RU2785662C2 - Dosing schemes for administration of glucagon-like peptide 2 (glp-2) analogues - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, in particular to the use of glucagon-like peptide 2 (hereinafter – GLP-2) analogues; it can be used in medicine. The invention discloses a new peptide GLP-2 analogue, as well as dosing schemes for its administration in case of medical use in the treatment and/or prevention of disorders related to the stomach and the intestine, including for reduction in side effects of chemotherapy or radiation therapy. The present invention also describes dosing schemes for administration of GLP-2 analogues for induction of the longitudinal growth of the intestine, for example, for the treatment of patients with the short bowel syndrome (SBS). Options of the medical use for correction of a volume of parenteral nutrition (hereinafter – PN) provided to subjects receiving the treatment, using GLP-2 analogues, in response to the treatment, and algorithms for determination of changes in PN volume are also described.

EFFECT: obtainment of GLP-2 analogues for use in medicine.

31 cl, 7 tbl, 7 ex

Description

Technical field

The present invention relates to dosing regimens for the administration of glucagon-like peptide-2 (GLP-2, GLP-2) analogues and their medical use, for example, for the treatment and/or prevention of disorders associated with the stomach and intestines, as well as to reduce the side effects of chemotherapy and radiation therapy. The present invention also relates to dosing regimens for administering glucagon-like peptide-2 (GLP-2) analogs to induce longitudinal intestinal growth, for example, for the treatment of patients with short bowel syndrome (SBS). The present invention also relates to medical applications for adjusting the volume of parenteral nutrition (PN) provided to subjects receiving treatment with GLP-2 analogs in response to treatment, as well as algorithms for determining changes in PN volume.

State of the art

Human GLP-2 is a 33 amino acid peptide having the following sequence: Hy-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu -Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH. It is formed as a result of specific post-translational processing of proglucagon in enteroendocrine L-cells of the intestine and in certain areas of the brainstem. GLP-2 binds to a single G-protein coupled receptor belonging to the class II glucagon-secretin family.

GLP-2 has been reported to induce significant growth of the small intestinal mucosal epithelium by promoting stem cell proliferation in the crypts and inhibiting apoptosis in the villi (Drucker et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7911-7916) . GLP-2 also affects colon growth. In addition, GLP-2 inhibits gastric emptying and gastric acid secretion (Wojdemann et al., 1999, J. Clin. Endocrinol. Metab. 84: 2513-2517), enhances intestinal barrier function (Benjamin et al., 2000, Gut 47 : 112-119), stimulates intestinal hexose transport by activating glucose transporters (Cheeseman, 1997, Am. J. Physiol. R1965-71) and increases intestinal blood flow (Guan et al., 2003, Gastroenterology, 125: 136- 147). An overview of GLP-2 and its properties is available at: http://jn.nutrition.org/content/131/3/709.full.

Glucagon-like peptide-2 receptor analogs are recognized in the art as having therapeutic potential for the treatment of bowel diseases. However, native hGLP-2, a 33 amino acid gastrointestinal tract peptide, is not suitable for clinical use due to its very short human half-life of approximately 7 minutes for full-length GLP-2 [1-33] and 27 minutes for truncated GLP-2 [3-33]. The short half-life is largely due to degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). Accordingly, attempts have been made in the art to develop GLP-2 receptor agonists with better pharmacokinetic characteristics, in particular to improve the half-life of GLP-2 molecules. For example, GLP-2 analogs with substitutions have been proposed, such as, for example, GLP-2 analogs containing a Gly substitution at position 2 ([hGly2] GLP-2, teduglutide), which increases the half-life from seven minutes (native GLP-2 ) up to about two hours. Acylation of peptide drugs with fatty acid chains has also been shown to be beneficial in increasing the duration of circulation in the systemic circulation, as well as in increasing enzymatic stability without compromising biological efficacy. However, while these attempts have improved the pharmacokinetics of GLP-2 analogs, and are sometimes described in the art as "long-acting", it should be kept in mind that this refers to a comparison with native hGLP-2, which has a half-life of several hours, not minutes. In turn, this means that GLP-2 analogs still need to be administered to patients one or more times a day.

There is considerable interest on the part of patients and medical professionals in the development of low-cost, long-acting, "user-friendly" therapeutic peptides. However, a major difficulty in delivering such therapeutic peptides is their short plasma half-life, mainly due to rapid serum clearance and proteolytic degradation by plasma proteases, requiring frequent, usually daily, injections. Patients experience aversion to injections, which leads to a decrease in adherence to treatment and a decrease in the effectiveness of the drug.

WO 2006/117565 (Zealand Pharma A/S) describes GLP-2 analogs which contain one of several substitutions compared to [hGly2]GLP-2 and which have improved in vivo biological activity and/or improved chemical stability, for example, as assessed in in vitro stability assays . In particular, GLP-2 analogs are described which contain substitutions at one or more positions 8, 16, 24 and/or 28 of the wild-type GLP-2 sequence, optionally in combination with additional substitutions at position 2 and one or more at positions 3, 5, 7, 10 and 11 and/or deletion of one or more amino acids 31 to 33. These substitutions can also be combined with the addition of an N-terminal or C-terminal stabilizing peptide sequence. Administration of said GLP-2 analogs once daily or twice daily is also described. Among the molecules described in WO 2006/117565 is glepaglutide (ZP1848), which has been designed to be stable in liquid formulations and is typically administered by daily dosing using a pen.

Brief description of the invention

In general terms, the present invention is based on the surprising observation that the pharmacokinetic (PK) and/or pharmacodynamic (PD) properties of GLP-2 analogs, for example of the type disclosed in WO 2006/117565 and WO 2008/056155, allow less frequent administration than previously suggested. Previous experiments indicated that GLP-2 analogs such as ZP1848 had a half-life of approximately 17 hours, which is significantly longer than the half-life of GLP-2 or other GLP-2 analogs, which implies that patients should be treated daily. introduction. However, in a phase 2 human clinical study using the GLP-2 analog ZP1848, it has now been found that the terminal plasma half-life of the molecule is actually 5 to 17 days. The terminal plasma half-life is the time required for a two-fold decrease in plasma concentration after reaching pseudo-equilibrium. In turn, this unexpectedly indicates that significantly less frequent dosing of the GLP-2 analogs of the present invention would be effective, including weekly and twice weekly dosing. The content of WO 2006/117565 and WO 2008/056155 is expressly included in its entirety for all purposes and in particular with respect to the GLP-2 analogs described in these documents.

The results of further studies also confirmed that both metabolites of ZP1848, i.e. ZP2711 and ZP2469 were present after in vivo administration and are pharmacologically active, with comparable in vitro efficacy to ZP1848. The plasma level of ZP2711 is within the same range as that of ZP1848, while the plasma level of ZP2469 is many times higher than that of ZP1848. Therefore, both metabolites can be included in the systemic exposure (exposure) assessment as they both contribute to in vivo efficacy. Due to the high plasma levels of ZP2469, this compound has plasma levels that exceed the limit of bioanalytical detection (50 pM for ZP2469, 25 pM for ZP1848 and ZP2711), therefore, it seems possible to determine the half-life of ZP2469. A terminal plasma half-life of 5 to 17 days, as shown in the examples, indicates that dosing ZP1848 once or twice a week would be effective.

In turn, this allows the GLP-2 analogs of the present invention to be administered, for example, once or twice a week. Alternatively or additionally, the GLP-2 analog dosing regimen of the present invention may include a plurality or course of doses separated in time by 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days. According to a preferred embodiment of the present invention, the doses are time-divided into 3 days, 3.5 days, 4 days, 5 days, 6 days, 7 days, or 8 days. According to a preferred embodiment of the present invention, the doses are time-divided into 3 days, 3.5 days, 4 days, or 7 days. As will be appreciated in the art, the time between doses may vary to some extent such that any and every dose is not separated by exactly the same time interval. The interval will often be set at the discretion of the physician. Thus, the doses may be separated in time by a clinically acceptable time interval, for example, from about 2 days to about 10 days, or from about 3 or 4 days to about 7 or 8 days. Clinical study results supporting these findings are included in the examples below.

Without wishing to be bound by theory, it is currently believed that the unexpected long half-life of GLP-2 analogs, which allows dosing once or twice a week, may result from a combination of subcutaneous depot formation and the formation of metabolites that are slowly released from subcutaneous depot and which are also GLP-2 receptor agonist. The authors of the present invention believe that the subcutaneous depot is formed when the GLP-2 analogs are administered as a result of the reaction between the analogs, i. lysine tail, and hyaluronic acid in the subcutaneous compartment. This type of depot has not previously been observed for other compounds with a lysine tail, such as lixisenatide, which has a half-life of 2-4 hours.

Accordingly, according to a first aspect of the present invention, there is provided a glucagon-like peptide-2 (GLP-2) analog for use in a method of treating and/or preventing a gastro-intestinal disorder in a human patient, said GLP-2 analog being represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

In this aspect of the present invention, a glucagon-like peptide-2 (GLP-2) analog can be used to treat and/or prevent disorders associated with the stomach and intestines, such as ulcers, digestive disorders, malabsorption syndromes, short bowel syndrome, blind pouch syndrome , inflammatory bowel disease, celiac disease (eg, caused by gluten-induced enteropathy or celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, small bowel injury, or short bowel syndrome (SBS). Alternatively or additionally, a glucagon-like peptide-2 (GLP-2) analog can be used to treat and/or prevent disorders associated with the stomach and intestines, such as radiation enteritis, infectious or post-infectious enteritis, or damage to the small intestine caused by toxic or other chemotherapeutic agents. . In this case, treatment with the GLP-2 analog may optionally be combined with one or more anti-cancer therapies, and therefore may include administering one or more chemotherapeutic agents to the patient or treating the patient with radiation therapy.

According to a further aspect of the present invention, there is provided a glucagon-like peptide-2 (GLP-2) analog for use in a method of treating and/or preventing a side effect of chemotherapy or radiation therapy in a human patient, said GLP-2 analog being represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

According to a further aspect of the present invention, there is provided a glucagon-like peptide-2 (GLP-2) analog for use in a method for increasing longitudinal intestinal growth in a human patient, said GLP-2 analog being represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ² ,

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

According to a further aspect of the present invention, there is provided a glucagon-like peptide-2 (GLP-2) analog for use in a method of treating short bowel syndrome (SBS) in a human subject receiving a parenteral nutrition (PN) regimen, said method comprising:

(i) administering to the subject a GLP-2 analogue represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

wherein said GLP-2 analog is administered once or twice a week for a period of time sufficient to lengthen and improve the function of the subject's small intestine;

(ii) after step (i), reducing the frequency or volume of the parenteral nutrition (PN) regimen in response to improvement in small bowel function; and

(iii) optionally repeating steps (i) and (ii) in response to the subject demonstrating improved function over time as a result of additional longitudinal growth of the small intestine.

According to a further aspect of the present invention, there is provided a method for treating short bowel syndrome (SBS) in a human subject receiving a parenteral nutrition (PN) regimen, said method comprising:

(i) administering to the subject a GLP-2 analogue represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

wherein said GLP-2 analog is administered once or twice a week for a period of time sufficient to lengthen and further improve the function of the subject's small intestine;

(ii) after step (i), reducing the frequency or volume of the parenteral nutrition (PN) regimen in response to improvement in small bowel function; and

(iii) optionally repeating steps (i) and (ii) in response to the subject demonstrating continued improvement in small bowel function.

For example, the treatment period during which the GLP-2 analog is administered to a subject may be 18 to 150 weeks, such as 18 to 100 weeks or 18 to 52 weeks. However, in other cases, if SBS is a chronic condition, patients may require treatment for the rest of their lives.

As used herein, the term "parenteral nutrition" or "PN" includes the administration of nutrients and/or fluids to a subject receiving GLP-2 therapy as a means of providing the subject with nutrients and/or fluids that the subject needs but cannot completely absorbed due to his condition. Determining the correct amount or volume of PN to give to subjects with SBS who are receiving GLP-2 therapy is difficult because if PN volume is not adjusted in a timely and appropriate manner, patients may experience fluid overload, be at risk of dehydration, and may not achieve optimal clinical responses. for therapy. This situation is further complicated by the fact that the amount of PN required by a subject tends to change over the course of GLP-2 therapy depending on the subject's response to treatment. In general, the estimate of the amount of PN required by a subject during GLP-2 therapy depends on how long the therapy is continued and on the susceptibility of individual patients to it. Considering this change, an initial assessment of PN volume can be made during the first few days of GLP-2 therapy, and then, as a rule, followed by weekly assessment during the first month, monthly assessment for the next 1-3 months, and then assessment every 3-6 months before completion of treatment. This is important because subjects may have a rapid initial response to GLP-2 therapy with improvement in small bowel function, for example, even before any increase in bowel length is observed. In turn, this allows the volume of PN to be reduced to avoid the risk of side effects such as fluid overload.

Accordingly, in the above method, step (ii) may include the step of (a) determining the volume of PN required by the subject at a given point in treatment, (b) comparing it with the initial volume of PN determined at the start of therapy using the GLP-2 analogue, and (c ) reducing the frequency or volume of PN if the subject demonstrates improved small bowel function. Optionally, reducing the frequency or volume of parenteral nutrition (PN) in step (ii) may be performed using the algorithm described below.

As an illustration of the relationship between the amount of parenteral nutrition required by patients and the degree of improvement in bowel function, it is currently believed that increasing the length of the small intestine by 40% will improve the function or absorption capacity of the small intestine by at least another 10%. Typically, GLP-2 therapy according to the present invention results in an improvement in small intestinal function or absorption capacity of at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40% and most preferably at least 50%. Additionally or alternatively, the amount by which parenteral nutrition is reduced during a course of GLP-2 therapy is at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, and most preferably at least 50%. According to a preferred embodiment of the present invention, the reduction in parenteral nutrition is at least 20%.

According to a further related aspect of the present invention, a solution is provided to one of the problems that patients and clinicians face when starting GLP-2 therapy, namely the appropriate and individual adjustment of the amount of parenteral nutrition (PN) provided to the patient. This is important because, if PN volume is not adjusted in a timely and appropriate manner, patients may experience fluid overload, be at risk of dehydration, and may not achieve optimal clinical responses to therapy.

For example, in a previous phase 3 study of 24 weeks of treatment with the GLP-2 analog, teduglutide, an attempt was made to reduce parenteral nutrition by 10% per month no earlier than 4 weeks after the start of treatment if urine output increased by at least by 10% relative to the initial level. Many of the patients in this study suffered from fluid overload and stopped drinking (Jeppesen et al. 2011, Gut 2011; 60: 902-914). In a subsequent 24-week phase 3 study in patients with SBS who received teduglutide subcutaneously (Jeppesen et al. more than 30%, after 2 weeks of treatment, with an increase in diuresis of at least 10% relative to the initial level. However, in this study, patients also suffered from fluid overload, particularly at the start of treatment.

Therefore, this aspect of the present invention provides an early assessment of altered PN fluid requirements (eg, within days of initiation of GLP-2 therapy) and proposes algorithms for adjusting PN volume during a course of GLP-2 therapy. Those of skill in the art will appreciate that this PN volume adjustment algorithm approach provides individual PN volume adjustment for each patient. This aspect of the present invention is applicable to GLP-2 therapies using the GLP-2 analogs described herein or using GLP-2 analogs known in the art, such as teduglutide.

Accordingly, according to a further aspect of the present invention, there is provided a method for adjusting the volume of parenteral nutrition (PN) in a human subject receiving a certain amount or volume of parenteral nutrition each week, said method comprising calculating a new volume of PN using an algorithm in which the volume of PN per week is calculated by subtracting the seven-fold absolute increase in daily urine volume from the initial volume, i.e. volume of PN at the start of GLP-2 therapy, from the current weekly volume of parenteral nutrition, to provide a new volume of PN for the subject. Thus, the subject's new PN volume (weekly) is equal to the subject's current PN volume (weekly) minus 7 × the absolute increase in daily urine volume from the initial volume.

According to a further aspect of the present invention, there is provided a method for determining the amount of parenteral nutrition (PN) for a human subject receiving GLP-2 therapy, said method comprising:

optionally determining the initial daily urine volume at the start of GLP-2 therapy;

calculation of a new PN volume (weekly) for the subject for the upcoming week based on the current PN volume (weekly) and daily urine volume (for example, at a doctor's visit) using an algorithm in which the new PN volume (weekly) for the subject is equal to the current PN volume ( weekly) minus 7 × absolute increase in daily urine volume relative to the initial volume.

In some cases, the calculation step is carried out if the daily urine volume is at least 10% higher than the initial urine volume. This method may include the additional step of determining the current volume of PP (weekly) for use in the calculation of the new amount of PP using the described algorithm.

It should be understood that the method may further include the step of administering the PN volume to the subject. In addition, the method can be repeated, for example, every week, month, or at a visit to the doctor, which allows you to reduce the amount of PN, if the subject shows continued improvement in the function of the small intestine. In one embodiment of the present invention, adjusting the PN volume results in a urine volume of at least 800 ml/day (24 hours), such as at least 1000 ml/day (24 hours) or more. A particular advantage of this method is that the effects of treatment and subsequent adjustment of PN volume can be achieved very early in GLP-2 therapy, eg in the first few days of treatment, which is significantly faster compared to prior art approaches. For example, in accordance with the present invention, subjects may undergo an optimization phase of 0-4 weeks followed by a stabilization phase of 2-4 weeks. The PN volume reduction algorithm may be based on changes in urine volume measured over a 48 hour period.

In some embodiments of this aspect of the present invention, GLP-2 therapy may include administering a GLP-2 analog represented by the formula described herein, specifically using examples of these GLP-2 analogs. According to a preferred embodiment of the present invention, the GLP-2 analogue is ZP1848.

Preferably, the method includes the initial steps of determining the initial volume and/or determining the daily volume of urine. Some or all of the steps of the method may be repeated during the course of GLP-2 therapy received by the subject. Preferably, the first assessment and determination of changes in PN volume adjustments is performed early in GLP-2 therapy, e.g., several days after initiation of GLP-2 therapy, e.g., 2, 3, 4, 5, 6, 7 days after initiation of GLP therapy. -2. Preferably, the time interval between retests to determine changes in PN volume adjustments is initially a week, and then approximately every 1-3 months. For example, according to one implementation variant of the present invention, the method can be applied after 1, 2, 4, 8 and 12 weeks after the start of treatment using a GLP-2 analogue, adjusting the volume of PN in response to the effects of treatment. The weekly volume of PN should be monitored and adjusted throughout treatment with a GLP-2 analog (eg, glepaglutide) to avoid fluid overload. Generally, as intestinal absorption capacity increases after treatment with a GLP-2 analog, typically as a result of small intestine lengthening or thickening, PN volume can be reduced to avoid fluid overload and improve patient treatment needs.

Other conditions that may be treated with the GLP-2 analogs of the present invention, or for which GLP-2 analogs may be used prophylactically or therapeutically, include radiation enteritis, infectious or post-infectious enteritis, and injury to the small intestine caused by toxic or other chemotherapeutic agents. . This may require the administration of a GLP-2 analog before, concomitantly, or after a course of chemotherapy or radiation therapy to reduce the side effects of chemotherapy such as diarrhea, abdominal cramps, and vomiting, and to reduce subsequent structural and functional damage to the intestinal epithelium from chemotherapy or radiation therapy. . Preferably, administration of the GLP-2 analog according to the present invention is started 1, 2, 3, 4, 5, 6, or 7 days before the start of the chemotherapy or radiation cycle. Preferably, administration of the GLP-2 analog of the present invention is started the day before or on the day of initiation of chemotherapy or radiation treatment, and once or twice a week thereafter.

According to a further aspect of the present invention, there is provided the use of a glucagon-like peptide-2 (GLP-2) analog in the manufacture of a medicament for the treatment and/or prevention of a gastro-intestinal disorder in a human patient, said GLP-2 analog being represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

According to a further aspect of the present invention, there is provided the use of a glucagon-like peptide-2 (GLP-2) analogue in the manufacture of a medicament for the treatment and/or prevention of a side effect of chemotherapy or radiation therapy in a human patient, said GLP-2 analogue being represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

According to a further aspect of the present invention, there is provided a method for treating and/or preventing a gastro-intestinal disorder in a human patient, said method comprising administering to the patient a therapeutically effective amount of a glucagon-like peptide-2 (GLP-2) analogue represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

According to a further aspect of the present invention, there is provided a method for treating and/or preventing a side effect of chemotherapy or radiation therapy in a human patient, said method comprising administering to the patient a therapeutically effective amount of a glucagon-like peptide-2 (GLP-2) analogue represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

According to a further aspect of the present invention, there is provided the use of a glucagon-like peptide-2 (GLP-2) analog in the manufacture of a medicament for increasing longitudinal intestinal growth in a human patient, said glucagon-like peptide-2 (GLP-2) analog being represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

According to a further aspect of the present invention, there is provided a method for increasing longitudinal intestinal growth in a human patient, said method comprising administering to the patient a therapeutically effective amount of a glucagon-like peptide-2 (GLP-2) analogue represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof; and

said method comprising administering said GLP-2 analog to a patient once or twice a week.

In all aspects of the present invention, the method of administering a glucagon-like peptide-2 (GLP-2) analog optionally comprises administering multiple doses of the GLP-2 analog to the patient, with the doses separated in time by one week or half a week. In some cases, it may be desirable to divide the total dose into multiple (e.g., two or three) individual doses, e.g., for administration at spatially separated injection sites, e.g., injection sites that are at least 5 cm apart.

Preferably, the doses of GLP-2 analogs used in accordance with the present invention are in the range of 0.5 mg to 25 mg inclusive per patient once or twice a week, optionally from 1 mg to 20 mg inclusive per patient once or twice a week. week, optionally 1 mg to 10 mg inclusive per patient once or twice a week, optionally 2 mg to 7 mg inclusive per patient once or twice a week, optionally 5 mg to 7 mg inclusive per patient once or twice per week or optionally from 2 mg to 5 mg inclusive per patient once or twice a week. According to one implementation variant of the present invention, the dose of GLP-2 analogs used in accordance with the present invention is 10 mg per patient, inclusive, once or twice a week. During treatment, the doses taken by the patient may be the same or different in accordance with the instructions of the physician.

Preferably, the glucagon-like peptide-2 (GLP-2) analogue is administered to patients by injection, most typically by subcutaneous injection or intramuscular injection. According to some preferred embodiments of the present invention, the GLP-2 analog can be administered using a pen that allows patients to self-administer the analog. In some aspects, administration of the GLP-2 analog causes the formation of a subcutaneous depot from which the GLP-2 analog or its metabolites are released. Without regard to any particular explanation, the subcutaneous depot may be formed by the interaction of GLP-2 analogs administered in accordance with the present invention, in particular if the analogs contain a lysine tail (i.e. a Z ¹ group and/or a Z ² group) , resulting from a reaction between analogs and hyaluronic acid in the subcutaneous compartment.

In some embodiments of the present invention, in the formula above, X5 is Thr and/or X11 is Ala. Examples of these glucagon-like peptide-2 (GLP-2) analogues include:

ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ₂ (SEQ ID NO: 1)

ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH (SEQ ID NO: 2)

ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH (SEQ ID NO: 3)

ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 4)

ZP1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH ₂ (SEQ ID NO: 5)

ZP2530 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH (SEQ ID NO: 6).

In some embodiments of the present invention, in the formula above, X5 is Ser and/or X11 is Ser. Examples of these glucagon-like peptide-2 (GLP-2) analogues include:

ZP1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH ₂ (SEQ ID NO: 7)

ZP1855 H-HGEGSFSELSTILDALAARDFIAWLIATKITD-NH ₂ (SEQ ID NO: 8)

ZP2242 H-HGEGSFSELSTILDALAARDFIAWLIATKITDK-OH (SEQ ID NO: 9).

Embodiments of the present invention will be further described by way of example and are not limiting. However, various additional aspects and embodiments of the present invention will be apparent to those skilled in the art in light of the present disclosure.

In this application, "and/or" should be considered as a specific description of each of the two specified features or components, with or without the other. For example, "A and/or B" should be considered as a specific description of each of (i) A, (ii) B and (iii) A and B, as if each of them were specified in this application separately.

Unless the context requires otherwise, the descriptions and definitions of the features set forth above are not limited to any particular aspect or embodiment of the present invention and are equally applicable to all aspects and embodiments that are described.

Detailed description of the invention

Definitions

Unless otherwise noted, the following definitions are provided for specific terms used in the above written description.

Throughout the description and claims for naturally occurring amino acids, conventional one-letter and three-letter codes are used. All amino acid residues in the peptides of the present invention preferably have an L-configuration, however amino acids with a D-configuration may also be present.

Preferred compounds of the present invention have at least one GLP-2 biological activity, in particular causing intestinal growth. This can be assessed in in vivo assays, for example, as described in the examples, in which the weight of the intestine or portion thereof is determined after the test animal has been treated with or exposed to a GLP-2 analogue.

The GLP-2 analogs of the present invention contain one or more amino acid substitutions, deletions, inversions or additions compared to native GLP-2 and as defined above. This definition also includes the synonymous terms "GLP-2 mimetics" and/or "GLP-2 agonists". In addition, the analogue according to the present invention may further contain a chemical modification of one or more of the side groups of its amino acids, α-carbon atoms, terminal amino group or terminal carboxyl group. Chemical modification includes, but is not limited to, the addition of chemical moieties, the creation of new bonds, and the removal of chemical moieties. Amino acid pendant modifications include, but are not limited to, acylation of the ε-amino groups of lysine, N-alkylation of arginine, histidine, or lysine, alkylation of carboxyl groups of glutamic acid or aspartic acid, and deamidation of glutamine or asparagine. Amino terminal modifications include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications to the terminal carboxyl group include, but are not limited to, amidation, modifications such as lower alkylamide, dialkylamide, and lower alkyl ester. Preferably, in the present application, the lower alkyl is C ₁ -C ₄ alkyl. In addition, one or more side groups or end groups may be protected with protecting groups known to those of ordinary skill in the art of peptide chemistry. The α-carbon of the amino acid may be mono- or dimethylated.

It should be understood that the peptides of the present invention may also be provided in the form of a salt or other derivative. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts include hydrochloride salts, citrate salts and acetate salts. Examples of basic salts include salts in which the cation is selected from alkali metals such as sodium and potassium, alkaline earth metals such as calcium, and ammonium ions ⁺ N (R ³ ) ₃ (R ⁴ ), where R ³ and R ⁴ are independently optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are described in Remington's Pharmaceutical Sciences, 17th ed. ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 and later editions, and in the Encyclopaedia of Pharmaceutical Technology.

Other derivatives of the GLP-2 analogs of the present invention include coordination complexes with metal ions such as Mn ²⁺ and Zn ²⁺ , esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids. Esters can be formed between the hydroxyl or carboxyl groups present in the compound and the appropriate carboxylic acid or alcohol reactive partner using techniques well known in the art. Prodrug derivatives of the compounds can be converted to one of the parent compounds under in vivo conditions or under in vitro conditions. Typically, in a prodrug form of a compound, at least one biological activity of the compound will be reduced and can be activated by converting the prodrug to release the compound or its metabolite. Examples of prodrugs include the use of protecting groups that can be removed in situ to release the active compound or serve to inhibit drug clearance in vivo .

Z ¹ and Z ² are independently present and/or absent or represent a peptide sequence of 1-6 amino acid units of Lys, i. e. 1, 2, 3, 4, 5 or 6 Lys residues. Residues of Lys may have a D- or L-configuration, but have an L-configuration. Particularly preferred Z sequences are sequences of four, five or six consecutive lysine residues, and in particular six consecutive lysine residues. Exemplary Z sequences are provided in WO 01/04156. In certain embodiments, Z ¹ is absent. In such cases, Z ² may either be present or absent.

Pharmaceutical compositions and administration

The GLP-2 analogs of the present invention, or salts or derivatives thereof, may be formulated into pharmaceutical compositions prepared for storage or administration, which contain a therapeutically effective amount of a GLP-2 peptide of the present invention, or a salt or derivative thereof, in a pharmaceutically acceptable carrier. According to the dosing regimens of the present invention, the GLP-2 analogs are administered to patients, eg, a human patient, once or twice a week. Thus, a course of administration in accordance with the present invention may include administering to a patient a course of doses divided in time into 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days, and more preferably divided in time into 3 days, 4 days, 5 days, 6 days, 7 days or 8 days. In some specific situations, the present invention may involve administering a fixed dose to the patient, such as 10 mg once or twice a week.

A therapeutically effective amount of a compound of the present invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the particular mammal in question. These factors and their relationship to the determination of such an amount are well known to those skilled in the art of medicine. This amount and route of administration can be tailored to achieve optimal efficacy so as to deliver the peptide to the colon, but will depend on factors such as weight, diet, concomitant drug therapy, and other factors well known to those skilled in the medical arts.

The present invention provides a pharmaceutical composition wherein a GLP-2 analog, or salt thereof, is present in an amount effective to treat or prevent disorders associated with the stomach and intestines, or to induce longitudinal growth of the intestine.

Pharmaceutically acceptable salts of the compounds of the present invention containing an acid moiety can be prepared using organic and inorganic bases. Suitable salts formed with bases include metal salts such as alkali metal or alkaline earth metal salts, eg sodium, potassium or magnesium salts; ammonia salts and salts of organic amines, for example, salts formed with morpholine, thiomorpholine, piperidine, pyrrolidine, lower mono-, di- or trialkylamine (for example, ethyl tert-butyl, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethylpropylamine) or a lower mono-, di- or trihydroxyalkylamine (eg mono-, di- or triethanolamine). Internal salts may also form. Similarly, if the compound of the present invention contains a basic moiety, salts can be formed using organic and inorganic acids. For example, salts can be formed from the following acids: acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, naphthalenesulfonic, benzenesulfonic, toluenesulfonic and camphorsulfonic, as well as other known pharmaceutically acceptable acids. Amino acid addition salts can also be formed with amino acids such as lysine, glycine or phenylalanine.

One of ordinary skill in the art of medicine will appreciate that a "therapeutically effective amount" of the peptides or pharmaceutical compositions of the present invention will vary with the age, weight, and species of the mammal being treated, the particular compounds used, the particular route of administration, and the intended effects and therapeutic indication. Since these factors and their relationship to determining this amount are well known in medicine, the determination of therapeutically effective dosage levels, the amount necessary to achieve the goal of preventing and/or treating diseases associated with the intestines and stomach described in this application, as well as other medical indications described in this application for the induction of longitudinal growth of the intestine, will be within the competence of a person skilled in the art.

In the present application, the term "therapeutically effective amount" means an amount that reduces the symptoms of a particular condition or pathology, and which preferably normalizes physiological responses in an individual suffering from a condition or pathology. Reduction of symptoms or normalization of physiological responses can be determined using conventional methods in the art and may vary depending on the specific condition or pathology. In one aspect, a therapeutically effective amount of one or more GLP-2 analogs, or a pharmaceutical composition comprising one or more GLP-2 analogs, is an amount that restores a measurable physiological parameter to a substantially similar value (preferably within +30%, more preferably within +20% and even more preferably within 10% of the value) of the parameter in an individual not suffering from the condition or pathology.

In one embodiment of the present invention, the administration of the compounds or pharmaceutical composition of the present invention is initiated at lower dosage levels, with dosage levels being increased until the target effect of preventing/treating relevant medical indications such as intestinal and stomach, or until an increase in longitudinal intestinal growth is achieved. This will determine the therapeutically effective amount. Whether used alone or as part of a pharmaceutical composition, doses of GLP-2 analogs used in accordance with the present invention typically range from 0.5 mg to 25 mg inclusive per patient once or twice a week, optionally from 1 mg to 20 mg inclusive per patient once or twice a week, optionally from 1 mg to 10 mg inclusive per patient once or twice a week, optionally from 2 mg to 7 mg inclusive per patient once or twice a week, optionally from 5 mg to 7 mg inclusive per patient once or twice a week, or optionally 2 to 5 mg inclusive per patient once or twice a week. According to one embodiment of the present invention, a therapeutically effective amount of GLP-2 analogs used in accordance with the present invention is about 10 mg per patient once or twice a week. However, the person skilled in the art will be able to adjust these doses in the event that an alternative dosing regimen is chosen in accordance with the present invention.

In aspects of the present invention relating to achieving increased longitudinal intestinal growth, preferably treatment with the GLP-2 analogs of the present invention increases intestinal growth by at least 30% over control, more preferably at least 40% over control. and most preferably at least 50% as compared to the control. In general, it is preferable that the increase in longitudinal growth is accompanied by an increase in suction capacity during treatment and to some extent even after treatment is stopped. In the treatment of a human patient who is in need of increased longitudinal intestinal growth, such as patients with short bowel syndrome (SBS), treatment usually lasts at least 1-3 years and may optionally include treatment once or twice a week or according to other treatment regimens according to the present invention, described in this application.

The class of human patients suffering from SCM includes patients suffering from SCM-intestinal failure syndrome (intestinal failure) (SCM-SKS) and patients whose condition is intermediate between SCM-intestinal failure (SCM-FI) and SCM-intestinal failure syndrome (SKK-SKN). In some cases, patients suffering from HCM bowel insufficiency syndrome (HCM-KIS) are also referred to as HCM-IB if they are dependent on parenteral nutrition, and patients suffering from HCM-colon insufficiency (HCM-CI) are also referred to as HCM without RI. if they are not dependent on parenteral nutrition.

The spectrum of patient types with SBS is reviewed in Jeppensen, Journal of Parenteral and Enteral Nutrition, 38(1), 8S-13S, May 2014, doi: 10.1177/0148607114520994. Further separation of types of patients with SBS can be done according to the principles described in Schwartz et al., Clinical and Translational Gastroenterology (2016) 7, e142; doi:10.1038/ctg.2015.69. It allows the division of patients with SBS into those with an early response and those with a late/slow response. Without wishing to be bound by any theory, the present inventors believe that early response patients are those who show an early effect on treatment with a GLP-2 analog caused by, among other effects, an increase in the width/diameter of the small intestine while as patients with a late or slow response are patients who primarily or primarily benefit from treatment with a GLP-2 analogue as a result of an increase in the length of the small intestine. Determining whether a subject is an early responder or late responder can be used to determine the duration of a GLP-2 analog treatment regimen, the timing of any clinical decision to reduce parenteral nutrition, and the interval between tests to determine if a reduction in parenteral nutrition is possible. . Accordingly, according to one embodiment of the present invention, the patient has a late or slow response. The length of the small intestine can be measured, for example, using CT (computed tomography), MRI (magnetic resonance imaging), histological, laparoscopic or other measurements or techniques known in the art.

According to a further aspect of the present invention, there is provided a glucagon-like peptide-2 (GLP-2) analogue for use in a method for treating and/or preventing HCM-intestinal failure (HCM-FI) in a patient, said treatment preventing or delaying the development of HCM-intestinal failure syndrome ( SKK-SKN), while the specified analogue of GLP-2 is represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl; and

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof.

In some embodiments, the method may include administering the GLP-2 analog to the patient once or twice a week and/or in accordance with any of the other treatment regimens described herein.

According to other related aspects of the present invention, the use of a GLP-2 analogue represented by the formula is provided:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof in the manufacture of a medicament for the treatment and/or prevention of a patient suffering from HCM-intestinal insufficiency (HCM-CI), said treatment preventing or delaying the development of HCM-intestinal insufficiency syndrome (HCM-KIS).

According to other related aspects of the present invention, there is provided a method for treating and/or preventing a patient suffering from HBS bowel failure, said method preventing or delaying the development of HBS bowel failure syndrome, said GLP-2 analog being represented by the formula:

R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ²

wherein:

R ¹ is hydrogen, C _1-4 alkyl (eg methyl), acetyl, formyl, benzoyl or trifluoroacetyl;

X5 is Ser or Thr;

X11 is Ala or Ser;

R ² is NH ₂ or OH; and

Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys;

or a pharmaceutically acceptable salt or derivative thereof.

For therapeutic use, the selected GLP-2 analog is formulated with a carrier that is pharmaceutically acceptable and suitable for delivery of the peptide via the chosen route of administration. For the purpose of the present invention, peripheral parenteral routes include intravenous, intramuscular, subcutaneous and intraperitoneal routes of administration. According to one embodiment of the present invention, the route of administration is subcutaneous or subcutaneous. The pharmaceutical composition of the present invention comprises the GLP-2 analog of the present invention, or a salt or derivative thereof, and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include those commonly used with peptide-based drugs, such as diluents, excipients, and the like. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit., 1985). For example, sterile saline and phosphate buffered saline at slightly acidic or physiological pH can be used. pH buffering agents can be phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine which is the preferred buffer, arginine, lysine or acetate, or mixtures thereof. Preferred buffer ranges include pH 4-8, pH 6.5-8, more preferably pH 7-7.5. Preservatives such as para-, meta- and ortho-cresol, methyl- and propylparaben, phenol, benzyl alcohol, sodium benzoate, benzoic acid, benzyl benzoate, sorbic acid, propanoic acid, p-hydroxybenzoic acid esters may be included in the pharmaceutical composition. . Stabilizers can be provided in the pharmaceutical composition to prevent oxidation, deamidation, isomerization, racemization, cyclization, hydrolysis of peptides, such as, for example, ascorbic acid, methionine, tryptophan, EDTA, asparagine, lysine, arginine, glutamine and glycine. Anti-aggregation, fibrillation and sedimentation stabilizers such as sodium dodecyl sulfate, polyethylene glycol, carboxymethyl cellulose, cyclodextrin may be provided in the pharmaceutical composition. Organic modifiers for solubilizing or preventing aggregation, such as ethanol, acetic acid or acetate, and salts thereof, may be provided in the pharmaceutical composition. Isotonicity agents may be provided in the pharmaceutical composition, such as salts, eg sodium chloride, or most preferably carbohydrates, eg dextrose, mannitol, lactose, trehalose, sucrose, or mixtures thereof.

Detergents such as Tween 20, Tween 80, SDS, poloxamers such as Pluronic F-68, Pluronic F-127 can be provided in the pharmaceutical composition. Coloring agents and even flavoring agents may be included in the pharmaceutical composition. In another embodiment, a pharmaceutically acceptable acid addition salt of a GLP-2 peptide analog is provided. Suspending agents may be used.

Organic modifiers such as ethanol, t-butanol, 2-propanol, ethanol, glycerol, polyethylene glycol may be provided in the pharmaceutical composition for lyophilization of the lyophilized product. Filling agents and isotonicity agents such as salt, such as sodium chloride, carbohydrates, such as dextrose, mannitol, lactose, trehalose, sucrose, or mixtures thereof, amino acids, such as glycine, glutamate, or excipients, such as cysteine, lecithin or human serum albumin, or mixtures thereof, may be provided in the pharmaceutical composition for lyophilization.

Pharmaceutical compositions according to the present invention can be prepared and used in the form of tablets, capsules or elixirs for oral administration; suppository for rectal administration; preferably sterile solutions or sterile powder or suspensions for injection; and the like. The dose and route of administration may be adapted for optimal efficacy, but will depend on factors such as weight, diet, concomitant drug therapy, and other factors to be determined by those skilled in the art.

In case the administration is to be parenteral, such as intravenous, subcutaneous or intramuscular, injectable pharmaceutical compositions may be prepared in the form of conventional forms, or in the form of aqueous solutions or suspensions; lyophilized, solid forms suitable for reconstitution immediately prior to use, either suspension in liquid prior to injection, or as emulsions.

Diluents for reconstitution of the lyophilized product may be a suitable buffer from the list above, water, saline, dextrose, mannitol, lactose, trehalose, sucrose, lecithin, albumin, monosodium glutamate, cysteine hydrochloride; or water for injection with added detergents such as Tween 20, Tween 80, poloxamers such as pluronic F-68 or pluronic F-127, polyethylene glycol, and/or with added preservatives such as para-, meta- and ortho-cresol , methyl and propyl paraben, phenol, benzyl alcohol, sodium benzoate, benzoic acid, benzyl benzoate, sorbic acid, propanoic acid, p-hydroxybenzoic acid esters, and / or with the addition of an organic modifier such as ethanol, acetic acid, citric acid, lactic acid or their salts.

In addition, if necessary, injectable pharmaceutical compositions may contain minor amounts of non-toxic excipients such as wetting agents or pH buffering agents. Absorption enhancers (eg, liposomes, detergents, and organic acids) may be used.

According to one embodiment of the present invention, the compounds are formulated for administration by infusion, for example, when used as liquid nutritional supplements for patients receiving total parenteral nutrition therapy (for example, neonates or patients suffering from cachexia or anorexia), or by injection, for example , subcutaneously, intraperitoneally or intravenously, and are respectively administered as aqueous solutions in sterile and pyrogen-free form and optionally buffered to a physiologically tolerable pH, eg slightly acidic or physiological pH. The composition for intramuscular administration may be based on solutions or suspensions in vegetable oil, for example canola oil, corn oil or soybean oil. These oil-based formulations can be stabilized with antioxidants such as BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene).

Thus, the peptide compounds of the present invention can be administered in vehicles such as distilled water or saline, phosphate buffered saline, 5% dextrose solutions or oils. The solubility of the GLP-2 analog can be increased, if necessary, by the inclusion of a solubility enhancer, such as detergents and emulsifiers.

For injectable use, the aqueous carrier or vehicle may be supplemented with some gelatin which serves to deposit the GLP-2 analog at or near the injection site for slow release to the target site of action. Other gelling agents such as hyaluronic acid can also be used as depot agents.

According to one embodiment of the present invention, the composition contains

a. L-histidine dissolved in water to give final concentrations of 0.5 mM to 300 mM, preferably 3 to 200 mM, most preferably 20 to 100 mM;

b. mannitol to give up to 350 mM, preferably 30 to 300 mM, most preferably 100 mM to 230 mM; and

c. acetic acid to give up to 200 mM, preferably 0.05 to 100 mM, most preferably 0.5 to 50 mM in solution.

An appropriate amount of therapeutic compound is added to give concentrations of 1 to 100 mg/ml, preferably 5 to 50 mg/ml, most preferably 10 to 30 mg/ml.

The pH is adjusted to a final pH of 4 to 8, preferably 6.5 to 7.5, most preferably 6.7 to 7.3. The resulting solution is adjusted to the target weight, sterile filtered and distributed into appropriate aliquots in vials for pharmaceutical use. The formulation is further processed as appropriate for a liquid product or a lyophilized product.

According to another embodiment of the present invention, the composition contains

a. L-histidine dissolved in water to give final concentrations of 0.5 mM to 300 mM, preferably 3 to 200 mM, most preferably 20 to 100 mM L-histidine;

b. L-arginine to obtain up to 200 mM, preferably 0.5 to 100 mM, most preferably 5 to 50 mM;

c. mannitol to give up to 350 mM, preferably 30 to 300 mM, most preferably 100 mM to 230 mM; and

d. acetic acid to give up to 200 mM, preferably 0.05 to 100 mM, most preferably 0.5 to 50 mM in solution.

An appropriate amount of therapeutic compound is added to give concentrations of 1 to 100 mg/ml, preferably 5 to 50 mg/ml, most preferably 10 to 30 mg/ml.

The pH is adjusted to a final pH of 4 to 8, preferably 6.5 to 7.5, most preferably 6.7 to 7.3. The resulting solution is adjusted to the target weight, sterile filtered and distributed into appropriate aliquots in vials for pharmaceutical use. The formulation is further processed as appropriate for a liquid product or a lyophilized product.

According to another embodiment of the present invention, the composition contains

a. L-histidine dissolved in water to give final concentrations up to 200 mM, preferably 3 to 100 mM, most preferably 5 to 50 mM L-histidine;

b. L-arginine to obtain up to 200 mM, preferably 0.5 to 100 mM, most preferably 5 to 50 mM;

c. mannitol to give up to 350 mM, preferably 30 to 300 mM, most preferably 100 mM to 230 mM; and

d. acetic acid to give up to 200 mM, preferably 0.05 to 100 mM, most preferably 0.5 to 50 mM in solution.

An appropriate amount of therapeutic compound is added to give concentrations of 1 to 100 mg/ml, preferably 5 to 50 mg/ml, most preferably 10 to 30 mg/ml.

The pH is adjusted to a final pH of 4 to 8, preferably 6.5 to 7.5, most preferably 6.7 to 7.3. The resulting solution is adjusted to the target weight, sterile filtered and distributed into appropriate aliquots in vials for pharmaceutical use. The formulation is further processed as appropriate for a liquid product or a lyophilized product.

According to another embodiment of the present invention, the composition contains

a. N-acetate dissolved in water to give final concentrations up to 200 mM, preferably 0.5 to 100 mM, most preferably 5 to 50 mM L-histidine;

b. mannitol to obtain up to 350 mM, preferably 30 to 300 mM, most preferably 100 mM to 230 mM.

An appropriate amount of therapeutic compound is added to give concentrations of 1 to 100 mg/ml, preferably 5 to 50 mg/ml, most preferably 10 to 30 mg/ml.

The pH is adjusted to a final pH of 4 to 8, preferably 6.5 to 7.5, most preferably 6.7 to 7.3. The resulting solution is adjusted to the target weight, sterile filtered and distributed into appropriate aliquots in vials for pharmaceutical use. The formulation is further processed as appropriate for a liquid product or a lyophilized product.

The GLP-2 analogs of the present invention can also be formulated into a slow release implantable device for sustained and sustained administration of a GLP-2 peptide analog. Such sustained release formulations may be in the form of a patch placed externally on the body. Examples of sustained release formulations include composites of biocompatible polymers such as polylactic acid, lactic acid-glycolic acid copolymer, methylcellulose, hyaluronic acid, sialic acid, silicate, collagen, liposomes, and the like. Sustained release formulations may be of particular interest if a high local concentration of the GLP-2 analog of the present invention is desired.

The GLP-2 analog can be administered in the form of a sterile filled vial or ampoule containing an enterotrophic amount of the peptide, either as a single dose or as multiple doses. The vial or ampoule may contain the GLP-2 analog and the desired carrier in a ready-to-administer formulation. Alternatively, the vial or ampoule may contain the GLP-2 peptide in a form, such as a lyophilized form, suitable for reconstitution in a suitable vehicle such as sterile water or phosphate buffered saline.

The peptide compounds of the present invention can be used alone or in combination with compounds having an anti-inflammatory effect. Without wishing to be bound by any theory, it is believed that such combination treatment may enhance the beneficial therapeutic effects of the peptide analogs of the present invention.

The therapeutic dosage and regimen most suitable for treating a patient will, as is known, vary with the disease or condition being treated and according to the parameters of the patient. Without wishing to be bound by any particular theory, it is expected that doses of 0.1 to 25 mg per patient and shorter or longer duration or frequency of treatment may result in therapeutically beneficial results, such as a statistically significant increase, in particular, in small intestine mass. In some cases, the therapeutic regimen may include the administration of maintenance doses appropriate to prevent tissue regression that occurs after cessation of the initial treatment. Dosage rates and dosing regimens most suitable for use in humans may be based on the results obtained with the present invention and may be confirmed in further clinical studies.

An effective dosage and treatment protocol can be determined using conventional methods, starting with a low dose in laboratory animals and then increasing the dosage while monitoring effects, as well as systematically changing the dosing regimen. In determining the optimal dosage for a particular subject, the clinician may take into account many factors. Such considerations are known to the person skilled in the art.

In humans, the GLP-2 peptide of the present invention may be administered at a dose of about 0.01 mg/kg to 100 mg/kg body weight, such as about 0.01 mg/kg to 10 mg/kg body weight, such as 10 -100 mcg/kg body weight. According to additional embodiments of the present invention, the dose (total dose) of the GLP-2 peptide according to the present invention in humans may be in the range or be from about 0.1 mg to 25 mg per patient, may be in the range or be from 0.5 mg to 20 mg per patient, for example, may be in the range or amount to 1 mg to 15 mg per patient, for example, may be in the range or amount to 1 mg to 10 mg per patient, once or twice a week or as multiple doses as defined herein, separated in time by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days. In some instances, a fixed dose of the GLP-2 peptide of the present invention may be administered according to the dosing pattern described herein, i.e. a dose that is the same regardless of the patient's body weight and is administered once or twice a week. For example, the fixed dose may be 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, or 15 mg. Convenient for use is a fixed dose of 10 mg. The advantage of using a fixed dosage is to increase patient adherence to treatment and to reduce the risk of dosing errors in the patient, including the risk of miscalculating the dose to be administered based on weight.

Medical indications

The peptides of the present invention can be used as a pharmaceutical agent for the prevention or treatment of an individual suffering from gastrointestinal disorders, including the upper gastrointestinal tract in the region of the esophagus, by administering an effective amount of a GLP-2 analog or salt thereof, as described herein. Disorders associated with the stomach and intestines include ulcers of any etiology (eg, peptide ulcers, drug-induced ulcers, ulcers associated with infections or other pathogens), digestive disorders, malabsorption syndromes, short bowel syndrome, blind pouch syndrome, inflammatory bowel disease, sprue-celiac disease (eg, caused by gluten-induced enteropathy or celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, ulcerative colitis, damage to the small intestine, and chemotherapy-induced diarrhea/mucositis (CID).

As generally mentioned above, individuals who would benefit from an increase in small intestinal mass and results thereof and/or maintenance of normal structure and function of the small intestinal mucosa are candidates for treatment with the GLP-2 analogs of the present invention. Specific conditions that can be treated with a GLP-2 analog include various forms of sprue, including sprue celiac disease, which develops as a result of a toxic reaction to wheat alpha-gliadin and can be caused by gluten-induced enteropathy or celiac disease, and is characterized by significant loss of villi of the small intestine; tropical sprue resulting from infection and characterized by partial flattening of the villi; hypogammaglobulinemic sprue, which is commonly seen in patients with unclassifiable variable immunodeficiency or hypogammaglobulinemia and is characterized by a significant reduction in villus height. Therapeutic efficacy of treatment with a GLP-2 analogue can be monitored by intestinal biopsy for villus morphology, biochemical assessment of nutrient absorption, weight gain in the patient, or reduction in symptoms associated with these conditions.

Another particular condition that can be treated with the GLP-2 analogs of the present invention, or for which GLP-2 analogs can be used therapeutically and/or prophylactically, is short bowel syndrome (SBS), also known as short bowel syndrome or simply short bowel that results from surgical resection, a birth defect, or disease-associated malabsorption in the gut in which patients subsequently cannot maintain fluid, electrolyte, and nutrient balance with a normal diet. Despite the adaptation that usually occurs two years after resection, patients with SBS have reduced alimentary intake and fluid loss.

Other conditions that may be treated with the GLP-2 analogs of the present invention, or for which GLP-2 analogs may be used prophylactically, include, in addition to the above, radiation enteritis, infectious or post-infectious enteritis, and damage to the small intestine caused by anticancer chemotherapeutic or toxic agents.

GLP-2 analogs can also be used to treat malnutrition such as cachexia and anorexia.

A specific embodiment of the present invention relates to the use of the peptides of the present invention for the prevention and/or treatment of intestinal injury and dysfunction. Such damage and dysfunction is a well-known side effect of cancer chemotherapy treatments. Administration of chemotherapy is often associated with adverse gastrointestinal side effects such as mucositis, diarrhoea, bacterial translocation, malabsorption, abdominal cramps, gastrointestinal bleeding and vomiting. These side effects are clinical consequences of structural and functional damage to the intestinal epithelium and often lead to the need to reduce the dose and frequency of chemotherapy. Administration of the GLP-2 peptide analogs of the present invention can enhance the trophic effect in intestinal crypts and rapidly provide new cells to replace damaged intestinal epithelium after chemotherapy. The ultimate goal achieved by administering the peptides of the present invention is to reduce the morbidity associated with gastrointestinal injury in patients undergoing chemotherapy treatment while providing the most optimal chemotherapy regimen for cancer treatment. Concomitant prophylactic or therapeutic treatment may be provided in accordance with the present invention to patients undergoing or about to undergo radiation therapy.

Stem cells of the small intestinal mucosa are particularly sensitive to the cytotoxic effects of chemotherapy due to their high proliferation rate (Keefe et al., Gut 2000; 47: 632-7). Chemotherapy-induced damage to the small intestinal mucosa is clinically often referred to as gastrointestinal mucositis and is characterized by impaired absorption and barrier function of the small intestine. For example, the commonly used chemotherapeutic agents 5-FU, irinotecan and methotrexate have been shown to enhance apoptosis leading to villous atrophy and crypt hypoplasia in the rodent small intestine (Keefe et al., Gut 47: 632-7, 2000; Gibson et al. , J Gastroenterol Hepatol Sep 18(9): 1095-1100, 2003 Tamaki et al, J Int Med Res 31(1): 6-16, 2003). In humans, chemotherapeutic agents have been shown to increase apoptosis in intestinal crypts 24 hours post-administration and subsequently decrease villus area, crypt length, mitotic index per crypt, and enterocyte height three days post-chemotherapy (Keefe et al., Gut 2000; 47: 632-7). Thus, structural changes in the small intestine lead directly to intestinal dysfunction and, in some cases, diarrhea.

Gastrointestinal mucositis after cancer chemotherapy is an increasing problem that is essentially untreatable once it occurs, although it is gradually subsiding. Studies conducted with the commonly used cytotoxic anti-cancer drugs, 5-FU and irinotecan, have shown that effective chemotherapy with these drugs predominantly negatively affects the structural integrity and function of the small intestine, while the large intestine is less sensitive and mainly responds to increased mucus production (Gibson et al., J Gastroenterol Hepatol. Sep; 18(9): 1095-1100, 2003; Tamaki et al., J Int Med Res. 31(1): 6-16, 2003).

The novel GLP-2 analogs of the present invention can be used to prevent and/or treat gastrointestinal injury and side effects of chemotherapeutic agents. This potentially important therapeutic application is applicable to currently used chemotherapeutic agents such as, but not limited to: 5-FU, altretamine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisanthaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, leucovorin, lomustine, melphalan, mercaptopurine, mesna, metholipacin, climitotrexate, mitotrexate, mesna , pemetrexed, pentostatin, procarbazine, raltitrexide, streptozocin, tegafururacil, temozolomide, thiotepa, thioguanine/thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine , crisanthaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dauno rubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, liposomal doxorubicin, leucovorin, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxed , raltitrexide, streptozocin, tegafururacil, temozolomide, thiotepa, thioguanine/thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine.

An additional embodiment of the present invention relates to the use of the peptides of the present invention to increase the longitudinal growth of the intestine in a patient, for example, in a human patient. As shown in the examples below, the GLP-2 analogs of the present invention are able to increase longitudinal intestinal growth relative to controls. Without wishing to be bound by theory, it is currently believed that a longer half-life results in more efficient and sustained stimulation of GLP-2 receptors, resulting in increased longitudinal growth compared to controls. It is also possible that the longer terminal plasma half-life of the GLP-2 analogs used in accordance with the present invention results in more effective stimulation of longitudinal intestinal growth compared to other prior art GLP-2 analogs known to have relatively more short half-lives.

This possibility is of particular importance in patients with SBS, as it will lead to an increase in absorption capacity even after treatment is stopped. Such a patient will receive treatment for at least 1 to 3 years, such as at least 1 to 4 years, such as 1 to 10 years, such as 1 to 20 years, such as 1 to 35 years, to induce longitudinal growth of the intestine.

As already described in this application, patients with SCM whose condition is intermediate between intestinal failure (SCM-CI) and SCM-KI and bowel failure syndrome (SKS-SKI) or SCM without CI, respectively, can specifically benefit from lengthening their intestines during a course of treatment of 1 to 3 years, after which their risk of developing intestinal failure syndrome is reduced, for example, when administered once a week or twice a week during the treatment period. This includes a lower risk associated with the need for a central catheter and the risk of sepsis associated with its use.

Dosing regimens disclosed herein can be used to treat disorders associated with the stomach and intestines, such as HBS, in which the treated subject is provided with parenteral nutrition (PN) during treatment with GLP-2 analogs in accordance with this invention. Parenteral nutrition involves the administration of nutrients and fluids to patients whose gastrointestinal tract is damaged or compromised while they are receiving treatment with GLP-2 analogues. However, one of the problems that patients and clinicians face during GLP-2 therapy is related to determining the amount or volume of PN given to the subject being treated and adjusting the amount or amount of PN provided to the patient accordingly and individually. In particular, this appears to be important because if the amount or volume of PN is not properly adjusted, treated subjects may experience fluid overload, be at risk of dehydration, and may not achieve optimal clinical responses to therapy. Therefore, according to the present invention, algorithms are provided for adjusting the amount or volume of PN during the course of treatment using the GLP-2 analog described in this application.

Accordingly, in a further treatment-related aspect, the present invention provides a glucagon-like peptide-2 (GLP-2) analog for use in a method of treating short bowel syndrome (SBS) in a human subject receiving a parenteral nutrition (PN) regimen, said method comprising:

(i) administering to a subject a GLP-2 analog as defined herein, said GLP-2 analog being administered once or twice a week for a period of time sufficient to elongate the small intestine and improve its function in the subject; and

(ii) after step (i), reducing the frequency or amount in the parenteral nutrition (PN) regimen in response to improvement in small bowel function.

(iii) optionally repeating steps (i) and (ii) in response to the patient demonstrating continued improvement in small bowel function.

For example, the time interval during which the GLP-2 analog is administered in step (i) of the method is 18 to 150 weeks, 18 to 100 weeks, or 18 to 52 weeks.

Typically, the degree of improvement in the function or absorption capacity of the small intestine is at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%. Additionally or alternatively, the amount by which parenteral nutrition is reduced is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. According to a preferred embodiment of the present invention, parenteral nutrition is reduced by at least 20%.

In order to facilitate determining the amount or volume of PN provided to subjects receiving GLP-2 therapy, according to a further aspect of the present invention, a method is provided for adjusting parenteral nutrition (PN) in a human subject receiving an amount or volume of parenteral nutrition each week, said method comprising calculating a new amount or volume of PN using an algorithm in which the amount or volume of PN per week is calculated by subtracting a seven-fold absolute increase in daily urine volume from baseline from the current weekly amount of parenteral nutrition to provide a new amount or volume of PN for the subject. Preferably, the method includes the initial steps of determining the initial volume and/or determining the daily volume of urine. Some or all of the steps of the method may be repeated during the course of GLP-2 therapy received by the subject. Preferably, the time interval between retests to determine changes in PP volume is about 1 to 3 months. For example, according to one embodiment of the present invention, the method can be applied at 1, 2, 4, 8 and 12 weeks after the start of treatment with a GLP-2 analogue, adjusting the amount or volume of PN in response to the effects of treatment. The weekly amount or volume of PN administered should be monitored and adjusted throughout treatment with a GLP-2 analog (eg, glepaglutide) to avoid fluid overload. Since intestinal absorption capacity generally increases after treatment with a GLP-2 analogue, typically by lengthening or thickening of the small intestine, the amount or volume of PN may be reduced to avoid fluid overload and improve patient treatment needs.

Examples

The following examples are given to illustrate the preferred aspects of the present invention and are not intended to limit the scope of the present invention. GLP-2 analogs administered according to the dosing regimens described herein can be prepared according to methods such as solid phase peptide synthesis as described in WO 2006/117565, the contents of which are expressly incorporated by reference in their entirety.

Example 1 Synthesis of ZP1848 Metabolites

Solid phase peptide synthesis was performed on a CEM Liberty peptide synthesizer using standard Fmoc chemistry. TentaGel S Ram S resin (1.33 g; 0.25 mmol/g) was swollen in DMF (10 mL) before use and transferred between tube and reaction vessel using DCM and DMF.

Binding

Fmoc-amino acid in DMF/DCM (2:1; 0.2 M; 5 ml) was added to the resin in a CEM Discover microwave unit along with COMU/DMF (0.5 M; 2 ml) and DIPEA/DMF (2.0 M; 1 ml). The binding mixture was heated to 75° C. for 5 minutes while nitrogen was bubbled through the mixture. The resin was then washed with DMF (4×10 ml). Pseudoproline Fmoc-Phe-Ser(Psi Me,Me,Pro)-OH was used for amino acids six and seven.

Removal of protection

Piperidine/DMF (20%; 10 ml) was added to the resin for initial deprotection, and the mixture was heated with microwaves (30 s; 40°C). The reaction vessel was emptied, a second portion of piperidine/DMF (20%; 10 ml) was added and heated again (75°C; 3 min). The resin was then washed with DMF (6×10 ml).

The resin was washed with EtOH (3×10 ml) and Et ₂ O (3×10 ml) and dried to constant weight at room temperature (RT). The crude peptide was cleaved from the resin by treatment with TFA/DODT (95/5; 60 ml, 2 h; rt). Most of the TFA was removed under reduced pressure and the crude peptide was precipitated, washed three times with diethyl ether and dried to constant weight at room temperature.

Purification of the crude peptide by HPLC

The crude peptide was first purified to 45% by preparative reverse phase HPLC using a PerSeptive Biosystems VISION workstation equipped with a Gemini NX 5μ C-18 110A, 10x250 mm column and fraction collector and analyzed at 35 ml/min with a gradient buffer A (0.1% TFA, aq.) and buffer B (0.1% TFA, 90% MeCN, aq.). Fractions were analyzed by analytical HPLC and MS and the respective fractions were pooled and lyophilized. The product (143 mg) was purified a second time on a C4 Jupiter 2, 12×25 cm column, with a yield of 27 mg and a purity of 89% determined by HPLC and MS. Calculated monoisotopic molecular weight = 3377.61, detected molecular weight 3377.57.

Table 1: Synthesized compounds

Compound Subsequence Teduglutide H-HGDGSFSDEMNTILDNLAARDFINWLIQTKITD-OH ZP1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ₂ ZP2949 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH ZP2711 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH ZP2469 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH

Example 2: EC measurements _fifty GLP-2R

Generation of a Cell Line Expressing Human GLP-2 Receptors

hGLP2-R was purchased from MRC-geneservice, Babraham, Cambridge as a clone Image: 5363415 (11924-I17). For subcloning into a mammalian expression vector, subcloning primers were obtained from DNA-Technology, Risskov, Denmark. The 5' and 3' primers used for the PCR reaction included end restriction sites for cloning, and the 5' primer sequence was modified to include the Kozak consensus sequence without altering the sequence of the ORF-encoded product. A standard PCR reaction was performed using Image clone 5363415 (11924-I17) as template with the above primers and Herculase II Fusion polymerase in a total volume of 50 µl. The resulting PCR product was purified using the GFX™ PCR and Gel Strip Purification Kit, digested with restriction enzymes, and cloned into a mammalian expression vector using a rapid DNA ligation kit. The ligation reaction was transformed into XL10 Gold ultracompetent cells and colonies were selected for DNA production using the ^EndoFree® Plasmid maxi kit. Subsequent sequence analysis was performed by MWG Eurofins, Germany. The clone was confirmed to be the hGLP-2 receptor splicing variant rs17681684.

HEK293 cells were transfected using the Lipofectamine PLUS transfection method. The day before transfection, HEK293 cells were seeded in two T75 flasks at a density of 2×10 ⁶ cells/T75 flask in cell culture medium without antibiotics. On the day of transfection, the cells were washed with 1×DPBS and the medium was replaced with Optimem to a volume of 5 ml/T75 flask, then Lipofectamine-plasmid complexes were added carefully dropwise to the cells in T75 flasks, after 3 hours the medium was replaced with fresh growth medium, and after 24 hours with growth medium with the addition of 500 µg/ml G418. After 4 weeks of G418 pressure selection, clones were selected and tested in a functional assay. One clone was selected for use in compound profiling.

GLP-2 Receptor Efficiency Analysis

Perkin Elmer's AlphaScreen® cAMP assay was used to quantify the cAMP response to ^GLP2 receptor activation. Teduglutide was used as a reference compound for GLP2 receptor activation. Data for test compounds that cause an increase in intracellular cAMP levels were normalized against positive (teduglutide) and negative controls (vehicle) to calculate EC ₅₀ and maximum response based on the concentration-response curve. The results are shown in table 2.

Table 2: EC measurements _fifty GLP-2R

ZP No. EC ₅₀ _mean (nM) EC ₅₀ _stand. off avg. (nM) Teduglutide 0.03 0.019 ZP1848 0.3 0.14 ZP2949 0.11 0.013 ZP2711 0.072 0.0076 ZP2469 0.052 0.0057 ZP1846 0.26

Example 3 Pharmacokinetic and Pharmacodynamic Profiling of GLP-2 Analogs

A phase 2 clinical study was conducted to determine the FA profile of ZP1848 and metabolites in human patients with SBS, among other things.

Way

The study was designed as a randomized, double-blind, crossover study using three different doses of ZP1848 (10 mg, 1 mg, 0.1 mg) in 16 patients with SBS.

The study protocol was approved by the Danish Medical Agency and the Danish Medical Research Ethics Committee.

Eighteen patients with SBS (16 patients completed the study) were randomized equally and double-blind to each of six crossover dose level combinations: 10 mg/1 mg, 10 mg/0.1 mg, 1 mg/10 mg, 1 mg/0, 1 mg, 0.1 mg/10 mg, 0.1 mg/1 mg ZP1848. The above doses of ZP1848 were administered to patients by subcutaneous injection once a day (QD).

Blood samples for PK analysis were collected at several study visits during the study period: the study included 1 balance study at baseline (4 days), 1 treatment period (21 days), including 1 balance study during treatment (last 4 days 1 treatment period) followed by a washout period (at least 4 weeks); and 2 balance study at baseline (4 days), 2 treatment period (21 days), including 2 balance study during treatment (last 4 days 2 treatment periods) and observation period (at least 4 weeks).

PK sampling was performed at visit 11 (treatment period 1, balance study) and visit 17 (second day 2 baseline study - after washout period), and again at visit 25 (treatment period 2 balance study) and visit 29 (last day of observation period).

On days 11 and 25 of the visit, the complete FC profile was studied at steady state. PK samples were taken prior to ZP1848 injection and at the following times (+/- 10 minutes) after test product administration: 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours and 24 hours. Each PK sample contained 2 ml of blood resulting in a total of 46 ml of blood collected for PK purposes. The plasma sample immediately after collection was stored at -80°C.

определяли как ln(2)/k_e, где k_e представляет собой величину наклона логарифмической линейной регрессии на графике зависимости логарифма концентрации от времени в конечной фазе.Plasma samples were analyzed after immunoaffinity extraction and then by liquid chromatography with mass spectrometry (LC-MS/MS). For each patient, plasma concentrations were used to calculate pharmacokinetic parameters in Excel using a non-compartmental approach. Terminal plasma half-life

was defined as ln(2)/k _e , where k _e is the magnitude of the slope of the logarithmic linear regression on the plot of the logarithm of concentration versus time in the final phase.

results

When ZP1848 is injected into the sc (subcutaneous) compartment, two functionally active metabolites are formed (ZP2469 and ZP2711, both C-terminally truncated analogs of glepaglutide, ZP1848). Thus, the overall FA profile of ZP1848 includes the effect of ZP1848 and its two major metabolites.

Both metabolites are pharmacologically active as shown above, with in vitro efficacy comparable to that of ZP1848. The plasma level of ZP2711 is in the same range as the plasma level of ZP1848, while the plasma level of ZP2469 is many times higher than that of ZP1848. Therefore, when evaluating systemic exposure, both metabolites must be considered as they both contribute to in vivo efficacy.

Due to the high plasma level of ZP2469, this compound has a plasma level that is above the bioanalytical limit of detection (50 pM for ZP2469, 25 pM for ZP1848 and ZP2711), therefore the half-life of ZP2469 can be determined. A terminal plasma half-life of 5 to 17 days, as shown in the table, indicates that once weekly dosing of ZP1848 would be effective.

Table 3: Results of PK assessment from the phase 2 clinical trial

Subject Compound Day visit number Conc. (pM) Half-life (days) 1003 ZP2469 0 25 4490 7.2 1003 ZP2469 32 29 209 1007 ZP2469 0 25 14700 7.2 1007 ZP2469 36 29 460 1008 ZP2469 0 eleven 6770 9.5 1008 ZP2469 56 17 116 1010 ZP2469 0 eleven 4340 eleven 1010 ZP2469 56 17 112 1013 ZP2469 0 25 4940 9.6 1013 ZP2469 35 29 394 1014 ZP2469 0 eleven 2220 5.2 1014 ZP2469 28 17 52.8 1015 ZP2469 0 eleven 556 7.9 1015 ZP2469 28 17 56.4 1015 ZP2469 0 25 3640 8.7 1015 ZP2469 32 29 288 1016 ZP2469 0 eleven 4750 eleven 1016 ZP2469 49 17 236 1018 ZP2469 0 25 4660 8.7 1018 ZP2469 40 29 195 1020 ZP2469 0 eleven 1040 12 1020 ZP2469 35 17 133 1013 ZP2711 0 eleven 131 fourteen 1013 ZP2711 28 17 32.6 1015 ZP2711 0 25 543 10 1015 ZP2711 32 29 61.4

Example 4: Longitudinal growth following subcutaneous administration of ZP1848 to Beagle dogs for 39 weeks followed by a 6-week recovery period

Way

ZP1848 (0.25, 1 or 5 mg/kg/day) was administered subcutaneously for 39 weeks to beagle dogs (22 to 24 weeks). The filler was used as a control. Recovery from any effect was assessed over a 6 week recovery period. The study was conducted in accordance with the applicable sections of the Animals (Scientific Procedures) Act 1986 of the United Kingdom, Amendment Regulations 2012 (the Act).

After 39 weeks, the dogs were sacrificed and all animals were subjected to a detailed autopsy. The length of the small and large intestines was measured separately and recorded in centimeters. The entire gastrointestinal tract (from the duodenum to the rectum) was removed and trimmed of any excess mesentery, connective tissue, or adipose tissue, measurements of the small and large intestines were recorded, laying the small intestine and colon on a clean surface, and measuring with a tape measure. Sections required for histopathology were left unopened and washed with water. The remaining parts were opened, washed with water and thoroughly dried by blotting before weighing. Upon completion of the length and weight measurements, the gastrointestinal tract was processed and fixed in 10% neutral buffered formalin in accordance with standard dissection practice for this species.

results

An increase in the length of the small intestine was observed after 39 weeks of treatment at all doses and in both sexes, with the degree of increase being greatest in males and females treated with 5 mg/kg/day, with increases of 46% and 37% observed in males and females. respectively. This result partially recovered by the end of the six week recovery period, with a 16% and 19% increase observed in males and females, respectively, compared to controls. In the same study, an increase in small bowel weight was observed after 39 weeks of treatment at all doses and in both sexes, with the degree of increase being greatest in males and females treated with 5 mg/kg/day, with 103% and 80% increases observed in males. and females, respectively. This result partially recovered by the end of the six-week recovery period, with a 78% and 34% increase observed in males and females, respectively, relative to controls.

Example 5: An open-label, partially randomized, 3-part, parallel-group, phase 1 clinical trial to evaluate the pharmacokinetic profile of glepaglutide (ZP1848) after a single intravenous injection and after multiple subcutaneous injections in healthy human subjects

A phase 1 clinical study was conducted to characterize the pharmacokinetic (PK) profiles of glepaglutide (ZP1848) and its major active metabolites after subcutaneous (SC) injections once a week and once a day and after a single intravenous (IV) infusion in healthy subjects. subjects. The study included an evaluation of the pharmacodynamic effect on plasma levels of citrulline following subcutaneous dosing in healthy subjects. The study confirmed the feasibility of dosing regimens in which GLP-2 analogs were administered to patients on weekly dosing regimens and compared weekly and daily dosing regimens.

Study Design

The study was designed as a single-centre, open-label, partially randomized, 3-part, parallel-group study to characterize the PK profile of glepaglutide administered by SC injection once daily for 7 days (Part 1), by SC injection once a week for 6 weeks (Part 2) and by a single IV infusion (Part 3) in healthy subjects.

In Part 1, eligible subjects were randomized 1:1 to Group A or B; in part 2, eligible subjects were randomized 1:1 to group C or D; all subjects in Part 3 (Group E) were assigned the same dose level.

Number of subjects

Approximately 75 male and female subjects were enrolled in the study, 15 subjects in each of the 5 dose groups, to ensure that at least 12 subjects completed treatment in each group.

Diagnosis and main criteria for inclusion:

Healthy male and female subjects aged 18 to 60 inclusive, with a body mass index (BMI) of 18.0 to 30.0 kg/m ² inclusive, and preferably subjects with a BMI≤25.0 kg/m ² .

Test products, dose and route of administration

Glepaglutide was supplied as an aqueous solution at concentrations of 2 and 10 mg/mL.

Group A: 1 mg glepaglutide once daily given as single s.c. injections from days 1 to 7.

Group B: 5 mg glepaglutide once daily given as single s.c. injections from days 1 to 7.

Group C: 5 mg glepaglutide once a week given as single s.c. injections on days 1, 8, 15, 22, 29 and 36.

Group D: 10 mg glepaglutide once a week given as single s.c. injections on days 1, 8, 15, 22, 29 and 36.

Group E: 1 mg glepaglutide given as an IV infusion at a rate of 4 mg/h over 15 minutes on day 1.

Duration of treatment

Planned duration of screening: approximately 28 days

Total duration (from screening to end of study):

Groups A and B: 71 days

Groups C and D: 100 days

Group E: 51 days

Criteria for evaluation:

Pharmacokinetics:

Blood samples were taken to analyze plasma concentrations of glepaglutide and its major active metabolites, ZP2469 (ZP1848 _1-34 ) and ZP2711 (ZP1848 _1-35 ); samples were analyzed using approved analytical methods. The following estimates of PK parameters were calculated using standard non-compartmental methods: terminal half-life (t _1/2 ), total drug clearance from the body after intravenous administration (CL; i.v. dose, parent drug only), apparent total clearance (CL/ F; s/c dose, parent drug only), volume of distribution at steady state (V _ss ; iv dose, parent drug only), apparent volume of distribution at steady state (V _ss /F; s/c dose, parent drug only), volume of distribution (V _z ; i.v. dose, parent drug only), apparent volume of distribution (V _z /F; s.c. dose, parent drug only), maximum observed plasma concentration (Cmax ), area under the plasma concentration-time curve (AUC) over the dosing interval (AUC _τ ), AUC from time zero to infinity (AUC _inf ), AUC from time zero eni to the last time point with a measurable concentration (AUC _last ) and the time to reach the maximum observed plasma concentration (t _max ). Metabolite exposure (based on C _max , AUC _τ and AUC _inf ) was determined for each group as the ratio of the 2 primary active metabolites, ZP2469 (ZP1848 _1-34 ) and ZP2711 (ZP1848 _1-35 ), to the parent drug, glepaglutide.

Pharmacodynamics

Blood samples were collected to measure plasma citrulline (for s.c. doses only).

Statistical Methods

Pharmacokinetics

PK parameter estimates for glepaglutide, ZP2469 (ZP1848 _1-34 ) and ZP2711 (ZP1848 _1-35 ) are listed and summarized using standard descriptive statistics. Primary PK parameters include t _1/2 for glepaglutide, ZP2469 (ZP1848 _1-34 ) and ZP2711 (ZP1848 _1-35 ); and CL (IV dose), CL/F (SC dose), V _ss (IV dose), V _ss /F (SC dose), V _z (IV dose), and V _z /F (sc dose) for glepaglutide. The half-life was calculated as LN(2)/k _e , where ÷k _e is an estimate of the slope of the terminal portion of the LN (concentration) vs. time curve, regressing the LN (concentration) values using the last three non-zero concentration points, then the last four dots, last five dots, etc. Points with a zero concentration value were not taken into account. Do not use points up to C _max . For each regression, an adjusted R ² was calculated: adjusted R ² =1-((1-R ² ) × (n-1))/(n-2) ; where n is the number of data points in the regression and R ² is the square of the correlation coefficient. K _e was applied using the highest adjusted R ² regression and, 1) if the adjusted R ² does not improve but is within 0.0001 of the highest adjusted R ² , regression with more points. 2) k _e must be calculated from at least three data points. 3) The design slope must be negative for a negative value of k _e to be positive for it. The results of the calculations performed on the preliminary data are presented in Table 4, and the results of the calculations with the complete final data set are presented in Table 5.

Pharmacodynamics

Plasma citrulline concentrations and changes from baseline are listed and summarized using descriptive statistics.

results

The initial results and analysis for the selection of subjects included in groups A, B, C and D, revealed that the half-life of ZP2469 is calculated in hours, as shown in table 4 below. The half-life found when using a once weekly dosing confirms the possibility of using the dosing of the GLP-2 peptides according to the present invention once a week or twice a week.

Table 4: Preliminary results of half-life calculations

Number of doses one 6 7 Group subject number Half-life of ZP2469 (h) A 101 twenty Once a day 104 95 105 76 109 36 110 33 112 87 B 102 94 Once a day 103 75 106 108 107 209 108 59 111 139 C 201 38 eleven Once a week 203 100 17 204 17 16 208 38 32 209 48 40 212 twenty 22 D 202 56 60 Once a week 205 52 55 206 38 74 207 43 34 211 34 58

After the completion of the study, a final, more complete data set was made available, which was also used to calculate the half-life. The half-life calculated in a specific time period from 72 to 168 hours after dosing (regardless of the value of the adjusted R ² and the number of data points) for the ZP2469 metabolite is presented in table 5 below.

Table 5: Final half-life calculation results

Number of doses one 6 7 Group Subject Half-life of ZP2469 between 72 and 168 hours post-dose (h) A 105 75 1 mg 112 87 Once a day 115 94 119 80 125 54 126 57 B 102 42 5 mg 103 168 Once a day 106 77 107 101 108 53 111 101 114 34 116 46 122 100 123 94 127 40 128 84 129 fifty C 203 329 26 5 mg 204 41 35 Once a week 208 38 59 209 48 94 212 59 75 213 48 57 214 61 43 216 There is no data 52 219 217 107 221 45 33 222 39 68 225 There is no data 47 226 91 65 229 75 452 D 202 66 60 10 mg 205 54 55 Once a week 206 120 74 207 36 58 210 46 There is no data 211 35 70 215 60 59 217 26 thirty 218 158 102 220 53 53 223 43 60 224 33 42 227 45 48 228 43 56 230 38 17

Example 6 Trophic Effects of Glepaglutide on the Gut After Long-Term Exposure in Rats

Ways

Wistar rats were injected daily subcutaneously (sc) with vehicle, 1, 3, and 10 mg/kg ZP1848 for 26 weeks. Additional subgroups of control animals and animals treated with high doses, provided a 6-week recovery period after completion of the dosing period. Subgroups of control animals and animals treated with high doses, provided a 6-week recovery period after completion of the dosing period. The length and weight of the small intestine and large intestine were measured at autopsy as indicators of trophic effects on the intestine, and the intestinal tract was assessed histologically.

results

ZP1848 induced a significant dose-dependent (p<0.01) increase in the length and mass of the small intestine in rats (see table 5). In addition, the length and mass of the large intestine also slightly increased. ZP1848 caused a dose-dependent increase in duodenal, jejunal, and ileal mucosal hyperplasia. It is interesting to note that at the end of the recovery period, significant trophic effects on the gut were still present in the high dose groups, although partial recovery was observed.

Table 6: Percentage (%) increase relative to the control group (male/female)

Animals in the main study Animals in recovery Dose (mg/kg) 10 10 Small intestine length 38/47 39/45 Weight of the small intestine 190/238 85/101

SI = small intestine.

Output

In rats, after 26 weeks of exposure to ZP1848, a significant dose-dependent trophic effect on the gut was observed. A similar effect was also observed in rats after 7 days of dosing. At all doses in the 26-week study, an increase in length and mass, as well as macroscopic thickening and hypertrophy of the villi, was noted in all segments of the small intestine. These observations still persisted after a 6-week recovery period, indicating long-term trophic effects of glepaglutide on the gut.

Example 7: Absolute Changes in Absolute Wet Faecal Excretion in Patients with SCM-KD/SCM-CI

Way

The study was designed as a randomized, double-blind, crossover study using three different doses of ZP1848 (10 mg, 1 mg, 0.1 mg) in 16 patients with SBS. The study protocol was approved by the Danish Medical Agency and the Danish Medical Research Ethics Committee.

Eighteen patients with SBS were randomized equally and double-blindly to each of six crossover dose level combinations: 10 mg/1 mg, 10 mg/0.1 mg, 1 mg/10 mg, 1 mg/0.1 mg, 0.1 mg/10 mg, 0.1 mg/1 mg ZP1848. The group of patients with SBS included 9 women, 9 men; of these, 13 patients with SCM-KD and 5 patients with SCM-CI. The median age was 62 years, the median short bowel length was 110 cm, and two patients with HCM-KD had >50% intact colon. Patients were administered the above doses of ZP1848 by subcutaneous injection once a day.

The aim of this study was to evaluate the effectiveness of ZP1848 on wet fecal excretion in patients with MCM-CI and MCM-CD and, accordingly, the absolute change in wet fecal excretion from baseline measured in studies. metabolic balance before and during the last 3 days of each treatment period.

results

Of the 18 patients randomized to receive ZP1848, 16 completed the study. The results of changes in absolute wet faecal excretion (g/day) are presented in Table 7 below. Improvements were similar in magnitude in all patient groups, ie. SCM-KN, SCM-KD and in patients with intact colon integrity. This suggests that ZP1848 is effective in treating both classes of patients with SBS. In addition, the present inventors believe that these results confirm that GLP-2 analog therapy using ZP1848 is effective in preventing or treating patients with MCM-CI that worsens to MCM-CD.

Table 7: Changes in absolute wet faecal excretion, SKK-KN vs. SKK-KD

Changes in absolute wet fecal excretion (g/day)
(means [95% CI]) 0.1 mg 1 mg 10 mg 1 mg + 10 mg KN+KD (n=16) 173
[-160, 506]
p=0.274 -592
[-913, -272]
p=0.002 -833
[-1152, -515]
p=0.0002 -713
[-935, -490]
p<0.0001 CD (n=11) 196
[-301, 693]
p=0.3575 -587
[-1080, -95]
p=0.0280 -894
[-1420, -368]
p=0.0072 -741
[-1079, -402]
p=0.0025 KN (n=5) -37
[-873, 800]
p=0.9094 -303
[-1310, 704]
p=0.4503 -837
[-1495, -178]
p=0.0243 -570
[-1459, -45]
p=0.0393

***

Although the present invention has been described with respect to the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art upon review of the present description. Accordingly, the exemplary embodiments of the present invention set forth are intended to be illustrative and not restrictive. Various changes to the described embodiments may be made without departing from the spirit and scope of the present invention. All documents cited in this application are expressly incorporated by reference.

--->

Sequence listing

<110> ZEALAND PHARMA A/S

<120> DOSING SCHEMES FOR ANALOGUE ADMINISTRATION

GLUCAGON-LIKE PEPTIDE-2 (GLP-2)

<130> SJK/FP7369903

<150>GB 1709643.9

<151> 2017-06-16

<150>GB 1714203.5

<151> 2017-09-05

<150>GB 1800873.0

<151> 2018-01-19

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 1

His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

Asp Lys Lys Lys Lys Lys Lys

35

<210> 2

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 2

His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

Asp Lys Lys Lys

35

<210> 3

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 3

His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

Asp Lys Lys

35

<210> 4

<211> 34

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 4

His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

Asp Lys

<210> 5

<211> 33

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 5

His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

asp

<210> 6

<211> 33

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 6

His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

asp

<210> 7

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 7

His Gly Glu Gly Ser Phe Ser Ser Glu Leu Ser Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

Asp Lys Lys Lys Lys Lys Lys

35

<210> 8

<211> 33

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 8

His Gly Glu Gly Ser Phe Ser Ser Glu Leu Ser Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

asp

<210> 9

<211> 34

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 9

His Gly Glu Gly Ser Phe Ser Ser Glu Leu Ser Thr Ile Leu Asp Ala

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr

20 25 30

Asp Lys

<---

Claims (101)

1. The use of a glucagon-like peptide 2 (GLP-2) analog for the treatment and/or prevention of a gastro-intestinal disorder in a human patient, said GLP-2 analog being represented by the formula: R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ² where: R ¹ is hydrogen, C _1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser; R ² is NH ₂ or OH; and Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys; or its pharmaceutical salt or derivative; and wherein said use comprises administering said GLP-2 analog to a patient once or twice a week. 2. Use according to claim 1, wherein said gastro-intestinal disorder is ulcers, indigestion, malabsorption syndromes, short bowel syndrome, blind pouch syndrome, inflammatory bowel disease, sprue celiac disease, in particular caused by gluten-induced enteropathy or celiac disease, tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, small bowel injury, or short bowel syndrome (SBS). 3. Use according to claim 2, wherein said stomach and intestinal disorder is short bowel syndrome. 4. Use according to claim 1, wherein said gastro-intestinal disorder is radiation enteritis, infectious or post-infectious enteritis, or damage to the small intestine caused by toxic or other chemotherapeutic agents, or a side effect of chemotherapy or radiation therapy. 5. The use of a glucagon-like peptide 2 (GLP-2) analogue for the treatment and/or prevention of a side effect of chemotherapy or radiation therapy in a human patient, said GLP-2 analogue being represented by the formula: R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ² where: R ¹ is hydrogen, C _1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser; R ² is NH ₂ or OH; and Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys; or its pharmaceutical salt or derivative; and wherein said use comprises administering said GLP-2 analog to a patient once or twice a week. 6. Use according to claim 5, wherein the side effect of chemotherapy is diarrhea, abdominal cramps, vomiting, or structural and functional damage to the intestinal epithelium as a result of chemotherapy treatment. 7. Application according to any one of paragraphs. 5, 6, in which: (a) said administration of the GLP-2 analog is started 1, 2, 3, 4, 5, 6, or 7 days prior to the start of chemotherapy or radiation therapy; or (b) said administration of the GLP-2 analog is carried out the day before or on the day of the start of chemotherapy or radiation therapy and once or twice a week thereafter. 8. The use of a glucagon-like peptide 2 (GLP-2) analogue to increase longitudinal intestinal growth in a human patient, said GLP-2 analogue being represented by the formula: R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ² where: R ¹ is hydrogen, C _1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser; R ² is NH ₂ or OH; and Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys; or its pharmaceutical salt or derivative; and wherein said use comprises administering said GLP-2 analog to a patient once or twice a week. 9. Application according to claim 8, where: (a) intestinal growth is increased by at least 30% compared to control; and/or (b) after cessation of treatment, an increased absorption capacity is achieved; and/or (c) said human patient is treated once or twice a week for at least 1 to 3 years; and/or (d) said human patient is a patient suffering from HCM-intestinal failure syndrome; and/or (e) said human patient is a patient intermediate between HCM bowel failure and HCM bowel failure syndrome. 10. Use according to any one of the preceding claims, wherein said use comprises administering said GLP-2 analog to a patient once a week. 11. Use according to any one of the preceding claims, wherein said use comprises administering said GLP-2 analog to a patient twice a week. 12. Use according to any one of the preceding claims, wherein said use comprises administering multiple doses of the GLP-2 analog to the patient, said doses being separated in time by one week, optionally the doses being administered to the patient over a period of 1 to 3 years. 13. Use according to any one of the preceding claims, wherein said use comprises administering the GLP-2 analog at a dose that is in the range of 0.5 mg to 25 mg inclusive per patient once or twice a week, optionally from 1 mg to 20 mg inclusive per patient once or twice a week, optionally from 1 mg to 10 mg inclusive per patient once or twice a week, optionally from 2 mg to 7 mg inclusive per patient once or twice a week, optionally from 5 mg to 7 mg inclusive per patient once or twice a week, or optionally 2 mg to 5 mg inclusive per patient once or twice a week. 14. Use according to any one of the preceding claims, wherein said use comprises administering said GLP-2 analog at a fixed dose of 2 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg or 15 mg once or twice a week. 15. Use according to any one of the preceding claims, wherein said use comprises administering said GLP-2 analog at a fixed dose of 10 mg once or twice a week. 16. Application according to any one of paragraphs. 1-13, wherein said use comprises administering said GLP-2 analog at a fixed dose of 2 mg once or twice a week. 17. Use according to any one of the preceding claims, wherein said use comprises administering said GLP-2 analog by injection. 18. Application according to clause 17, where: (a) said dose of the GLP-2 analog is divided into two or three doses administered at spatially separated injection sites; and/or (b) said use comprises administering said GLP-2 analog by intravenous injection, subcutaneous injection, or intramuscular injection; and/or (c) said administration of the GLP-2 analog causes the formation of a subcutaneous depot from which the GLP-2 analog or its metabolites is released; and/or (d) said use comprises administering said GLP-2 analog using a pen. 19. Use according to any one of the preceding claims, wherein X5 is Thr and/or X11 is Ala. 20. Use according to any one of the preceding claims, wherein said glucagon-like peptide 2 analog (GLP-2) is: H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ₂ , H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKK-OH, H-HGEGTFSSELATILDALAARDFIAWLIATKITDKK-OH, H-HGEGTFSSELATILDALAARDFIAWLIATKITDK-OH, H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH ₂ or H-HGEGTFSSELATILDALAARDFIAWLIATKITD-OH. 21. Use according to claim 20, wherein said glucagon-like peptide 2 (GLP-2) analogue is: H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH ₂ . 22. Application according to any one of paragraphs. 1-8, where X5 is Ser and/or X11 is Ser. 23. Application according to any one of paragraphs. 1-18 or 22, wherein said glucagon-like peptide 2 analog (GLP-2) is: H-HGEGSFSELSTILDALAARDFIAWLIATKITDKKKKKK-NH ₂ , H-HGEGSSFSSELSTILDALAARDFIAWLIATKITD-NH ₂ or H-HGEGSFSELSTILDALAARDFIAWLIATKITDK-OH. 24. Use according to claim 23, wherein said glucagon-like peptide 2 (GLP-2) analogue is: H-HGEGSFSELSTILDALAARDFIAWLIATKITDKKKKKK-NH ₂ . 25. Use of a glucagon-like peptide 2 analog (GLP-2) for the treatment of short bowel syndrome (SBS) in a human subject receiving a parenteral nutrition (PN) regimen, said treatment comprising: (i) administering to said subject a GLP-2 analogue represented by the formula: R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ² , where: R ¹ is hydrogen, C _1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser; R ² is NH ₂ or OH; and Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys; or its pharmaceutical salt or derivative; and wherein said GLP-2 analog is administered once or twice a week for a period of time sufficient to lengthen and improve the function of the subject's small intestine; (ii) after step (i), reducing the frequency or volume of the parenteral nutrition (PN) regimen in response to improvement in small bowel function; and (iii) optionally repeating steps (i) and (ii) if the subject shows an improvement in function over time as a result of further longitudinal growth of the small intestine. 26. Use according to claim 25, wherein the subject is treated for 18 to 150 weeks, 18 to 100 weeks, or 18 to 52 weeks. 27. Use according to claim 25 or 26, where the treatment includes an assessment of the initial volume of PN during the first few days of GLP-2 therapy, followed by weekly assessment during the first month of GLP-2 therapy, monthly assessment during the next 1-3 months of GLP therapy -2 and/or evaluation every 3-6 months until completion of GLP-2 therapy. 28. The use of claim 27, wherein the subject's reduced weekly PN volume is equal to the subject's current weekly PN volume minus 7 x the absolute increase in daily urine volume from baseline. 29. Application according to any one of paragraphs. 25-28, where step (ii) includes the steps of: (a) determining the volume of PN required by the subject at a given treatment point, (b) comparing it with the initial volume of PN determined at the start of GLP-2 therapy, and (c) reducing the frequency or PN volume if the subject demonstrates improvement in small bowel function. 30. The use of a glucagon-like peptide 2 analog (GLP-2) for the treatment and/or prevention of HCM-intestinal failure (HCM-FI) in a patient, where said treatment prevents or delays the development of HCM-intestinal insufficiency syndrome (HCM-SKI), while said GLP-2 analog is represented by the formula: R ¹ -Z ¹ -His-Gly-Glu-Gly-X5-Phe-Ser-Ser-Glu-Leu-X11-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe- Ile-Ala-Trp-Leu-IIe-Ala-Thr-Lys-Ile-Thr-Asp-Z ² -R ² , where: R ¹ is hydrogen, C _1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl; X5 is Ser or Thr; X11 is Ala or Ser; R ² is NH ₂ or OH; and Z ¹ and Z ² are independently absent or represent a peptide sequence of 1-6 amino acid units of Lys; or its pharmaceutical salt or derivative; and said use comprises administering said GLP-2 analog to a patient once or twice a week. 31. The use of claim 30, wherein said use comprises administering said GLP-2 analog to a patient once or twice a week.