Patent Description:
The invention relates to the GLP-<NUM> agonist semaglutide in a pharmaceutical composition for use in the prevention or treatment of type <NUM> diabetes, wherein said GLP-<NUM> agonist is administered once weekly in an amount of <NUM>, and wherein said GLP-<NUM> agonist is administered by parenteral administration.

SE: Standard error. FAS: Full analysis set. LOCF: Last observation carried forward.

Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy. The present invention relates to an improved use of GLP-<NUM> agonists in therapy. In one embodiment the invention relates to certain dosage regimes of GLP-<NUM> agonists which provide improved effect in diseases or conditions, such as prevention and/or treatment of type <NUM> diabetes and obesity. In one embodiment the methods of the present invention provides surprisingly showed improved reduction of HbA1c and reduction of body weight. In one embodiment the GLP-<NUM> agonist is administered in an amount which provides an improved a) reduction in HbA1c or b) reduction in body weight compared to administration of <NUM> liraglutide or less, such as <NUM> liraglutide or less, per day.

In one embodiment the invention relates to a method for reduction of HbA1c or for prevention or treatment of type <NUM> diabetes, hyperglycemia, impaired glucose tolerance, or non-insulin dependent diabetes, said method comprising administration of a GLP-<NUM> agonist to a subject in need thereof in an amount of at least <NUM> per week. In one embodiment the method is for reduction of HbA1c. In one embodiment the method is for prevention or treatment of type <NUM> diabetes. In one embodiment the method is for prevention or treatment of hyperglycemia. Described herein is a method for prevention or treatment of impaired glucose tolerance. Described herein is a method for prevention or treatment of non-insulin dependent diabetes. Described herein is a method comprising delaying or preventing diabetic disease progression. In one embodiment a HbA1c level below <NUM>% is achieved. In one embodiment the level of HbA1c is determined according to the method defined by the Diabetes Control and Complications Trial (DCCT). In one embodiment the level of HbA1c is determined according to the method defined by the International Federation of Clinical Chemistry (IFCC).

In one embodiment the GLP-<NUM> agonist is administered in an amount per week of <NUM>.

The GLP-<NUM> agonist is administered by parenteral administration, such as subcutaneous injection.

Described herein is a GLP-<NUM> agonist for use in the reduction of HbA1c or for use in the prevention or treatment of type <NUM> diabetes, hyperglycemia, impaired glucose tolerance, or non-insulin dependent diabetes comprising administering a GLP-<NUM> agonist in an amount of at least <NUM> per week. In one embodiment the GLP-<NUM> agonist and/or administration is as defined herein.

In one embodiment the invention relates to a composition comprising a GLP-<NUM> agonist and one or more pharmaceutically acceptable excipients for use in reduction of HbA1c or for prevention or treatment of type <NUM> diabetes, hyperglycemia, impaired glucose tolerance, or non-insulin dependent diabetes, wherein said GLP-<NUM> agonist is administered in an amount of at least <NUM> per week. In one embodiment the GLP-<NUM> agonist and/or administration is as defined herein.

In one embodiment the GLP-<NUM> agonist is administered with another therapeutic agent. Administration with another therapeutic agent may be carried out as administration of the GLP-<NUM> agonist and the other therapeutic agent within the same therapeutic window (e.g. withinin a period of two weeks, a period of one week, or in a <NUM>, <NUM>, or <NUM> hour period, etc.). The treatment with a GLP-<NUM> agonist according to the present invention may be combined with one or more additional therapeutic agents, e.g. selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity; examples of these therapeutic agents are: sulphonylureas, thiazolidinediones, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, and DPP-IV (dipeptidyl peptidase-IV) inhibitors.

In one embodiment an "effective amount" of a GLP-<NUM> agonist as used herein means an amount sufficient to cure, alleviate, or partially arrest the clinical manifestations of a given disease or state and its complications. An amount adequate to accomplish this is defined as "effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinary.

In one embodiment the term "treatment" or "treating" as used herein means the management and care of a patient for the purpose of combating a condition, such as a disease or a disorder. In one embodiment the term "treatment" or "treating" is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications; to delay the progression of the disease, disorder, or condition; to alleviate or relieve the symptoms and complications; and/or, to cure or eliminate the disease, disorder, or condition as well as to prevent the condition. In one embodiment prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

In one embodiment the term "hydrophilic spacer" as used herein means a spacer that separates a peptide and an albumin binding residue with a chemical moiety which comprises at least <NUM> non- hydrogen atoms where <NUM>-<NUM>% of these are either N or O.

In one embodiment the term "analogue" as used herein referring to a polypeptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide. A simple system is used to describe analogues: For example Arg<NUM>GLP-<NUM> (<NUM>-<NUM>) Lys designates a GLP-<NUM> analogue wherein the naturally occurring lysine at position <NUM> has been substituted with arginine and a lysine residue has been added to the C-terminal (position <NUM>).

In one embodiment the term "GLP-<NUM> peptide" as used herein means GLP-<NUM> (<NUM>-<NUM>), a GLP-<NUM> analogue, a GLP-<NUM> derivative or a derivative of a GLP-<NUM> analogue.

In one embodiment the term "DPP-IV protected" as used herein referring to a polypeptide means a polypeptide which has been chemically modified in order to render said compound resistant to the plasma peptidase dipeptidyl aminopeptidase-<NUM> (DPP-IV). The DPP-IV enzyme in plasma is known to be involved in the degradation of several peptide hormones, e.g. GLP-<NUM>, Exendin-<NUM> etc. Thus a considerable effort is being made to develop GLP-<NUM> agonists less susceptible to DPP-IV mediated hydrolysis in order to reduce the rate of degradation by DPP-IV.

The present invention also relates to a GLP-<NUM> agonist of the invention, for use as a medicament. Described herein, the GLP-<NUM> agonist may be used for the following medical treatments:.

In another particular embodiment, the indication is (i). In a further particular embodiment the indication is (ii). In a still further particular embodiment the indication is (iii). In one embodiment the indication is type <NUM> diabetes and/or obesity.

In one embodiment the method comprises prevention, treatment, reduction and/or induction in one or more diseases or conditions defined herein. In one embodiment the indication is (i) and (iii). In one embodiment the indication is (ii) and (iii).

In one embodiment the invention relates to administration of an effective amount of a GLP-<NUM> agonist.

In one embodiment as used herein, specific values given in relation to numbers or intervals may be understood as the specific value or as about the specific value.

In a first functional aspect, the GLP-<NUM> agonists of the invention have a good potency. Also, or alternatively, in a second functional aspect, the GLP-<NUM> agonists of the invention have a protracted pharmacokinetic profile. Also, or alternatively, in a third functional aspect, the GLP-<NUM> agonists of the invention are stable against degradation by gastro intestinal enzymes.

According to the first functional aspect, the GLP-<NUM> agonists of the invention are biologically active, or potent. In a particular embodiment, "potency" and/or "activity" refers to in vitro potency, i.e. performance in a functional GLP-<NUM> receptor assay, more in particular to the capability of stimulating cAMP formation in a cell line expressing the cloned human GLP-<NUM> receptor.

The stimulation of the formation of cAMP in a medium containing the human GLP-<NUM> receptor may preferably be determined using a stable transfected cell-line such as BHK467-12A (tk-ts13), and/or using for the determination of cAMP a functional receptor assay, e.g. based on competition between endogenously formed cAMP and exogenously added biotin-labelled cAMP, in which assay cAMP is more preferably captured using a specific antibody, and/or wherein an even more preferred assay is the AlphaScreen cAMP Assay, such as the one described in Assay (I).

In one embodiment the term half maximal effective concentration (EC<NUM>) generally refers to the concentration which induces a response halfway between the baseline and maximum, by reference to the dose response curve. EC<NUM> is used as a measure of the potency of a compound and represents the concentration where <NUM>% of its maximal effect is observed.

The in vitro potency of the GLP-<NUM> agonists of the invention may be determined as described above, and the EC<NUM> of the GLP-<NUM> agonist in question determined. The lower the EC<NUM>, the better the potency.

In a particular embodiment, the medium has the following composition (final in-assay concentrations): <NUM> TRIS-HCl; <NUM> HEPES; <NUM> MgCl<NUM>, <NUM><NUM>O; <NUM> NaCl; <NUM>% Tween; <NUM>% BSA ; <NUM> IBMX; <NUM> ATP; <NUM> GTP; pH <NUM>.

In a further particular embodiment, the GLP-<NUM> agonist of the invention has an in vitro potency corresponding to an EC<NUM> at or below 3000pM, such as below 2000pM, below 1000pM, or below 500pM, or such as below <NUM> pM or below <NUM> pM.

In another particular embodiment the GLP-<NUM> agonist of the invention are potent in vivo, which may be determined as is known in the art in any suitable animal model, as well as in clinical trials.

The diabetic db/db mouse is one example of a suitable animal model, and the blood glucose lowering effect may be determined in such mice in vivo, e.g. as described in Assay (III), or as described in Example <NUM> of <CIT>.

Also, or alternatively, the effect on food intake in vivo may be determined in pharmacodynamic studies in pigs, e.g. as described in Assay (IV).

According to the second functional aspect, the GLP-<NUM> agonists of the invention are protracted. In a particular embodiment protraction may be determined as half-life (T½) in vivo in minipigs after i. administration. In additional embodiments, the half-life is at least <NUM> hours, such as at least <NUM> hours, at least <NUM> hours, at least <NUM> hours, or such as at least <NUM> hours, at least <NUM> hours, or at least <NUM> hours.

A suitable assay for determining half-life in vivo in minipigs after i. administration is disclosed in Assay (II).

According to the third functional aspect, the GLP-<NUM> agonists of the invention are stable, or stabilised, against degradation by one or more gastro intestinal enzymes.

Gastro intestinal enzymes include, without limitation, exo and endo peptidases, such as pepsin, trypsin, chymotrypsin, elastases, and carboxypeptidases. The stability may be tested against these gastro intestinal enzymes in the form of purified enzymes, or in the form of extracts from the gastrointestinal system.

In a particular embodiment, the GLP-<NUM> agonist of the invention has an in vitro half-life (T½), in an extract of rat small intestines, divided by the corresponding half-life (T½) of GLP-<NUM>(<NUM>-<NUM>), of at least <NUM>, such as above <NUM>, at least <NUM>, at least <NUM>, or such as at least <NUM>, or at least <NUM>. In other words, a ratio(SI) may be defined for each GLP-<NUM> agonist, viz. as the in vitro half-life (T½) of the GLP-<NUM> agonist in question, in an extract of rat small intestines, divided by the corresponding half-life (T½) of GLP-<NUM>(<NUM>-<NUM>).

A suitable assay for determining in vitro half-life in an extract of rat small intestines is disclosed in Assay (V).

In one embodiment the GLP-<NUM> agonists of the invention have GLP-<NUM> activity. In one embodiment "a GLP-<NUM> agonist" is understood to refer to any compound, including peptides and non-peptide compounds, which fully or partially activate the human GLP-<NUM> receptor. In one embodiment the "GLP-<NUM> agonist" is any peptide or non-peptide small molecule that binds to a GLP-<NUM> receptor, preferably with an affinity constant (KD) or a potency (EC<NUM>) of below <NUM>, e. below <NUM> as measured by methods known in the art (see e. In one embodiment methods for identifying GLP-<NUM> agonists are described in <CIT> (Novo Nordisk A/S) and examples of suitable GLP-<NUM> agonists which can be used according to the present invention includes those referred to in <CIT> (Novo Nordisk A/S), the teachings of which are both incorporated by reference herein. "GLP-<NUM> activity" refers to the ability to bind to the GLP-<NUM> receptor and initiate a signal transduction pathway resulting in insulinotropic action or other physiological effects as is known in the art. For example, the GLP-<NUM> agonists of the invention can be tested for GLP-<NUM> activity using the assay described in Assay (I) herein.

In yet another embodiment the GLP-<NUM> agonist is a stable GLP-<NUM> agonist. As used herein a "stable GLP-<NUM> agonist" means a GLP-<NUM> agonist which exhibits an in vivo plasma elimination half-life of at least <NUM> hours in man, optionally determined by the method described below. Examples of stable GLP-<NUM> agonists can be found in <CIT>.

In one embodiment the method for determination of plasma elimination half-life of a compound in man may be carried out as follows: The compound is dissolved in an isotonic buffer, pH <NUM>, PBS or any other suitable buffer. The dose is injected peripherally, preferably in the abdominal or upper thigh. Blood samples for determination of active compound are taken at frequent intervals, and for a sufficient duration to cover the terminal elimination part (e. , Pre-dose, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> (day <NUM>), <NUM> (day <NUM>), <NUM> (day <NUM>), <NUM> (day <NUM>), <NUM> (day <NUM>) and <NUM> (day <NUM>) hours post dose). Determination of the concentration of active compound is performed as described in <NPL>. Derived pharmacokinetic parameters are calculated from the concentration-time data for each individual subject by use of non-compartmental methods, using the commercially available software WinNonlin Version <NUM> (Pharsight, Cary, NC, USA). The terminal elimination rate constant is estimated by log-linear regression on the terminal log-linear part of the concentration-time curve, and used for calculating the elimination half-life.

In one embodiment the GLP-<NUM> agonist is formulated so as to have a half-life in man of at least <NUM> hours. This may be obtained by sustained release formulations known in the art.

In one embodiment the GLP-<NUM> agonist is a GLP-<NUM> peptide. The GLP-<NUM> peptide is semaglutide. <CIT> discloses semaglutide (Example <NUM>), a mono-acylated GLP-<NUM> agonist for once weekly administration.

In one embodiment the present invention encompasses pharmaceutically acceptable salts of the GLP-<NUM> agonists. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium, and alkylated ammonium salts. Also intended as pharmaceutically acceptable acid addition salts are the hydrates which the present GLP-<NUM> agonists are able to form.

In one embodiment medicaments or pharmaceutical compositions comprising the GLP-<NUM> agonist semaglutide may be administered parenterally to a patient in need thereof. In one embodiment parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe.

Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of a GLP-<NUM> agonist in the form of a nasal or pulmonal spray. As a still further option, the GLP-<NUM> agonist can also be administered transdermally, e.g., from a patch, optionally an iontophoretic patch, or transmucosally, e.g., bucally. The above-mentioned possible ways to administer GLP-<NUM> agonists are not considered as limiting the scope of the invention.

In one embodiment the GLP-<NUM> agonist is co-administered together with a further therapeutically active agent used in the treatments defined herein.

In one embodiment the GLP-<NUM> peptide comprises [Aib8,Arg34]GLP-<NUM>-(<NUM>-<NUM>).

Human Glucagon-Like Peptide-<NUM> is GLP-<NUM>(<NUM>-<NUM>) and has the sequence HAEGTFTSDVSSYLEGQAAKEFI AWLVKGRG (SEQ ID NO: <NUM>). GLP-<NUM>(<NUM>-<NUM>) may also be designated "native" GLP-<NUM>. In the sequence listing, the first amino acid residue of SEQ ID NO: <NUM> (histidine) is assigned no. However, in what follows - according to established practice in the art - this histidine residue is referred to as no. <NUM>, and subsequent amino acid residues are numbered accordingly, ending with glycine no. Therefore, generally, any reference herein to an amino acid residue number or a position number of the GLP-<NUM>(<NUM>-<NUM>) sequence is to the sequence starting with His at position <NUM> and ending with Gly at position <NUM>. A non-limiting example of a suitable analogue nomenclature is [Aib<NUM>,Arg<NUM>,Lys<NUM>]GLP-<NUM>(<NUM>-<NUM>), which designates a GLP-<NUM>(<NUM>-<NUM>) analogue, in which the alanine at position <NUM> has been substituted with α-aminoisobutyric acid (Aib), the lysine at position <NUM> has been substituted with arginine, and the glycine at position <NUM> has been substituted with lysine.

The concentration in plasma of the GLP-<NUM> agonists of the invention may be determined using any suitable method. For example, LC-MS (Liquid Chromatography Mass Spectroscopy) may be used, or immunoassays such as RIA (Radio Immuno Assay), ELISA (Enzyme-Linked Immuno Sorbent Assay), and LOCI (Luminescence Oxygen Channeling Immunoasssay). General protocols for suitable RIA and ELISA assays are found in, e.g., <CIT> on p. <NUM>-<NUM>. A preferred assay is the LOCI (Luminescent Oxygen Channeling Immunoassay), generally as described for the determination of insulin by <NPL> - briefly blood samples may be collected at desired intervals, plasma separated, immediately frozen, and kept at -<NUM> until analyzed for plasma concentration of the respective GLP-<NUM> agonist; the donor beads are coated with streptavidin, while acceptor beads are conjugated with a monoclonal antibody recognising a mid-/C-terminal epitope of the peptide; another monoclonal antibody, specific for the N-terminus, is biotinylated; the three reactants are combined with the analyte and formed a two-sited immuno-complex; illumination of the complex releases singlet oxygen atoms from the donor beads, which are channeled into the acceptor beads and triggered chemiluminescence which may be measured in an Envision plate reader; the amount of light is proportional to the concentration of the compound.

In one embodiment the term "Aib" as used herein refers to α-aminoisobutyric acid.

An administered dose may contain from <NUM> - <NUM> of the GLP-<NUM> agonist, or from <NUM>-<NUM>, or from <NUM>-<NUM>, or from <NUM>-<NUM> of the GLP-<NUM> agonist.

Pharmaceutical compositions comprising a GLP-<NUM> agonist of the invention or a pharmaceutically acceptable salt, amide, or ester thereof, and a pharmaceutically acceptable excipient may be prepared as is known in the art.

In one embodiment the term "excipient" broadly refers to any component other than the active therapeutic ingredient(s). The excipient may be an inert substance, an inactive substance, and/or a not medicinally active substance. The formulation of pharmaceutically active ingredients with various excipients is known in the art, see e.g. <NPL>), and any later editions). Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity regulating agents (e. g isotonic agents), chelating agents, stabilisers (e.g. oxidation inhibitors, aggregation inhibitors, surfactants, and/or protease inhibitors).

Examples of formulations include liquid formulations, i.e. aqueous formulations comprising water. A liquid formulation may be a solution, or a suspension. An aqueous formulation typically comprises at least <NUM>% w/w water, or at least <NUM>%, <NUM>%, <NUM>%, or even at least <NUM>% w/w of water.

Alternatively, a pharmaceutical composition may be a solid formulation, e.g. a freeze-dried or spray-dried composition, which may be used as is, or whereto the physician or the patient adds solvents, and/or diluents prior to use.

The pH in an aqueous formulation may be anything between pH <NUM> and pH <NUM>, for example from about <NUM> to about <NUM>; or from about <NUM> to about <NUM>.

A composition may further be compounded in a drug carrier or drug delivery system, e.g. in order to improve stability, bioavailability, and/or solubility. In a particular embodiment a composition may be attached to such system through covalent, hydrophobic, and/or electrostatic interactions. The purpose of such compounding may be, e.g., to decrease adverse effects, achieve chronotherapy, and/or increase patient compliance.

A composition may also be used in the formulation of controlled, sustained, protracting, retarded, and/or slow release drug delivery systems.

The composition is administered by parenteral administration. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection by means of a syringe, optionally a pen-like syringe, or by means of an infusion pump.

In one embodiment GLP-<NUM> peptides can be produced by appropriate derivatisation of an appropriate peptide backbone which has been produced by recombinant DNA technology or by peptide synthesis (e.g., Merrifield-type solid phase synthesis) as known in the art of peptide synthesis and peptide chemistry.

In one embodiment the production of peptides like GLP-<NUM>(<NUM>-<NUM>) and GLP-<NUM> analogues is well known in the art. The GLP-<NUM> moiety of the GLP-<NUM> peptide of the invention (or fragments thereof) may for instance be produced by classical peptide synthesis, e.g., solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see, e.g., <NPL>, <NPL>, and "<NPL>.

In one embodiment GLP-<NUM> agonists may be produced by recombinant methods, viz. by culturing a host cell containing a DNA sequence encoding the GLP-<NUM> agonist and capable of expressing the peptide in a suitable nutrient medium under conditions permitting the expression of the peptide. Non-limiting examples of host cells suitable for expression of these peptides are: Escherichia coli, Saccharomyces cerevisiae, as well as mammalian BHK or CHO cell lines.

In one embodiment GLP-<NUM> agonists of the invention which include non-natural amino acids and/or a covalently attached N-terminal mono- or dipeptide mimetic may e.g. be produced as described in the experimental part. Or see e.g., <NPL>; and <CIT> entitled "Semi-recombinant preparation of GLP-<NUM> analogues".

The following abbreviations are used in the following, in alphabetical order:.

Semaglutide is a unique acylated GLP-<NUM> peptide with a half-life of <NUM> hours. The aim was to investigate HbA1c dose-response of once-weekly doses of semaglutide (five dose-levels) in subjects with type <NUM> diabetes. Safety, tolerability and pharmacodynamics of semaglutide versus placebo and open-label once-daily liraglutide were also investigated.

Liraglutide may be prepared as described in Example <NUM> of <CIT>. Semaglutide may be prepared as described in Example <NUM> of <CIT>. The composition of the GLP-<NUM> agonists administered may be formulated as isotonic aqueous solutions with a phosphate buffer, such as a sodium dihydrogen phosphate buffer, having a pH in the range <NUM>-<NUM>, such as pH <NUM> or pH <NUM>, for example further comprising the excipients propylene glycol and phenol. The composition of the GLP-<NUM> agonists administered may be as described in <CIT> or <CIT>. The placebo composition may be identical to the composition of the GLP-<NUM> agonists, but not containing a GLP-<NUM> agonist.

In a <NUM>-week, randomised, double-blind, placebo-controlled trial, <NUM> human subjects (n=<NUM>-<NUM> per group) with type <NUM> diabetes were exposed. Participants (male/female <NUM>/<NUM>%; baseline HbA1c (mean±SD) <NUM>±<NUM>%; baseline body weight <NUM>±<NUM>; duration of diabetes <NUM>±<NUM> years; metformin only/diet and exercise alone <NUM>/<NUM>%) received subcutaneous injection of one of five semaglutide doses (<NUM>-<NUM>) once weekly, open-label liraglutide (<NUM>, <NUM>) once daily, or placebo once weekly. Two of the semaglutide doses were titrated (T) in weekly increments of <NUM>. The primary endpoint was change in HbA1c from baseline. Secondary efficacy endpoints included proportion of subjects reaching ADA HbA1c target (<<NUM>%) and change in body weight. Change and percentage to target were analysed by ANOVA and logistic regression, respectively. Comparisons between semaglutide and liraglutide were not corrected for multiplicity. Baseline characteristics of the subjects are shown in Table <NUM>.

In the full analysis set, semaglutide (≥<NUM>) dose-dependently reduced HbA1c from baseline (<FIG>), and increased the likelihood of achieving HbA1c <<NUM>% (p<<NUM> vs. placebo for doses ≥<NUM>). The results with respect to change in HbA1c are shown in <FIG>. The change in HbA1c in <FIG> is from baseline at week <NUM>. <FIG> shows the change in HbA1c over time with the different treatments. Treatment with semaglutide ≥<NUM> numerically brought more patients to target than liraglutide <NUM> (<NUM> T <NUM>%, <NUM> <NUM>%, <NUM> T <NUM>% vs. liraglutide <NUM> <NUM>%). The results (see e.g. <FIG>) shows that treatment with semaglutide <NUM>, <NUM> T, or <NUM> T improved reduction of HbA1c compared to treatment with liraglutide <NUM> or <NUM>; furthermore, treatment with semaglutide <NUM> T was statistically superior to treatment with liraglutide <NUM> or <NUM> with respect to reduction of HbA1c (based on unadjusted means). <FIG> shows the percentage and the number of subjects reaching the AACE or ADA criteria for glycaemic control with the different treatments. The results (see <FIG>) shows that treatment with semaglutide <NUM>, <NUM> T, or <NUM> T improved the percentage and the number of subjects reaching the AACE or ADA criteria for glycaemic control compared to treatment with liraglutide <NUM> or <NUM>.

Body weight was dose-dependently reduced from baseline by up to <NUM> vs. placebo <NUM> (p<<NUM> for doses ≥<NUM>). <FIG> and <FIG> shows mean body weight change versus time and body weight change from baseline at week <NUM>, respectively, with the different treatments. The results (see e.g. <FIG>) shows that treatment with semaglutide <NUM>, <NUM> T, or <NUM> T increased reduction of body weight compared to treatment with liraglutide <NUM> or <NUM>. Furthermore, the results (see e.g. <FIG>) shows that treatment with semaglutide <NUM> T or <NUM> T was statistically superior to treatment with liraglutide <NUM> with respect to reduction of body weight; and that treatment with semaglutide <NUM>, <NUM> T, or <NUM> T was statistically superior to liraglutide <NUM> with respect to reduction of body weight (based on unadjusted means).

There were no reports of pancreatitis or treatment-related changes in blood calcitonin. Proportion of subjects with nausea and vomiting increased with dose, but were generally mild or moderate and ameliorated by titration. Withdrawals due to gastrointestinal adverse events were <NUM>%-<NUM>% for semaglutide and <NUM>%-<NUM>% for liraglutide. Few subjects reported minor hypoglycaemia (semaglutide n=<NUM>, liraglutide n=<NUM>); no major hypoglycaemia. Injection site reactions were reported by <NUM> subjects: semaglutide n=<NUM>; liraglutide n=<NUM>. One subject (semaglutide <NUM> T) developed low titre non-neutralising anti-semaglutide antibodies (no cross-reaction to native GLP-<NUM>).

Over <NUM> weeks, semaglutide dose-dependently reduced HbA1c and body weight. The effect of semaglutide <NUM> on glycaemic control and body weight was comparable to that of liraglutide <NUM>, while semaglutide ≥<NUM> appeared to bring more subjects to target and provided better weight loss than liraglutide <NUM>. No semaglutide safety concerns were identified. Dose escalation was not a major focus of this trial and it will be optimised in future clinical trials.

The purpose of this example is to test the activity, or potency, of GLP-<NUM> agonists in vitro. The potencies of GLP-<NUM> agonists may be determined as described below, i.e. as the stimulation of the formation of cyclic AMP (cAMP) in a medium containing membranes expressing the human GLP-<NUM> receptor.

Purified plasma membranes from a stable transfected cell line, BHK467-12A (tk-ts13), expressing the human GLP-<NUM> receptor are stimulated with the GLP-<NUM> agonist in question, and the potency of cAMP production is measured using the AlphaScreenTM cAMP Assay Kit from Perkin Elmer Life Sciences. The basic principle of The AlphaScreen Assay is a competition between endogenous cAMP and exogenously added biotin-cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to acceptor beads.

A stable transfected cell line and a high expressing clone are selected for screening. The cells are grown at <NUM>% CO<NUM> in DMEM, <NUM>% FCS, <NUM>% Pen/Strep (Penicillin/Streptomycin) and <NUM>/ml of the selection marker G418.

Cells at approximate <NUM>% confluence are washed 2X with PBS and harvested with Versene (aqueous solution of the tetrasodium salt of ethylenediaminetetraacetic acid), centrifuged <NUM> at <NUM> rpm and the supernatant removed. The additional steps are all made on ice. The cell pellet is homogenised by the Ultrathurax for <NUM>-<NUM> sec. in <NUM> of Buffer <NUM> (<NUM> Na-HEPES, <NUM> EDTA, pH=<NUM>), centrifuged <NUM> at <NUM>,<NUM> rpm and the pellet resuspended in <NUM> of Buffer <NUM> (<NUM> Na-HEPES, <NUM> EDTA, pH=<NUM>). The suspension is homogenised for <NUM>-<NUM> sec and centrifuged <NUM> at <NUM>,<NUM> rpm. Suspension in Buffer <NUM>, homogenisation and centrifugation is repeated once and the membranes are resuspended in Buffer <NUM>. The protein concentration is determined and the membranes stored at -<NUM> until use.

The assay is performed in ½-area <NUM>-well plates, flat bottom (e.g. Costar cat. The final volume per well is <NUM>µl.

Exemplary solutions and reagents are given below.

AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences (cat. No: <NUM>); containing Anti-cAMP Acceptor beads (<NUM> U/µl), Streptavidin Donor beads (<NUM> U/µl) and Biotinylated-cAMP (<NUM> U/µl).

AlphaScreen Buffer, pH=<NUM>: <NUM> TRIS-HCl (Sigma, cat. no: T3253); <NUM> HEPES (Sigma, cat. no: H3375); <NUM> MgCl<NUM>, <NUM><NUM>O (Merck, cat. no: <NUM>); <NUM> NaCl (Sigma, cat. no: S9625); <NUM>% Tween (Merck, cat. no: <NUM>). The following was added to the AlphaScreen Buffer prior to use (final concentrations indicated): BSA (Sigma, cat. A7906): <NUM>%; IBMX (Sigma, cat. I5879): <NUM>; ATP (Sigma, cat. A7699): <NUM>; GTP (Sigma, cat. G8877): <NUM>.

cAMP standard (dilution factor in assay = <NUM>): cAMP Solution: <NUM>µL of a <NUM> cAMP-stock + <NUM>µL AlphaScreen Buffer.

Suitable dilution series in AlphaScreen Buffer are prepared of the cAMP standard as well as the GLP-<NUM> agonist to be tested, e.g. the following eight concentrations of the GLP-<NUM> agonist: <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>M, and a series from, e.g., <NUM>-<NUM> to 3x10-<NUM> of cAMP.

As the beads are sensitive to direct light, any handling is in the dark (as dark as possible), or in green light. All dilutions are made on ice.

The EC<NUM> [pM] values may be calculated using the Graph-Pad Prism software (version <NUM>). If desired, the fold variation in relation to GLP-<NUM> may be calculated as EC<NUM> (GLP-<NUM>)/ EC<NUM> (analogue) - <NUM>.

The purpose of this study is to determine the protraction in vivo of GLP-<NUM> agonists after i. administration to minipigs, i.e. the prolongation of their time of action. This is done in a pharmacokinetic (PK) study, where the terminal half-life of the GLP-<NUM> agonist in question is determined. By terminal half-life is generally meant the period of time it takes to halve a certain plasma concentration, measured after the initial distribution phase.

Male Göttingen minipigs are obtained from Ellegaard Göttingen Minipigs (Dalmose, Denmark) approximately <NUM>-<NUM> months of age and weighing from approximately <NUM>-<NUM> are used in the studies. The minipigs are housed individually and fed restrictedly once or twice daily with SDS minipig diet (Special Diets Services, Essex, UK). After at least <NUM> weeks of acclimatisation two permanent central venous catheters are implanted in vena cava caudalis or cranialis in each animal. The animals are allowed <NUM> week recovery after the surgery, and are then used for repeated pharmacokinetic studies with a suitable wash-out period between dosings.

The animals are fasted for approximately <NUM> before dosing and for at least <NUM> after dosing, but have ad libitum access to water during the whole period.

The GLP-<NUM> agonist is dissolved in <NUM> sodium phosphate, <NUM> sodium chloride, <NUM>% tween <NUM>, pH <NUM> to a concentration of usually from <NUM>-<NUM> nmol/ml. Intravenous injections (the volume corresponding to usually <NUM>-<NUM> nmol/kg, for example <NUM>/kg) of the compounds are given through one catheter, and blood is sampled at predefined time points for up till <NUM> days post dosing (preferably through the other catheter). Blood samples (for example <NUM>) are collected in EDTA buffer (<NUM>) and then centrifuged at <NUM> and <NUM> for <NUM> minutes. Plasma is pippetted into Micronic tubes on dry ice, and kept at -<NUM> until analyzed for plasma concentration of the respective GLP-<NUM> compound using ELISA or a similar antibody based assay or LC-MS. Individual plasma concentration-time profiles are analyzed by a non-compartmental model in WinNonlin v. <NUM> (Pharsight Inc. , Mountain View, CA, USA), and the resulting terminal half-lives (harmonic mean) determined.

The purpose of the study is to verify the effect of GLP-<NUM> agonists on blood glucose (BG) and body weight (BW) in a diabetic setting. GLP-<NUM> agonists may be tested in a dose-response study in an obese, diabetic mouse model (db/db mice) as described in the following.

Fifty db/db mice (Taconic, Denmark), fed from birth with the diet NIH31 (NIH <NUM> Rodent Diet, commercially available from Taconic Farms, Inc. , US, see www. com), are enrolled for the study at the age of <NUM>-<NUM> weeks The mice are given free access to standard chow (e.g. Altromin <NUM>, Brogaarden, Gentofte, Denmark) and tap water and kept at <NUM>. After <NUM>-<NUM> weeks of acclimatisation, the basal blood glucose is assessed twice on two consecutive days (i.e. at <NUM> am). The <NUM> mice with the lowest blood glucose values may be excluded from the experiments. Based on the mean blood glucose values, the remaining <NUM> mice may be selected for further experimentation and allocated to <NUM> groups (n=<NUM>) with matching blood glucose levels. The mice may be used in experiments with duration of <NUM> days for up to <NUM> times. After the last experiment the mice are euthanised.

The seven groups may receive treatment as follows:.

Vehicle: <NUM> sodium phosphate, <NUM> sodium chloride, <NUM>% tween <NUM>, pH <NUM>.

The GLP-<NUM> agonist is dissolved in the vehicle, e.g. to concentrations of <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> nmol/ml. Animals are dosed subcutaneous with a dose-volume of <NUM>/kg (i.e. <NUM>µl per <NUM> mouse).

On the day of dosing, blood glucose is assessed at time -½h (<NUM> am), where after the mice are weighed. The GLP-<NUM> agonist is dosed at approximately <NUM> am (time <NUM>). On the day of dosing, blood glucose is assessed e.g. at times <NUM>, <NUM>, <NUM> and <NUM> (<NUM> am, <NUM> am, <NUM> pm and <NUM> pm).

On the following days, the blood glucose is assessed e.g. at time <NUM>, <NUM>, <NUM>, and <NUM> after dosing (i.e. at <NUM> am on day <NUM>, <NUM>, <NUM>, <NUM>). On each day, the mice are weighed following blood glucose sampling.

The mice are weighed individually on a digital weight.

Samples for the measurement of blood glucose are obtained from the tail tip capillary of conscious mice. Blood, <NUM>µl, is collected into heparinised capillaries and transferred to <NUM>µl glucose buffer (EKF system solution, Eppendorf, Germany). The glucose concentration is measured using the glucose oxidase method (glucose analyser Biosen <NUM>, EKF Diagnostic, GmbH, Barleben, Germany). The samples are kept at room temperature for up to <NUM> until analysis. If analysis has to be postponed, samples are kept at <NUM> for a maximum of <NUM>.

ED<NUM> is the dose giving rise to half-maximal effect in nmol /kg. This value is calculated on the basis of the ability of the GLP-<NUM> agonists to lower body weight as well as the ability to lower blood glucose, as explained below.

ED<NUM> for body weight is calculated as the dose giving rise to half-maximum effect on delta BW <NUM> hours following the subcutaneous administration of the GLP-<NUM> agonist. For example, if the maximum decrease in body weight after <NUM> hours is <NUM>, then ED<NUM> bodyweight would be that dose in nmol/kg which gives rise to a decrease in body weight after <NUM> hours of <NUM>. This dose (ED<NUM> body weight) may be read from the dose-response curve.

ED<NUM> for blood glucose is calculated as the dose giving rise to half-maximum effect on AUC delta BG <NUM> hours following the subcutaneous administration of the GLP-<NUM> agonist.

The ED<NUM> value may only be calculated if a proper sigmoidal dose-response relationship exists with a clear definition of the maximum response. Thus, if this would not be the case the GLP-<NUM> agonist in question is re-tested in a different range of doses until the sigmoidal dose-response relationship is obtained.

The purpose of this experiment is to investigate the effect of GLP-<NUM> agonists on food intake in pigs. This is done in a pharmacodynamic (PD) study as described below, in which food intake is measured <NUM>, <NUM>, <NUM>, and <NUM> days after administration of a single dose of the GLP-<NUM> agonist, as compared to a vehicle-treated control group.

Female Landrace Yorkshire Duroc (LYD) pigs, approximately <NUM> months of age, weighing approximately <NUM>-<NUM> are used (n=<NUM>-<NUM> per group). The animals are housed in a group for <NUM>-<NUM> weeks during acclimatisation to the animal facilities. During the experimental period the animals are placed in individual pens from Monday morning to Friday afternoon for measurement of individual food intake. The animals are fed ad libitum with pig fodder (Svinefoder, Antonio) at all times both during the acclimatisation and the experimental period. Food intake is monitored on line by logging the weight of fodder every <NUM> minutes. The system used is Mpigwin (Ellegaard Systems, Faaborg, Denmark).

The GLP-<NUM> agonists are dissolved in a phosphate buffer (<NUM> phosphate, <NUM>% tween <NUM>, pH <NUM>) e.g. at concentrations of <NUM>, <NUM>, <NUM>, <NUM> or <NUM> nmol/ml corresponding to doses of <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> nmol/kg. The phosphate buffer served as vehicle. Animals are dosed with a single subcutaneous dose of the GLP-<NUM> agonist or vehicle (dose volume <NUM>/kg) on the morning of day <NUM>, and food intake is measured for <NUM> days after dosing. On the last day of each study, <NUM> days after dosing, a blood sample for measurement of plasma exposure of the GLP-<NUM> agonist is taken from the heart in anaesthetised animals. The animals are thereafter euthanised with an intra-cardial overdose of pentobarbitone. Plasma content of the GLP-<NUM> agonist is analysed using ELISA or a similar antibody based assay.

Food intake is calculated as mean ± SEM <NUM> food intake on the <NUM> days. Statistical comparisons of the <NUM> hour food intake in the vehicle vs. GLP-<NUM> agonist group on the <NUM> days are done using one-way or two-way-ANOVA repeated measures, followed by Bonferroni post-test.

The purpose of this example is to test the stability against degradation by intestinal enzymes. GLP-<NUM>(<NUM>-<NUM>) may be used in the assay as a comparative compound. The strongest proteolytic activities in the intestine are of pancreatic origin and include the serine endopeptidases trypsin, chymotrypsin, and elastase as well as several types of carboxypeptidases. An assay with small intestine extract from rats was developed and used as described in the following.

Small intestines are prepared from rats and flushed with <NUM> of <NUM> NaCl, <NUM> Hepes pH <NUM>. The solutions are centrifuged for <NUM> at <NUM>,<NUM> rpm in a Heraeus Multifuge <NUM>-R centrifuge with a <NUM> rotor. The supernatants are removed and filtered through a <NUM> Millipore Millex GV PVDF membrane. Filtrates of several animals are pooled to average out individual differences.

The protein content of the obtained extracts is determined by Bradford Assay (see e.g. <NPL>, and <NPL>).

<NUM> nmol of the GLP-<NUM> agonists to be tested are incubated with the intestinal extract in a volume of <NUM>µl at <NUM> over a period of one hour. Intestinal samples are assayed in presence of <NUM> Hepes at pH <NUM>. The concentration of the intestinal extract is titrated in pilot experiments so that the half-life (t½) of GLP-<NUM>(<NUM>-<NUM>) is in the range of <NUM>-<NUM> minutes. The small intestine extract is used at a concentration of <NUM>µg/ml. All components except for the intestinal extract are mixed and pre-warmed for ten minutes at <NUM>. Immediately after addition of the intestinal extract a sample of <NUM>µl is taken and mixed with the same volume of <NUM>% trifluoroacetic acid (TFA). Further samples are taken accordingly after <NUM>, <NUM>, and <NUM> minutes.

UPLC analysis: <NUM>µl of the samples are analysed by UPLC using a Waters Acquity system with a BEH C18 <NUM> <NUM> x <NUM> column and a <NUM> to <NUM>% gradient of <NUM>% TFA and <NUM>% TFA in acetonitrile over <NUM> minutes at a flow rate of <NUM>/min. After baseline subtraction the peak integrals of the intact compounds in the HPLC chromatogram recorded at a wavelength of <NUM> are determined.

MALDI-TOF analysis: <NUM>µl of each sample is transferred to a Bruker/Eppendorf PAC HCCA <NUM> MALDI target. Analysis is performed with a Bruker Autoflex matrix-assisted laser desorption and ionisation - time of flight (MALDI-TOF) mass spectrometer using the pre-defined method "PAC_measure" with an extended detection range of <NUM> to <NUM> Da and the pre-defined calibration method "PAC_calibrate".

Claim 1:
A composition comprising a GLP-<NUM> agonist and one or more pharmaceutically acceptable excipients for use in the prevention or treatment of type <NUM> diabetes, wherein said GLP-<NUM> agonist is semaglutide, wherein said GLP-<NUM> agonist is administered once weekly in an amount of <NUM> and wherein said GLP-<NUM> agonist is administered by parenteral administration.