WO2012160212A1 - Formulation de peptides à libération contrôlée - Google Patents

Formulation de peptides à libération contrôlée Download PDF

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Publication number
WO2012160212A1
WO2012160212A1 PCT/EP2012/059916 EP2012059916W WO2012160212A1 WO 2012160212 A1 WO2012160212 A1 WO 2012160212A1 EP 2012059916 W EP2012059916 W EP 2012059916W WO 2012160212 A1 WO2012160212 A1 WO 2012160212A1
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WIPO (PCT)
Prior art keywords
formulation
glp
cmpd
administration
amylin
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English (en)
Inventor
Catalin Nistor
Markus Johnsson
Fredrik Joabsson
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Camurus AB
Amylin Pharmaceuticals LLC
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Camurus AB
Amylin Pharmaceuticals LLC
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Publication of WO2012160212A1 publication Critical patent/WO2012160212A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the present invention relates to formulation precursors (pre-formulations) for the in situ generation of compositions for the controlled release of certain hormone active agents and methods of treatment with such formulations.
  • the invention relates to high-loading pre-formulations of amphiphilic components and at least one hybrid dual agonist polypeptide hormone active agent for parenteral application, which undergo phase transition upon exposure to aqueous fluids, such as body fluids, thereby forming a controlled release composition.
  • bioactive agents including pharmaceuticals, nutrients, vitamins and so forth have a "functional window”. That is to say that there is a range of concentrations over which these agents can be observed to provide a beneficial or desirable biological effect. Where the concentration in the appropriate part of the body (e.g. locally or as demonstrated by serum concentration) falls below a certain level, no beneficial effect can be attributed to the agent. Similarly, there is generally an upper concentration level above which no further benefit is derived by increasing the concentration. In some cases increasing the concentration above a particular level results in undesirable or even dangerous effects.
  • bioactive agents have a long biological half-life and/or a wide functional window and thus may be administered occasionally, maintaining a functional biological concentration over a substantial period of time (e.g. 6 hours to several days). In other cases the rate of clearance is high and/or the functional window is narrow and thus to maintain a biological concentration within this window regular (or even continuous) doses of a small amount are required. This can be particularly difficult where non-oral routes of administration (e.g. parenteral administration) are desirable or necessary, since self-administration may be difficult and thus cause inconvenience and/or poor compliance. Agents for treatment or maintenance in chronic conditions also benefit considerably from slow-release formulations because the dose is potentially more stable and the disruption to the lifestyle of a subject living with a chronic or long-term condition is minimised. In such cases it would be advantageous for a single administration to provide active agent at a therapeutic level over the whole period during which activity is needed or over a period of days or weeks in chronic conditions.
  • Peptides and proteins that are administered to the mammalian body are subject to degradation by various proteolytic enzymes and systems present throughout the body.
  • Well known sites of peptidase activity include the stomach (e.g. pepsin), and the intestinal tract (e.g. trypsin, chymotrypsin, and others) but other peptidases (e.g. aminopeptidases, carboxypeptidases, etc.) are found throughout the body.
  • gastric and intestinal degradation reduces the amount of peptide or protein which potentially could be absorbed through the intestinal surface lining and thereby decreases their bioavailability.
  • free peptides and proteins in the mammalian blood stream are also subject to enzymatic degradation (e.g. by plasma proteases etc.).
  • a depot system allowing loading and controlled release of a larger dose over a longer period would offer a considerable advantage over conventional delivery systems. Diabetes is one such condition that may benefit by long-term or chronic treatment, as well as by use of a depot formulation.
  • GLP-1 glucagon-like peptide
  • GLP-1 is a 37-amino acid peptide with a MW of 4.2 KDa, having a sequence highly conserved between different species.
  • GLP-1 is involved in modification of glucose homeostasis through actions that include potentiation of glucose-stimulated insulin secretion and biosynthesis and suppression of glucagon secretion, gastric emptying, and food intake.
  • GLP-1 The abilities of GLP-1 to stimulate insulin secretion and inhibit glucagon release are glucose-dependent; thus, the risk of hypoglycemia with GLP-1 administration is low. GLP-1 also increases beta-cell mass in preclinical models of diabetes through mechanisms that include stimulation of beta-cell proliferation and neogenesis and inhibition of beta-cell apoptosis.
  • GLP-1 may also play a protective role in the cardiovascular system.
  • GLP-1 glycopeptidyl peptidase-IV
  • DPP dipeptidyl peptidase
  • GLP-1 mimetics bind to GLP-1 receptors with similar affinity and produce biological actions overlapping to those of native GLP-1 but are resistant to DPP-IV-mediated inactivation and, in some cases more resistant to renal clearance. These compounds are able to exert more sustained GLP-l-like activity for longer periods of time in vivo.
  • An alternative therapeutic approach for prolonging the action of native GLP-1 is to inhibit DPP-IV activity, thereby preventing GLP-1 degradation.
  • Several orally active agents that inhibit DPP-IV activity are also being evaluated for the treatment of type 2 diabetes.
  • GLP-1 therapies are typically injectables which require administration around twice a day for the period of treatment. This will generally be by patient self-administration. Since frequent injection over a long period is not an optimal administration strategy, there is clearly scope for GLP-1 users to benefit from long-acting, sustained formulations, which might be administered much less frequently.
  • DPP-IV inhibitors are often associated with undesirable side-effects. Furthermore, controlling or reducing obesity, a co-morbidity often associated with diabetes, e.g. type II diabetes, is extremely desirable, but is not generally obtained by DPP-IV inhibitors. On the other hand while GLP-1 receptor agonists can provide some weight control benefit, their primary benefit is glucose control, and further weight control is desirable.
  • the present invention provides a pharmaceutical formulation comprising an appropriate combination of lipid excipients, organic alcoholic solvent, aqueous solvent, hybrid polypeptide hormone active agent and certain optional components, that can be used as a depot-precursor formulation (referred to herein for brevity as a pre-formulation) to address one or more of the needs described above.
  • the invention provides a pre-formulation comprising a low viscosity mixture of: a) 20-80 wt.% of at least one diacyl glycerol and/or a tocopherol;
  • PC phosphatidyl choline
  • a preservative e.g. antimicrobial or microbial-static agent
  • the preservative may be an additional agent to the formulations herein or, preferably a preservative activity is provided by one of or any combination of c), d) or f) above.
  • component a) comprises glycerol dioleate (GDO).
  • component a) is present at a level of 30-40% by weight.
  • component b) comprises PC.
  • Component b) preferably is present at a level of 30-40% by weight.
  • the PC is derived from soy.
  • the PC comprises 18:2 fatty acids as the primary fatty acid component with 16:0 and/or 18: 1 as the secondary fatty acid components. These are preferably present in the PC at a ratio of between 1.5: 1 and 6: 1.
  • PC having approximately 60-65% 18:2, 10 to 20% 16:0, 5-15% 18: 1, with the balance predominantly other 16 carbon and 18 carbon fatty acids is preferred and is typical of soy PC.
  • the ratio of components a:b is in the range 40:60 to 70:30
  • component c) comprises at least one mono-ol, diol or triol.
  • component c) comprises ethanol or propylene glycol or mixtures thereof.
  • Component c) can be in the range of 5 to 35%), even 10-30%.
  • component c) is present at a level of 10-25%) by weight.
  • component d) comprises water.
  • component d) is present at a level of 1.2 to 15% by weight.
  • the antioxidant (component f)) is ascorbic acid, EDTA or citric acid, more preferably EDTA.
  • the ratio of components d:f is in the range 100: 1 to 10000: 1, preferably 1000: 1 to 5000: 1.
  • the dual amylin receptor/GLP-1 receptor agonist active agent is as defined herein below.
  • the pre-formulation according to the invention has an L 2 phase structure.
  • the pre-formulation of the invention comprises or consists of at least one formulation selected from those listed in Table 1.
  • Each composition listed in Table 1 of Example 1 below forms a separate and independent preferred embodiment of the present invention.
  • the invention provides a process for the formation of a pre- formulation suitable for the administration of a hybrid polypeptide hormone bioactive agent to a (preferably mammalian) subject, said process comprising forming a low viscosity mixture of: a) 20-80 wt.% of at least one diacyl glycerol and/or a tocopherol;
  • PC phosphatidyl choline
  • a further aspect of the invention is the use of a low viscosity mixture of: a) 20-80 wt.% of at least one diacyl glycerol and/or a tocopherol;
  • PC phosphatidyl choline
  • the invention provides a method for the treatment of a human or non-human mammalian subject comprising administering to said subject a pre- formulation according to the invention.
  • said method is for the treatment of a human or non-human mammalian subject in need thereof to combat at least one condition selected from diabetes, type I diabetes, type II diabetes, abnormal glucose control, excess bodyweight, obesity, hypertension, arthrosclerosis, dyslipidemia, congestive heart failure, stroke, hypercholesterolemia, cardiovascular disease, myocardial ischemia, myocardial reperfusion, eating disorders, gestational diabetes, diabetic neuropathy, pulmonary hypertension, or a metabolic disease or disorder associated with insufficient pancreatic beta cell mass, wherein the pre-formulation preferably comprises a dual- functional GLP-1 receptor/ amylin receptor agonist, more preferably Cmpd 1, described herein.
  • the subject is in need of treatment for at least one of the above conditions that benefits by glucose lowering, delay of gastric emptying, insulin secretion, glucagons suppression and/or HbAlc lowering (as may be provided by a GLP-1 receptor agonist) and for at least one of the above conditions that benefits by further reducing body weight, inhibiting food intake or further delaying gastric emptying (as may be provided by an amylin receptor agonist).
  • the treatments provide improved patient compliance due to reduced adverse events and side effects, such as flushing, nausea, and/or to a more convenient, patient friendly administration regimen such as by once weekly dosing.
  • the invention provides a method of cosmetic treatment of a human or non-human mammalian subject comprising administering to said subject a pre- formulation according to the invention
  • the invention provides the use of: a) 20-80 wt.% of at least one diacyl glycerol and/or a tocopherol;
  • PC phosphatidyl choline
  • a low viscosity pre-formulation medicament for use in the in vivo formation of a depot for treatment of at least one of type I diabetes, type II diabetes, abnormal glucose control, excess bodyweight, obesity, hypertension, arthrosclerosis, dyslipidemia, congestive heart failure, stroke, hypercholesterolemia, cardiovascular disease, myocardial ischemia, myocardial reperfusion, eating disorders, gestational diabetes, diabetic neuropathy, pulmonary hypertension, or a metabolic disease or disorder associated with insufficient pancreatic beta cell mass.
  • the subject is in need of treatment for at least one of the above conditions that benefits by glucose lowering, delay of gastric emptying, insulin secretion, glucagons suppression and/or HbAlc lowering (as may be provided by a GLP-1 receptor agonist) and for at least one of the above conditions that benefits by further reducing body weight, inhibiting food intake or further delaying gastric emptying (as may be provided by an amylin receptor agonist).
  • the treatments provide improved patient compliance due to reduced adverse events and side effects, such as flushing, nausea, and/or to a more convenient, patient friendly administration regimen such as by once weekly dosing.
  • the invention provides a pre-filled administration device containing a pre-formulation according to the invention.
  • the device may be single- or multi-use.
  • the pre-formulation according to the invention delivers a dosage of active agent in the range of 1 ⁇ g to 10 mg/day, or about 0.5 mg to 5 mg/day, or alternatively as described herein, in the range for example of 3 mg to 60 mg/week or 5 to 40 mg/week, and even 10 to 30 mg/week, whether from a single use or multi-use device.
  • each administered dose is a total volume for administration of no more than or equal to 3 ml, preferably no more than or equal to 1 ml, more preferably no more than or equal to 0.5 ml, and even more preferably no more than or equal to 0.1 ml. Further, the volume is preferably no less or equal to than 0.03 ml, more preferably no less than or equal to 0.06 ml, and even no less than or equal to 0.1 ml.
  • a preferred volume for administration is approximately 0.5 ml.
  • kits comprising said administration device according to the invention.
  • the kit can optionally contain instructions for subcutaneous or intramuscular administration of said composition. All compositions described herein are suitable for use in such a kit and may thus be contained therein.
  • the kits of the invention can optionally include additional administration
  • kits may additionally contain other devices and/or medicaments relating to blood glucose control in a diabetic subject, such as blood- or urine-glucose testing devices (e.g. strips or sticks), insulin formulations, carbohydrate preparations etc.
  • blood- or urine-glucose testing devices e.g. strips or sticks
  • insulin formulations e.g. insulin formulations
  • carbohydrate preparations etc.
  • the kit may additionally contain glucose and/or other carbohydrate preparation(s) and/or blood glucose testing devices.
  • the kit is for routine administration, it can optionally be combined with the subject's routine insulin medication, such that the kit contains compositions of the invention plus one or more insulin preparations and/or one or more blood or urine glucose testing devices.
  • the formulations can be provided as single use or multi-use formats, which can include for example, vials with syringes, prefilled syringes, prefilled cartridges, prefilled injection pens and/or needle-free injection pens and devices.
  • Multi-use is meant as providing for more than one injection, for example 2, 3, 4, 5, 6 or more injections, from the same device. These may be from the same reservoir of pre- formulation or by means of multiple smaller reservoirs.
  • the injections are preferably made over a period of at least at or about two weeks, three weeks, four weeks and even 8 weeks, more preferably over at least 4 weeks, most preferably as once weekly injections over any of those periods.
  • the injections are made at appropriate dosing intervals as discussed herein.
  • Multi-dose formats must remain stable and potent in storage prior to use, but are disposable after the single use.
  • Multi-dose formats must not only remain stable and potent in storage prior to use, but must also remain stable, potent and relatively free of bacteria over the multiple-dose use regimen administration period after the first use in which a seal has been compromised. For this reason multi-dose formats often require a anti-microbial or microbial-static agent, e.g. bacteriostatic agent, preservative.
  • bacteriostatic agent e.g. bacteriostatic agent
  • antimicrobial or microbial-static agent which includes bacteriostatic agents and preservative.
  • bacteriostatic agents include benzalkonium chloride, m-cresol, benzyl alcohol or other phenolic preservatives. Typical concentrations as known in the art can be used.
  • the present formulations with dual receptor agonist do not require an additional preservative, anti-microbial or microbial-static agent, e.g. bacteriastatic or bacteriacide or additional amount of such agent to provide a multi-use format.
  • the formulations as described herein provide a preservative effect with an acceptable peptide stability and formulation stability. They can be used for single-dose as well as for multiple-dose use.
  • preferred formulations herein for multi-use format can contain ethanol, propylene glycol, citric acid and/or EDTA as described, preferably in sufficient concentrations to not only provide their primary benefit as taught herein but also at sufficient concentration, either alone or in any combination, to provide the preservative effect while maintaining stability of the active and the formulation.
  • Figure 1 shows the change in peptide concentration (vs. start value measured directly after manufacturing) after storage of the formulations with compositions presented in Table 1 for 3 months (formulation 16: 2 months) under different conditions.
  • Figures 2 A and 2B Figure 2 A is a graph depicting change in body weight in DIO (diet induced obese) rats administered Cmpd 2 or the other compounds in the amounts shown in Figure 1 A as described in the Examples.
  • Figure 2B is a graph depicting change in body weight in DIO rats administered Cmpd 1 or the compounds in the amounts shown in Figure IB as described in the Examples.
  • Figures not sharing a superscript are significantly different from each other; p ⁇ 0.05, e.g. the vehicle controls of Figures 1 A and IB are not significantly different from each other.
  • Figures 3 A and 3B Figure 3 A is a graph depicting total cumulative food intake for DIO rats administered Cmpd 2 or the other compounds in the amounts shown in Figure 2A as described in the Examples.
  • Figure 3B is a graph depicting total cumulative food intake for DIO rats administered Cmpd 1 or the other compounds in the amounts shown in Figure 2B as described in the Examples.
  • Groups not sharing a superscript are significantly different from each other; p ⁇ 0.05, e.g. the vehicle controls of Figures 2 A and 2B are not significantly different from each other.
  • Figure 4 shows mean body weights of male rats following administration of a single subcutaneous dose of Cmpdl formulated as set out in the Examples below.
  • Figure 5 shows the variation in plasma concentration over time from a Single Dose Subcutaneous Pharmacokinetic Study in Rats using Cmpdl .
  • Figure 5 A shows results over a timescale up to 312 hours.
  • Figure 5B shows results over the first 24 hours.
  • Figures 6A to 6C depict Pharmacokinetic (Fig. 6A and 6B) and Pharmacodynamics (Fig. 6C) profiles in dogs following a single subcutaneous administration of two exemplary and preferred sustained release formulations containing Cmpd 1 over a 312 hour (two week) time course.
  • Fig. 6A depicts the Cmpd 1 plasma concentration in pg/ml of plasma over the 312 hour time course for formulation B10 and formulation B12.
  • Fig. 6B is a plot expanding the 0 to 36 hour period of the pharmacokinetic profile.
  • Fig. 6C depicts percent change in body weight for each individual animal over the 312 hour time course.
  • BIO closed circle
  • B12 closed square
  • open symbols and star represent BIO and closed symbols represent B12. DETAILED DESCRIPTION OF THE INVENTION
  • the formulations of the present invention generate a non-lamellar liquid crystalline phase following administration.
  • the use of non-lamellar phase structures (such as liquid crystalline phases) in the delivery of bioactive agents is now relatively well established.
  • the present inventors have now established that the combination of particular lipid controlled-release compositions with certain peptide active agents serves to provide a still further and unexpected improvement in performance in one or more key criteria.
  • a pre-formulation comprising at least one diacyl glycerol, at least one phosphatidyl choline, at least one biocompatible, organic alcohol solvent, at least one active agent as described herein and optionally at least one antioxidant in a low viscosity phase, such as molecular solution
  • a pre-formulation may be generated addressing many of the shortfalls of known depot formulations.
  • Such a formulation may be applied to provide a depot of the active agent in situ.
  • the pre-formulation is easy to manufacture, may be sterile-filtered, has low viscosity (allowing easy and less painful administration typically through a narrow needle), allows a higher level of bioactive agent to be incorporated than has previously been demonstrated (thus potentially allowing a smaller amount of composition to be used), contains a highly effective bioactive agent (again reducing the necessary volume), requires shallow injection and/or forms a desired non-lamellar depot composition in vivo having a controllable "burst" or "non-burst” release profile.
  • the compositions are also formed from materials that are non-toxic, biotolerable and biodegradable, which can be administered by i.m., or s.c. and are suitable for self-administration. Preferably once weekly administration.
  • Certain of the formulations of the present invention generate a non-lamellar liquid crystalline phase following administration.
  • the use of non-lamellar phase structures (such as liquid crystalline phases) in the delivery of bioactive agents is now relatively well established.
  • a most effective lipid depot system is described in WO2005/117830, and a highly preferred lipid depot for use in the present invention is a development from that described in that document, the full disclosure of which is hereby incorporated herein by reference.
  • compositions have numerous advantages over polymer based formulations, such as those using PLGA microspheres.
  • Hybrid, dual agonist polypeptides have many advantages in terms of activity and may be delivered by systems consisting of microspheres of biodegradable polymers.
  • polymer microsphere formulations must generally be administered by means of a sizable needle, typically of 20-gauge or wider. This is necessary as a result of the nature of the polymeric dosing systems used, which are typically polymer suspensions.
  • such systems typically require suspension or multi- step preparation at the point of care prior to administration, making such
  • the present formulations advantageously provide a system of low viscosity, such as a homogeneous solution, dispersion of fine particles, or L 2 phase, which could be administered easily through a narrow needle, thus decreasing the discomfort of the patient during the procedure.
  • a system of low viscosity such as a homogeneous solution, dispersion of fine particles, or L 2 phase
  • this ease and comfort of administration is particularly significant because most patients will typically be on a self-administration regime.
  • Providing a sustained formulation with a duration of a few days, but which is sufficiently complex to administer that it requires treatment by a healthcare professional will not be an advantage to all patients over twice-daily or daily self-administration, and is likely to be more costly.
  • compositions which give sufficiently long duration to justify a visit to a health professional for administration and/or a preparation which can be self-administered, and reducing preparation time of health-care professionals or patients prior to the actual administration are all important issues.
  • the present formulations surprisingly provide these advantages by means of a sterile, ready-to-administer composition requiring little or no preparation and which can be provided in pre-filled
  • compositions suitable for use by heathcare professionals or patients further allow for the formation of a depot in-situ by the administration of a suitable active agent, as described herein, in an administration system formed from low-toxicity, non-immunogenic, non-inflammatory lipids and biotolerable solvents. Not only does this formulation (in combination with low viscosity) provide minimal pain at the injection site but no irritant degradation products are generated as the active agent is released and thus very little build-up of inflammation or scar tissue is observed even following repeated administration (for example in treating a chronic condition such as diabetes).
  • polymer depot compositions generally have the disadvantage of accepting only relatively low drug loads and having a "burst/lag" release profile.
  • the nature of the polymeric matrix especially when applied as a solution or pre-polymer, causes an initial burst of drug release when the composition is first administered. This is followed by a period of low release, while the degradation of the matrix begins, followed finally by an increase in the release rate to the desired sustained profile.
  • This burst/lag release profile can cause the in vivo concentration of active agent to burst above the functional window immediately following administration, and then drop back through the bottom of the functional window during the lag period before reaching a sustained functional concentration for a period of time.
  • this burst/lag release profile is undesirable and could be dangerous. It may also limit the equilibrium concentration which can be provided due to the danger of adverse effects at the "peak" point.
  • the presence of a lag phase may furthermore require supplementary dosing with repeat injections during the start-up period of depot treatment in order to maintain a therapeutic dose while the concentrations of active provided from the depot are sub-functional.
  • the manufacture of PLGA microbeads and suspensions is additionally a
  • the beads are particulate, and polymers clog membranes, they cannot generally be sterile-filtered and furthermore, since the PLGA copolymer melts at around 40°C, they cannot be heat-treated for sterility. As a result, a complex manufacturing process must all be conducted under conditions of high sterility.
  • biodegradable polymer microspheres include complex reconstitution prior to injection and limited storage stability, due both to aggregation and degradation of the delivery system and/or active.
  • the present formulations surprisingly addresses these disadvantages by providing a formulation that is a low- viscosity fluid and can thus be readily sterile-filtered, thereby providing a straightforward method of sterile manufacture.
  • compositions of the present invention over polymer formulations, such as PLGA spheres, is the low initial release ("non-burst profile") of active agent, as demonstrated herein.
  • This may be defined such that the area under a plasma concentration against time the curve during an initial period (e.g. the first 6 hours) is less than a certain proportion of the area under the curve for the entire curve (measured or extrapolated from time 0 to infinity or from time 0 to the last sampling time point, such as day 7 (about 168 hours) for a once weekly injection or day 1 (about 24 hours) for a once daily injection, and so forth).
  • This amount might be, for example, less than 15%, particularly less than 10% in the first 6 hours of a 7-day composition.
  • compositions of the invention have an AUC of less than 15% in the first 6 hours of a 7 day composition and five of the six compositions (those with chloride counter-ion) have a AUC of less than 11%.
  • the two lowest- burst compositions which form highly preferred embodiments have an AUC of less than 6% in the first 6 hours of a 7-day formulation.
  • the Cmax/Cave ratio is less than 15, preferably less than or equal to 12, more preferably less than or equal to 10, even more preferably less than or equal to 5.
  • Cmax is defined as is known in the art, as the peak or maximal plasma concentration observed during the period of release before the next dose is adminsitered and Cave is defined as the average plasma concentration during that period of release. Cave can be calculated by calculating the drug present in the plasma as area under the curve (AUC) over the selected period of time, generally the entire period of release before the
  • the initial burst and/or Cmax can be minimized by use of the present formulations, which minimizes initial dilution and/or diffusion of the active agent prior to transition to solid phase, as well as by their relatively rapid time to phase transition to solid, as discussed herein.
  • the formulations of the present invention are advantageous even to previously reported lipid formulations. Compared to previously reported lipid-based, slow- release composition described in WO2006/131730 for GLP-1, the present formulations provide a higher concentration of active agent that allows for the possibility of longer duration depot products, products maintaining a higher systemic concentration, and products having a smaller injection volume, all of which factors are of considerable advantage under appropriate circumstances.
  • the present formulations can have the advantage to provide a slow- release composition comprising a highly effective active agent which may show longer residence times in vivo and may furthermore provide more functional effect per unit mass of injected active, as well as providing dual receptor pharmacology to provide multiple biological effects with improved benefits to the patient as described herein.
  • a highly effective active agent which may show longer residence times in vivo and may furthermore provide more functional effect per unit mass of injected active, as well as providing dual receptor pharmacology to provide multiple biological effects with improved benefits to the patient as described herein.
  • % by weight indicated is the % of the total pre-formulation including all of the components indicated herein.
  • the pre-formulations can optionally consist of essentially only the components indicated herein (including where appropriate additional optional components indicated herein below and in the attached claims) and in one aspect consist entirely of such components.
  • the lipid-based systems described herein comprise lipid components a) and b), plus organic solvent (c), aqueous solvent (d), dual amylin receptor/GLP-1 receptor agonist active agent e) and optional antioxidant (f) components.
  • Preferable ranges for component a) are 20-80 wt.%, preferably 25-70 wt.%, more preferably 28-60% (e.g. 30-55%), particularly 30-45% (e.g. 30-40%).
  • Preferable ranges of component b) are 20-80 wt.%, preferably 25-70 wt.%, more preferably 28- 60% (e.g. 30-55%), particularly 30-45% (e.g. 30 to 40%).
  • Ratios of a:b are typically 40:60 to 70:30, preferably 45:55 to 60:40 and more preferably 48:52 to 55:45. Ratios of around 50:50 are highly effective.
  • Component "a” as indicated herein is preferably at least one diacyl glycerol (DAG) and thus has two non-polar "tail” groups.
  • the two non-polar groups may have the same or a differing number of carbon atoms and may each independently be saturated or unsaturated.
  • Examples of non-polar groups include C6-C32 alkyl and alkenyl groups, which are typically present as the esters of long chain carboxylic acids. These are often described by reference to the number of carbon atoms and the number of unsaturations in the carbon chain.
  • CX:Z indicates a hydrocarbon chain having X carbon atoms and Z unsaturations.
  • typical non-polar chains are based on the fatty acids of natural ester lipids, including caproic, caprylic, capric, lauric, myristic, palmitic, phytanic, palmitolic, stearic, oleic, elaidic, linoleic, linolenic, arachidonic, behenic or lignoceric acids, or the corresponding alcohols.
  • Preferable non-polar chains are palmitic, stearic, oleic and linoleic acids, particularly oleic acid.
  • glycerol dioleate A highly preferred example is DAG comprising at least 50%, preferably at least 80% and even comprising substantially 100% GDO.
  • GDO and other diacyl glycerols are products derived from natural sources, there is generally a certain proportion of "contaminant" lipid having other chain lengths etc.
  • GDO as used herein is thus used to indicate any commercial grade of GDO with concomitant impurities (i.e. GDO of commercial purity). These impurities may be separated and removed by purification but providing the grade is consistent this is rarely necessary.
  • GDO may be essentially chemically pure GDO, such as at least 80% pure, preferably at least 85% pure and more preferably at least 90% pure GDO.
  • Component "b” in the preferred lipid matrices of the present invention is at least one phosphatidyl choline (PC).
  • this component comprises a polar head group and at least one non-polar tail group.
  • the difference between components a) and b) lies principally in the polar group.
  • the non-polar portions may thus suitably be derived from the fatty acids or corresponding alcohols considered above for component a.
  • the PC will contain two non-polar groups.
  • the phosphatidyl choline portion even more suitably than any diacyl glycerol portion, may be derived from a natural source. Suitable sources of phospholipids include egg and plant sources including soybean. Such sources may provide one or more constituents of component b, which may comprise any mixture of
  • the PC component preferably contains at least 50% soy PC or egg PC, more preferably at least 75% soy PC or egg PC and most preferably essentially pure soy PC or egg PC.
  • the PC component may comprise synthetic PC, such as synthetic dioleoyl PC (DOPC).
  • synthetic PC such as synthetic dioleoyl PC (DOPC).
  • DOPC synthetic dioleoyl PC
  • the PC component preferably contains at least 50% synthetic dioleoyl PC, more preferably at least 75% synthetic dioleoyl PC and most preferably essentially pure synthetic dioleoyl PC. Any remaining PC is preferably soy PC as above.
  • the synthetic dioleoyl PC is most preferably l,2-dioleoyl-sn-glycero-3- phosphocholine, and other synthetic PC components include DDPC (1,2- Didecanoyl-sn-glycero-3-phosphocholine); DEPC(l,2-Dierucoyl-sn-glycero-3- phosphocholine); DLOPC(l,2-Dilinoleoyl-sn-glycero-3-phosphocholine);
  • the pre-formulations of the invention are to be administered to a subject for the controlled release of a hybrid polypeptide active agent, it is important that the components are biocompatible.
  • the preferred lipid matrices for use in the pre-formulations of the present invention are highly advantageous since both PC and DAGs are well tolerated and are broken down in vivo into components that are naturally present in the mammalian body.
  • a particularly favoured combination of components a) and b) are GDO with PC, especially GDO with soy PC.
  • Appropriate amounts of each component suitable for the combination are those amounts indicated herein for the individual components in any combination. This applies also to any combinations of components indicated herein, where context allows.
  • Component c) of the pre-formulations of the invention is an organic alcoholic solvent. Since the pre-formulation is to generate a depot composition following administration (e.g. in vivo), typically upon contact with excess aqueous fluid, it is desirable that this solvent be tolerable to the subject and be capable of mixing with the aqueous fluid, and/or diffusing or dissolving out of the pre-formulation into the aqueous fluid. Solvents having at least moderate water solubility are thus preferred.
  • component c) comprises or consists of ethanol or propylene glycol or mixtures thereof.
  • the solvent is such that a relatively small addition to a mixture comprising a) and b) (i.e. preferably below 25%) gives large viscosity reductions, of one order of magnitude or more.
  • a relatively small addition to a mixture comprising a) and b) i.e. preferably below 25%
  • the addition of 25%, 20%, 15% or even 10% solvent can give a reduction of two or more orders of magnitude in viscosity over the solvent- free composition, or over a depot containing a viscosity-increasing solvent such as glycerol.
  • the amount of solvent component c) in the pre-formulation will have a considerable effect upon several features.
  • the solubility of the active substance, viscosity and the rate (and duration) of release may alter significantly with the solvent level.
  • the amount of solvent will thus be at least sufficient to provide the required solubility of the drug substance, a low viscosity mixture but will additionally be determined so as to provide the desired release rate. This may be determined by routine methods in view of the Examples below.
  • a level of 0.1 to 35%), particularly 5 to 35% solvent will provide suitable release and viscosity properties, as will 5 to 25%. This will preferably be 8 to 28% (e.g.
  • the amount of component c) in the pre-formulations of the invention will be at least sufficient to provide the required drug load in solution, a low viscosity mixture (e.g. a molecular solution, see above) of components a), b), c) and d) and optionally f) and will be easily determined for any particular combination of components by standard methods.
  • a low viscosity mixture e.g. a molecular solution, see above
  • the solubility of the active agents described herein is significantly affected by the presence of water and/or propylene glycol in the pre-formulations that relate to all aspects of the invention.
  • the solvent component c) comprises propylene glycol (PG).
  • PG may be present, for example, in an amount of 1 to 15% by weight, preferably 2 to 15% (e.g. 10 to 15%).
  • the amount of PG may be selected to achieve an appropriate level of solubility in combination with the water content, which also increases active agent solubilisation, as discussed below. In any event, the amount of PG will be consistent the total amount of c) as described above and herein.
  • phase behaviour may be analysed by techniques such as visual observation in combination with polarized light microscopy, nuclear magnetic resonance, and cryo- transmission electron microscopy (cryo-TEM) to look for solutions, L 2 or L 3 phases, or liquid crystalline phases or as in the case of cryoTEM, dispersed fragments of such phases. Viscosity may be measured directly by standard means. As described above, an appropriate practical viscosity is that which can effectively be syringed and particularly sterile filtered. This will be assessed easily as indicated herein.
  • Typical solvents suitable for use in the invention include at least one solvent selected from mono-ols, diols and triols.
  • suitable alcohols include ethanol, propylene glycol, isopropanol, benzyl alcohol and glycerol formal, particularly ethanol.
  • Monools are preferred to diols and polyols. Most preferred is a mixture of at least one diol and at least one mono-ol. Ethanol and/or propylene glycol are most preferred.
  • a highly preferred combination for the lipid matrix aspect is PC, GDO and ethanol/propylene glycol or mixtures thereof. As indicated above, appropriate amounts of each component suitable for the combination are those amounts indicated herein for the individual components, in any combination.
  • component c) contains halogen substituted hydrocarbons since these tend to have lower biocompatibility. Where a portion of halogenated solvent such as dichloromethane or chloroform is necessary, this proportion will generally be minimised.
  • Component c) as used herein may be a single solvent or a mixture of suitable solvents but will generally be of low viscosity. This is important because one of the key aspects of the present invention is that it provides pre-formulations that are of low viscosity and one of the primary roles of a suitable solvent is to reduce this viscosity. This reduction will be a combination of the effect of the lower viscosity of the solvent and the effect of the molecular interactions between solvent and lipid composition.
  • One observation of the present inventors is that the oxygen-containing solvents of low viscosity described herein have highly advantageous and unexpected molecular interactions with the lipid parts of the composition, thereby providing a non-linear reduction in viscosity with the addition of a small volume of solvent.
  • some embodiments preferably contain propylene glycol and further surprisingly can contain PG and substantial water.
  • WO2006/131730 which discloses lipid-based, slow-release compositions containing GLP-1, reported that the formulations should contain glycerol, ethylene glycol or propylene glycol and contain no more than a trace of water.
  • depot compositions based upon lipid precursors having substantial amounts of water (e.g. greater than 2% water) or showing the benefits attributable to this water content as described herein.
  • the pre-formulations of the present invention must also contain an aqueous solvent, component d).
  • a suitable amount will typically be greater than 1% by weight of the pre-formulation, for example 1-15 wt.%, particularly 1.2-15 wt.%, especially 2 to 15%, and even more as 2-13 wt.%.
  • Component d) is preferably water, such as purified water for injection.
  • the pre-formulations of the invention contain ethanol and/or propylene glycol as component c) with water as component d).
  • aqueous component d) does not include any organic solvent (that solvent being present in component c).
  • the presence of a certain amount of water in the pre-formulations increases the solubility of the dual agonist peptide active agent (as described herein).
  • This function is also served to some extent by the propylene glycol component and thus the amount of water necessary may be reduced when greater amounts of PG are employed.
  • the total concentration of water and PG in the formulations may be in the range of 8 to 20% by weight, with 10 to 18%, and 10 to 17% being preferred. This is believed to provide a loading of active agent that may be 2% or more.
  • a highly preferred combination for the lipid matrix aspect is soy PC and/or DOPC, GDO, ethanol/propylene glycol or mixtures thereof, and water.
  • appropriate amounts of each component suitable for the combination are those amounts indicated herein for the individual components, in any combination.
  • the formulations of the present invention comprise as an active agent one or more dual amylin receptor/GLP-1 receptor agonist compounds—compounds having a component that is an amylin receptor agonist linked to a component that is a GLP-1 receptor agonist.
  • the GLP-1 receptor agonist component refers to a compound that has GLP-1 receptor binding and activation activity and elicits a biological activity of an exendin reference peptide (e.g. exendin-4) or a GLP-1 (7-37) reference peptide when evaluated by art-known measures including receptor binding studies, in vitro cAMP generation or insulin secretion, or in vivo blood glucose control, insulin secretion, delay of gastric emptying and/or glucagon suppression assays as known in the art.
  • GLP-1 receptor agonist compounds include, for example, native exendins and exendin analogues and native GLP-1, for example GLP-1 (7-37) and GLP-1 analogues.
  • the amylin receptor agonist component refers to a compound that has amylin receptor binding and activation and elicits a biological activity of an amylin reference peptide (preferably rat amylin or davalintide), when evaluated by art- known measures including receptor binding, amylin agonist activity in soleus muscle assays, in vivo blood glucose control, body weight loss and gastric emptying delay as known in the art.
  • Amylin receptor agonist compounds include, for example, native amylins, e.g. human and rat amylins, and amylin analogues, e.g. pramlintide and davalintide.
  • the dual amylin receptor/GLP-1 receptor agonist polypeptide comprises an amylin or amylin analogue, including davalintide and pramlintide, conjugated or fused either to an exendin or exendin analogue or to a GLP-1 or GLP-1 analogue.
  • the two peptides can be conjugated or fused directly or through a linker.
  • Either or both peptides can be conjugated to the other through a side-chain of an internal or terminal amino acid or through the peptide's backbone, i.e. via the terminal alpha amino or carboxy groups.
  • the N-terminal amino acid of the amylin or amylin analogue e.g. davalintide
  • the linkage is via the N-terminal alpha amino group of the amylin or amylin analogue and the C-terminal alpha carboxy group of the exendin or exendin analogue or the GLP-1 or GLP-1 analogue.
  • a peptide linker fuses the two peptides creating a fusion polypeptide that can be recombinantly produced.
  • Exendin-4 and exendin-4 analogues are preferred as the GLP-1 receptor agonist. Exendin analogues having a leucine substitution for methionine at position 14 are even more preferred.
  • the most preferred dual amylin receptor/GLP-1 receptor agonist polypeptide is a fusion polypeptide comprising the C-terminally amidated polypeptide having the sequence
  • the polypeptide is a fusion of davalintide covalently attached inframe through its peptide backbone to the peptide backbone of Leu(14) exendin-4 via a glycine-glycine- glycine peptide linker. Less preferred is its C-terminal free acid form.
  • Another dual receptor agonist is a fusion polypeptide comprising C-terminally amidated polypeptide having the sequence
  • the polypeptide is a fusion of davalintide covalently attached in-frame to
  • Cmpd 5 is the amide form of the preceding exendin analog:
  • GLP-1 Receptor Agonist Component GLP-1 and GLP-1 Analogues.
  • GLP-1 is meant a GLP-1 receptor agonist that is a naturally-occurring form of glucagon like peptide- 1, either human or from any other species.
  • a straightforward system is used to describe fragments and analogues of GLP-1, which is based on full-length GLP-1 : GLP-l(l-37).
  • Arg34-GLP- 1(7-37) designates an analogue of GLP-1 formally derived from full-length GLP-1 by deleting the amino acid residues Nos. 1 to 6 and substituting the naturally occurring amino acid residue in position 34 (Lys) by Arg.
  • Native human GLP-l(7-37) has the sequence 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-Gly.
  • Native human GLP-1 (7-36)amide has the sequence 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-NH2. Also included are their C-terminal amide and acid forms, and physiologically acceptable salts, esters and derivatives.
  • GLP-1 analogue as used herein is meant a GLP-1 receptor agonist that has at least 70% sequence identity to naturally occurring forms of GLP-1, either human or from any other species, and is derived from GLP-1 by modifications including insertions, substitutions, extensions, and/or deletions of the reference GLP-1 amino acid sequence. More preferably the analogue sequence has at least 75%, 80%, 90%>, or 95% amino acid sequence identity with the reference GLP-1 peptide. In one aspect the analogue has no more than 10, 6, 5, 4, 3, 2, and/or 1 insertions, substitutions, extensions, and/or deletions relative to the reference compound.
  • An extension at the C-terminal end can be all or part of the exendin-4 "tail" (i.e.
  • PSSGAPPPS or frog GLP-1 "tail" (PSKEIIS or PKKIRYS) or can be positively charged amino acids, e.g. Lys, Arg, of 1 to 10 amino acids long; such exemplary extensions include KNGGPSSGAPPPS, PSSGAPPPS, FIEWLKNGGPSSGAPPPS, PKKIRYS, KKKKKK, and their analogs.
  • the GLP-1 analogue may comprise conservative or non-conservative amino acid substitutions (including non-natural amino acids and L and D forms). These analogues are preferably peptides, peptide derivatives or less preferably peptide mimics.
  • GLP-1 is a peptide hormone
  • typical GLP-1 analogues will be peptides, especially of around 30 amino acids, e.g. 27 to 45, especially 28 to 38.
  • Peptide derived GLP-1 agonists are most preferred, such as those indicated herein, especially Val8-GLP- 1(7-37), Val8- GLP-l(7-36)amide, Aib8-GLP 1(7-37), Aib8-GLP- 1(7-37) and liraglutide.
  • GLP-1 analogues include 9Gln-GLP- 1(7-37) , D-9Gln GLP-l(7-37), 16Thr-18 Lys GLP-l(7-37), 18Lys-GLP- 1(7-37), 8Gly-GLP-l (7-36), 9Gln-GLP-l (7-37), D- 9Gln-GLP-l (7-37), acetyl-9Lys-GLP-l(7-37), 9Thr-GLP-l(7-37), D-9Thr-GLP-l (7-37), 9Asn-GLP-l (7-37), D- 9 Asn-GLP-l (7-37), 22Ser23Arg24Arg26Gln-GLP- 1(7-37), 16Thrl8Lys-GLP- 1(7-37), 18Lys-GLP- 1(7-37), 23Arg-GLP-l(7-37), and 24Arg-GLP- 1(7-37).
  • An analogue may have superior chemical stability, physical stability, protease resistance (e.g. DPPIV resistance), solubility, efficacy, half-life, and the like.
  • GLP-1 Receptor Agonist Component Exendin and Exendin Analogues.
  • exendin is meant the sequences of naturally-occurring exendin peptides, exendin-3 and exendin-4, that are found in the salivary secretions of the Gila monster. Exendins are not the species homolog of mammalian GLP-1 (Chen & Drucker J Biol. Chem. 272:4108-15 (1997)); the Gila monster has separate genes for proglucagons (from which GLP-1 is processed). Exenatide (exendin-4) is a 39 amino acid GLP-1 receptor agonist currently indicated for the treatment of type 2 diabetes, and also exerts glucose lowering, glucagon suppression, delay of gastric emptying, body weight loss and other metabolic actions. The sequence of exendin-4 is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH 2 , where -NH2 indicates the presence of an amidated C-terminal alpha-carboxy amino acid.
  • Exendin-4 is also active in its free acid form.
  • Exendin-3 is identical to exendin-4 but for Ser and Asp at positions 2 and 3 respectively. Also included are C-terminal amide and acid forms, and physiologically acceptable salts, esters and derivatives.
  • exendin analogue as used herein is meant a GLP-1 receptor agonist that has at least 70% sequence identity to naturally occurring forms of exendin, either exendin- 3 or exendin-4, and is derived from the exendin by modifications including insertions, substitutions, extensions, and/or deletions of the reference exendin amino acid sequence. More preferably the analogue sequence has at least 75%, 80%, 90%, or 95% amino acid sequence identity with the reference exendin peptide. In one aspect the analogue has no more than 10, 6, 5, 4, 3, 2, and/or 1 insertions, substitutions, extensions, and/or deletions relative to the reference compound.
  • An extension at the C-terminal end can be all or part of the exendin-4 "tail" (i.e.
  • PSSGAPPPS or frog GLP-1 "tail" (PSKEIIS or PKKIRYS) or can be positively charged amino acids, e.g. Lys, Arg, of 1 to 10 amino acids long; such exemplary extensions include KNGGPSSGAPPPS, PSSGAPPPS, FIEWLKNGGPSSGAPPPS, PKKIRYS, KKKKKK, and their analogs.
  • exendin analogues may comprise conservative or non-conservative amino acid substitutions (including non-natural amino acids and L and D forms). These analogues are preferably peptides, peptide derivatives or less preferably peptide mimics.
  • exendin-4 is a peptide
  • typical exendin analogues will be peptides, especially of around 39 amino acids, e.g. 27 to 45, especially 28 to 39.
  • Peptide derived GLP-1 receptor agonists are most preferred, such as those indicated herein, especially 14 Leu-exendin-4, despite Hargrove et al. having reported that this exendin-4 analogue is remarkably inferior to exendin-4 with respect to its delaying of gastric emptying, anti-obesity properties and half-life.
  • this analogue is surprisingly effective in its exendin activities.
  • Additional analogues include a chimera of the first 32 amino acids of exendin-4 having amino acid substitutions at positions 14 and 28 followed by a 5 amino acid sequence from the C-terminus of a non-mammalian (frog) GLP1, with the HGEGTFTSDLSKQLEEEAV LFIEWLKQGGPSKEIIS, truncated biologically active forms of exendin-4 including exendin-4(l-27), exendin-4(l-28), exendin-4(l-29), exendin-4(l-30), exendin-4(l-28), 14 Leu, 25 Phe-exendin-4(l-28), 5 Ala, 14 Leu, 25 Phe-exendin-4(l-28), exendin(7-15) and its Ser2 analog, HSEGTFTSD, 14 Leu, 25 Phe-exendin-4, 5 Ala, 14 Leu, 25 Phe-exendin-4, desPro38 exendin-4, and AVE- 010 (also known as ZP
  • analogues described below are their C-terminal amide and acid forms, and physiologically acceptable salts, esters and derivatives.
  • An analogue may have superior chemical stability, physical stability, protease resistance (e.g. DPPIV resistance), solubility, efficacy, half-life, and the like.
  • Exendin analogues include compounds comprising the structure of Formula (I) following:
  • Exendin analogues also include those described in U.S. Patent No. 7,223,725 (incorporated herein by reference and for all purposes), such as compounds of Formula (II) following:
  • Xaai is His, Arg or Tyr;
  • Xaa 2 is Ser, Gly, Ala or Thr;
  • Xaa 3 is Ala, Asp or Glu;
  • Xaa 5 is Ala or Thr;
  • Xaa 6 is Ala, Phe, Tyr;
  • Xaa 7 is Thr or Ser;
  • Xaa 8 is Ala, Ser or Thr;
  • Xaag is Asp or Glu;
  • Xaaio is Ala, Leu, He, Val, or Met;
  • Xaan is Ala or Ser;
  • Xaai 2 is Ala or Lys;
  • Xaai 3 is Ala or Gin;
  • Xaai 4 is Ala, Leu, He, , Val or Met;
  • Xaais is Ala or Glu;
  • Xaai 6 is Ala or Glu;
  • Xaan is Ala or Glu;
  • the C-terminus of the peptide is modified by -Zi is -OH or -NH 2 , or is attached to the linker or directly to the amylin receptor agonist, or the C-terminus of the peptide further includes Gly-Z 2 , Gly Gly-Z 2 , Gly Gly Xaa 3 i-Z 2 , Gly Gly Xaa 3 i Ser- Z 2 , Gly Gly Xaa 3 i Ser Ser-Z 2 , Gly Gly Xaa 3 i Ser Ser Gly-Z 2 , Gly Gly Xaa 3 i Ser Ser Gly Ala-Z 2 , Gly Gly Xaa 3 i Ser Ser Gly Ala-Z 2 , Gly Gly Xaa 3 i Ser Ser Gly Ala Xaa 36 -Z 2 , Gly Gly Xaa 3 i Ser Ser Gly
  • exendin analogues specifically contemplated are those wherein a replacement for the histidine corresponding to Xaai is made with any of D- histidine, desamino-histidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine.
  • D- histidine desamino-histidine
  • 2-amino-histidine 2-amino-histidine
  • beta-hydroxy-histidine homohistidine.
  • exendin analogues include those described in U.S. Patent 6528486 (incorporated herein by reference and for all purposes).
  • exendin analogues include those consisting of an exendin or exendin analogue having at least 90% homology to exendin-4 having optionally between one and five deletions at positions 34-39, and a C-terminal extension of a peptide sequence of 4- 20 amino acid units covalently bound to said exendin wherein each amino acid unit in said peptide extension sequence is selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, and Met.
  • the extension is a peptide sequence of 4-20 amino acid residues, e.g., in the range of 4- 15, more preferably in the range of 4-10 in particular in the range of 4-7 amino acid residues, e.g., of 4, 5, 6, 7, 8 or 10 amino acid residues, where 6 amino acid residues are preferred.
  • the extension peptide contains at least one Lys residue, and is even more preferably from 3 to 7 lysines and even most preferably 6 lysines.
  • one analogue is
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPP SKKKKKK also designated ([des- 36 Pro]exendin-4(l-39)-Lys6 or lixisenatide).
  • Additional exemplary analogues include Lys 6 -His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln- Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Ser-(Lys) 6 (H-Lys 6 -des Pro 36 exendin-4(l-39)-Lys 6 ); His- Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu
  • repetition of an amino acid can be indicated by a subscripted number setting forth the number of repetitions; i.e., Lys 6 , (Lys) 6 and the like refer to hexalysyl.
  • Lys 6 a replacement for the histidine corresponding to position 1 is made with any of D-histidine, desamino-histidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine.
  • exendin analogues suitable for use in the methods described herein are described in published PCT application WO2004035623 (incorporated herein by reference and for all purposes), particularly those comprised of naturally- occurring amino acids, which describes exendin analogues having at least one
  • Amylin is a peptide hormone co-secreted with insulin by pancreatic beta-cells after nutrient ingestion whose primary physiological roles involve the inhibition of feeding behavior and gastric emptying, and subsequently reduced body weight, as well as lowering meal-related blood glucose levels.
  • Amylin also referred to islet (or insulinoma) amyloid polypeptide
  • islet (or insulinoma) amyloid polypeptide is a 37-residue, amidated peptide hormone.
  • Amylin also acts in bone metabolism, along with the related peptides calcitonin and calcitonin gene related peptide.
  • the human form of IAPP has the amino acid sequence
  • KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH2 with a disulfide bridge between cysteine residues 2 and 7. Both the amidated C-terminus and the disulfide bridge are necessary for the full biological activity of amylin.
  • Amylin compounds including rat amylin and pramlintide and davalintide are reported to reduce body weight in animals and/or humans, and thus have been proposed for treating obesity and obesity-related disorders.
  • amylin is meant an amylin receptor agonist that is a naturally-occurring form of amylin, either rat, human or from any other species.
  • the sequence of amylin is highly preserved across mammalian species, with structural similarities to calcitonin.
  • the sequence of rat amylin (amidated) is
  • amylin analogue as used herein is meant an amylin receptor agonist that has at least 50% sequence identity, preferably at least 70% sequence identity, to a naturally-occurring form of amylin, either rat or human or from any other species, or to davalintide, and is derived from them by modifications including insertions, substitutions, extensions, and/or deletions of the reference amino acid sequence.
  • Davalintide is an amylin agonist peptide chimera of amylin and sCT, having the sequence KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH2 (Cmpd 6).
  • the amylin analogue sequence can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% amino acid sequence identity with the reference amylin or davalintide.
  • the analogue has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or even 16 amino acid substitutions, insertions, extensions, and/or deletions relative to the reference compound.
  • the amylin analogue may comprise conservative or non-conservative amino acid substitutions (including non- natural amino acids and L and D forms). These analogues are preferably peptides, peptide derivatives or less preferably peptide mimics. Since amylin and davalintide are peptides, typical amylin analogues will be peptides, especially of 32-37 amino acids, e.g. 27 to 45, especially 28 to 38, and even 31-36.
  • Amylin analogues with identity to rat and human amylin include 25 ' 28 ' 29 Pro-h-amylin (pramlintide); des ⁇ Lys-h-amylin; 25 Pro, 26 Val, 28 ' 29 Pro-h-amylin; 18 Arg, 25 ' 28 Pro-h- amylin; des- 1 Lys, 18 Arg, 25 ' 28 Pro-h amylin; 18 Arg, 25 ' 28 ' 29 Pro-h-amylin; des- 1 Lys, 18 Arg, 25 ' 28 ' 29 Pro-h-amylin; des- 1 ,Lys 25 ' 28 ' 29 Pro-h-amylin; 25 Pro, 26 Val, 28 ' 29 Pro-h- amylin; 28 Pro-h-amylin, 2,7-Cyclo-[ 2 Asp, 7 Lys]-h-amylin; 2 " 37 h-amylin; 1 Ala-h- am
  • amylin analogue component includes an amino acid sequence of residues 1-37 of Formula (III) following, wherein up to 25% of the amino acids set forth in Formula (III) may be deleted or substituted with a different amino acid:
  • X' is hydrogen, an N-terminal capping group, a bond to an exendin
  • Xaa is Lys or a bond
  • Xaa is Lys
  • Xaa is Lys, Cys, or Gly
  • Xaa is Lys, Cys, or Pro
  • Xaa is Lys, Cys,
  • Xaa is Lys, Cys, or Leu
  • Xaa is Lys, Cys, or Pro
  • Xaa is Lys, Cys, or Pro
  • Xaa is Lys, Cys, or Asn.
  • the variable X represents a C-terminal functionality (e.g., a C-terminal cap).
  • X is substituted or unsubstituted amino, substituted or unsubstituted alkylamino, substituted or unsubstituted dialkylamino, substituted or unsubstituted cycloalkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted aralkylamino, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, substituted or unsubstituted aralkyloxy, or hydroxyl.
  • the exendin or exendin analogue is covalently linked, optionally through a linker, to a side chain of an internal amino acid residue of the amylin analogue. If the C- terminal of the polypeptide component with the sequence of residues 1-37 of Formula (III) is capped with a functionality X, then X is preferably amine thereby forming a C-terminal amide. In some embodiments, up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or even 50% of the amino acids of residues 1-37 of Formula (III) are deleted or substituted in a polypeptide component according to Formula (III).
  • the amylin analogue component has 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or even 16 amino acid substitutions relative to the amino acid sequence set forth in Formula (III).
  • the amylin analogue has a sequence which has a defined sequence identity with respect to the residues 1-37 of the amino acid sequence according to Formula (III).
  • the sequence identity between a polypeptide component described herein and residues 1-37 of Formula (I) is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even higher.
  • amino acids set forth in residues 1-37 of Formula (III) may be deleted or substituted with a different amino acid.
  • the sequence identity is within the range 75%-100%. In some embodiments, the sequence identity is within the range 75%-90%. In some embodiments, the sequence identity is within the range 80%-90%. In some embodiments, the sequence identity is at least 75%.
  • the polypeptide component of the conjugate has the sequence of residues 1-37 of Formula (III).
  • the amylin analogue of Formula (III) has the sequence of pramlintide, in others the sequence of davalintide. It has reported that davalintide, a second-generation analogue of amylin for the treatment of obesity, did not provide an improved weight loss efficacy and tolerability profile over pramlintide in a Phase 2 human clinical study.
  • the dual receptor polypeptides of the present formulations preferably comprise davalintide or closely related analogue thereof, surprisingly providing superior potency and efficacy, with potentially fewer side effects, than those containing amylin or pramlintide, particularly when compared as a fusion polypeptide with exendin-4 or analogue including Leul4 exendin-4 (Cmpd 4).
  • amylin analogues which are derivable, from davalintide include those disclosed in publications WO 2006/083254 and WO 2007/114838, each of which is incorporated by reference herein in its entirety and for all purposes.
  • amylin analogues also include 32 amino acid human calcitonin (hCT) having the sequence
  • CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP and species variants thereof, including salmon calcitonin (sCT) having the sequence
  • An analogue may have superior chemical stability, physical stability, protease resistance, solubility, efficacy, half-life, and the like
  • the amylin or amylin analogue can be conjugated or fused to the exendin or exendin analogue or to the GLP-1 or GLP-1 analogue by a covalent linker in any manner known in the art.
  • the linker structure which since serves primarily as a spacer, is preferably flexible, chemically and proteolytically stable, capable of little or no interaction with amino acids in either the exendin or amylin component, and more preferably the linker is also a peptide comprising naturally-occurring amino acids compatible with recombinant production.
  • the linker can affect and be useful in optimizing pharmacological activity in some embodiments as described herein.
  • a linker can be one or more amino acid residues, typically from about 1 to about 50 amino acid residues, more typically 1-30 amino acid residues, preferably about 1-20 amino acid residues, more preferably about 1-10, more preferred about 3-10, and even more preferably 3 amino acids, with Gly-Gly-Gly being most preferred, as used in the most preferred dual receptor agonist described above.
  • the amino acid residues in the linker are from among the twenty canonical (i.e., physiologic, naturally-occurring) amino acids, more preferably, cysteine, glycine, alanine, proline, asparagine, glutamine, and /or serine.
  • a peptidyl linker is made up of a majority of amino acids that are sterically unhindered, such as glycine, serine, and alanine linked by a peptide bond. It is also desirable that, if present, a peptidyl linker be selected that avoids rapid proteolytic turnover in circulation in vivo.
  • preferred linkers include polyglycines, polyserines, and polyalanines, or combinations of any of these.
  • beta-amino acids e.g., ⁇ -ala
  • linkers include (P-ala) n , where n is 1 to 20, preferably 1 to 10, more preferably 1 to 4.
  • linkers or moieties for use in a linker include a short or medium chain alkyl; 1-10 PEG repeating units; bifunctional linker (see, e.g., Pierce catalog, ockford, II); aminocaproyl ("Aca”) and 8-amino-3,6-dioxaoctanoyl.
  • Some additional exemplary peptidyl linkers are poly(Gly)i -8 , particularly (Gly) 3 , (Gly) 4 , (Gly) 5 (Gly) 6 and (Gly) 7 , as well as, poly(Gly) 4 Ser, poly(Gly-Ala) 2-4 and poly(Ala) 2-8 .
  • Other specific examples of peptidyl linkers include (Gly) 5 Lys and
  • linkers include: (Gly) 3 Lys(Gly) 4j
  • the linkers include the following peptide linker sequences: GGEGGG, GGEEEGGG, GEEEG,
  • the linker contains a cysteine or homocysteine residue, or other 2-amino- ethanethiol or 3-amino-propanethiol moiety for conjugation to maleimide, iodoacetaamide or thioester, functionalized half-life extending moiety.
  • Another useful peptidyl linker is a large, flexible linker comprising a random Gly/Ser/Thr sequence, for example:
  • GSGSATGGSGSTASSGSGSATH or HGSGSATGGSGSTASSGSGSAT that is estimated to be about the size of a 1 kDa polyethylene glycol (PEG) molecule.
  • a useful peptidyl linker may be comprised of amino acid sequences known in the art to form rigid helical structures (e.g., Rigid linker: -
  • a peptidyl linker can also comprise a non-peptidyl segment such as a 6 carbon aliphatic molecule of the formula - (CH 2 )6-.
  • non-peptidyl linkers are also useful for conjugating the two components.
  • alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., Ci-C 6 ) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc.
  • non-peptidyl linkers are PEG linkers as known in the art and/or described herein.
  • Either or both peptides in the dual receptor agonist can be conjugated to the other through a side-chain of an internal or terminal amino acid or through the peptide's backbone, i.e. via the terminal alpha amino or carboxy groups.
  • the N-terminal amino acid of the amylin or amylin analogue e.g.
  • davalintide is conjugated or fused at the C-terminal amino acid of the exendin or exendin analogue or to the GLP-1 or GLP-1 analogue. More preferably the linkage is via the N-terminal alpha amino group of the amylin or amylin analogue and the C- terminal alpha carboxy group of the exendin or exendin analogue or the GLP-1 or GLP-1 analogue, even more preferably through a peptide linker creating a fusion polypeptide that can be recombinantly produced.
  • the dual receptor agonist compound will have a peptide backbone from 50 to 120 amino acids, preferably from 55 to 105, more preferably 60 to 100, even more preferably 65 to 80, and most preferably 74 to 76 amino acids.
  • the dual receptor agonist compound will have a GLP1 receptor agonism potency from 0.1 to 10 times that of the most preferred dual receptor agonist Cmpd 1, preferably from 0.2 to 5 times, more preferably from 0.4 to 3 times, even more preferably 0.5 to 2 times.
  • the dual receptor agonist compound will have an amylin receptor agonism potency from 0.1 to 10 times that of the most preferred dual receptor agonist Cmpd 1, preferably from 0.2 to 5 times, more preferably from 0.4 to 3 times, even more preferably 0.5 to 2 times.
  • the dual receptor agonist compound will have potency from 0.1 to 10 times, preferably from 0.2 to 5 times, more preferably from 0.4 to 3 times, even more preferably 0.5 to 2 times that of the most preferred dual receptor agonist Cmpd 1 for at least one or more in vivo biological activities of body weight loss, acute plasma glucose lowering (as in an oral glucose tolerance test), insulin secretion, delaying of gastric emptying, HbAlc lowering and/or inhibition of food intake.
  • Additonal dual receptor agonists include previously described conjugates of an exendin analog and an amylin analogue include Cmpd 3 having the sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGGKCNTATCVLGRLSQELHRLQ TYPRTNTGSNTY-NH2 (Cmpd 3) where the polypeptide is C-terminally amidated, and Cmpd 7 having the sequence
  • HGEGTFTSDLSKQMEEEAVRLFIEWLKN(beta-A)(beta- A)KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH2 (Cmpd 7) where the polypeptide is C-terminally amidated and contains a beta-alanine, beta-alanine unnatural amino acid dipeptide linker.
  • the above compounds comprise an active C- terminally truncated form of exendin-4: exendin-4(l-28) amide (Cmpd 10).
  • Cmpd 1 is the most preferred dual amylin receptor/GLP-1 receptor agonist. Surprisingly, despite nausea associated with exendin-4 and with davalintide, Cmpd 1 displayed no significant kaolin intake in a rat study, which is accepted as a predictor of nausea in humans. In addition Cmpd 1 displays a high potency for both the GLP- 1 receptor and the amylin and calcitonin receptors, demonstrating that their exendin- like and davalintide moieties retain their biological activities. The activities for these target receptors are only moderately attenuated compared with the parent compounds, exendin-4 and davalintide.
  • Cmpd 1 binds the CGRP receptor poorly if at all, and thus displays better selectivity than prior dual receptor agonist compounds for the amylin receptor (> 600 fold versus > 100 fold, respectively) and calcitonin receptors (> 1600 fold versus > 280 fold) against the
  • Cmpd 1 did not display functional activation or antagonism of the adrenomedullin receptor at concentrations up to 10 uM. Accordingly, Cmpd 1 presents a surprisingly different
  • Cmpd 1 has fewer off-target activities than the parent peptide.
  • This improved pharmacological profile for Cmpd 1 is expected to result in decreased side-effects, such as reduced severe flushing, nausea and/or vomiting, particularly with human subjects, as compared to davalintide.
  • side-effects such as reduced severe flushing, nausea and/or vomiting, particularly with human subjects, as compared to davalintide.
  • CGRP and CGRP agonists have been reported to induce severe flushing, and even nausea and vomiting, in human subjects, which is believed in part due to activation of CGRP receptors and which is relieved by CGRP antagonists. It is expected that Cmpd 1 will have increased patient compliance and/or allow increased dosing as needed compared to previous compounds, for example compared to davalintide, resulting in improved commercial success.
  • Dual receptor agonists are peptide compounds having at least two distinct domains wherein one domain serves as an agonist for amylin receptor and another serves as an agonist for GLP-1 receptor. Such dual agonists are distinct from a single non-specific agonist in that, although the domains may and preferably will be covalently bound together, the domain serving as amylin receptor agonist resides on a distinct portion of the peptide sequence from the domain serving at the GLP-1 receptor agonist.
  • the dual agonist is a compound in which a peptide sequence having amylin receptor function and substantially no GLP-1 agonist function is chemically linked (directly or indirectly) to a sequence having GLP-1 agonist function and substantially no amylin receptor agonist function.
  • the dual amylin receptor/GLP-1 receptor agonist active agent (e.g. Cmpd 1) will generally be formulated as 0.02 to 12% by weight of the total formulation. Typical values will be 0.1 to 10%, preferably 0.2 to 8% and more preferably 0.5 to 6%, and most preferably 1 to 3%, by weight of the formulation. These levels may be applied to all aspects of the invention, where context allows.
  • the dual amylin receptor/GLP-1 receptor agonist active agent e.g. Cmpd 1
  • the dual amylin receptor/GLP-1 receptor agonist active agent (e.g. Cmpd 1) will be formulated at a level which cannot easily be achieved in the absence of water and/or propylene glycol.
  • the dual amylin receptor/GLP-1 receptor agonist active agent (e.g.
  • Cmpd 1) content is typically at least 0.7%, preferably at least 1%, more preferably at least 2% by weight of formulation, and even more preferably at 2.5%, such that also preferable load of active is 1 to 3 % by weight. Levels of at least 3% and at least 4% are achievable with the present invention, as are loading levels up to 8, 10 or 12%.
  • Such compositions of the present invention typically not only contain a very high level of dual amylin receptor/GLP-1 receptor agonist active agent (especially Cmpd 1), as indicated, but are additionally stable to storage with minimal degradation of the active agent for considerable periods, as indicated herein.
  • Such periods will generally be at least a month at 25°C or at 5°C, preferably at least 3 months, and more preferably at least 6 months at 5°C or alternatively at 25°C. These degrees of stability are applicable to all aspects of the invention, where context allows.
  • compositions of the present invention rely upon the effect of the water and/or propylene glycol to allow for a loading of dual amylin
  • receptor/GLP-1 receptor agonist active agent e.g. Cmpd 1
  • a high loading level is highly advantageous and has been surprisingly established by the present inventors that by including the water and/or PG as specified herein, in the amounts indicated, a much higher loading of hybrid polypeptide active agents (particularly Cmpd 1) can be obtained (see examples).
  • the level of dual amylin receptor/GLP-1 receptor agonist active agent (e.g. Cmpd 1) which could be loaded in a composition is easily established by equilibration of the composition with excess active agent (e.g. by slow end-over-end rotation for 5 days at 25°C ).
  • compositions can and preferably do contain a greater amount of dual amylin receptor/GLP-1 receptor agonist active agent(p articular ly Cmpd 1) than can be achieved by equilibration in the absence of the water and/or PG component. This can apply in all aspects of the present invention, where context allows.
  • Suitable doses of dual amylin receptor/GLP-1 receptor agonist active agent (such as Cmpd 1) for inclusion in the formulation, and thus the volume of formulation used, will depend upon the release rate (as controlled, for example by the solvent type and amount used, and so forth) and release duration, as well as the desired therapeutic level, the activity of the specific agent, and the rate of clearance of the particular active chosen.
  • an amount of active around 0.05 to 60 mg per week of depot duration typically 0.5 mg to 60 mg per week, 0.05 to 40 mg per week, or 5 to 35 mg per week duration, and even 5 to 25 mg per week, for a period of administration of at least 1 to 24 weeks, preferably at least 2 to 16 (e.g. 8, 10 or 12) weeks.
  • the stability of the active and linearity of the release rate will mean that the loading to duration may not be a linear relationship.
  • a depot administered every 7 days might have, for example 0.05 to 60 mg of active agent (e.g.
  • Cmpd 1 while a 14 day depot may have more than twice the amount in the event that not 100% is released or may have less than twice that amount in the event that higher release in the earlier part of the cycle consumes additional active agent.
  • the biological half-life of the specific active will be particularly important.
  • the dose of dual amylin receptor/GLP-1 receptor agonist active agent may be insufficient to provide a therapeutic concentration of active agent during the dosing period when administered in isolation but sufficient to provide a therapeutic concentration when a regime of regular dosing is undertaken. Such a regime will typically be
  • Cmax ⁇ Cther, and optionally Caver ⁇ Cther where Cmax is the maximum plasma concentration generated by a single administration and Cther is the desired therapeutic concentration.
  • CmaxM > Cther and CaverM > Cther, where CmaxM is the maximum plasma concentration achieved after multiple periodic dosings (once every dosing period) and CaverM is the average plasma concentration achieved after multiple periodic dosings (once every dosing period).
  • lipids and biologically active agents are thermodynamically unstable to oxidation.
  • lipid formulations including those comprising bioactive agents such as APIs are susceptible to degradation upon storage, especially by oxidation.
  • the lipid matrix aspect is soy PC, GDO, ethanol/propylene glycol or mixtures thereof, and water, and the hybrid polypeptide hormone active agent.
  • appropriate amounts of each component suitable for the combination are those amounts indicated herein for the individual components, in any combination.
  • Component f) is an antioxidant. In a most preferred embodiment, component f) is present (i.e. it is mandatory not optional) and is selected from ascorbic acid, ethylenediaminetetraacetic acid (EDTA) and citric acid.
  • EDTA ethylenediaminetetraacetic acid
  • component f) is typically present at a molar ratio of antioxidant to dual amylin receptor/GLP-1 receptor agonist active agent of 1 : 10 to 1 : 10000 (e.g. 1 :50 to 1 :5000), preferably 1 : 100 to 1 : 1300, and most preferably 1 : 150 to 1 : 1250.
  • typical antioxidants are of lower molecular weight that the hybrid polypeptide active agents, the proportion by weight of antioxidant may be relatively small.
  • EDTA and similar agents e.g. less than 500 amu
  • 0.001 to 5% of the composition may be antioxidant, preferably 0.002 to 2%, more preferably 0.002 to 0.15%, e.g. 0.002 to 0.15%.
  • antioxidants are not highly compatible with lipid systems. Indeed, the present inventors have previously established that some antioxidants commonly used in previous systems can cause increased degradation of active agents in a lipid system. This applies particularly to peptide active agents. The present inventors have therefore analysed a variety of potential antioxidant compounds and classes for use with lipid based matrix systems and have surprisingly found that one particular class of antioxidants is unusually well suited for use in these systems.
  • the antioxidant component is generally included in the range 0.001 to 5% by weight of the total pre-formulation. Around 0.002 to 0.15% of antioxidant (particularly
  • EDTA is particularly preferred, especially in combination with the other preferred components and ranges indicated herein above and below.
  • EDTA as antioxidant can also show a synergistic effect in combination with the antioxidants of the present invention, in maintaining the chemical and physical stability of the dual amylin receptor/GLP-1 receptor agonist active agent and complete pre-formulation.
  • EDTA has a stabilising effect on the active agent. This is demonstrated by the examples, particularly figure 1, in which the stability of formulations B10, B 12 and B16 are demonstrably better than those of the corresponding formulations not containing EDTA. It is notable that composition B09 and B10 differ only by the presence or absence of EDTA and compositions Bl 1 and B12 differ from each other in the same way.
  • stabilising is indicated an increase in the stability of the composition, especially with regard to the physical and chemical stability of the dissolved or dispersed active agent. Such an increase in stability may be demonstrated by the chemical and/or physical stability of a dual amylin receptor/GLP-1 receptor agonist active agentin a lipid formulation for a greater period than would be observed in the absence of an antioxidant. This would preferably be tested under conditions of typical storage, such as 0-5°C, 25°C and/or ambient temperature. This is further described herein below.
  • the pre-formulations of the present invention are generally formulated to be administered parenterally.
  • This administration will generally not be an intra- vascular method but will preferably be subcutaneous (s.c), intracavitary or intramuscular (i.m.).
  • the administration will be by injection, which term is used herein to indicate any method in which the formulation is passed through the skin, such as by needle, catheter, or needle-less (needle-free) injector.
  • Preferred parenteral administration is by i.m or s.c. injection, most preferably by deep s.c. injection.
  • An important feature of the composition of the invention is that it can be administered both by i.m. and s.c. and other routes without toxicity or significant local effects. It is also suitable for intracavital administration.
  • the deep s.c. injection has the advantage of being less deep and less painful to the subject than the (deep) i.m. injection used for some current depots and is technically most suitable in the present case as it combines ease of injection with low risk of local side effects. It is a surprising observation of the present inventors that the formulations provide sustained release of active agent over a predictable time period by subcutaneous injection. This therefore allows the site of injection to be varied widely and allows the dose to be administered without detailed consideration of the tissue depth at the site of injection.
  • non-lamellar is used to indicate a normal or reversed liquid crystalline phase (such as a cubic or hexagonal phase) or the L3 phase or any combination thereof.
  • liquid crystalline indicates all hexagonal, all cubic liquid crystalline phases and/or all mixtures thereof.
  • Hexagonal as used herein indicates "normal” or “reversed” hexagonal (preferably reversed) and "cubic” indicates any cubic liquid crystalline phase unless specified otherwise.
  • ratio of components a:b are in the region of equality (e.g. ranges around 30:40 to 40:30, more preferably 33:38 to 38:33). Accordingly, in the present formulations the ratio of components a:b are in the region of equality (1 : 1), including 1 :2 to 2: 1 ratios, but each of a) and b) are also preferably expressed as the range of absolute weight percentages of 30 to 40%, more preferably 33 to 38%, and even specifically 33.7, 34.8, 35.0 and 37.7%.
  • preferred formulation ratios of a:b that are in the region of equality when expressed as the desired absolute weight percentages are ratios of a:b where component a) is 30-40% and b) is 30-40%), where a) is 33 to 38% and b) is 33 to 38%, and even more specifically where a) and b) are each any one of 33.7, 34.8, 35.0 and 37.7%, for example more preferably where ratio of a:b is 37.7:37.7, 35.0:35.0, 34.8:34.8 and 33.7:33.7. It is important to appreciate that the pre-formulations of the present invention are of low viscosity.
  • these pre-formulations must not be in any bulk liquid crystalline phase since all liquid crystalline phases have a viscosity significantly higher than could be administered by syringe or spray dispenser.
  • the pre- formulations of the present invention will thus be in a non-liquid crystalline state, such as a solution, L 2 or L 3 phase, particularly solution or L 2 .
  • the L 2 phase as used herein throughout is preferably a "swollen" L 2 phase containing greater than 10 wt% of solvent (component c) having a viscosity reducing effect.
  • the term "low viscosity mixture” is used to indicate a mixture which may be readily administered to a subject and in particular readily administered by means of a standard syringe and needle arrangement. This may be indicated, for example by the ability to be dispensed from a 1 ml disposable syringe through a small gauge needle.
  • the low viscosity mixtures can be dispensed through a needle of 19 awg, preferably smaller than 19 awg, more preferably 23 awg (or most preferably even 27 awg) needle by manual pressure.
  • the low viscosity mixture should be a mixture capable of passing through a standard sterile filtration membrane such as a 0.22 ⁇ syringe filter.
  • a typical range of suitable viscosities would be, for example (all at 20°C), 0.1 to 5000 mPas, (e.g. 5 to 2000 mPas) preferably 1 to 1000 mPas, more preferably 1 to 500 mPas and most preferably 1 to 350 mPas at 20°C, for example 250-350 mPas at 20 °C.
  • the preferred lipid-based pre-formulations of the present invention undergo a phase structure transition from a low viscosity mixture to a high viscosity (generally tissue adherent) depot composition.
  • a phase structure transition from a low viscosity mixture to a high viscosity (generally tissue adherent) depot composition.
  • this will be a transition from a molecular mixture, swollen L 2 and/or L 3 phase to one or more (high viscosity) liquid crystalline phases such as normal or reversed hexagonal or cubic liquid crystalline phases or mixtures thereof.
  • Further phase transitions may also take place following administration.
  • complete phase transition is not necessary for the functioning of the invention but at least a surface layer of the administered mixture will form a liquid crystalline structure.
  • this transition will be rapid for at least the surface region of the administered formulation (that part in direct contact with air, body surfaces and/or body fluids). This will most preferably be over a few seconds or minutes (e.g. from 1 second up to 30 minutes, preferably up to 10 minutes, more preferably 5 minutes of less). The remainder of the composition may change phase to a liquid crystalline phase more slowly by diffusion and/or as the surface region disperses.
  • the pre-formulations of the invention upon exposure to excess aqueous fluid, lose some or all of the organic solvent included therein (e.g. by diffusion) and take in aqueous fluid from the bodily environment (e.g. the in vivo environment).
  • the bodily environment e.g. the in vivo environment
  • at least a part of the formulation preferably generates a non-lamellar, particularly liquid crystalline phase structure. In most cases these non-lamellar structures are highly viscous and are not easily dissolved or dispersed into the in vivo environment. The result is a monolithic "depot" generated in vivo with only a limited area of exposure to body tissue.
  • the lipid depot is highly effective in solubilising and stabilising active agents such as peptides and protecting these from degradation mechanisms.
  • active agents such as peptides and protecting these from degradation mechanisms.
  • the depot composition formed from the pre-formulation gradually degrades over a period of days, weeks or months, the active agent is gradually released and/or diffuses out from the composition. Since the environment within the depot composition is relatively protected, the pre-formulations of the invention are highly suitable for active agents with a relatively low biological half-life (see above).
  • the depot systems formed by the formulations of the present invention are highly effective in protecting the active agent from degradation and thus allow an extended release period.
  • the formulations of the present invention give very low degradation under simulated in vivo conditions.
  • the formulations of the invention thus may provide in vivo depots of dual amylin receptor/GLP-1 receptor agonist active agents which require administration only once every 3 to 60 days (e.g. 5 to 30 days), preferably 5 to 14 days (e.g. 7 to 14 days), preferably once every 7 days (once every "dosing period").
  • a longer stable release period is desirable for patient comfort and compliance, as well as demanding less time from health professionals if the composition is not to be self-administered.
  • composition is to be self- administered
  • patient compliance may be aided by a weekly (e.g. every 7 days, optionally ⁇ 1 day) or monthly (e.g. every 28 or 30 days (optionally ⁇ 7 days)) administration so that the need to administer is not forgotten.
  • weekly e.g. every 7 days, optionally ⁇ 1 day
  • monthly e.g. every 28 or 30 days (optionally ⁇ 7 days)
  • the administration is once weekly.
  • a considerable advantage of the depot precursors of the present invention is that they are stable homogeneous phases. That is to say, they may be stored for considerable periods (preferably at least 6 months) at refrigerator temperature (e.g. 0-4°C, 0-5°C, and even 2-8°C ), without phase separation.
  • refrigerator temperature e.g. 0-4°C, 0-5°C, and even 2-8°C
  • this allows for the dose of hybrid polypeptide hormone active agent (e.g. Cmpd 1) to be selected by reference to the species, age, sex, weight, and/or physical condition of the individual subject, by means of injecting a selected volume.
  • the present invention thus provides for methods comprising the selection of a dosing amount specific to an individual, particularly by subject weight.
  • the means for this dose selection is the choice of administration volume.
  • the composition may be necessary for the composition to equilibrate repeatedly at differing temperatures.
  • the device will be refrigerated, then equilibrated, then re-refrigerated several times over the lifetime of the device.
  • Such devices therefore will have a longer time at high temperature and the high temperature stability of the formulation must be sufficient for the relevant number of equilibration cycles, for example, up to 12, preferably up to 6.
  • the active agent counterion is a halide, particularly chloride, as this has been observed to give improved high temperature stability, as demonstrated in the current examples.
  • the present invention provides a pre-formulation comprising components a), b), c), d), f) and at least one dual amylin receptor/GLP-1 receptor agonist active agent (e.g. Cmpd 1) as indicated herein.
  • the amounts of these components will typically be in the range 30-70% a), 30-60% b), 5-35% c) and 0.1- 20% d), with the dual amylin receptor/GLP-1 receptor agonist active agent (e.g. Cmpd 1) present at 0.01% to 10%.
  • a highly preferred combination is 30-40% a), 30-40%) b), 15-25%) c) and 2-15% d), with the hybrid polypeptide hormone active agent (e.g. Cmpd 1) present at 1% to 3%.
  • component f) is present at an antioxidant to dual amylin receptor/GLP-1 receptor agonist active agent molar ratio of 1 : 10 to 1 : 10000, preferably 1 : 100 to 1 : 1300, and most preferably 1 : 150 to 1 : 1250.
  • typical antioxidants are of lower molecular weight than hybrid polypeptide hormone active agent (e.g. Cmpd 1), the proportion by weight of antioxidant may be relatively small.
  • 0.001 to 5% of the composition may be antioxidant, preferably 0.002 to 2%, more preferably 0.002 to 0.15%, e.g. 0.002 to 0.015%.
  • the pre-formulations of the present invention are highly advantageous in that they are stable to prolonged storage in their final "administration ready” form. As a result, they may readily be supplied for administration either by health professionals or by patients or their carers, who need not be fully trained health professionals and may not have the experience or skills to make up complex preparations. This is particularly important in long-duration, slow-effecting diseases such as diabetes.
  • the present invention provides a disposable administration device (which is also to include a device component) pre-loaded with a measured dose of a pre-formulation of the present invention.
  • a disposable administration device which is also to include a device component
  • Such a device will typically contain a single dose ready for administration, and will generally be sterile-packed such that the composition is stored within the device until administration.
  • Suitable devices include cartridges, ampoules and particularly syringes and syringe barrels, either with integral needles or with standard (e.g. luer) fittings adapted to take a suitable disposable needle, or prefilled syringes with preattached needles.
  • kits for the administration of at least one hybrid polypeptide active agent may also suitably be included in an administration kit, which kit also forms a further aspect of the invention.
  • the invention thus provides a kit for the administration of at least one hybrid polypeptide active agent, said kit containing a measured dose of a
  • kits may include additional administration components such as needles, swabs, etc. and will optionally and preferably contain instructions for administration. Such instructions will typically relate to administration by a route as described herein and/or for the treatment of a disease indicated herein above.
  • the device may be single- or multi-use.
  • the multi- use device may provide once-weekly doses for a period of at least 2, 3, 4 or even 8 weeks.
  • each administered dose is a total volume for administration of no more than 3 ml, preferably no more than 1 ml more preferably no more than 0.5 ml, and more preferably no more than 0.1 ml. Further, the volume is preferably no less than 0.04 ml, more preferably no less than 0.06 ml, and even not less than 0.1 ml.
  • the pre- formulations of the invention may have one or more of the following preferred features independently or in combination:
  • Component a) comprises, consists essentially of or preferably consists of GDO;
  • Component b) comprises, consists essentially of or preferably consists of soy PC;
  • Component c) comprises, consists essentially of or preferably consists of a 1, 2, 3 or 4 carbon alcohol, preferably isopropanol or more preferably ethanol, propylene glycol or mixtures thereof;
  • Component f) comprises, consists essentially of or preferably consists of
  • EDTA ethylenediaminetetraacetic acid
  • component f is present.
  • the pre-formulation contains CMPD 1 as defined herein.
  • the pre-formulation has a low viscosity as indicated herein.
  • the pre-formulation forms a liquid crystalline phase as indicated herein upon in vivo administration.
  • the pre-formulation generates a depot following in vivo administration, which depot releases at least one active agent at a therapeutic level over a period of at least 3 days, preferably at least 5 days, more preferably at least 7 days
  • the pre-formulation has a higher loading of dual amylin receptor/GLP-1 receptor agonist active agent (e.g. Cmpd 1) than is stable in the same formulation in the absence of component e).
  • the pre-formulation has a higher loading of dual amylin receptor/GLP-1 receptor agonist active agent (e.g. Cmpd 1) than is obtainable by equilibration at 25°C of the same formulation in the absence of water and propylene glycol.
  • the method(s) of treatment of the present invention may have one or more of the following preferred features independently or in combination:
  • the method comprises the administration of at least one formulation with one or more preferred features as indicated above;
  • the method comprises the administration of at least one formulation as indicated herein by i.m., s.c. or preferably deep s.c. injection;
  • the method comprises administration by means of a pre-filled administration device as indicated herein;
  • the method comprises administration through a needle no larger than 20 gauge, preferably smaller than 20 gauge, and most preferably 23 gauge or smaller, including 25 gauge;
  • the method comprises a single administration each dosing period, such as every 3 to 30 days, preferably 5 to 14 days, more preferably 7 ⁇ 1 days.
  • the use(s) of the pre-formulations indicated herein in the manufacture of medicaments may have one or more of the following preferred features independently or in
  • the use comprises the use of at least one formulation with one or more preferred features as indicated above;
  • the use comprises the manufacture of a medicament for administration of at least one formulation as indicated herein by i.m., s.c. or preferably deep s.c. injection;
  • the use comprises the manufacture of a medicament for administration by means of a pre-filled administration device as indicated herein;
  • the use comprises the manufacture of a medicament for administration through a needle no larger than 20 gauge, preferably smaller than 20 gauge, and most preferably 23 gauge or smaller, including 25 gauge;
  • the use comprises the manufacture of a medicament for administration once every dosing period of 3 to 30 days, preferably 5 to 14 days, more preferably 7 ⁇ 1 days.
  • the pre- filled devices of the invention may have one or more of the following preferred features independently or in combination:
  • They contain a preferred formulation as indicated herein; They comprise a needle smaller than 20 awg, preferably no larger than 23 awg; They contain a single dose of 0.05 to 250 mg of hybrid polypeptide hormone active agent (e.g. Cmpd 1), preferably 0.1 to 100 mg and more preferably 1-50 mg
  • hybrid polypeptide hormone active agent e.g. Cmpd 1
  • compositions of the invention contain a homogeneous mixture of a composition of the invention in ready-to- inject form.
  • They contain a total volume for administration of no more than 3 ml, preferably no more than 1 ml more preferably no more than 0.5 ml, and more preferably no more than 0.1 ml. Further, the volume is no less than 0.04 ml, and preferably no less than 0.06 ml.
  • the at least one diacyl glycerol and/or a tocopherol is present at 33- 38% by weight or a value from Table 1.
  • the at least one diacyl glycerol is GDO.
  • the at least one PC is present at 33-38% by weight or a value from Table 1.
  • the at least one PC is soy PC or DOPC.
  • the at least one biocompatible, organic alcoholic solvent is present at 33-38% by weight or a value from table 1.
  • the at least one biocompatible, organic alcoholic solvent is ethanol and/or PG.
  • the at least one biocompatible, organic alcoholic solvent is ethanol and is present at 15% and PG is absent; or the at least one biocompatible, organic alcoholic solvent is a mixture of ethanol and PG wherein ethanol is present at 5% and PG is present at 15%.
  • the total aqueous solvent is present in a 15% to 20% range and/or a value from table 1.
  • the antioxidant is present in a 0.002 % to 0.015 % range and/or a value from Table 1.
  • the active agent is Cmpd 1.
  • pre-formulation selected from the group B9, B10, Bl 1, B12, B15 and B16.
  • the invention comprises a pre-formulation selected from B12 or B16.
  • Example 1 Depot precursors with the compositions presented in Table 1 were prepared by first preparing an excess of lipid stock solution, by mixing all the components except Cmpd 1 until a homogeneous solution was obtained; when EDTA was included in the samples, it was first dissolved in the required amount of water (final concentration 0.5 - 1 mg EDTA/mL) prior to mixing with the other excipients.
  • the final formulations were obtained by adding 14.625 g of the corresponding lipid stock solution to 0.375 g Cmpd 1 powder, mixing by brief vortex followed by end- over-end rotation for up to 24h giving clear, homogeneous and low viscosity lipid/Cmpd 1 formulations.
  • Table 1 Sample compositions according to the invention.
  • Formulations with compositions presented in Table 1 were filled into 0.5 mL glass syringes, which were then placed in cabinets at controlled storage conditions (5°C; 25°C/60% RH, or 40°C/75% RH). The content and purity of Cmpd 1 in the samples were evaluated (after equilibration of the samples to ambient temperature, when necessary) directly after manufacturing, and after 1; 2; and 3 months of storage in the mentioned condition, using an HPLC-UV/DAD method based on a Polysulfoethyl A (PolyLC Inc.) analytical column (cation-exchange) and a gradient of NaC10 4 in a mixture of acetonitrile, methanol, water and HC1.
  • Example 3 Methods for In Vivo Studies in DIO Rats The present examples characterized the metabolic actions of Cmpd 1, parent compounds and other dula receptor agonists, demonstrating activity of the dual receptor agonists and surprising superiority of Cmpd 1. As disclosed in the
  • mice Male Sprague Dawley rats (C L:CD rats, Charles River Laboratories, Wilmington, MA) were individually housed and maintained on high fat diet (32% kcal from fat; D12266B Research Diets, Brunswick, NJ) for approximately 8 weeks prior to the study. At the start of testing (day 0) average body weight of the rats was 545 ⁇ 3.8 g.
  • Cmpds used in the examples include the following:
  • Cmpd 4 Leul4 exendin-4 amide.
  • Cmpd 5 a chimera of the first 32 amino acids of exendin-4 having amino acid substitutions at positions 14 and 28 followed by a 5 amino acid sequence from the C-terminal of a non-mammalian (frog) GLP1;
  • Body composition was assessed on day -1 and day 28 using an NM instrument (Echo Medical Systems, Houston, TX). Adiposity (percent fat mass) was defined as the amount of fat mass relative to body weight (fat mass/body weight x 100). Blood was collected via tail vein on day 14. On day 28 a sample of blood was drawn via the jugular vein and animals were euthanized by isoflurane overdose. Mini-pumps were immediately removed, animals were subjected to a brief NMR scan, and tissues were collected for future histological examination and preliminary toxicological assessment. Statistical Analysis. Data were analyzed using one-way analysis of variance
  • Plasma levels of study drug were measured at day 14 and termination by an ELISA.
  • Whole blood percent hemoglobin Ale (%HbAlc), plasma triglyceride, total cholesterol, HDL cholesterol and plasma glucose at day 14 and at termination were measured using an Olympus bioanalyzer (Olympus America Diagnostics).
  • Plasma insulin levels at day 14 and at termination were analyzed by an ELISA kit (Rat/Mouse Insulin ELISA, Linco Diagnostics).
  • Example 4 Compound 1 Provides Superior Body Weight Loss
  • Cmpd 1 and Cmpd 2 dose-dependently reduced body weight significantly (p ⁇ 0.05 relative to vehicle) at 3, 10, 30 and 100 nmol/kg/d over the 28 day treatment (see Figures 2A and 2B).
  • Co-administration of the parent compounds significantly reduced body weight compared to vehicle controls and compared to Cmpd 6 alone (p ⁇ 0.05). See Figures 2A and 2B.
  • Cmpd 2 there was no dose-effect beyond 10 nmol/kg/d; further weight loss was not observed with doses of 30 or 100 nmol/kg/d. Maximal weight loss was achieved in the 100 nmol/kg/d group, at -27.8 ⁇ 5.3%. See Figure 2A.
  • Cmpd 2 was also several fold more potent at reducing body weight via sustained infusion than Cmpd 3.
  • Plasma parameters The effects of the parent peptides and the dual receptor agonist compounds on plasma parameters including lipids (triglycerides, total and HDL cholesterol) and glucose and concentration of the dual receptor agonist in plasma, at 14 and 28 days.
  • Cmpd 1 decreased HDL cholesterol at 10 and 30 nmol/kg/d, e.g. as compared to Cmpd 6.
  • Cmpd 1 did not alter total cholesterol. See Table 4. All doses of Cmpd 1, and parent peptides, decreased triglycerides relative to vehicle, and lowered glucose at 10 and 30 nmol/kg/d, as did Cmpd 4A+Cmpd 6A (see Table 4).
  • Dual receptor agonists and parent compounds were analyzed in an in vivo basal glucose lowering assay.
  • This assay reflects the conjugate polypeptide's ability to enhance insulin-mediated glucose clearance of orally administered glucose challenge (Oral Glucose Tolerance Test; OGTT).
  • OGTT Oral Glucose Tolerance Test
  • IP intraperitoneally
  • Pica behavior is a marker of nausea in rodents, typically associated with reduced food intake and weight loss. Pica can be assessed by measuring intake of the synthetic clay kaolin.
  • Cmpd 7 a previously known dual receptor agonist conjugate, at a dose that inhibited food intake, induced significant kaolin intake similar to the positive control, a sign of nausea in rats.
  • Cmpd 3 at doses that suppressed food intake acutely similar to cisplatin injection, had only a modest, if any, effect on kaolin consumption.
  • Cmpd 1 and Cmpd 2 at doses that elicited reductions in food intake even greater than the positive control, Cmpd 3 or 7, did not induce significant increases in kaolin consumption.
  • Cmpd 1 and Cmpd 2 displayed no significant kaolin intake in this study.
  • Cmpd 1 displays a high potency for both the GLP-1 receptor and the amylin and calcitonin receptors, demonstrating that their exendin-like and davalintide moieties retain their biological activities. The activities for these target receptors are only moderately attenuated compared with the parent compounds. Interestingly, Cmpd 1 binds the CGRP receptor with very low affinity, displaying better selectivity than
  • Cmpd 3 for amylin receptor and calcitonin receptors against the CGRP receptor, and even better than davalintide selectivity for binding to calcitonin and amylin receptors against the CGRP receptor.
  • davalintide being a potent adrenomedullin receptor antagonist, Cmpd 1 did not display functional activation or antagonism of the adrenomedullin receptor at concentrations up to 10 uM.
  • Cmpd 1 presents a surprisingly different pharmacological profile compared to davalintide with respect to cellular receptors that recognize amylin and amylinomimetics.
  • Cmpd 1 has fewer off-target activities than the parent peptide.
  • This improved pharmacological profile for Cmpd 1 is expected to result in decreased side-effects, such as reduced severe flushing, nausea and/or vomiting, particularly with human subjects, as compared to the parent peptide Cmpd 6.
  • side-effects such as reduced severe flushing, nausea and/or vomiting, particularly with human subjects, as compared to the parent peptide Cmpd 6.
  • CGRP and CGRP agonists have been reported to induce severe flushing, and even nausea and vomiting, in human subjects, which is believed in part due to activation of CGRP receptors and which is relieved by CGRP antagonists.
  • Table 6 presents percent inhibition of gastric emptying.
  • Cmpd 1 and Cmpd 2 were as efficacious as Cmpd 6 at inhibiting gastric emptying up to six hours after a single injection.
  • Cmpd 3 and Cmpd 7 did not significantly inhibit gastric emptying at the time point and doses tested.
  • Cmpd 1 provided a longer duration of action compared to Cmpd 2.
  • Cmpd 1 which comprises Cmpd4, in the assays provided herein is surprising. Hargrove et al. 2007 teaches that exendin analogue Cmpd 4 displays a markedly less potent delay of gastric emptying (4 fold less), a markedly less potent inhibition of food intake (8 fold less), a shorter half-life and a shorter duration of action compared to exendin-4.
  • Cmpd 1 presents surprisingly superior pharmacological properties compared to Cmpd 2 and previously known conjugates, such as robust and longer acting inhibition of gastric emptying activity as well as a surprisingly robust and long acting reduction of food intake and reduction in body weight.
  • Cmpd 1 and Cmpd 2 have a more stable (at least two fold) metabolic profile in vitro in human plasma and with human kidney brush border membrane matrices over a 5 hr incubation period compared to exendin-4, Cmpd 4 and Cmpd 6 (data not shown). No metabolites were detected for Cmpd 1 or Cmpd 2 during that period, which indicates that any unidentified metabolite would have been presents at levels below 10%.
  • Cmpd 1 and Cmpd 2 have a longer half-life with similar bioavailability (subcutaneous injection in rats) compared to either exendin-4, Cmpd 4 or Cmpd 6 (data not shown).
  • Compound 1 has a half-life of 72 minutes given subcutaneously (in male Sprague-Dawley rats) compared to exendin-4 of 20 minutes and davalintide of about 30 minutes, with similar absolute bioavailibility.
  • These superior pharmacological properties coupled with an excellent PK profile and other favorable drug properties such as fewer off-target activities and reduced nausea, provide a surprisingly useful dual receptor agonist (Cmpd 2 and even more so Cmpd 1) for controlling glucose with further improvement in controlling body weight and composition in subjects in need of such treatment having diseases and conditions where such treatment is beneficial.
  • Such conditions include subjects, e.g.
  • test article formulations of Cmpdl- were prepared according to Example 1 above.
  • Compositions B09, BIO, B11, B12, B15 and B16 as set out in Table 1 were used in the following protocol.
  • the dose formulations were stored at approximately -20°C and protected from light.
  • Formulations were not heated, sonicated, or vortexed. Care was taken to ensure that no air bubbles were present in the suspension contained in the dosing syringe.
  • the animals were not fasted prior to dosing. Individual doses were calculated based on body weights taken on the day of dose administration.
  • the back of each animal was clipped free of hair, and the injeciton site was prepared by carefully cleaning the area with an alcohol wipe.
  • the injection site (on the dorsum, mid-scapular region) was marked with a permanent marker prior to injection to permit embarkations and scoring.
  • the marking of the injection site was done by drawing a circle around the injection site, taking care not to obscure the injection site itself or the immediate surrounding area of the injection site.
  • the subcutaneous doses were administered using a 0.5 mL (1 ⁇ 2 cc) syringe with an attached 27 G, 3/8-inch needle. A new syringe was used for each animal. Animals were restrained in a manner that minimized irritation or pressure to the injection sites. To administer the dose, the total length of the needle was inserted and the plunger was pushed gently and slowly to administer the dose to the mid-scapular region.
  • Injection site irritation was evaluated once daily beginning on Day 1 using a modified Draize technique.
  • Serine Protease Inhibitor Cocktail I Prior to each sample collection, 4 ⁇ ⁇ of reconstituted Serine Protease Inhibitor Cocktail I (SPIC1 from A.G. Scientific; Fisher Catalog Number P1516 or NC9600474, reconstituted by Covance) was added directly into the K2EDTA collection tube. Prepared tubes were stored at approximately 5°C no more than 24 hours prior to each blood sample collection. Blood (approximately 0.4 mL) was collected from 3 animals/group predose and at 0.25, 1, 6, 24, 48, 72, 96 (Day 5), 144 (Day 7), 168 (Day 8), 216 (Day 10), and 312 (Day 14) hours postdose. Samples were collected according to the following table.
  • Injection-site scoring (modified Draize technique) data are presented in Table 9. Edema scores generally ranged from none to slight. Erythema was not evident in any animals.
  • Test site A Area of observation (mid-scapular region surrounding injection site).
  • Example 11 Solubility of Cmpd 1 in lipid formulations with different compositions
  • Formulations with the compositions presented in Table 11 were prepared by adding 975 mg of a lipid stock solution (previously manufactured by mixing all the components except Cmpd 1 until a homogeneous solution was obtained) over 25 mg Cmpd 1 powder, mixing by brief vortex, followed by end-over-end rotation for up to 24h.
  • Table 11 Composition of samples used for solubility evaluation.
  • Example 12 Pharmacokinetic and Pharmacodynamic Study in Dogs. Pharmacokinetic and pharmacodynamic profiles were determined for a single dose subcutaneous administration of two exemplary, preferred sustained release formulations, B10 and B12, both containing Cmpd 1. The injectability of these compositions was studied in Example 10 with 2.5 wt% active agent. The results are shown in the relevant rows of Table 10 above. This study was conducted in accordance with generally recognized good laboratory practices, and all procedures were in compliance with the U.S. Animal Welfare Act Regulations (9
  • Cmpd 1 was present in each formulation at a concentration of 25 mg/ml.
  • a dose volume of 0.02 ml per kg of subject was administered subcutaneously, providing a Cmpd 1 dosage of 0.5 mg/kg, to each of four male dogs for each formulation.
  • Eight male purebred beagles were acclimated and quarantined to the test site and study room for approximately two weeks prior to dose administration.
  • animals were young adult/adult in age, approximately 9.2 kg at the start of the study. Animals were drug-naive prior to the first dose administration.
  • animals were individually housed in stainless steel cages with plastic-coated flooring.
  • the frozen formulations Prior to use, the frozen formulations were allowed to thaw at ambient temperature for approximately 1 hour. Formulations were vortex mixed for approximately 10 seconds to ensure uniformity. Formulations for both groups were clear and yellow. Dose administration was completed within 8 hours after the formulations were removed from the freezer. For each group, the animals were fasted overnight through approximately 4 hours post dose. Individual doses were calculated based on body weights taken on the day of dosing. The subcutaneous doses were administered via sterile syringe and needle in the dorsal thoracic region. The dosing site was identified with a marker.
  • Blood was collected from a jugular vein into collection tubes containing potassium EDTA plus Serine Protease Inhibitor Cocktail I (SPIC1 from A. G. Scientific; Fisher Catalog Number P1516). After the addition of the whole blood, the collection tubes were gently mixed and placed immediately on wet ice, in chilled cryoracks, or at approximately 5°C prior to centrifugation to obtain plasma. Upon completion of the in-life portion of the study, animals were transferred to the stock colony. Blood was maintained on wet ice prior to centrifugation to obtain plasma. Centrifugation (3000 rpm for 10 minutes in a refrigerated centrifuge) began within 30 minutes of collection. Separated plasma was transferred into individual tubes for storage and shipment. Plasma samples were placed on dry ice prior to storage at approximately -70°C.
  • Fig. 6C Percent change in body weight from baseline Day 1 is plotted in Fig. 6C. Body weights generally decreased from Day 1 to Days 3-5, leveled off by days 6-8, then increased to approximately Day 1 levels by the end of the 14-day post-dose study periods. Fig. 6C presents the percent body weight change induced in subjects by Cmpd 1 delivered in sustained release formulations B10 and B12.
  • Figures 6A and 6B provide the pharmacokinetic profile of Cmpd 1 plasma concentration over the 312 hour study.
  • Dog subjects provide a PK profile and parameters, including Cmax, Cave and Cmax/Cave ratio, that more closely predicts and approximates human parameters than do rats.
  • Rats provide a relatively rapid and less expensive means to compare and rank profiles and parameters.
  • FIG. 6A presents the 0 to 36 hour time points from Fig. 6 A, which expands the period of initial release ("initial burst") of drug.
  • the T max for BIO and B12 are 2 and 3 hours, respectively.
  • the C max for BIO and B12 are 62,309 and 30,639 pg/mL, respectively.
  • Table 12 presents an analysis of the pharmacokinetic results for the single, subcutaneous administration of the two exemplary and preferred formulations in dog.
  • Maximum drug plasma concentration is C max , average drug plasma
  • formulation B10 yielding a Cmax/Cave ratio of 10.0 and B12 a more desirable ratio of 7.1.
  • the SR portion AUC (from 30 to 312 hours) of the total AUCo-iast (from 0 to 312 hours) was 72%> and 80%> for B10 and B 12, respectively.

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Abstract

La présente invention concerne des compositions formant un mélange de faible viscosité de composants à base de lipides avec un agent actif agoniste des peptides, la pré-formulation formant, ou étant capable de former, au moins une structure de phase cristalline liquide lors de la mise en contact avec un fluide aqueux en excès. L'invention concerne également des procédés de traitement comprenant l'administration de telles compositions, et des dispositifs d'administration pré-remplis et des kits contenant les formulations.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019529470A (ja) * 2016-09-27 2019-10-17 カムルス エービー アルキルアンモニウムｅｄｔａ塩を含む混合物及び製剤
WO2020240018A1 (fr) * 2019-05-29 2020-12-03 Camurus Ab Dispositif & régime d'administration
WO2021160887A1 (fr) 2020-02-14 2021-08-19 Immunor As Vaccin contre le coronavirus
KR20220002140A (ko) 2020-06-30 2022-01-06 주식회사 종근당 GnRH 유도체를 포함하는 주사용 조성물
WO2022129526A1 (fr) * 2020-12-18 2022-06-23 Novo Nordisk A/S Co-agonistes des récepteurs du glp 1 et de l'amyline

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528486B1 (en) 1999-07-12 2003-03-04 Zealand Pharma A/S Peptide agonists of GLP-1 activity
WO2004035623A2 (fr) 2002-10-02 2004-04-29 Zealand Pharma A/S Composes d'exendine-4 stabilises
WO2005117830A1 (fr) 2004-06-04 2005-12-15 Camurus Ab Formulations de depot lipidique
WO2006083254A1 (fr) 2004-02-11 2006-08-10 Amylin Pharmaceuticals, Inc. Peptides de la famille amyline et procedes de production et d'utilisation desdits peptides
WO2006131730A1 (fr) 2005-06-06 2006-12-14 Camurus Ab Formulations d'analogues de glp-1
US7223725B1 (en) 1997-11-14 2007-05-29 Amylin Pharmaceuticals, Inc. Exendin agonist compounds
WO2007114838A1 (fr) 2006-03-31 2007-10-11 Amylin Pharmaceuticals, Inc. Amyline et agonistes d'amyline pour traiter les maladies et troubles psychiatriques
WO2009024795A1 (fr) * 2007-08-22 2009-02-26 Camurus Ab Formulations lipidiques à teneur en acide
EP2052716A1 (fr) * 2007-10-24 2009-04-29 Camurus AB Formulations à libération contrôlée

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7223725B1 (en) 1997-11-14 2007-05-29 Amylin Pharmaceuticals, Inc. Exendin agonist compounds
US6528486B1 (en) 1999-07-12 2003-03-04 Zealand Pharma A/S Peptide agonists of GLP-1 activity
WO2004035623A2 (fr) 2002-10-02 2004-04-29 Zealand Pharma A/S Composes d'exendine-4 stabilises
WO2006083254A1 (fr) 2004-02-11 2006-08-10 Amylin Pharmaceuticals, Inc. Peptides de la famille amyline et procedes de production et d'utilisation desdits peptides
WO2005117830A1 (fr) 2004-06-04 2005-12-15 Camurus Ab Formulations de depot lipidique
WO2006131730A1 (fr) 2005-06-06 2006-12-14 Camurus Ab Formulations d'analogues de glp-1
WO2007114838A1 (fr) 2006-03-31 2007-10-11 Amylin Pharmaceuticals, Inc. Amyline et agonistes d'amyline pour traiter les maladies et troubles psychiatriques
WO2009024795A1 (fr) * 2007-08-22 2009-02-26 Camurus Ab Formulations lipidiques à teneur en acide
EP2052716A1 (fr) * 2007-10-24 2009-04-29 Camurus AB Formulations à libération contrôlée

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN; DRUCKER, J. BIOL. CHEM., vol. 272, 1997, pages 4108 - 15
HARGROVE ET AL., REGULATORY PEPTIDES, vol. 141, 2007, pages 113 - 119
MACK ET AL., INT. J. OBESITY, vol. 34, 2009, pages 385 - 395
SHAH JAYMIN C ET AL: "Cubic phase gels as drug delivery systems", ADVANCED DRUG DELIVERY REVIEWS, ELSEVIER BV, AMSTERDAM, NL, vol. 47, no. 2-3, 25 April 2001 (2001-04-25), pages 229 - 250, XP002320651, ISSN: 0169-409X, DOI: 10.1016/S0169-409X(01)00108-9 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7138626B2 (ja) 2016-09-27 2022-09-16 カムルス エービー アルキルアンモニウムｅｄｔａ塩を含む混合物及び製剤
JP2019529470A (ja) * 2016-09-27 2019-10-17 カムルス エービー アルキルアンモニウムｅｄｔａ塩を含む混合物及び製剤
WO2020240018A1 (fr) * 2019-05-29 2020-12-03 Camurus Ab Dispositif & régime d'administration
WO2021160887A1 (fr) 2020-02-14 2021-08-19 Immunor As Vaccin contre le coronavirus
KR20220002140A (ko) 2020-06-30 2022-01-06 주식회사 종근당 GnRH 유도체를 포함하는 주사용 조성물
WO2022005169A1 (fr) 2020-06-30 2022-01-06 주식회사 종근당 Composition injectable comprenant des dérivés de gnrh
WO2022129526A1 (fr) * 2020-12-18 2022-06-23 Novo Nordisk A/S Co-agonistes des récepteurs du glp 1 et de l'amyline
KR20230019499A (ko) * 2020-12-18 2023-02-08 노보 노르디스크 에이/에스 Glp-1 수용체 및 아밀린 수용체의 공동-작용제
JP2023553591A (ja) * 2020-12-18 2023-12-25 ノヴォ ノルディスク アー／エス Ｇｌｐ－１およびアミリン受容体の共作動薬
KR102667196B1 (ko) 2020-12-18 2024-05-27 노보 노르디스크 에이/에스 Glp-1 수용체 및 아밀린 수용체의 공동-작용제
KR20240074867A (ko) * 2020-12-18 2024-05-28 노보 노르디스크 에이/에스 Glp-1 수용체 및 아밀린 수용체의 공동-작용제
US12054528B2 (en) 2020-12-18 2024-08-06 Novo Nordisk A/S Co-agonists of the GLP-1 and amylin receptors
JP7554928B2 (ja) 2020-12-18 2024-09-20 ノヴォ ノルディスク アー／エス Ｇｌｐ－１およびアミリン受容体の共作動薬
AU2021403851B2 (en) * 2020-12-18 2025-09-25 Novo Nordisk A/S Co-agonists of the glp-1 and amylin receptors
KR102884383B1 (ko) 2020-12-18 2025-11-14 노보 노르디스크 에이/에스 Glp-1 수용체 및 아밀린 수용체의 공동-작용제

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