20 L EDTA+50 L Chymotrypsin Stock+50 L BTPNA Stock (3) 50 l copper stock+20 l C8+20 L Tween 20 (1)+20 Lbuffer+50 L Chymotrypsin Stock+50 L BTPNA Stock (4) 50 l copper stock+20 l C8+20 L Tween 20 (2)+20 Lbuffer+50 L Chymotrypsin Stock+50 L BTPNA Stock (5) 25 l EDTA+25 l copper gluconate+50 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (6) 50 l sodium caprylate+25 l copper gluconate+25 l buffer (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA (turbid, could not be measured) (7) 25 l EDTA+25 l copper gluconate+50 l sodium caprylate (10 mM TRIS, pH=7)+50 l Chymotrypsin Stock+50 l BTPNA

(349) Results:

(350) Addition of EDTA to a solution of copper gluconate, Tween 20 and sodium caprylate has a positive effect on chymotrypsin inhibition (see also FIG. 10).

Example 20

Trypsin Inhibition by Copper Gluconate, Copper Lysinate and Iron Gluconate

(351) Stock Solutions:

(352) Benzoyl-Arginine p-nitroanilide (BAPNA): 0.5 mg/ml in 50 mM TRIS pH 7 (centrifuged and supernatant used as stock solution)

(353) Trypsin: 0.2mg/mL in 50 mM TRIS pH 7

(354) 1 mg/mL iron gluconate in 10 mM TRIS pH 7 (iron content=12%), 0.12 mg/ml iron

(355) 1 mg/mL copper gluconate in 10 mM TRIS pH 7 (copper content=14%), 0.14 mg/ml copper

(356) 12 mg/mL copper lysinate in 10 mM TRIS pH 7 (copper content=3%), 0.12 mg/ml copper

(357) Solution was centrifuged (to remove the white precipitate) and the blue, clear supernatant was used for the experiments

(358) Study:

(359) 100 l of the respective salt solution+50 L Trypsin Stock+50 L BAPNA Stock

(360) Results:

(361) In the used concentrations, trypsin can be inhibited by copper(II)gluconate, iron(II)gluconate and copper(II)lysinate (see FIG. 11).

Example 21

Trypsin Inhibition by Copper Gluconate, Lauryl-Glutamate and a Combination thereof

(362) Final Concentrations:

(363) Experiments were carried out at pH 7, 50 mM TRIS.

(364) 0.025 mg/ml Trypsin, 0.125 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA.) Lauryl glutamate:

(365) 0.25 mg/ml; copper content (as copper(II)gluconate): 0.035 mg/ml.

(366) Study:

(367) 50 L lauryl-glutamate stock (or buffer)+50 L copper gluconate stock (or buffer)+50 L

(368) Trypsin Stock+50 L BAPNA Stock

(369) Adsorption measurement at 405 nm

(370) Results:

(371) Trypsin can be inhibited by copper gluconate and lauryl-glutamate; a combination of lauryl-glutamate and copper gluconate is a more potent trypsin inhibitor system than the lauryl-glutamate or copper gluconate alone (see FIG. 12).

Example 22

Trypsin Inhibition by Iron Gluconate, Lauryl-Glutamate and a Combination thereof

(372) Final Concentrations:

(373) Experiments were carried out at pH 7, 50 mM TRIS.

(374) 0.025 mg/ml Trypsin, 0.125 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA). lauryl-glutamate:

(375) 0.25 mg/ml; iron (as iron gluconate): 0.03 mg/ml.

(376) Study:

(377) 50 L lauryl-glutamate stock (or buffer)+50 L iron gluconate stock (or buffer)+50 L Trypsin Stock+50 L BAPNA Stock

(378) Absorption measurement at 405 nm

(379) Results:

(380) Trypsin can be inhibited by iron gluconate and lauryl-glutamate; a combination of lauryl-glutamate and iron gluconate is a more potent trypsin inhibitor system than the lauryl-glutamate or iron gluconate alone (see FIG. 13).

Example 23

Chymotrypsin Inhibition by Copper Gluconate, Lauryl-Glutamate and a Combination thereof

(381) Final Concentrations:

(382) Experiments were carried out at pH 7, 50 mM TRIS.

(383) 0.025 mg/ml Chymotrypsin, 0.05 mg/mL Benzoyl-Tyrosine p-nitroanilide (BTPNA). Lauryl glutamate: 0.25 mg/ml; copper (as copper gluconate): 0.035 mg/ml.

(384) Study:

(385) 50 L lauryl-glutamate stock (or buffer)+50 L copper gluconate stock (or buffer)+50 L Trypsin Stock+50 L BTPNA Stock

(386) Absorption measurement at 405 nm

(387) Results:

(388) Chymotrypsin can be inhibited by copper gluconate; a combination of lauryl-glutamate and copper gluconate is a more potent chymotrypsin inhibitor system than lauryl-glutamate or copper gluconate alone (see FIG. 14).

Example 24

Chymotrypsin Inhibition by Iron Gluconate, Lauryl-Glutamate and a Combination thereof

(389) Final Concentrations:

(390) Experiments were carried out at pH 7, 50 mM TRIS.

(391) 0.025 mg/ml Chymotrypsin, 0.05 mg/mL Benzoyl-Tyrosine p-nitroanilide (BTPNA). Lauryl glutamate: 0.25 mg/ml; iron (as iron gluconate): 0.03 mg/ml.

(392) Study:

(393) 50 L lauryl-glutamate stock (or buffer)+50 L iron gluconate stock (or buffer)+50 L Trypsin Stock+50 L BTPNA Stock

(394) Absorption measurement at 405 nm

(395) Results:

(396) Chymotrypsin can be inhibited by iron gluconate; a combination of lauryl-glutamate and iron gluconate is a more potent chymotrypsin inhibitor system than lauryl-glutamate or iron gluconate alone (see FIG. 15).

Example 25

Trypsin Inhibition by Iron Gluconate, Capryl-Glucoside and a Combination thereof

(397) Final Concentrations:

(398) Experiments were carried out at pH 7, 50 mM TRIS.

(399) 0.025 mg/ml Trypsin, 0.125 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA). Capryl-glucoside:

(400) 0.25 mg/ml; iron (as iron gluconate): 0.03 mg/ml.

(401) Study:

(402) 50 L capryl-glucoside (or buffer)+50 L iron gluconate stock (or buffer)+50 L Trypsin Stock+50 L BAPNA Stock

(403) Absorption measurement at 405 nm

(404) Results:

(405) Trypsin can be inhibited by iron gluconate and a combination of iron gluconate .sub.+capryl-glucoside (see FIG. 16).

Example 26

Trypsin Inhibition by Copper Sulfate, Copper Tartrate and Zinc Orotate

(406) Stock Solutions:

(407) Copper sulfate stock solution: 1 mg/ml of copper sulfate was dissolved in 50 mM TRIS pH 7. Copper tartrate stock solution: 1 mg/ml of copper tartrate was dispersed in 50 mM TRIS pH 7 and moderately stirred for 5 minutes. The suspension was then centrifuged at 3000 rpm for 10 minutes, to separate the insoluble salt. The supernatant was used as stock solution for the experiments.

(408) Zinc orotate stock solution: 1 mg/ml of zinc orotate was dispersed in 50 mM TRIS pH 7 and moderately stirred for 5 minutes. The suspension was then centrifuged at 3000 rpm for 10 minutes, to separate the insoluble salt. The supernatant was used as stock solution for the experiments.

(409) Study:

(410) 100 L of the respective salt solution stock+50 L Trypsin Stock+50 L BAPNA Stock

(411) Control: 100 L of 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(412) Experiments were carried out at pH 7, 50 mM TRIS.

(413) Final concentrations of protease and substrate were 0.025 mg/ml Trypsin, 0.125 mg/mL

(414) Benzoyl-Arginine p-nitroanilide (BAPNA)

(415) Absorption measurement at 405 nm

(416) Results:

(417) Trypsin can be strongly inhibited by copper sulfate. Despite their poor solubilities in water, copper tartrate and zinc orotate show trypsin inhibitory properties in vitro (see FIG. 17).

Example 27

Chymotrypsin Inhibition by Copper Gluconate, Beta-Cyclodextrin and a Combination thereof

(418) Stock Solutions:

(419) 0.1 mg/ml Chymotrypsin

(420) 1 mg/mL Benzoyl-Tyrosine p-nitroanilide (BTPNA)

(421) 1mg/m1 Beta-cyclodextrin

(422) 2.5 mM copper(II)gluconate

(423) Study:

(424) 100 l 50 mM TRIS pH 7+50 L Chymotrypsin Stock+50 L BTPNA Stock

(425) 50 l Beta-cyclodextrin+50 l 50 mM TRIS pH 7+50 L Chymotrypsin Stock+50 L BTPNA Stock

(426) 50 l copper gluconate+50 l 50 mM TRIS pH 7+50 L Chymotrypsin Stock+50 L BTPNA Stock

(427) 50 l copper gluconate+50 l Beta-cyclodextrin+50 L Chymotrypsin Stock+50 L BTPNA Stock

(428) Results:

(429) Chymotrypsin can be inhibited by copper gluconate and a combination of copper gluconate+beta-cyclodextrin (see FIG. 18).

Example 28

Trypsin Inhibition by Copper Gluconate and a Combination of Copper Gluconate+Manganese Sulfate

(430) Stock Solutions:

(431) 0.1 mg/ml Trypsin

(432) 0.5 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA)

(433) 1 mg/ml copper(II)gluconate

(434) 1 mg/ml manganese(II)sulfate

(435) Study:

(436) Experiments were carried out at pH 7 in 50 mM TRIS buffer.

(437) Final concentrations of protease and substrate were 0.25 mg/ml Trypsin, 0.125 mg/mL BAPNA.

(438) 100 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(439) 50 l copper gluconate+50 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(440) 50 l manganese sulfate+50 l copper gluconate+50 L Trypsin Stock+50 L BAPNA Stock

(441) Absorption measurement at 405 nm; final pH in all solutions was monitored to be pH 7.

(442) Results:

(443) Trypsin can be inhibited by copper gluconate and a combination of copper gluconate and manganese sulfate (see FIG. 19).

Example 29

Trypsin Inhibition by Combinations of Copper Gluconate+SiO.SUB.2 .and Iron Gluconate+SiO.SUB.2

(444) Stock Solutions:

(445) 0.1 mg/ml Trypsin

(446) 0.5 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA)

(447) 3 mg/ml SiO.sub.2suspended in 50 mM TRIS pH 7 and centrifuged; supernatant was used for experiments.

(448) 1 mg/ml copper(II)gluconate

(449) 1 mg/ml iron(II)gluconate

(450) Study:

(451) 100 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(452) 50 l SiO.sub.2+50 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(453) 50 l copper gluconate+50 l SiO.sub.2+50 L Trypsin Stock+50 L BAPNA Stock

(454) 50 l iron gluconate+50 l SiO.sub.2+50 L Trypsin Stock+50 L BAPNA Stock

(455) Absorption measurement at 405 nm; final pH in all solutions was monitored to be pH 7.

(456) Experiments were carried out at pH 7 in 50 mM TRIS buffer.

(457) Results:

(458) Trypsin can be inhibited by combinations of copper gluconate+SiO.sub.2 and iron gluconate+SiO.sub.2 (see FIG. 20).

Example 30

Chymotrypsin Inhibition by Combinations of Copper Gluconate+Trisodium Phosphate and Iron Gluconate+Trisodium Phosphate

(459) Stock Solutions:

(460) 0.1 mg/ml Chymotrypsin

(461) 0.5 mg/mL Benzoyl-Tyrosine p-nitroanilide (BTPNA) in Acetone

(462) 2.5 mg/ml trisodium phosphate

(463) 1 mg/ml copper(II)gluconate

(464) 1 mg/ml iron(II)gluconate

(465) Study:

(466) 100 l 50 mM TRIS pH 7+50 L Chymotrypsin Stock+50 L BTPNA Stock

(467) 50 l copper gluconate+50 l trisodium phosphate+50 L Chymotrypsin Stock+50 L BTPNA Stock

(468) 50 l iron gluconate+50 l trisodium phosphate+50 L Chymotrypsin Stock+50 L BTPNA Stock

(469) Experiments were carried out at pH 7 in 50 mM TRIS buffer.

(470) Absorption measurement at 405 nm; final pH in the control solution was monitored to be pH 7;

(471) pH in all other solutions was between 7 and 7.5.

(472) Results:

(473) Chymotrypsin can be inhibited by combinations of copper gluconate+trisodium phosphate and iron gluconate+trisodium phosphate (see FIG. 21).

Example 31

Chymotrypsin Inhibition by Copper Gluconate, EDTA and a Combination thereof

(474) Stock Solutions:

(475) 0.1 mg/ml Chymotrypsin

(476) 0.5 mg/mL Benzoyl-Tyrosine p-nitroanilide (BTPNA) in Acetone

(477) 2.5 mM copper(II)gluconate

(478) 1.25 mM EDTA

(479) Study:

(480) 100 l 50 mM TRIS pH 7+50 L Chymotrypsin Stock+50 L BTPNA Stock

(481) 50 l copper gluconate+50 l 10 mM TRIS pH 7+50 L Chymotrypsin Stock+50 L BTPNA Stock

(482) 50 l EDTA+50 l 10 mM TRIS pH 7+50 L Chymotrypsin Stock+50 L BTPNA Stock

(483) 50 l copper gluconate+50 l EDTA+50 L Chymotrypsin Stock+50 L BTPNA Stock

(484) Experiments were carried out at pH 7 in 10 mM TRIS buffer.

(485) Adsorption measurement at 405 nm; final pH in the control solution was monitored to be pH 7.

(486) Results:

(487) Chymotrypsin can be inhibited by EDTA and copper gluconate. A combination of copper gluconate and EDTA is a more potent inhibitor system than EDTA or copper gluconate only (see FIG. 22).

Example 32

Solubility of Cu(II)-Bisglycinate in the Presence of Sorbitol

(488) Stock Solutions:

(489) A solution of 80 mg/mL Sorbitol in Aqua dest. was prepared

(490) A supersaturated solution of Cu(II)-bisglycinate was prepared by adding 24 mg of copper salt to 500 l of Aqua dest. (1) To 250 l of the supersaturated copper solution, 250 l of Aqua dest. was added (2) To 250 l of the supersaturated copper solution, 250 l of the Sorbitol solution was added

(491) Study:

(492) Both dispersions (1) and (2) were centrifuged at room temperature at 2'000 rpm for 5 minutes. Then the absorption of the supernatant was measured at 450 nm. Blank values of Aqua dest. and the Sorbitol solution were the same and were subtracted from the measured values. Calibration curve with copper in Aqua dest. and copper in Sorbitol solution were prepared, demonstrating linear behaviour.

(493) Results:

(494) The solubility of copper-bisglycinate in the presence of sorbitol increased (+50%) in comparison to the solubility of copper-bisglycinate in Aqua dest.

Example 33

Trypsin Inhibition by Copper Gluconate, Iron Gluconate and Combinations thereof

(495) Stock Solutions:

(496) 0.1 mg/ml Trypsin

(497) 0.5 mg/mL Benzoyl-Arginine p-nitroanilide (BAPNA)

(498) 1 mg/ml copper(II)gluconate

(499) 1 mg/ml iron(II)gluconate

(500) Study:

(501) 120 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(502) 80 l copper gluconate+40 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(503) 40 l copper gluconate+80 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(504) 80 l iron gluconate+40 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(505) 40 l iron gluconate+80 l 50 mM TRIS pH 7+50 L Trypsin Stock+50 L BAPNA Stock

(506) 80 l iron gluconate+40 l copper gluconate+50 L Trypsin Stock+50 L BAPNA Stock

(507) 40 l iron gluconate+80 l copper gluconate+50 L Trypsin Stock+50 L BAPNA Stock

(508) Experiments were carried out at pH 7 in 50 mM TRIS buffer.

(509) Absorption measurement at 405 nm

(510) Results:

(511) Trypsin can be inhibited by copper gluconate, iron gluconate and any combination thereof. Combinations of iron gluconate and copper gluconate are the most potent inhibitor systems (see FIG. 23).

Example 34

Pharmacokinetic Profile of Teriparatide Formulations after Administration into Proximal Jejunum

(512) Teriparatide formulations were dosed into proximal jejunum in volume of 0.4 ml/kg (final concentration 0.42 mg/ml teriparatide) to anaesthetized rats. Blood was taken from tail vessels at the time points 0, 10, 20, 40, 60, 90, 120 and 180 min after dosing. The teriparatide plasma concentrations were determined using commercial high sensitivity teriparatide ELISA kit (Immutopics Inc., USA, cat.number 60-3900).

(513) TER092

(514) 0.42 mg/ml Teriparatide

(515) 60 mg/ml Sucrose laurate

(516) 5 mg/ml Copper(II)gluconate

(517) (pH of final formulation=4.4)

(518) TER093

(519) 0.42 mg/ml Teriparatide

(520) 60 mg/ml Sucrose laurate

(521) 5 mg/ml Iron(11)gluconate

(522) (pH of final formulation=4.5)

(523) TER095

(524) 0.42 mg/ml Teriparatide

(525) 40 mg/ml SNAG

(526) 20 mg/ml SDS

(527) 2.5 mg/ml EDTA

(528) 5 mg/ml Copper(II)glycinate

(529) (pH of final formulation=7.0)

(530) Results: The formulation TER095 was rapidly absorbed and exhibited the highest Cmax and AUC. The formulations TER092 and TER093 were more slowly absorbed and had longer elimination half-life. The results are shown in the following table:

(531) Pharmacokinetic Parameters:

(532) TABLE-US-00011 AUC.sub.(0-180) (ng/ml min) C.sub.max (ng/ml) Half-life (min) TER092 17898 8328 0.173 0.085 71.4 13.6 TER093 15327 11588 0.174 0.119 61.4 26.2 TER095 48307 16335 0.670 0.164 56.9 9.6

Example 35

Leuprolide Acetate Formulations for Oral administration

(533) LEU007

(534) HPMC capsule

(535) 3.5 mg Leuprolide acetate

(536) 300 mg Sodium caprylate

(537) 200 mg Sodium citrate

(538) 10 mg Copper(II)gluconate

(539) LEU008

(540) AR capsule (acid resistant capsule)

(541) 3.5 mg Leuprolide

(542) 100 mg Sodium dodecyl sulfate

(543) 100 mg Mannitol

(544) 10 mg Copper(II)gluconate

(545) 5 mg EDTA