Definitions

• GLP-1 is known to gel and aggregate under numerous conditions, making it difficult to make stable soluble peptide formulations. See EP 0978565 Al .
• a variety of pharmaceutical formulations comprising GLP-1, PTH and GRF have been described in the art.
• Such peptides have generally been administered by dissolving the peptide in water containing albumin or other adjuvants and injecting it into a human (Creutzfeldt et al.
• PCT Application WO 98/19698 describes a combination of 100 nmol GLP-1 (7-36)amide and 0.025 mL human albumin solution (20%), with the pH adjusted to 4 using 5 M acetic acid. The volume of this formulation was brought to 1 mL using normal saline for administration to the abdomen of a human making the concentration of GLP-1 100 ⁇ M (about 0.3 mg/mL). However, as noted above, it is desirable to not use albumin in pharmaceutical formulations.
• the 1999 Physician's Desk Reference (pp. 532-539) describes NEUPOGEN, commercially available from Amgen Inc., California.
• NEUPOGEN is the name of the drug product that is a formulation of filgrastim, a human granuloctye colony stimulating factor (G-CSF) produced by recombinant DNA technology, suitable for pharmaceutical use in stimulating white blood cell production.
• G-CSF human granuloctye colony stimulating factor
• the PDR entry states that NEUPOGEN is formulated in a 10 mM sodium acetate buffer at pH 4.0, containing 5% sorbitol and 0.004% TWEEN 80.
• TWEEN 80 is an emulsifying, wetting, and dispersing agent (i.e. , detergent), commercially available from Atlas Powder Company, Delaware.
• the PDR entry further states that the quantitative composition (per mL) of NEUPOGEN is: filgrastim 300 meg., acetate 0.59 mg, sorbitol 50 mg, TWEEN 80 0.004 %, sodium 0.035 mg, water for injection USP q.s. in 1.0 mL.
• G-CSF is a protein that is 175 amino acids long, and, as noted, the NEUPOGEN formulation contains detergent. Accordingly, there is a need in the art for stable pharmaceutical formulations of relatively small peptides, such as GLP-1, PTH and GRF, that contain minimal levels of non-therapeutic adjuvants (such as albumin, detergents, and solvents) because this can cause adverse side effects.
• a unit dose of a pharmaceutical composition for administration to a mammal includes a therapeutically effective amount of a peptide; the composition also includes a buffer comprising an acid having a pKa less than about 5, or less than 5.
• the inventive formulations comprise acetic acid.
• the formulations also include a diluent to make the composition isotonic.
• the inventive formulations comprise D-mannitol.
• the composition consists essentially of a peptide, a buffer comprising an acid having a pKa less than about 5, or less than 5, and a diluent such as D- mannitol.
• the composition consists of peptide, a buffer comprising an acid having a pKa less than about 5 or less than 5, and a diluent.
• the inventive formulations comprise a peptide, acetic acid, and D-mannitol.
• the inventive formulations consist essentially of a peptide, acetic acid, and D-mannitol.
• the unit dose includes a therapeutically effective amount of a peptide having a molecular weight of between about 200 to 50,000 atomic mass units, including, for example, a GLP-1 molecule, a GRF molecule, or a PTH molecule.
• a method for the treatment of a disease in a mammal having the disease includes administering to the mammal an effective amount of a pharmaceutical composition of the invention. Further objects include the following.
• a pharmaceutical composition comprising (1) a molecule selected from the group consisting of a GLP1 molecule, and GRF molecule, and a PTH molecule; (2) an acid having a dissociation constant value of greater than lxlO "5 ; and (3) an excipient, wherein the pH of the composition is between about 3.0 and 5.0.
• the above composition, wherein the acid comprises acetic acid.
• the above composition, wherein the excipient is D-mannitol.
• the above composition wherein the acid is acetic acid and the excipient is D-mannitol.
• the above composition, wherein the composition comprises GLP-1 (7-36)amide.
• the above composition, wherein the composition comprises GRF(l-44)amide.
• composition wherein the composition comprises PTH(l-34)OH.
• composition wherein the composition is in unit dosage form.
• composition wherein the composition is sterile.
• a system for administering a pharmaceutical composition comprising: an infusion pump for administering a unit dose of the above composition.
• the above system, wherein the composition is diluted up to about 40-fold with isotonic saline prior to administration.
• a method for the treatment of a disease or condition in a mammal comprising administering to the mammal a pharmaceutically effective amount of an above composition.
• composition is administered to the mammal by a method selected from the group consisting of intravenous, subcutaneous, continuous, intermittent, parenteral, and combinations thereof.
• the above composition wherein the composition has a pH of about 4.5.
• the above composition wherein the composition has a pH of about 4.7.
• the above composition wherein the composition has a pH of between about 4.5 and 4.7.
• the above composition wherein the composition has a pH of 4.5.
• composition, wherein the composition has a pH of 4.7.
• composition consisting essentially of acetic acid, D-mannitol, and a molecule selected from the group consisting of a GLP1 molecule, and GRF molecule, and a PTH molecule, wherein the composition is in liquid form.
• composition consisting of acetic acid, D-mannitol, and a molecule selected from the group consisting of a GLP1 molecule, and GRF molecule and a PTH molecule, wherein the composition is in liquid form.
• the above composition comprising acetate (about 10 mM) and D-mannitol (about 50.7 mg/mL).
• the above composition consisting essentially of acetate (about 10 mM), D-mannitol (about 50.7 mg/mL), and a molecule selected from the group consisting of a GLP1 molecule, and GRF molecule, and a PTH molecule.
• the above composition comprising acetate (about 10 mM), D-mannitol (about 50.7 mg/mL), and GLP-1 (7-36)amide (about 1 mg/mL).
• the above composition consisting essentially of acetate (about 10 mM), D-mannitol (about 50.7 mg/mL), and GLP-l(7-36)amide (about 1 mg/mL).
• composition wherein the composition comprises acetate (about 10 mM), D-mannitol (about 50.7 mg/mL), and GRF(l-44)amide (about 4 mg/ml).
• the above composition consisting essentially of acetate (about 10 mM), D-mannitol (about 50.7 mg/mL), and GRF(l-44)amide (about 4 mg/ml).
• the above composition wherein the composition comprises acetate (about 10 mM), D-mannitol (about 50.7 mg/mL), and PTH(l-34)OH (about 50 mg/mL).
• composition wherein the composition consists essentially of acetate (about 10 mM), D-mannitol (about 50.7 mg/mL), and PTH(l-34)OH (about 50 mg/mL).
• the pump is programmed to release the molecule at a rate of about 10 or more ⁇ L per hour.
• FIG. 1 Examples of the use of reverse phase HPLC for peptide purity analysis and illustrating the capacity to monitor the degradation of peptides.
• Samples were analyzed by reversed phase HPLC by elution with water/acetonitrile gradients in 0.1 %> trifluoroacetic acid.
• the HPLC system used was an HP 1100 chromatography system.
• Top Panel GLP-1 stored at -20° C (dotted line) and 50° C (solid line) for one month in 10 mM acetic acid, 5.07%> D-mannitol, adjusted to pH 4.5. Elution is with a gradient of from 33%> to 95% acetonitrile in 22 min. with a Waters Symmetry Reverse Phase C18 column, 4.6x250 mm.
• Bottom panel GRF stored at - 20° C (dotted line) and 37° C (solid line) for one month in 10 mM acetic acid, 5.07% D- mannitol, adjusted to pH 4.7.
• the compositions of the HPLC buffers A and B were 20% and 50%(v/v) acetonitrile, respectively, and elutzon was with a gradient of f om 25% to 55% B in 25 min. 5 using a Zorbax 5 micron, 4.6x250mm column.
• Drawing 8 Total GRF detected in the plasma of a rat following intravenous administration of 20 ⁇ g of GRF in the preferred formulation (4 mg/mL GRF dissolved in 10 mM sodium acetate, 5.07% D-mannitol, adjusted to pH 4.7).
• a peptide may have a molecular weight of between about 200 to 50,000 atomic mass units.
• the peptide is a GLP-1 molecule, a PTH molecule, a GRF molecule, or a combination thereof.
• the peptide may be a derivative or an analog of GLP-1, PTH, GRF, or a combination thereof.
• the peptide is GLP-1 (7-36)amide, PTH(1 -34)OH, or GRF( 1 -44)amide.
• the peptide concentration(s) (whether GLP-1, PTH, GRF, or combinations thereof) of the formulations are preferably in the range of about 25 ⁇ g to 5 mg per 1 mL of the combination of buffer and diluent.
• the peptide is a glucagon- like peptide- 1 (7-36)amide.
• This molecule is a natural incretin hormone secreted from the L-cells of the ileum. It assists in the regulation of insulin secreatory rates and has a profound effect on glucose homeostasis.
• GLP-1 also acts systemically to suppress free fatty acids and to facilitate normalization of blood glucose levels tlirough a large number of endocrine functions, including the control and expression of insulin from the pancreatic ⁇ -cells, and the suppression of glucagon.
• GLP-1 molecule as used in the context of the present invention includes glucagon-like peptides, analogs of glucagon-like peptide- 1, and derivatives of glucagon-like peptide-1, that bind to glucagon-like peptide-1 receptor proteins.
• GLP-1 (7-36)amide (Seq. 1): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH 2 -
• GLP-1 derivatives an analog of GLP-1 may be used such as the GLP-1 derivatives:
• GLP-l(7-36)OH (Seq. 2): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Gm-Phe-Ile-Ala-Trp-Leu-Val-Lys-Giy-Arg-OH Sequence of GLP-1 (7-34)OH (Seq.3): His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys- Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-OH
• GLP-1 analogs are known in the art.
• U.S. Pat. No.5,958,409 describes suitable GLP-1 analogs.
• the peptide may be a GLP-1 derivative such as alkylated or acylated GLP-1 derivatives or other analogs.
• Analogs of GLP-1 that are homologous, including the exendins, such as exendin 3 and 4, and GLP-2, are also included in the invention.
• the GLP-1 molecule is GLP-1 (7-36)amide, having the amino acid sequence Seq 1.
• a factor that may play a role in the stability of the GLP-1 formulations is the concentration of the GLP-1 molecule.
• the solubility profile as a function of pH of GLP-1 is shown in Drawing 2.
• pH values below about 5.0 the solubility of GLP-1 in 10 mM sodium acetate, 5.07% D-mannitol is generally above 1 mg/mL, allowing effective doses for s.c. and i.v. injections.
• the present inventors have determined that a GLP-1 (7-36)amide concentration of about 1 mg/mL was stable in the inventive formulations at pH 4.5, for up to 6 months at 25°C with ⁇ 4%> degradation. This stability was evidenced by the minimal amount of breakdown products (e.g., acid cleavage and beta shifts at aspartic acid) over time determined by HPLC methods. See Drawing 4.
• a particularly stable formulation includes about 0.1 to 4 mg/mL of a GLP-1 molecule.
• GLP-1 molecules also included in “GLP-1 molecules” of the present invention are six peptides in Gila monster venoms that are homologous to GLP1. Their sequences are compared to the sequence of GLP1 in the following table. TABLE Position 1 a. HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (NH2) b. HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(NH2) c. DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH2) d. HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (NH2) e.
• c exendin 4(9-39)(NH 2 ).
• d exendin 4.
• e helospectin I.
• f helospectin II.
• g helodermin.
• h Q8, Q9 helodermin.
• exendins 3 and 4 are identical in 15 positions and equivalent in 5 additional positions. The only positions where major structural changes are evident are at residues 16, 17, 19, 21, 24, 27, 28 and 30. Exendins also have 9 extra residues at the carboxyl terminus.
• the peptide is a PTH molecule.
• PTH molecule includes parathyroid hormones, analogs of parathyroid hormones, and derivatives of parathyroid hormones. PTHs are regulatory factors in the homeostatic control of calcium and phosphate metabolism. The principal sites of PTH activity are believed to be the skeleton, kidneys, and gastrointestinal tract.
• the peptide is GRF(l-44)amide (GRF).
• GRF is a peptide of 44 amino acids.
• GRF is one of a group of peptides secreted by the hypothalamus, and is believed to stimulate pituitary growth hormone release. GRF may be important in normal growth and development during childhood, and may mediate (together with somatostatin) the neuroregulation of GH secretion.
• GRF is an attractive molecule for the treatment of postmenopausal osteoporosis, and other indications because it is relatively small, and therefore can be effective when given by nasal insufflation using an appropriate vehicle.
• GRF molecule as used in the context of the present invention includes growth hormone releasing factor, analogs of growth hormone releasing factor, and derivatives of growth hormone releasing factor, that bind to a growth hormone releasing factor receptor protein. Sequence of GRF(l-44) amide (Seq.
• an analog of GRF may be used.
• GRF analogs that have biological activity are known in the art and generally contain about 27 to about 44 amino acids, but such analogs may be somewhat less potent than GRF.
• Kubiak et al, J. Med. Chem. 36, 888 (1993) describes suitable GRF analogs.
• GRF analogs examples include GRF(l-44)-OH, GRF(1-40)-OH, GRF(l-40)- NH 2j GRF(l-32)-NH 2j GRF(l-39)-NH 2 , GRF(l-40)-Phe-NH 2 , GRF(1-40)-Phe-OH, GRF(l-40)- Phe-Gln-NH 2 , GRF(l-29)-NH 2 , and GRF(l-27)-NH 2 , and combinations thereof.
• the peptide may be a GRF derivative such as detailed by Kubiak et al. above.
• the peptide is GRF (1-44) amide having the amino acid sequence of Seq. 6.
• a particularly stable formulation for GRF includes about 1.0 to 10.0 mg/mL of GRF.
• the buffer of the formulations should have a pH that is slightly acidic. Without intending to be limited by any particular theory, it is known to those skilled in the art that acidic conditions increase the stability of the amide bond of the peptide. Acidic conditions are provided by a generally weak acid. An acid is a generally weak acid if it has an acid dissociation constant value of greater than about lxlO "5 , or greater than lxlO "5 , i.e., a pKa ⁇ about 5, or a pKa ⁇ 5. Such acids may include propionic, succinic, malic acids, and combinations thereof. According to a particularly preferred embodiment, the acid is acetic acid.
• the acid may have an acid dissociation constant value greater than about lxlO "5 , or greater than lxlO "5 , (such as proprionic or lactic acids).
• the buffer may have buffering capabilities and may be selected from the group consisting of acetates, borates, phosphates, phthalates, carbonates, and combinations thereof.
• the buffer is included in a solution including the peptide and excipient to establish a pH in the range of about 3.0 to about 5.0. It is well known in the art that pH can be adjusted to a desired range using well known reagents, such as weak acids, as described herein, and strong bases, such as sodium or potassium hydroxide.
• the pH of the buffer is in the range of 3.0 to 5.0. In more preferred embodiments, the pH of the buffer is in the range of about 4.0 to about 5.0 or 4.0 to 5.0. In more particularly preferred embodiments, the pH of the buffer is in the range of about 4.5 to about 5.0 or 4.5 to 5.0. In a most preferred embodiment, the pH of the buffer is in the range of about 4.5 to about 4.7 or 4.5 to 4.7. In yet other most preferred embodiments, the pH of the buffer is 4.5, 4.6 or 4.7.
• the buffer preferably has a molarity of between about 1 mM and 20 mM, more preferably in the range of between about 5 and 10 mM.
• Isotonic excipient The excipient assists in rendering the formulations approximately isotonic with body fluid (depending on the mode of administration).
• concentration of the excipient is selected in accordance with the known concentration of a tonicity modifier in a peptide formulation.
• Preferred excipients include saccharides, such as lactose or D-trehalose having a chemical composition of C 12 H 22 O 1 ⁇ .
• a particularly preferred excipient (also sometimes referred to as a "diluent" in this context) in the present invention is D-mannitol, having a chemical composition of C 6 H 1 O 6 .
• Other preferred excipients include alcohols having a Ci to C 12 chain.
• compositions of the present invention are novel peptide formulations that are well- suited for clinical therapeutic administration, because (1) they may be sterilized, (2) may have controlled tonicity, (3) may be pH-adjusted, and (4) are compatible with administration in a variety of ways.
• An unexpected property of embodiments of the inventive formulations is that despite their relatively low pH, they produce little or no adverse side effects in patients, when administered parenterally.
• both subcutaneous and intravenous injections of the peptides produce biological responses indicative of their function.
• the inventors of the present invention have found that an acceptable solubility of the peptide in the formulations is possible at a low pH range.
• At least about 2 mg of GLP-1, at least about 4 mg PTH, or at least about 10 mg of GRF peptide is soluble in about 1 mL of the buffer and the excipient combined, when the formulation has a pH in the range of about 4.0 to 5.0, or 4.0 to 5.0.
• inventive formulations preferably are substantially free of agents such as detergents, solvents, or other adjuvants or excipients, that would be required for adequate peptide solubility at higher pH values.
• the inventive formulations comprise acetic acid, D-mannitol, and a molecule selected from the group consisting of a GLP-1 molecule, a GRF molecule, and a PTH molecule, and have a pH of about 4.5 to about 4.7, or 4.5 to 4.7.
• the inventive formulations consist essentially of acetic acid, D-mannitol, and a molecule selected from the group consisting of a GLP-1 molecule, a GRF molecule, and a PTH molecule and have a pH of about 4.5 to about 4.7, or 4.5 to 4.7.
• the inventive formulations consist of acetic acid, D-mannitol, and a molecule selected from the group consisting of a GLP-1 molecule, a GRF molecule or a PTH molecule and have a pH of about 4.5 to about 4.7 or 4.5 to 4.7.
• the inventive formulations have apH of about 4.5, a pH of about 4.6, apH of about 4.7, a pH of 4.5, a pH of 4.6, or a pH of 4.7.
• a pH range of between about 4.0 to 5.0 has not presented problems with precipitation at the site of injection, even though the peptide may be rather insoluble at physiological pH.
• Test results show that blood glucose falls to euglycemic levels within 10 minutes of injection of GLP- 1 in a human subject, which indicates that generally none of the peptide precipitated at the site of injection.
• GLP-1 or GRF formulations were injected subcutaneously in the amount of about 1 mL into humans, they produced no apparent discomfort at the injection site and produced a rapid response, as assessed by the level of peptide drug appearing in the blood.
• the formulations of the present invention are surprisingly stable even when injected in a human subject.
• the biological half-life of peptide molecules is quite short. For example, the biological half-life of GLP-l(7-37) in blood is 3 to 5 minutes, according to U.S. Patent No. 5,118,666.
• inventive formulations results from a combination of the identity and pH of the buffer and the stabilizing effect of the excipient (e.g. , D-mannitol).
• excipient e.g. , D-mannitol
• the inventors of the present invention have developed HPLC methods capable of quantifying the degree of degradation of the peptide (See Drawing 1).
• the formulations of the present invention comprising GLP-1 were used in human patients in clinical trials and caused few adverse effects, hi excess of 10,000 vials of such formulations have been stable for at least a period of 9 months at -20°C, 4°C, and 25°C.
• the formulations of the present invention where the peptide is GRF or PTH also exhibit comparable stability (See Drawings 3, 5).
• a formulation of 1 mg/mL GLP-1 in 10 mM acetate, 5.07% (w/v) D-mannitol, and pH 4.5 showed a stability of at least 98% over 28 days at 25°C; at least 92%> over 28 days at 37°C, and at least 66% over 28 days at 50°C.
• this GLP-1 formulation showed no change in purity when stored for one month at 4°C or -20°C.
• An additional stability study showed at least 90% stability of GLP-1 in this formulation over 18 months at 4°C and 6 months at 25°C.
• Formulations of PTH(l-34) at 0.1, 1.0 and 10.0 mg/mL, pH 4.7, 5.07% D-mannitol, 10 mM acetate were highly stable, at least about 98% over 14 days at temperatures from -20°C to 25°C.
• PTH(l-34) was shown to be at least 90% stable for more than 6 months at -20°C and 5°C, and for three months at 25°C.
• GRF formulations at 4, 8, and 10 mg/mL, pH 4.7, 5.07% D-mannitol, 10 mM acetate, at temperatures from -20°C to 4°C showed a stability of at least 98% over 14 days, at least 96% at 25°C and 63% at 50°C. Additional formulations tested for extended periods of time showed stability of at least 90% for 12 months at 4°C, and 4-6 weeks at 25°C. Therefore, the formulations of the present invention include peptides that are very stable and storable, probably for years at -20°C. Also, their decomposition at higher temperatures yields fragments that have been identified and are predictable. There has been no detectable dimerization or aggregation of these formulations.
• the peptides of the present invention may be prepared by methods as are generally known in the art of peptide preparation.
• the peptides can be prepared by solid-state chemical peptide synthesis or by conventional recombinant techniques.
• recombinant means that a desired peptide or protein is derived from recombinant (e.g. , microbial or mammalian) expression systems.
• the peptides can be recovered and purified from recombinant cell cultures by methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.
• High performance liquid chromatography (HPLC) can be employed for final purification steps.
• the formulations of the present invention have a variety of uses for treating disease and illness in mammals.
• the skilled artisan will recognize that the present inventive formulations can be used for any disease or condition that requires parenteral administration of a GLP-1 molecule, a GRF molecule, or a PTH molecule.
• the formulations including GLP-1 may be useful for treating diabetes, excess appetite, and obesity.
• the formulations including PTH may be useful for treating bone growth deficiency and osteoporosis.
• the formulations including GRF may be useful for treating osteoporosis and wasting; patients who have been injected with formulations of the present invention have had minimal or no irritation at all upon injection and have experienced a growth hormone response, which indicates that the peptide gets into the circulation.
• the formulations of the present invention are preferably administered in unit dosage form.
• the formulations are subdivided into unit doses containing appropriate quantities of the peptide.
• the unit dose can be a packaged preparation, the package containing discrete quantities of peptide, such as liquid containing solubilized peptide in vials or ampoules, packeted tablets, capsules, and powders in vials or ampoules.
• the determination of the proper dose for a particular situation is within the skill of the art. In general, treatment is initiated with smaller doses, which are less than the optimum dose of the preparation. Thereafter, the dose is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dose may be divided and administered in portions during the day, if desired.
• a typical unit dose of a formulation including GLP-1 is about 0.1 to 2 mg or 0.1 to 2 mg, about 10 to 50 ⁇ g for a formulation including PTH, and about 1 to 8 mg or 1 to 8 mg for a formulation including GRF, though doses above and below these amounts may have application.
• the doses are liquid formulations of about 1 mg/mL of GLP-1, about 50 ⁇ g /mL of PTH, or 50 ⁇ g /mL, and about 1 to 4 mg/mL of GRF or 1 to 4 mg/mL, each dose is made up in standard 3 mL vials and filled at a commercial facility (e.g., SP Pharmaceuticals in New Mexico).
• the formulations of the present invention are primarily intended for administration to a human subject, but may also be administered to other mammalian subjects, such as dogs and cats (e.g., for veterinary purposes).
• the formulations can also be preferably administered for continuous subcutaneous delivery using, for example, a MiniMed® programmable medication infusion pump commercially available from Pacesetter Systems, Inc., of California. In vitro and in vivo studies show minimal adsorption of the formulations to components of the MiniMed pump.
• the formulations in the preferred embodiment can be diluted up to 40-fold with isotonic saline and delivered by pump, such as the Harvard pump, Harvard Apparatus , MA, without loss of biological activity nor adsorption of peptide.
• the formulations may be delivered by other means, including subcutaneous or micropressure injection, external or implant pump, depot injection, and other prolonged-application dispensing devices.
• a syringe can be used that comprises an inventive formulation of the present application.
• Such a syringe can be used for self-administration of a GLP-1 molecule.
• Such syringes are well known in the art. See, e.g., U.S. Pat. Nos. 5,980,491 and 5,984,900.
• the formulations may be sterile.
• sterile as used in the context of the present invention means aseptic or substantially free of microorganisms.
• the formulations may be made sterile by the destruction or removal of substantially all microorganisms by a variety of methods known in the art including, but not limited to, physical methods (e.g., heat, sound, light, radiation, adsorption, filtration) and chemical methods (e.g. , antiseptics).
• physical methods e.g., heat, sound, light, radiation, adsorption, filtration
• chemical methods e.g. , antiseptics.
• the present inventive formulations may be embodied in other specific forms without departing from its spirit or its central characteristics.
• the described embodiments are to be considered in all respects only as illustrative and not restrictive.
• the scope of the invention is, therefore, indicated by the appended claims, rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.
• the formulations of the present invention may include a pharmaceutically acceptable preservative, a tonicity modifier, an adjuvant or auxiliary drug to assist the action of the peptide, an excipient or an inert carrier for the peptide, a detergent such as TWEEN 80, or a solvent to increase the solubility of the peptide.
• a pharmaceutically acceptable preservative for example, a pharmaceutically acceptable preservative, a tonicity modifier, an adjuvant or auxiliary drug to assist the action of the peptide, an excipient or an inert carrier for the peptide, a detergent such as TWEEN 80, or a solvent to increase the solubility of the peptide.
• Example 1 GLP-1, PTH, and GRF, as their chloride salts, were dissolved in the formulation at the pH values indicated in Table 1, vialed in 1 mL tubing glass vials and stoppered with Helvoet Omniflex stoppers and metal crimp seals (SP Pharmaceuticals, NM). The vials were stored at the indicated temperatures for the indicated times. Samples were removed and assayed for the loss of parent peptide by HPLC, using a reversed phase C18 (1x15 cm) analytical column. Samples (10 ⁇ l) were injected directly and resolved with a gradient of acetonitrile in water, in the presence of 0.1% trifluoroacetic acid. Percent peptide remaining at the times indicated was calculated as the area of the intact peptide divided by the total area of the intact peptide plus that of the decomposition products times 100.
• GRF(l-44)amide was formulated as listed in Table 2 and the purity after 7 days at various temperatures was measured using a Beckman HPLC commercially available from Beckman Instruments, CA, using a reversed phase C18 analytical column with a gradient of increasing acetonitrile in water, in the presence of 0.1 %> trifluoroacetic acid.
• GLP-1, GRF, and PTH were formulated at SP Pharmaceuticals under cGMP guidelines in 10 mM acetate, 5.07% D-mannitol in 3 mL glass vials with Helvoet stoppers and metal seals.
• the vials containing 1 mL of formulated drug were put into thermostatted chambers and assayed for % peptide remaining as a function of time after storage at different temperatures. Bioactivity of the formulations at the time points was also measured.
• Drawings 3, 4, and 5 show results that demonstrate that the formulations are highly stable for at least 9 months at -20°C and 4°C as assessed by decomposition (measured by HPLC) and/or bioactivity.
• GLP-1 formulation stability data is presented in Drawing 4
• PTH formulation stability data is shown in Drawing 5.
• Example 5 To illustrate that the preferred formulations deliver peptide rapidly and effectively to animals, rats were injected subcutaneously with GLP-1 in the preferred formulation and the plasma was assayed for GLP-1 by conventional immunoassay for total GLP-1 as a function of time. The injected GLP-1 caused a rapid increase in plasma levels, shown in Drawing 7, indicating rapid and sigmficant delivery of the peptide. Similarly, Drawing 8 shows that when a rat is given an intravenous bolus of 20 ⁇ g of GRF formulated in 10 mM sodium acetate, 5.07% D-mannitol, pH 4.7, the peptide rapidly appears in the blood plasma.
• Example 6 GLP-1 formulated and delivered subcutaneously continuously over 24 hours produced plasma concentrations of GLP-1 about 6-fold above basal levels in man.
• GLP-1 dissolved at 1 mg/mL in 5.07%> ' D-mannitol and 10 mM sodium acetate at pH 4.5 was placed in a MiniMed 507 infusion pump and delivered subcutaneously to a human subject at a rate of 2.4 pmol/kg/min for 24 hours.

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