WO2005097831A2

WO2005097831A2 - Uses of isolated binding members capable of binding specifically to secretagogues

Info

Publication number: WO2005097831A2
Application number: PCT/DK2005/000241
Authority: WO
Inventors: Christian Hansen
Original assignee: Gastrotech Pharma AS
Current assignee: Gastrotech Pharma AS
Priority date: 2004-04-07
Filing date: 2005-04-07
Publication date: 2005-10-20
Anticipated expiration: 2006-10-07
Also published as: WO2005097831A3

Abstract

The present invention relates to pharmaceutical compositions comprising isolated binding members, such as antibodies and/or affibodies, capable of specifically binding a secretagogue, such as ghrelin or a ghrelin-like compound. In another aspect of the present invention, a kit and method are provided for detecting a disorder in appetite regulation in an individual. The present invention also relates to use of the isolated binding members disclosed herein for the production of a pharmaceutical composition, for the treatment of an individual in need thereof. There is further provided herein a method for treatment of an individual in need thereof, comprising administering to said individual an effective amount of one or more of the pharmaceutical compositions of the present invention. The present invention further relates to isolated nucleic acid molecules encoding at least a part of the binding members disclosed herein and vectors comprising said nucleic acid molecules. Host cells comprising said nucleic acids are also disclosed, and the present invention further relates to a cell line engineered to express one or more of the binding members disclosed herein.

Description

Uses of isolated binding members capable of binding specifically to secretagogues

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

Background of invention

Malnutrition or/and insufficient food intake is associated with severe health risk as well as loss of the sense of well- being and quality of life for the individual. In particular, individuals suffering from diseases such as HIV or cancer and individuals that have undergone surgical treatment, for example gastrectomy, are likely to experience health problems associated with malnutrition e.g. loss of body weight, body fat mass, lean body mass, bone mass as well as loss of appetite and well- being.

The terminal state of many different clinical conditions or chronic diseases such as cancer, infections, AIDS, congestive heart failure, rheumatoid arthritis, tuberculosis, cystic fibrosis and Crohn diseases is associated with cachexia. It can also occur in frail elderly people who do not have any obvious symptoms of disease. Although cachexia represents the complex metabolic syndrome that is seen in such patients it is commonly recognized as a progressive weight loss with depletion of host reserves of adipose tissue and skeletal muscle.

The core of cancer cachexia syndrome relates to the problem of progressive tumor growth and the catabolic side effects of conventional anti-neoplastic therapy. These two phenomena gives rise to alterations in the neuro-endocrine system, to the production of a variety of pro-inflammatory cytokines and to the release of cancer specific cachectic factors. In turn, these mediators cause either a reduction in food intake, abnormality in the metabolism or a combination of these two. Cachexia is associated with particular types of cancer eg. cancer in the upper gastrointestinal (Gl) tract and lung cancer. At the moment of diagnosis 80% of all patients with cancer in upper Gl tract and 60 % of all patients with lung cancer have already experienced substantial weight loss (Bruera 1219-22). Cachexia is more common in children and in elderly patients and becomes more pronounced as the cancer progresses. On average the prevalence of cachexia increases from 50 percent to more than 80% percent before death and in more than 20 % of the patients cachexia is the main cause of death (Bruera 1219-22).

Detection of cancer cachexia: The nutritional state is evaluated with a combination of clinical assessment, antropometric tests (body weight, skin fold thickness and mid arm circumference) and imaging (DEXA scan, MR scan, CT scan and bioelectric impedance meassuring). Cachexia is generally suspected if the involuntary weight loss of greater than 5% of the premorbid weight is observed within a six-month period - especially when combined with muscle wasting.

The most commonly used laboratory parameter is serum albumin. It is however an unspecific parameter. Other markers are proteins with a short half life transferrrin and transthyretin has also been used. Other markers of canchexia are IGF-1, IGFBP-3, ALP (alkaline phosphatase) and testosterone.

Anorexia: Energy intake has been shown to be substantially reduced among weight- loosing cancer patients. Cancer patients may frequently suffer from physical obstruction of the Gl tract, pain, depression, constipation, malabsorption, debility or the side effects of treatment such as opiates, radiotherapy or chemotherapy, which all may decrease food intake (Barber, Ross, and Fearon 133-41). Cancer associated hypercalcemia may also induce nausea, vomiting and appetite loss. However there remain a large number of patients with cancer in whom there is no obvious clinical cause of reduced food intake. It is important with this distinction as only the latter subset of patients may benefit from medication that acts centrally to stimulate appetite.

Ghrelin

Ghrelin is a bioactive peptide which originally was described to be involved in the control of GH secretion but later found to be a major regulator of appetite, food intake and energy homeostasis ( Kojima M et al., Trends Endocrinol Metab 12:118- 122; Nakazato M et al., 2001 , Nature 409:194-198). Similar to many other bioactive peptides, ghrelin probably act both as a hormone, a paracrine substance and as a neurotransmitter. The story of ghrelin, its receptor and synthetic compounds acting through this receptor unraveled in a unique "reverse" order. In the eighties a synthetic hexa-peptide from a series of opioid-like peptides was found to be able to release growth hormone (GH) from isolated pituitary cells (Bowers CY et al., 1980, Endocrinology 106:663-667). Since this action was independent of the growth hormone releasing hormone (GHRH) receptor, several pharmaceutical companies embarked upon drug discovery projects based on this hexa-peptide GH secretagogue (GHS) and its putative receptor. Several series of potent and efficient peptide as well as non-peptide GH secretagogues were consequently described in the mid nineties (Bowers CY et al., Endocrinology 114:1537-1545; Patchett AA et al., 1995; Proc Natl Acad Sci U S A 92:7001-7005; Smith RG et al., Science 260:1640-1643). However, it was only several years later that the receptor through which these artificial GH secretagogues acted was eventually cloned and shown to be a member of the 7TM G protein coupled receptor family (Howard AD et al., Science 273:974-977; Smith RG et al., 1997 Endocr Rev 18:621-645). In 1999, the endogenous ligand for this receptor the hormone ghrelin was finally discovered (Kojima M et al., 1999, Nature 402:656-660). The main site for ghrelin production is the stomach, where the peptide is found in classical endocrine cells in the gastric mucosa. From here, ghrelin is secreted in the pre-meal situation which results in a sharp, short-lived surge in plasma levels of ghrelin before the meal and starting 1-2 hours before and lasting a short while after initiation of the meal. Since ghrelin is the only peripherally produced orexigenic (appetite promoting) substance it is believed that the increase in plasma levels of ghrelin is crucial for the initiation of the meal.

In its role as a key initiator of appetite, ghrelin released from the endocrine cells in the mucosa of the Gl tract may act both locally as a paracrine substance and centrally as a hormone.

Ghrelin deficiency

One cause of dysregulation of appetite is ghrelin deficiency. An individual with ghrelin deficiency lacks sufficient levels of the peptide hormone ghrelin. Ghrelin deficiency is associated with a number of pathological causes, however until now was not deemed in itself to be a significant cause of further pathology. Indeed, a ghrelin-deficient mouse has been generated that showed that ghrelin is not a vital regulator of mouse bodily systems: the deficient mice had the same size, growth rate, food intake, body composition, reproduction, gross behaviours and tissue pathology as their healthy littermates Sun et al., Molecular and Cellular Biology, 23 (22): 7973-7981 , "Deletion of Ghrelin affects neither growth nor appetite"). Thus, ghrelin deficiency could be considered an effect of pathology rather than a cause of further pathology.

Summary of invention

In one aspect of the present invention are provided pharmaceutical compositions comprising an isolated binding member, said isolated binding member comprising at least one binding domain capable of specifically binding a secretagogue, such as ghrelin or a ghrelin-like compound, and said binding domain having furthermore a dissociation constant K_d for a secretagogue, such as ghrelin or a ghrelin-like compound, that is less that 1x 10^"4 M, such as less than 1x 10^"5 M, such as less than 1x 10^"6 M, such as less than 1x 10^"7 M. Said pharmaceutical composition preferably comprises, or is administered in combination with, a secretagogue compound, such as ghrelin, ghrelin-like compounds or a pharmaceutically acceptable salt thereof. Thus, the binding memberd of the present invention may be targeted to: a) a secretagogue administered to an individual, such as ghrelin synthesised outside the human body, and/or b) a natural secretagogue endogenously within the individual to be treated, such as endogenous human ghrelin.

Without being bound by theory, it is envisaged that a secretagogue such as ghrelin, or a ghrelin-like compound, may be used as a substance to increase the anabolic factors GH and IGF-1 , and that as a result leads to improvements such as increased body weight and/or prevention of loss of body weight and body fat. It is furthermore believed, without being bound by theory, that the binding member prolongs the half-life of a secretagogue, such as ghrelin or a ghrelin-like compound within the individual, thus prolonging the actions (such as appetite promotion) of said secretagogue, such as ghrelin or ghrelin-like compound. Thus, the antibody acts effectively to potentiate the effects of a secretagogue, such as ghrelin or a ghrelin- like compound. Without being bound by theory, it is believed that this potentiating effect is triggered by binding of the binding member to the secretagogue, leading to an effect such as reduced secretagogue degradation, and/or increased binding of the secretagogue to the ghrelin receptor, for example either by increased affinity of the secretagogue to the ghrelin receptor and/or secondary binding of the binding member to said receptor.

The present invention also relates to kits and methods for detecting a disorder in appetite regulation in an individual. Furthermore, the present invention relates to use of an isolated binding member for the production of the pharmaceutical compositions disclosed herein, for the treatment of an individual in need thereof. The present invention further relates to methods of treatment of an individual in need thereof, comprising administering to said individual an effective amount of one or more of the pharmaceutical compositions disclosed herein. Furthermore, the present invention provides isolated nucleic acid molecule encoding at least a part of the binding members disclosed herein and vectors comprising said nucleic acid molecule. The present invention further relates to host cells comprising said nucleic acids and to cell lines engineered to express the binding members disclosed herein. Definitions:

Affinity: The term refers to the binding strength between receptors and their ligands, for example an antigen and an antibody or an antigen and an affibody. Affibody: A recombinant immunologically active molecule, selected from a library constructed by combinatorial variegation of the Fc binding surface of of a protein that is not an antibody, preferably the 58 residue staphylococcal protein A (SPA). Amino acid: Entity comprising an amino terminal part (NH₂) and a carboxy terminal part (COOH) separated by a central part comprising a carbon atom, or a chain of carbon atoms, comprising at least one side chain or functional group. NH₂ refers to the amino group present at the amino terminal end of an amino acid or peptide, and COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide. The generic term amino acid comprises both natural and non- natural amino acids. Natural amino acids of standard nomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and adopted in 37 C.F.R., section 1.822(b)(2) belong to the group of amino acids listed in Table 1 herein below. Non-natural amino acids are those not listed in Table 1. Examples of non-natural amino acids are those listed e.g. in 37 C.F.R. section 1.822(b)(4), all of which are incorporated herein by reference. Further examples of non-natural amino acids are listed herein below. Amino acid residues described herein can be in the "D" or or "L" isomeric form.

Symbols Amino acid 1 -Letter 3-Letter

Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser serine I lie isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline K Lys lysine H His histidine Q Gin glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cys cysteine

Table 1. Natural amino acids and their respective codes.

Appetite: Appetite in an individual is assessed by measuring the amount of food ingested and by assessing the individual's desire to eat. Appetite (i.e., hunger) is typically assessed with a short questionnaire given to individuals on a random basis several times a week. Typically, subjects rate their hunger, preoccupation with food, and desire to eat greater quantities and different types of food by answering the questions using analogue scales ranging from 1 , not at all, to 5, extremely. Amino acid residue: the term "amino acid residue" is meant to encompass amino acids, either standard amino acids, non-standard amino acids or pseudo-amino acids, which have been reacted with at least one other species, such as 2, for example 3, such as more than 3 other species. In particular amino acid residues may comprise an acyl bond in place of a free carboxyl group and/or an amine-bond and/or amide bond in place of a free amine group. Furthermore, reacted amino acids residues may comprise an ester or thioester bond in place of an amide bond

Antagonist: A molecule that inhibits that action of its targetl Antibody: Are immunoglobulin molecules and active portions of immunoglobulinmolecules. Antibodies are for example intact immunoglobulin molecules or fragments there of retaining the immunologic activity. Antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'). Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Antigen: The molecule recognised by an antibody. Usually a peptide, polypeptide or a multimeric polypeptide. Antigens are preferably capable of eliciting an immune response.

Binding member: a molecule that can bind to a specific molecule. Here for example a molecule that binds an epitope on a ghrelin-like molecule. Binding domain: The binding domain is a region of a molecule that comprises the binding site.

Binding site: The binding site is the smallest region of a receptor molecule that mediates the interaction with the ligand.

BMI: Body mass index measures an individuals height/weight ration . It is determined by calculating weight in kilograms divided by the square of height in meters. The BMI "normal" range is 19-22.

Body fat mass: Body fat mass can be measured e.g. by the fat fold technique: In this technique, a pincer-type caliper is used to measure subcutaneous fat by determining skin fold thickness at representative sites on the body. These skin fold measurements are then used to compute body fat by either, adding the scores from the various measurements and using this value as an indication of the relative degree of fatness among individuals or by using the measurements in mathematical equations that have been developed to predict percent body fat.

Chimera : A molecule consisting of at least two parts not found together in nature. A chimeric peptide or protein is a peptide or protein constructed by the fusion of two peptides or proteins. A chimeric DNA molecule is a DNA molecule that encodes a chimeric protein.

Dissociation constant, Kd: a measure to describe the strength of binding (or affinity or avidity) between receptors and their ligands, for example an antibody and its antigen. The smaller the Kd, the stronger binding.

Concentration equivalent: A concentration equivalent is an equivalent dosage being defined as the dosage of binding member compound having in vitro or/and in vivo the same response (as evaluated e.g. from a dosage-response curve) as a known binding member. Epitope: A peptide segment or segments of a polypeptide recognized by the binding site of a binding member.

Functional equivalent: a functional equivalent of a protein or peptide has essentially the same biological activity. Fusion Polypeptide: A polypeptide comprised of at least two polypeptides and a linking sequence to operatively link the two polypeptides into one continuous polypeptide. The two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature. Gastrectomy: Herein, "gastrectomy" is defined as removal , loss or reduction in size of all , or part of, the stomach of an individual.

Gastrectomized individual: Herein, the term "Gastrectomized individual" refers to an individual who has undergone a gastrectomy, i.e. all or part of said indicudual's stomach has been subjected to removal, loss or reduction in size, preferably via surgical methods.

Ghrelin: a polypeptide as described in Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656-660. Human 28 aa ghrelin has the amino acid of SEQ ID NO: 1. Ghrelin analogues: The present invention also embraces the use of ghrelin analogues. In the context of the present application, analogues to ghrelin are to be understood as any peptide or non-peptide compound that essentially exerts the same biological effect as ghrelin in vivo, preferably by activating and/or binding the GHS-R1a receptor. Exemplary non-peptide ghrelin analogues are described in EP 0 869 974 and EP 1 060 190, which illustrate a number of ghrelin analogues and which documents are incorporated herein by way of reference. Ghrelin deficiency: The ghrelin deficiency syndrome in the human being has been found by the inventors of the present invention to be associated with one or more of the following symptoms: loss of fat mass, weight loss, cachexia, loss of appetite, immunosuppression and malnutrition, disrupted sleep pattern, sleepiness, malabsorption and motility problems with the intestine. Never before has it been realised that ghrelin deficiency has such side-effects in the human patient.

There are a number of methods for measuring ghrelin deficiency, and the "levels" of ghrelin calculated using these methods are not always directly comparable. For the purposes of this disclosure, "ghrelin deficiency" is defined using one of the following methods, or an equivalent method within the skill of one skilled in the art:

(a) The method of Marchesini et al., J. Clin. Endocrinol. Metab, 2003 Dec; 88(12): 5674-9

- this method calculates normal fasting ghrelin levels as 401 fmol/ml with a range of error of 130 fmol/ml. Using this assay method, it is to be understood herein that ghrelin deficiency is diagnosed when an individual is measured as having a fasting ghrelin level lower than 265 fmol/ml, such as lower than 255 fmol/ml, such as lower than 245 fmol/ml, such as lower than 235 fmol/ml, such as lower than 225 fmol/ml, such as lower than 215 fmol/ml, such as lower than 205 fmol/ml, such as lower than 195 fmol/ml, such as lower than 185 fmol/ml, such as lower than 175 fmol/ml, such as lower than 165 fmol/ml, such as lower than 155 fmol/ml, such as lower than 145 fmol/ml .

(b) The method ofAriyasυ et al., Endocrinology 2002, 143(9):3341-3351

- this method calculates normal fasting ghrelin levels as 150 fmol/ml with a range of error of 40 fmol/ml. Using this assay method, it is to be understood herein that ghrelin deficiency is diagnosed when an individual is measured as having a fasting ghrelin level lower than 105 fmol/ml, such as lower than 100 fmol/ml, such as lower than 95 fmol/ml, such as lower than 90 fmol/ml, such as lower than 85 fmol/ml, such as lower than 80 fmol/ml, such as lower than 75 fmol/ml, such as lower than 70 fmol/ml, such as lower than 65 fmol/ml, such as lower than 60 fmol/ml .

(c) The method of Enomoto et al., Clinical Science 105, 431-435, 2003

- this method calculates normal fasting ghrelin levels as 150 fmol/ml. Using this assay method, it is to be understood herein that ghrelin deficiency is diagnosed when an individual is measured as having a fasting ghrelin level lower than 130 fmol/ml, such as lower than 125 fmol/ml, such as lower than 120 fmol/ml, such as lower than 115 fmol/ml, such as lower than 110 fmol/ml, such as lower than 105 fmol/ml, such as lower than 100 fmol/ml, such as lower than 95 fmol/ml, such as lower than 90 fmol/ml, such as lower than 85 fmol/ml, such as lower than 80 fmol/ml, such as lower than 75 fmol/ml, such as lower than 70 fmol/ml, such as lower than 65 fmol/ml, such as lower than 60 fmol/ml . (d) The method of Cummings et al., New England Journal of Medicine, 2002, 346(21): 1623-30

- this method calculates normal ghrelin levels as 192 fmol/ml at breakfast peak. Using this assay method, it is to be understood herein that ghrelin deficiency is diagnosed when an individual is measured as having a ghrelin level lower than 175 fmol/ml at breakfast peak, such as lower than 170 fmol/ml, such as lower than 165 fmol/ml, such as lower than 160 fmol/ml, such as lower than 155 fmol/ml, such as lower than 150 fmol/ml, such as lower than 145 fmol/ml, such as lower than 140 fmol/ml, such as lower than 135 fmol/ml, such as lower than 130 fmol/ml, such as lower than 125 fmol/ml, such as lower than 120 fmol/ml, such as lower than 115 fmol/ml, such as lower than 110 fmol/ml, such as lower than 105 fmol/ml .

(e) The method of Arioso et al., J. Clin. Endocrinol Metab; 2003, 88(2):701-4

- this method calculates normal fasting ghrelin levels as 1967 fmol/ml. Using this assay method, it is to be understood herein that ghrelin deficiency is diagnosed when an individual is measured as having a fasting ghrelin level lower than 1800 fmol/ml, such as lower than 1700 fmol/ml, such as lower than 1600 fmol/ml, such as lower than 1500 fmol/ml, such as lower than 1400 fmol/ml, such as lower than 1300 fmol/ml, such as lower than 1200 fmol/ml, such as lower than 1100 fmol/ml, such as lower than 1000 fmol/ml, such as lower than 900 fmol/ml, such as lower than 800 fmol/ml, such as lower than 700 fmol/ml, such as lower than 600 fmol/ml, such as lower than 500 fmol/ml, such as lower than 400 fmol/ml .

(f) The method of Stoeckli et al., 2004, 12(2):346-50- this method calculates normal fasting ghrelin levels as 553 pg/ml, (164 fmol/mL) with a range of error of 105 pg/mL. Using this assay method, it is to be understood herein that ghrelin deficiency is diagnosed when an individual is measured as having a fasting ghrelin level lower than 400 pg/ml, such as lower than 380 pg/ml, such as lower than 360 pg/ml, such as lower than 340 pg/ml, such as lower than 320 pg/ml, such as lower than 300 pg/ml, such as lower than 280 pg/ml, such as lower than 260 pg/ml, such as lower than 240 pg/ml, such as lower than 220 pg/ml, such as lower than 200 pg/ml, such as lower than 180 pg/ml, such as lower than 160 pg/ml, such as lower than 140 pg/ml, such as lower than 120 pg/ml . It is most preferred for the purposes of the present invention that ghrelin deficiency is defined using the method of Cummings, Enomoto or Ariasu, most preferably the method of Cummings

Other indicators associated with ghrelin deficiency may also be taken into account when assessing ghrelin deficiency, such as lowered HDL cholesterol and increased insulin resistance, both correlated with ghrelin deficiency. It is also herein envisaged that one skilled in the art will also take other factors such as an individual's age, sex and physical size into consideration when making a diagnosis of ghrelin deficiency. GHS: growth hormone secretagogue

GHS-R 1a: the receptor for GHS. GHS-R 1a is also denoted GHS 1a. The receptor has GENBANK accession number NM_198407

Half-life (or T1/2): the half-life of a substance is defined herein in the usual meaning, i.e. the amount of taken taken for the concentration of a compound to decrease 50 %.

Immunoglobulin: The serum antibodies, including, IgG, IgM, IgA, IgE and IgD Immunologically active: Description referring to the ability of a molecule to distinguish between two of more polypeptide by preferentially binding to one of the polypeptide. "Immunologically active" may also refer to Functionality associated with an entity capable of eliciting an immunological response.

Individual: A living animal or human. In preferred embodiments, the subject is a mammal, including humans and non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. In the most preferred embodiment, the subject is a human. Isolated: is used to describe any of the various secretagogues, polypeptides and nucleotides disclosed herein, that have been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified.

Label: refers to single atoms or molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex Lean body mass: the compounds of the present invention preferably act to increase or maintain lean body mass. Lean body mass is defined herein as body weight minus body fat; primarily muscle, bone and other non-fat tissue. A number of methods may be used for calculating an individual's percentage lean body mass: 1) Body mass index (BMI) calculated as body weight (kg) divided by the square of height (m) is used to describe the severity of obesity. BMI correlates reasonably well with fat mass and using age- and sex- specific prediction equations, the relative fat mass can be predicted with an error of ~5% in most subjects (Gallagher.D., et al., 1996 Am J Epidemiol 143:228-239). 2) Waist-to-hip ratio (WHR): in the mid-eighties WHR was shown to be particularly well correlated to increased visceral adipose tissue as measured by computed tomography (CT) or magnetic resonance (MRI) waist scans (r=0.88, P<0.001) (Seidell.J.C., et al., 1989, INT.J.OBES. 13:289-303; Ashwell.M., et al., 1985,. Br Med J (Clin Res Ed) 290:1692-1694). Furthermore, WHR has been tightly correlated to total body fat as assessed by CT (r=0.97, p<0.0001) (Despres,J.P., et al., 1991 American journal of clinical nutrition 54:471-477). It has since been used as a valuable surrogate measure of visceral adipose tissue. WHR is a useful measure to quantify either inter-individual differences or individual changes over time (Gallagher.D., et al., 1996 Am J Epidemiol 143:228-239). 3) Computed Tomography (CT) provides unique direct information on body composition and permits quantification of all major tissue system level components (adipose tissue, skeletal muscle, visceral organs and bone) (Hemmingsson.A et al., 1982, Acta Radiol 23:149-151). CT moreover has the advantage of permitting the partition of the total adipose tissue (AT) mass into its subcutaneous and visceral components (Tokunaga.K., et al., INT.J.OBES. 7:437-445). This leaves CT with a significant advantage for body composition studies because of its potential for monitoring changes in the visceral AT and subcutaneous AT compartments separately, information, which is at present not available with any alternative in vivo technique except for MRI. CT scan for the assessment of body composition can be performed as either multi-slice covering the whole body and wherefrom calculations of whole body and regional AT can be derived. The CT method has been extensively validated. Animal and human cadaver studies have been used to verify the accuracy of the AT-estimate and its anatomical distribution showing a high correlation and therefore by some suggested as a "gold standard" against which to compare other non-invasive methods (Kvist, H., et al., 1988, INT.J.OBES. 12:249- 266). At present, the most accurate in vivo methods of measuring body composition are multi-slice MRI and CT (Rossner.S., 1990, INT.J.OBES. 14:893-902). The highest precision is obtained using multi-slice CT scans (CV<1%) (Jensen.M.D., et al., 1995, American journal of clinical nutrition 61 :274-278). However, due to radiation exposure, multi-slice CT is unsuitable for studies requiring repeated measurements on the same subject. A regional scan assessing the AT at the abdominal level is highly correlated with total AT mass in both men and women (r=0.92 and 0.97, respectively) (Despres.J.P., et al., American journal of clinical nutrition 54:471-477; Koester.R.S., et al., 1992, Int J Obes Relat Metab Disord 16:543-554; Lemieux.S., et al., 1993, American journal of clinical nutrition 58:463-

467).

4) Magnetic Resonance Imaging (MRI) has the same imaging properties as CT, but does not require the use of radiation and therefore may be preferred to CT.

5) Bioelectric Impedance Analysis (BIA): In BIA an alternating current at one or more frequencies is introduced via electrodes and the body impedance ^resistance) to the electrical flow is measured. Body water poses the least impedance to the electric current, whereas body fat and bone provides the most (Heymsfield.S.B., et al., 1997, Annu Rev Nutr 17:527-558). BIA is inexpensive, easy to use and portable, and is therefore frequently used in combination with anthropometric measurements to predict body composition. BIA was reviewed by an expert panel in 1996, evaluating the method to provide a reliable estimate under most conditions and to be useful in healthy individuals and diseases where no major disturbances of water distribution are prominent.

6) Dual energy X-ray Absorptiometry ("DXA" or "DEXA"): DXA is a direct, operator independent, non-invasive method to estimate body composition. Measurements are based on the differential attenuation of two X-rays as they pass through the body. It distinguishes bone mineral from soft tissue and subsequently divides the latter into FM and FFM (Pietrobelli.A., et al., 1996, Am J Physiol 271 : E941-E951). The analyzed data yield information about composition of the whole body but also permits regional body composition determination. DXA has been widely validated and appear to be a precise (CV~1%(bone), CV~3%(LBM and FM) and simple way of measuring total and regional FM and LBM. Exposure to radiation is minimal (2-5 μSv) in most DXA machines. In one embodiment of the invention described herein, it is preferred that the increase or maintenance of lean body mass is measured using DXA. In another preferred embodiment, said increase or maintenance of lean body mass is measured using MRI. "Increasing lean body mass" and variations on this phrase can mean e.g. either increasing total lean body mass in an individual and/or increasing an individual's overall percentage lean body mass (e.g. as compared to total body mass), such as increasing an individual's percentage lean body mass by more than 0.5 %, such as more than 0.75%, such as more than 1%, for example more than 1.25%, such as more than 1.5%, for example more than 1.75%, such as more than 2%, for example more than 2.25%, such as more than 2.5%, for example more than 2.75%, such as more than 3%, for example more than 3.25%, such as more than 3.5%, for example more than 3.75%, such as more than 4%, for example more than 4.25%, such as more than 4.5%, for example more than 4.75%, such as more than 5%, for example more than 5.25%. In one embodiment it is preferred that the increase lean body mass is caused by an increase in muscle mass, as measured using for instance MRI or CT. In another preferred embodiment, said increase of lean body mass may be with respect to a control group of individuals not treated with the GHS-R1a secretagogue. By "maintaining" lean body mass and grammatical variants thereof, is mean that said GHS-R1a secretagogue acts to counteract loss of an individual's lean body mass, by preventing or reducing a decrease in total amount of lean body mass (as measured using for instance DEXA scans). In all cases, by "increase or maintenance of lean body mass" herein is meant that the increase or maintenance of lean body mass is caused by the beneficial effects of the secretagogue itself on the individual thus treated, instead of being caused by e.g. increased exercise or other factors not directly related to a GHS-R1 A secretagogue's metabolic effects. Ligand: A molecule, for example a peptide, capable of specific binding to one or more cognate receptors. An antigen is, for example, a ligand to its cognate antibodies. "Loss of body weight": defined herein as a reduction in BMI.

"Loss of body fat": defined herein as either a reduction of an individual's overall fat mass or a reduction in the percentage of an individual's body fat. Pathological condition: by "pathological condition" is meant any disease or syndrome having a detrimental effect on an individual's physical and/or mental health. Said pathological condition may have a genetic cause. Preferably, said pathological condition leads to one or more undesirable symptoms including loss of fat mass, loss of weight, cachexia, loss of appetite, immunosuppression, malnutrition and/or causes a reduction in the individual's ghrelin levels. ultimeric: A polypeptide molecule comprising more than one polypeptide. A multimeric polypeptide may be di eric and contain two polypeptides etc. Mulitmers may be homomeric and contain two or more identical polypeptides or a multimer may be heteromeric and contain two or more non-identical polypeptides. Peptide: Plurality of covalently linked amino acid residues defining a sequence and linked by amide bonds. The term is used analogously with oligopeptide and polypeptide. The amino acids may be both natural amino acids and non-natural amino acids, including any combination thereof. The natural and/or non-natural amino acids may be linked by peptide bonds or by non-peptide bonds. The term peptide also embraces post-translational modifications introduced by chemical or enzyme-catalyzed reactions, as are known in the art. Such post-translational modifications can be introduced prior to partitioning, if desired. Amino acids as specified herein will preferentially be in the L-stereoisomeric form. Amino acid analogs can be employed instead of the 20 naturally-occurring amino acids. Several such analogs are known, including fluorophenylalanine, norleucine, azetidine-2- carboxylic acid, S-aminoethyl cysteine, 4-methyl tryptophan and the like.

Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to an amino-terminal group such as NH₂ or acetyl or to a carboxy-terminal group such as COOH. Receptor: A receptor is a molecule, such as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to another molecule Recombinant DNA (rDNA) molecule: A DNA molecule produced by operatively linking two DNA segments. Thus, a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.

Valency: The term valency refers to the number of potential antigen binding sites, i.e. binding domain, in a polypeptide. A polypeptide may be monovalent and contain one antigen binding site or a polypeptide may be bivalent and contain two antigen binding sites. Additionally, a polypeptide may be tetravalent and contain four antigen binding sites. Each antigen binding site specifically binds one antigen. When a polypeptide comprises more than one antigen binding site, each antigen binding site may specifically bind the same or different antigens. Thus, a polypeptide may contain a plurality of antigen binding sites and therefore be multivalent and a polypeptide may specifically bind the same or different antigens.

Detailed description of the invention

Pharmaceutical composition

In one aspect of the present invention, a pharmaceutical composition comprising an isolated binding member is provided, said isolated binding member comprising at least one binding domain capable of specifically binding a secretagogue compound, said binding domain having furthermore a dissociation constant K_d for said secretagogue compound that is less that 1x 10^"6 M. Said secretagogue is preferably ghrelin.

Binding member

The isolated binding member of the pharmaceutical compositions of the present invention is preferably a pure isolated binding member. The binding member of the present invention is capable of binding to a ligand. In a preferred embodiment, said ligand is a secretagogue, such as a secretagogue described herein, or human ghrelin itself. The binding domain of said binding member may be a single moiety, e.g., a polypeptide or protein domain, or it may include two or more moieties, e.g., a pair of polypeptides such as a pair of single chain antibody domains. The binding domain may be derived from a naturally occurring protein or polypeptide; it may be designed de novo, or it may be selected from a library. For example, the binding domain may be or derived from an antibody, a single chain antibody (scFv), a single domain antibody (VHH), a lipocalin, a single chain MHC molecule, an Anticalin.TM. (Pieris), an Affibody.TM. (Affibody), or a Trinectin.TM. (Phylos).

In one embodiment of the invention, the binding member is immunologically active, for example as an antibody or an affibody, such as being capable of binding to an antigen. In particular the invention relates to a binding member capable of specifically recognising and binding to a a secretagogue, such as ghrelin-like compound, and preferably able to bind said ghrelin-like compound specifically. Accordingly, the binding members according to the invention have a binding domain having a dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghrelin-like compound, which is less than 1x 10^"6 M. More preferably the dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghrelin-like compound, which is less than 5x 10^"7 M, more preferably a dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghrelin-like compound which is less than 1x 10^"7 M, more preferably a dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghrelin-like compound, which is less than 5x 10^"8 M, more preferably a dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghrelin-like compound, which is less than 1x 10^"8 M, more preferably a dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghrelin-like compound, which is less than 5x 10^"9 M, more preferably a dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghreliη-like compound, which is less than 1x 10^"9 M, more preferably a dissociation constant Kd for a secretagogue, such as ghrelin and/or a ghrelin-like compound, which is less than 5x 10^"10 M, such as 1 x10^"10 M.

Dissociation constants can be determined using methods well-known in the art, such as ELISA (e.g. as described in Orosz and Ovadi (2002) J. Immunol. Methods 270: 155-162) or surface plasmon resonance analysis.

The binding member is preferably an isolated binding member as defined above and more preferably an isolated pure binding member.

In one embodiment of the invention the binding member is an antibody, such as any suitable antibody known in the art, in particular antibodies as defined herein, such as antibodies or immunologically active fragments of antibodies, or single chain antibodies. Antibody molecules are typically Y-shaped molecules whose basic unit consist of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains. The N-terminal regions, also known as V-domains, are variable in sequence and are responsible for the antibody specificity. The antibody specifically recognizes and binds to an antigen mainly through six short complementarity- determining regions located in their V-domains.

In another embodiment of the invention the binding member is an affibody, such as any suitable affibody known in the art, in particular affibodies as defined herein, such as affibodies or immonologically fragments of affibodies. Affibodies are selected in vitro using phage display, using a affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA) (see for example Hansson M et al., "An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein", Immunotechnology. 1999 Mar; 4(3-4): 237-52). Thus, the size of an affibody is considerably less than of an antibody.

In yet another embodiment of the invention the binding member is a chimera of an antibody and an affibody. The chimera may be constructed by several methods, for example, but not restricted to, the affibody may be fused with an Fc fragment, thus potentially allowing dimers to form by homodimerisation, alternatively even dimeric affibodies may be fused to the Fc fragment resulting in a tetravalent binding member. An example of a method of producing chimeras of this type is found in Ronnmark J et al, Construction and characterization of affibody-Fc chimeras produced in Escherichia coli. J Immunol Methods. 2002 Mar 1 ; 261 (1-2): 199-211.

Thus, the present invention relates to a composition comprising a binding member as described above for the production of a pharmaceutical composition for the treatment of disorders in appetite regulation

It is preferred that said binding member binds to a site on said secretagogue, in a position that still allows full, or at least partial, activation of the GHS receptor by said secretagogue.

Said site is, in one preferred embodiment, within residues 9-28 of SEQ ID NO: 1 , or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 11-28 of SEQ ID NO: 1 , or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 16-28 of SEQ ID NO: 1 , or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 21- 28 of SEQ ID NO: 1 , or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 6-28 of SEQ ID NO:1 , or a variant, homologue or functional equivalent thereof said SEQ ID NO:1.

Said site is, in another preferred embodiment, within residues 1-9 of SEQ ID NO: 1 , or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 1-12 of SEQ ID NO: 1, or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 1 -15 of SEQ I D NO: 1 , or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 1- 20 of SEQ ID NO: 1 , or a variant, homologue or functional equivalent thereof. In another preferred embodiment, said site is within residues 1-5 of SEQ ID NO:1 , or a variant, homologue or functional equivalent thereof said SEQ ID NO:1.

Most preferred epitopes recognised by the binding member of the present invention

Most preferably, the ghrelin epitope recognised by the binding member of the present invention is selected from the group consisting of: • within residues 1-15 of SEQ ID NO; 1 • within residues 2-16 of SEQ ID NO: 1 • within residues 16-25 of SEQ ID NO: 1 • within residues 17-25 of SEQ ID NO:1 • within residues 16-23 of SEQ ID NO: 1 • within residues 18-25 of SEQ ID NO: 1

Thus, in one preferred embodiment, the ghrelin epitope recognised by the binding member of the present invention is an epitope within residues 1-15 of SEQ ID NO; 1. For example, said epitope is within residues 1-7 of SEQ ID NO; 1, or within residues 2-9 of SEQ ID NO; 1 , or within residues 6-15 of SEQ ID NO; 1 , or within residues 5-11 of SEQ ID NO; 1. Most preferably, said epitope consists of residues 1- 15 of SEQ ID NO; 1.

In another preferred embodiment of the present invention, the ghrelin epitope recognised by the binding member of the present invention is an epitope within residues 2-16 of SEQ ID NO; 1. For example, said epitope may be within residues 2-7 of SEQ ID NO; 1 , or within residues 6-16 of SEQ ID NO; 1 , such as within residues 9-16 of SEQ ID NO; 1. Most preferably, said epitope consists of residues 2- 15 of SEQ ID NO; 1.

In another preferred embodiment of the present invention, the ghrelin epitope recognised by the binding member of the present invention is an epitope within residues 16-25 of SEQ ID NO; 1. For example, said epitope may be within residues 16-20 of SEQ ID NO; 1 , or within residues 19-23 of SEQ ID NO; 1 , or within residues 21-25 of SEQ ID NO; 1. Most preferably, said epitope consists of residues

16-25 of SEQ ID NO; 1.

In another preferred embodiment of the present invention, the ghrelin epitope recognised by the binding member of the present invention is an epitope within residues 17-25 of SEQ ID NO; 1. For example, said epitope may be within residues

17-21 of SEQ ID NO; 1, or within residues 19-22 of SEQ ID NO; 1, or within residues 20-25 of SEQ ID NO; 1. Most preferably, said epitope consists of residues 17-25 of SEQ ID NO; 1.

In another preferred embodiment of the present invention, the ghrelin epitope recognised by the binding member of the present invention is an epitope within residues 16-23 of SEQ ID NO; 1. For example, said epitope may be within residues 16-20 of SEQ ID NO; 1 , or within residues 18-21 of SEQ ID NO; 1, or within residues 19-22 of SEQ ID NO; 1 , or within residues 20-23 of SEQ ID NO; 1. Most preferably, said epitope consists of residues 16-23 of SEQ ID NO; 1.

In another preferred embodiment of the present invention, the ghrelin epitope recognised by the binding member of the present invention is an epitope within residues 18-25 of SEQ ID NO; 1. For example, said epitope be within residues 19- 22 of SEQ ID NO; 1 , or within residues 20-23 of SEQ ID NO; 1 , or within residues

21-24 of SEQ ID NO; 1, or within residues 22-25 of SEQ ID NO; 1. Most preferably, said epitope consists of residues 18-25 of SEQ ID NO; 1.

In one preferred embodiment of the present invention, it is preferred that the epitope recognised by the binding member of the present invention has a length of 3-15 amino acids, such as 4-14 amino acids, such as 5-13 amino acids, such as 6-12 amino acids. In another preferred embodiment of the present invention, it is preferred that the epitope recognised by the binding member of the present invention has a length of fewer than 13 amino acids, such as fewer than 10 amino acids, such as fewer than 8 amino acids, such as fewer than 6 amino acids. In another preferred embodiment of the present invention, it is preferred that the epitope recognised by the binding member of the present invention has a length of 10 amino acids. In another preferred embodiment of the present invention, it is preferred that the epitope recognised by the binding member of the present invention has a length of 9 amino acids. In another preferred embodiment of the present invention, it is preferred that the epitope recognised by the binding member of the present invention has a length of 8 amino acids.

Multivalent In one preferred embodiment of the invention, the binding member is a multivalent binding member comprising at least two binding sites, thus in another embodiment the binding member comprises 4 binding sites, or more that 4 binding sites, such as

6, or such as 8 binding sites. The binding sites of the binding member may have identical or different binding sites

Multispecificity

In one embodiment of the invention the binding member has multiple binding sites.

The binding sites may have specificity for the same antigen or for different antigens, and thus an embodiment of the invention relates to a binding member with at least two binding sites with different specificity, thus a binding member capable of binding two different entites. The different entities may be two different antigens or two different epitopes of the same antigen.

Thus in another embodiment ot the invention the binding member may be able to bind a a secretagogue, such as ghrelin-like compound, and in addition a second, a third, and a fourth ligand. This ligand may be for example be another secretagogue compound. In another preferred embodiment, said second ligand is a secretagogue receptor: in one preferred embodiment this leads to the secretagogue staying in the vicinity of its cognate receptor for a longer period, most preferably docking or binding to its cognate receptor for a more prolonged period. Monoclonal/polvclonal antibodies

In one embodiment of the invention the binding member is an antibody, wherein the antibody may be a polyclonal or a monoclonal antibody derived from a mammal or mixtures of monoclonal antibodies. In a preferred embodiment the binding member is a monoclonal antibody or a fragment thereof. The antibody may be any kind of antibody, however it is preferably a IgG antibody. More preferably the antibody is a lgG1 antibody or a fragment thereof.

Monoclonal antibodies (Mab's) are antibodies, wherein every antibody molecule are similar and thus recognises the same epitope. Monoclonal antibodies are in general produced by a hybridoma cell line. Methods of making monoclonal antibodies and antibody-synthesizing hybridoma cells are well known to those skilled in the art. Antibody-producing hybridomas may for example be prepared by fusion of an antibody-producing B lymphocyte with an immortalized cell line.

A monoclonal antibody can be produced by the following steps. In all procedures, an animal is immunized with an antigen such as a protein (or peptide thereof) as described above for preparation of a polyclonal antibody. The immunization is typically accomplished by administering the immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response. Preferably, the mammal is a rodent such as a rabbit, rat or mouse. The mammal is then maintained on a booster schedule for a time period sufficient for the mammal to generate high affinity antibody molecules as described. A suspension of antibody-producing cells is removed from each immunized mammal secreting the desired antibody. After a sufficient time to generate high affinity antibodies, the animal (e.g., mouse) is sacrificed and antibody- producing lymphocytes are obtained from one or more of the lymph nodes, spleens and peripheral blood. Spleen cells are preferred, and can be mechanically separated into individual cells in a physiological medium using methods well known to one of skill in the art. The antibody-producing cells are immortalized by fusion to cells of a mouse myeloma line. Mouse lymphocytes give a high percentage of stable fusions with mouse homologous myelomas, however rat, rabbit and frog somatic cells can also be used. Spleen cells of the desired antibody-producing animals are immortalized by fusing with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol. Any of a number of myeloma cell lines suitable as a fusion partner are used with to standard techniques, for example, the P3- NS1/1-Ag4-1 , P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines, available from the American Type Culture Collection (ATCC), Rockville, Md.

Monoclonal antibodies can also be generated by other methods well known to those skilled in the art of recombinant DNA technology. An alternative method, referred to as the "combinatorial antibody display" method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies.

Polyclonal antibodies are a mixture of antibody molecules recognising a specific given antigen, hence polyclonal antibodies may recognise different epitopes within said antigen. In general polyclonal antibodies are purified from serum of a mammal, which previously has been immunized with the antigen. Polyclonal antibodies may for example be prepared by any of the methods described in Antibodies: A Laboratory Manual, By Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, 1988. Polyclonal antibodies may be derived from any suitable mammalian species, for example from mice, rats, rabbits, donkeys, goats, and sheep.

An embodiment of the invention relates to a composition wherein the binding member is selected from monoclonal antibodies, polyclonal antibodies or a mixture of monoclonal antibodies.

Further suitable binding molecules and/or antibodies include those disclosed in US patent 6,420,175 , incorporated herein by reference.

Antibody fragments

In one embodiment of the invention the binding member is a fragment of an antibody, preferably an antigen binding fragment or a variable region. Examples of antibody fragments useful with the present invention include Fab, Fab', F(ab')₂ and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'). Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site. Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the VH of an Fv (Cumber et al., 1992), or at the C-terminus of the VL of an scFv (Pack and Pluckthun, 1992), or through the hinge cysteine residues of Fab's (Carter et al.,

1992).

Preferred antibody fragments retain some or essential all the ability of an antibody to selectively binding with its antigen or receptor. Some preferred fragments are defined as follows:

(1) Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. A Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.

(2) Fab' is the fragment of an antibody molecule and can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.

(3) (Fab')₂ is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction. F(ab')₂ is a dimer of two Fab' fragments held together by two disulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V_H -V _L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V_H -V _L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

In one embodiment of the present invention the antibody is a single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also refered to as "single-chain Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the V_H and VL domains that enables the sFv to form the desired structure for antigen binding.

The antibody fragments according to the invention may be produced in any suitable manner known to the person skilled in the art. Several microbial expression systems have already been developed for producing active antibody fragments, e.g. the production of Fab in various hosts, such as E. coli (Better et al., 1988, Skerra and

Pluckthun, 1988, Carter et al., 1992), yeast (Horwitz et al., 1988), and the filamentous fungus Trichoderma reesei (Nyyssonen et al., 1993) has been described. The recombinant protein yields in these alternative systems can be relatively high (1-2 g/l for Fab secreted to the periplasmic space of E. coli in high cell density fermentation, see Carter et al., 1992), or at a lower level, e.g. about 0.1 mg/l for Fab in yeast in fermenters (Horwitz et al., 1988), and 150 mg/l for a fusion protein CBHl-Fab and 1 mg/l for Fab in Trichoderma in fermenters (Nyyssonen et al., 1993) and such production is very cheap compared to whole antibody production in mammalian cells (hybridoma, myeloma, CHO).

The fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a VH and a VL can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv. Humanised antibody

It is not always desirable to use non-human antibodies for human therapy, since the non-human "foreign" epitopes may elicit immune response in the individual to be treated. To eliminate or minimize the problems associated with non-human antibodies, it is desirable to engineer chimeric antibody derivatives, i.e.,

"humanized" antibody molecules that combine the non-human Fab variable region binding determinants with a human constant region (Fc). Such antibodies are characterized by equivalent antigen specificity and affinity of the monoclonal and polyclonal antibodies described above, and are less immunogenic when administered to humans, and therefore more likely to be tolerated by the individual to be treated.

Accordingly, in one embodiment the binding may be a humanised antibody.

Humanised antibodies are in general chimeric antibodies comprising regions derived from a human antibody and regions derived from a non-human antibody, such as a rodent antibody. Humanisation (also called Reshaping or CDR-grafting) is a well- established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent), increasing the homology to a human immunoglobulin, and for improving their activation of the human immune system. Thus, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

It is further important that humanized antibodies retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three- dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is maximized, although it is the the CDR residues that directly and most substantially influence antigen binding.

One method for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody are fused to constant domains derived from a second antibody, preferably a human antibody. Methods for carrying out such chimerisation procedures are for example described in EP-A-0 120 694 (Celltech Limited), EP-A-0 125 023

(Genentech Inc.), EP-A-0 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford University) and EP-A-0 194 276 (Celltech Limited). A more complex form of humanisation of an antibody involves the re-design of the variable region domain so that the amino acids constituting the non-human antibody binding site are integrated into the framework of a human antibody variable region (Jones et al.,

1986).

The humanized antibody of the present invention may be made by any method capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987), the contents of which is expressly incorporated by reference. The human CDRs may be replaced with non-human CDRs using oligonucleotide site-directed mutagenesis as described in the examples below.

As an example the humanized antibody of the present invention may be made as described in the brief explanation below. The humanized antibodies of the present invention may be produced by the following process: (a) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding an antibody heavy chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain antibody binding specificity are derived from a non-human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention; (b) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding a complementary antibody light chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention; (c) transfecting the expression vectors into a host cell by conventional techniques to produce the transfected host cell of the invention; and

(d) culturing the transfected cell by conventional techniques to produce the humanised antibody of the invention.

The host cell may be cotransfected with the two vectors of the invention, the first vector containing an operon encoding a light chain derived polypeptide and the second vector containing an operon encoding a heavy chain derived polypeptide. The two vectors contain different selectable markers, but otherwise, apart from the antibody heavy and light chain coding sequences, are preferably identical, to ensure, as far as possible, equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used, the vector including the sequences encoding both the light and the heavy chain polypeptides. The coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both.

The host cell used to express the altered antibody of the invention may be either a bacterial cell such as Escherichia coli, or a eukaryotic cell. In particular a mammalian cell of a well defined type for this purpose, such as a myeloma cell or a Chinese hamster ovary cell may be used.

The general methods by which the vectors of the invention may be constructed, transfection methods required to produce the host cell of the invention and culture methods required to produce the antibody of the invention from such host cells are all conventional techniques. Likewise, once produced, the humanized antibodies of the invention may be purified according to standard procedures as described below.

Human antibody framework In a more preferred embodiment the invention relates to a binding member, wherein the binding domain is carried by a human antibody framework, i.e. wherein the antibodies have a greater degree of human peptide sequences than do humanised antibodies.

Human mAb antibodies directed against human proteins can be generated using transgenic mice carrying the human immunoglobulin germ line. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741 ; Lonberg et al. International

Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21 ; Morrison, S. L et al. 1994 Proc. Natl. Acad. Sci. USA 81 :6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21 :1323-1326; all incorporated by reference herein).

Such transgenic mice are available from Abgenix, Inc., Fremont, Calif., and Medarex, Inc., Annandale, N.J. Suitable mice are disclosed in US patents 6,150,584 and 6,111 ,166, both incorporated by reference herein. It has been described that the homozygous deletion of the antibody heavy-chain joining region (IH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human antibodies can also be derived from phage-display libraries (Hoogenboom et al ., J. Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991); Vaughan, et al., Nature Biotech 14:309 (1996)). Non-immonoglobulin binding members

In one preferred embodiment, the present invention relates to binding members derived from a naturally occurring protein or polypeptide; said protein or polypeptide may for example be designed de novo, or may be selected from a library. The binding member may be a single moiety, e.g., a polypeptide or protein domain, or it may include two or more moieties, e.g., a pair of polypeptides such as a pair polypeptides. The binding member may for example, but not exclusively, be a lipocalin, a single chain MHC molecule, an Anticalin™ (Pieris), an Affibody^™, or a Trinectin™ (Phylos). The binding member may be selected or designed by recombinant methods known by people well known in the art.

Affibody

In one embodiment of the present invention, said binding member is an affibody. Affibodies are produced recombinantly by methods well known to those skilled in the art of recombinant DNA technology. Phage display techniques may be used to identify affibodies capable of specifically recognising a particular antigen. Affibodies can be produced in any suitable host, as for example, but not exclusively E. coli or S. cerevisiae (see below).

An embodiment of the invention relates to a composition wherein the binding member is an affibody or a mixture of affibodies.

Affibody-antibody chimeras

In another embodiment of the present invention, said binding member is an affibodyantibody chimera. According to the invention affibody-antibody chimeras can be constructed by several methods, for example by fusion of nucleotide sequences or fusion of polypeptide sequences. The nucleic acid sequence of an affibody maybe fused to a nucleic acid sequence of an antibody by DNA recombinant technology for the production of the binding member in a suitable host. The affibody nucleotide sequences may for example be fused to an antibody light chain nucleotide sequence or an antibody heavy chain nucleic acid sequence. In an embodiment of the invention the affibody sequence may be fused with a fragment of an antibody sequences. The affibody sequence may for example, but not exclusively, be fused with an Fc fragment of an antibody, thus potentially allowing dimers to form by homo-dimerisation. The affibody antibody chimeras may contain multiple affibody sequences, such as at least two, three, four of at least six affibody sequences. In an embodiment of the invention a fusion of two affibodies may be fused with an Fc fragment resulting in a tetravalent binding member upon dimerization.

Alternatively the chimeras may be obtained by linking of the two protein/polypeptide molecules together by methods known to the people skilled in the art.

In a further embodiment of the invention the binding member may be an antibody- affibody chimera or a mixture of antibody-affibody chimeras.

Other embodiments

In another preferred embodiment of the present invention, the backbone of the binding member may be derived from proteins other than antibodies, such as for instance cameloid antibodies. Other preferred embodiments comprise proteins that bind to the FcN receptor, since this may increase the half-life beyond that of proteins that do not bind FcN.

Purification of binding members

After production, the binding members of the present invention are preferably purified. The method of purification used is dependent upon several factors, including the purity required, the source of the binding member, the intended use for the binding member, the species in which the binding member was produced. In addition the method of purification is dependent on the type of binding member, selected from antibody, antibody fragment, affibody and antibody-affibody chimears.

Purification of antibodies is dependent of the class of the antibody, the subclass of the antibody and, whether the antibody is a monoclonal antibody. Purification of affibodies depends on there biochemical characteristics. Purification of antibody- affibodies chimeras are dependent on structure of the fusion, thus the molecules may be purified based on characteristics associated with the antibody part of the molecule or the affibody part of the molecule, or the total molecule. Any suitable conventional methods of purifying polypeptides comprising antibodies and affibodies include precipitation and column chromatography are well known to one of skill in the purification arts, including cross-flow filtration, ammonium sulphate precipitation, affinity column chromatography, gel electrophoresis and the like may be used.

The method of purifying an antibody with an anti-immunoglobulin antibody can be either a single purification procedure or a sequential purification procedure. Methods of single and sequential purification are well known to those in the purification arts. In a single-step purification procedure, the antibody is specifically bound by a single anti-immunoglobulin antibody. Non-specifically bound molecules are removed in a wash step and the specifically bound molecules are specifically eluted. In a sequential purification procedure, the antibody is specifically bound to a first anti- immunoglobulin antibody, non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted. The eluant from the first anti-immunoglobulin antibody is then specifically bound to a second anti- immunoglobulin antibody. The non-specifically bound molecules are removed in a wash step, and the specifically bound molecules are specifically eluted. In a preferred embodiment, the antibody is sequentially purified by a first and second anti-immunoglobulin antibody selected from the group consisting of antibodies which specifically bind heavy and light chain constant regions. In a more preferred embodiment, the antibody is sequentially purified by a first and second anti- immunoglobulin antibody selected from the group consisting of antibodies which specifically bind the heavy chain constant region of IgG and light chain constant regions of kappa and lambda. In an even more preferred embodiment, the anti- immunoglobulin antibody is selected from the group consisting of antibodies which specifically bind the light chain constant regions of kappa and lambda.

A commonly used method of purification is affinity chromatography in which the antibody to be purified is bound by protein A, protein G or by an anti-immunoglobulin antibody. Another method of affinity chromatography, which is well known to those of skill in the art, is the specific binding of the antibody to its respective antigen.

In particular for purifying a multispecific, including a bispecific antibody, a sequential purification procedure may be used, wherein the bispecific antibody comprising two or more variable domains is specifically bound to a first antigen and then to a second antigen.

In an alternative embodiment, a bispecific antibody comprising two or more variable regions is purified by sequential purification by specifically binding the antibody to a first antigen in a first purification step and to a second antigen in a second purification step.

The methods use for purification of antibodies may, when suitable, be applied for the purification of affibodies or antibody-affibody chimeras.

Functionality

The plasma concentration of an active (for example acylated) secretagogue, such as ghrelin can be measured in a number of ways known by those skilled in the art. In a preferred embodiment of the present invention, the binding member of the present invention is capable of increasing the plasma concentration of a secretagogue such as ghrelin, within an individual, preferably by binding to the secretagogue. Preferably, this increase is caused by an increase in the plasma half- life of a secretagogue, such as ghrelin, within an individual. For example, the binding of said binding member to said secretagogue may cause, or be associated with, slowed or reduced breakdown of said secretagogue molecule, such as a ghrelin molecule. Said breakdown is for example caused by a plasma protease, or a deacylase, thus in one preferred embodiment the binding member of the present invention prevents recognition and/or breakdown of the acyl group on an active secretagogue molecule, such as ghrelin, by a deacylase. Said prevention may for example be by sterically hindering the action of the deacylase. It is preferred that in all embodiments of the present invention, binding of the binding member to the secretagogue does not significantly reduce the capability of an individual secretagogue molecule to activate the GHS receptor. In another preferred embodiment, the affinity of the binding member to the secretagogue is less than the affinity of the secretagogue to the GHS receptor.

In another preferred embodiment, binding of the binding member to a secretagogue, such as ghrelin, results in increased biological activity of said secretagogue, increasing the ability of a secretagogue such as ghrelin to activate the corresponding receptors, for example by increasing the affinity of a secretagogue, such as ghrelin, for its cognate receptor, for example by altering the 3D structure of said secretagogue to make receptor binding more favourable and/or more frequent. GHS receptor activity can be measured using different techniques such as detecting a change in the intracellular conformation of the GHS receptor, in the G-protein coupled activities, and/or in the intracellular messengers:

The ghrelin receptor can either be expressed endogenously on primary cells cultures, for example pituitary cells, or heterologously expressed on cells transfected with the ghrelin receptor. Whole cell assays or assays using membranes prepared form either of these cell types can be used depending on the type of assay.

As the ghrelin receptor is generally believed to be primarily coupled to the Gq signalling pathway, any suitable assay which monitors activity in the Gq/G11 signalling pathway can be used to demonstrate the effectiveness of the binding member of the present invention to increase the biological activity of the secretagogue, for example:

1 ) an assay measuring the activation of Gq / G11 performed for example by measurement of GTPgS binding combined with, e.g., anti-Gαq or -11 antibody precipitation in order to increase the signal to noise ratio. This assay may also detect coupling to other G-proteins than Gq/11.

2) An assay which measure the activity of phopholipase C (PLC) one of the first down-stream effector molecules in the pathway, for example by measuring the accumulation of inositol phosphate which is one of the products of PLC.

3) More down stream in the signalling cascade is the mobilization of calcium from the intracellular stores. Intracellular calcium accumulation can be measured using many different methods known to the skilled person.

4) Further more down stream signalling molecules such as the activity of different kinds of MAP kinases (p38, jun, ect), NF-K-B translocation and CRE driven gene transcription may also be measured. 5) Alternatively binding of fluorescently tagged arrestin to the activated ghrelin receptor may also be used.

Examples of further suitable protocols for use in determining GHS-R1A ligand functionality are given in Example 5 of PCT application publication no. WO2005014032 (Gastrotech Pharma A/S).

In one embodiment the binding of a compound to the receptor GHS-R 1A, can be measured by the use of the assay described herein above.

In a further embodiment, the dissociation constant (Kd) of the binding member to its cognate secretagogue, such as ghrelin, is less than 500 nM. In a still further embodiment the dissociation constant (Kd) of the binding member to its cognate secretagogue, such as ghrelin, is less than 100 nM, such as less than 80 nM, for example less than 60 nM, such as less than 40 nM, for example less than 20 nM, such as less than 10 nM, for example less than 5 nM, such as less than 1 nM, for example less than 0.5 nM, such as less than 0.1 nM, for example less than 0.05 nM, such as less than 0.01 nM.

Binding assays can be performed using recombinantly produced GHS receptor polypeptides present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing the GHS receptor polypeptide expressed from recombinant nucleic acid or naturally occurring nucleic acid; and also include, for example, the use of a purified GHS receptor polypeptide produced by recombinant means or from naturally occurring nucleic acid which is introduced into a different environment.

Using a recombinantly expressed GHS receptor offers several advantages such as the ability to express the receptor in a defined cell system so that a response to a secretagogue at the GHS receptor can more readily be differentiated from responses at other receptors. For example, the GHS receptor can be expressed in a cell line such as HEK 293, COS 7, and CHO not normally expressing the receptor by using an expression vector, wherein the same cell line without the expression vector can act as a control. In another preferred embodiment, said binding causes an increase in the levels of activated (e.g. acylated) secretagogue, such as ghrelin, within the individual: this can be measured using specific antibodies to activated and inactivated secretagogue, such as ghrelin, such as in the methods of "Delayed short-term secretory regulation of ghrelin in obese animals: Evidensed by a specific RIA for the active form of ghrelin, Endocrinology 143(9):3341-3350, 2002, or othermethods for measuring ghrelin levels described earlier herein.

The efficacy of the binding member of the present invention in stimulating the biological activity of a secretagogue may also be measured indirectly - for example, by examining food intake or weight gain of an individual treated with the compounds of the present invention.

Secretagogue The binding members of the present invention are preferably administered in combination with a secretagogue, or a variant, homologue or functional equivalent therof. By the term "in combination" is meant herein that said binding member(s) may be co-formulated with one or more secretagogue compound in the same composition, and/or that said secretagogue compound(s) is administered before, during (including concurrently with) and/or after administration of the binding member(s) of the present invention.

The term "secretagogue" according to the invention is used in its normal meaning, i.e. a substance capable of stimulating growth hormone release. In the present context, a secretagogue is defined by its ability of binding GHS-R 1a, and more preferably activating the receptor. The secretagogues of the present invention may be acylated or non-acylated. One preferred secretagogue is a ghrelin-like compound. "Ghrelin analogue" and "ghrelin-like compound" are used interchangeably herein and preferably exert the same biological effect as ghrelin in vivo. Exemplary non-peptide ghrelin analogues are described in EP 0 869 974 and

EP 1 060 190, which illustrate a number of ghrelin analogues and which documents are incorporated herein by way of reference. Ghrelin-like compounds may be functional variants of ghrelin. One preferred type of ghrelin-like compound according to the invention described herein is a compound comprising a structure defined by formula I:

Formula I: Z¹ - (X¹)_m - (X²) - (X³)_n- Z², wherein

Z¹ is an optionally present protecting group

each X¹ is independently selected from an amino acid, wherein said amino acid is selected from naturally occurring and synthetic amino acids,

X² is any amino acid selected from naturally occurring and synthetic occurring amino acids, said amino acid being modified with a bulky hydrophobic group, preferably an acyl group, or a fatty acid,

each X³ is independently selected from an amino acid, wherein said amino acid is selected from naturally occurring and synthetic amino acids,

wherein one or more of X¹ and X³ optionally may be modified by a bulky hydrophobic group, preferably an acyl group, or a fatty acid,

Z² is an optionally present protecting group,

m is an integer in the range of from 1-10

n is 0 or an integer in the range of from 1-35.

Accordingly, the term "secretagogue" or "growth hormone secretagogue", or "GHS- R1a secretagogue" includes the naturally occurring 28 aa human ghrelin, the amino acid of which is shown in SEQ ID NO: 1 , as well as the naturally occurring 27 aa human ghrelin, the amino acid of which is shown in SEQ ID NO: 2. Thus, the present invention relates to the use of ghrelin or a peptide homologous thereto. Ghrelin is described by Kojima in Nature (1999), vol. 402,656-660.

The present invention includes diastereomers as well as their racemic and resolved enantiomerically pure forms. GHS-R1a secretagogues can contain D-amino acids, L-amino acids, alpha-amino acid, beta-amino acid, gamma-amino acid, natural amino acid and synthetic amino acid or the like or a combination thereof. Preferably, amino acids present in a ghrelin-like compound are the L-enantiomer.

Further suitable GHS-R1a secretagogues for use in the present invention are disclosed in PCT patent application no. PCT/DK2004/000529, Danish patent application no. PA 200401875, and PCT applications with publication numbers WO0192292 (Merck and Co. Inc), WO0134593 (Novo Nordisk AS) and WO0107475 ("Novel peptides", Kangawa et al.); said documents all being incorporated herein by reference.

Methods for production of GHS-R1a secretagogues are well known to thoese skilled in the art, for example in Example 2 of PCT patent application PCT/DK2004/000519 (Gastrotech Pharma), incorporated herein by reference.

Identity and homology

The term "identity" or "homology" shall be construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics

Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

A homologue of one or more of the secretagogue sequences specified herein may vary in one or more amino acids as compared to the sequences defined, but is capable of performing the same function, i.e. a homologue may be envisaged as a "functional equivalent" of a predetermined sequence. Homologues to SEQ ID NO: 1 may be 27 aa human ghrelin SEQ ID NO: 2, rat ghrelin SEQ ID NO: 3. Other homologues are the variants described in EP 1197496 (Kangawa) incorporated herein by reference.

Conjugates

The binding member and, optionally a secretagogue, may also be administered in a form, wherein the binding member and/or secretagogue is conjugated to another entity, in order for example, to prolong its half-life.

In one embodiment the conjugate is a a conjugate of the binding member and/or ghrelin or a derivative or homologue thereof and Ac-RYY(RK)(WI)RK)-NH₂, where the brackets show allowable variation of amino acid residues. Examples of peptides in the conjugate may also be found in US patent application 2003040472

Disorder in appetite regulation

In one preferred embodiment, the pharmaceutical composition of the present invention is suitable for use in the treatment of disorders in appetite regulation, preferably any disorder associated with lowered food intake.

Some disorders of appetite regulation are caused by a direct malfunction of the appetite regulating system, psychological disorders e.g. anorexia nervosa, others are secondary effects of illnesses such cancer and HIV or AIDS, or the treatment of such diseases. Disorders of appetite regulation may relate to post surgical symptoms. Such symptoms may occur after for example a gastrectomy. Anorexia may also be seen in frail elderly subjects. All such situations are considered disorders of appetite regulation.

Cancer cachexia

One embodiment of the invention relates to a pharmaceutical composition for the treatment or prevention of cancer cachexia caused by a catabolic disorder. This is particularly experienced, when the cancer is a lung cancer, a pancreatic cancer, liver cancer, other Gl tract cancers, in particular esophagus cancer, ventricle cancer, or other cancerous processes located in the mediastinum or the upper abdomen. In another embodiment of the invention relates to a pharmaceutical composition for the treatment or prevention of cancer cachexia caused by an anorectic disorder.

In yet another embodiment of the invention relates to a pharmaceutical composition for the treatment of or prevention of cancer cachexia independent of the cause of cachexia, as well as for cachexia caused by a combination of the catabolic disorder and the anorectic disorder.

One embodiment of the invention relates to a pharmaceutical composition for the treatment or prevention of anorexia caused by anorexia nervosa.

In another embodiment of the invention relates to a pharmaceutical composition for the treatment or prevention of caused by anorexia not caused by anorexia nervosa.

Gastrectomy

The surgical removal of parts of the stomach results in several post operation symptoms related to the well-being and quality of life of the individual as well as heath associated problems cause by loss of body weight, lean body mass, body fat and/or bone mass.

An embodiment of the invention relates to the production of a pharmaceutical composition for stimulation of appetite and prevention of malnutrition in a gastrectomized individual. A further embodiment of the invention relates to the treatment of loss of body weight, fat mass or lean body mass in a gastrectomized individual. A further embodiment of the invention relates to the treatment of loss of bone mass in a gastrectomized individual.

Ghrelin deficiency In one preferred embodiment of the present invention, said pharmaceutical composition is for the prophylaxis or treatment of undesirable symptoms associated with ghrelin deficiency. Preferably, said undesirable symptoms include one or more of: loss of fat mass, weight loss, cachexia, loss of appetite, immunological dysfunction, malnutrition, disrupted sleep pattern, sleepiness, malaborption and motility problems with the intestine. In particular the present invention relates to treatment and/or prevention of loss of body weight, lean body mass and body fat, or stimulation of weight gain, more preferably treatment and/or prevention of loss of body weight, lean body mass and body fat. Treatment and prevention is seen when an already arising weight loss is stopped from progressing and/or weight gain is initiated. This is probably due to the effect of ghrelin or its analogues to stimulate appetite, and thereby stimulate of food intake, and the ability of the binding member to potentiate the effects of ghrelin or a ghrelin-like compound, said secretagogue being either naturally synthesised within the individual's body and/or administered in combination with the binding member. The present invention also relates to stimulation of appetite and stimulation of food intake, more specifically to stimulation of appetite, in individuals at risk of acquiring partial or complete ghrelin deficiency. In one preferred embodiment of the present invention, said individual is at risk of acquiring partial or complete ghrelin deficiency resulting from a pathological condition. In another preferred embodiment of the present invention, said individual is at risk of acquiring partial or complete ghrelin deficiency resulting from a pathological condition. In one preferred embodiment, said pathological condition is associated with insulin resistance. Preferably, said condition associated with insulin resistance is selected from the group consisting of: polycystitic ovary syndrome, acromegaly, primary/secondary hypogonadism, Non-Alcoholic Fatty Liver Disease (NAFLD) and/or Type I Diabetes Mellitus. In another preferred embodiment, said pathological condition is hyperthyroidism. Preferably, said hyperthyroidism is caused by one or more of the following: Grave's disease, drugs containing a high level of iodine, thyroiditis, subacute thyroiditis, postpartum thyroiditis, loss of feedback control of thyroid hormone producing cells, toxic nodular goiter, excessive doses of thyroid hormone or thyroid medication.

In another preferred embodiment of the present invention, the individual in need of treatment with the compounds of the present invention is suffering from, or at risk of suffering from, ghrelin defiency associated with disrupted epithelium in the Gl tract. Said disruption of the epithelium is preferably caused by one or more of: a pathological condition, a genetic disease, or a medical treatment. It is envisaged that the compounds of the present invention may be adminstered to a patient that has been, will be, or is currently, treated using said medical treatment. Said medical treatment is preferably chemotherapy. In another preferred embodiment, said medical treatment is radiotherapy, which may be used in combination with chemotherapy treatment. In another preferred embodiment of the present invention, said disruption of epithelium in the Gl tract is caused by gastritis.

In another preferred embodiment of the present invention, said individual in need of treatment with the compounds of the present invention has a genetic mutation associated with low plasma ghrelin concentrations, such as the Arg51Gln ghrelin mutation.

In one preferred embodiment of the present invention, it is evisaged that individuals treated by the compounds of the present invention may be suffering from ghrelin deficiency or may be at risk of suffering from the disorder.

"The term "malnutrition" refers to a state whereby an individual does not consume, absorb, or maintain in their body sufficient levels of one or more macro- or micro- nutrients so as to remain fit and healthy. By "immunosuppressed" is meant that the individual has a lower than average immune function. An immunosuppressed person may have, for example, a lowered white blood cell count. Causes of immunosuppression are, for example, bone marrow reduction, immunosuppressive pharmaceutical agents, chemotherapeutic agents, and/or reduced protein intake (one form of malnutrition).

In another preferred embodiment of the present invention, the pharmaceutical composition of the present invention is for the prophylaxis or treatment of one or more of: loss of fat mass, loss of lean body mass, weight loss, cachexia, loss of appetite, immunological dysfunction in an individual subjected to one or more of chemotherapy or other anti-cancer treatments.

In one preferred embodiment of the present invention, it is preferred that administration of the compositions of the present invention aids in improving the sense of well-being and the quality of life in the individual. In another preferred embodiment, adminstration of said composition stimulates appetite and prevents malnutrition of the individual. Frailty

In one embodiment of the present invention, the individual treated is classified as suffering from the condition of frailty.

Frailty is a condition characterised by impaired strength, reduced endurance, and increased vulnerability. Recently an objective definition of frailty has been proposed and validated (Fried LP et al., J Gerontol A Biol Sci Med Sci. 2001 ;56:M146-56). According to this definition individuals are considered frail if they meet at least three of the following five criteria: • Unintentional weight loss. • General feeling of exhaustion. • Weakness. • Slow walking speed. • Low levels of physical activity.

These five criteria are defined herein as follows:

• Unintentional weight loss: subjects with an unintentional weight loss of more than 10 lb in the previous year are considered positive for this criterion.

• General feeling of exhaustion. Two items from the CES-D Scale are used to characterize exhaustion 1) "I felt that everything I do is an effort" and 2) "I cannot get going". Individuals are asked to indicate if they felt that way 0 (none of the time), 1 (some of the time [1-2 days a week]), 2 (a moderate amount of time [3-4 days], or 3 (most of the time). Subjects answering 2 or 3 to either of these questions are positive for the exhaustion criterion.

• Weakness. Weakness is assessed by measurement of grip strength in the dominant hand using a JAMAR handheld dynamometer set at level 2. Three attempts at maximal squeeze are recorded. The average value is adjusted by the individual's body mass index (BMI). Those with grip strength in the bottom 20%) are considered positive for the weakness criterion.

• Slow walking speed. Individuals are asked to walk 15 ft (4.5m) at their usual pace, following a standardized protocol; and time is measured by a trained examiner. Height- and sex-adjusted time points are used, with the slowest 20% being considered positive for this criterion.

• Low levels of physical activity. This criterion is based on the Modified Minnesota Leisure Time Activities questionnaire and involves self-report regarding whether a person performed any of 18 activities in the prior week, along with the frequency and duration of these activities. Kilocalories of energy expended in a week on leisure time activity are calculated. Those in the bottom quartile of physical activity are deemed positive for this criterion.

Using the above definition of frailty and excluding individuals with acute or chronic medical conditions, about 7% of the population older than 65 years of age and 20%o of the population older than 80 years are frail. Frailty is considered a distinct pathophysiological condition separate from the normal aging process. Frailty is an independent predictor for deteriorating mobility, disability, hospitalization and death with hazard ratios ranging from 1.82 to 4.46, unadjusted, and 1.29-2.24, adjusted for a number of risk factors ((Fried LP; Cardiovascular Health Study Collaborative Research Group. Frailty in older adults: evidence for a phenotype. J Gerontol A Biol Sci Med Sci. 2001 ;56:M146-56).

The main cause of unintentional weight loss in elderly subjects is reduced appetite and food intake. On average energy intake decreases by approximately 30% between 20 and 80 years of age (Chapman IM. Endocrinology of anorexia of ageing. Best Pract Res Clin Endocrin Metab 2004;18:437-52). In many elderly the decline in energy intake exceeds the decline in energy expenditure leading to weight loss. Aging also results in an impaired homeostatic regulation of food intake. Consequently, elderly are less likely to regain weight following an illness or an event leading to loss of body weight, such as surgery or trauma.

Weight loss in elderly subjects is characterised by a disproportionally high loss of muscle mass leading to sarcopenia, a relative deficiency of skeletal muscle. Sarcopenia reduces strength and contributes to impaired functional capacity. Sarcopenia may also occur in the absence of weight loss. Sarcopenic obese subjects have a low skeletal muscle mass with a high percent body fat. Sarcopenic obesity is associated with a particularly high risk of developing disability and dependence.

Endocrine factors may be causally involved in the development of frailty (Wilson JF. Frailty - and its dangerous effects - might be preventable. Ann Intern Med

2004;141 :489-492). With increasing age growth hormone (GH) and IGF-1 levels decrease, and in males testosterone levels decreases. These changes may aggravate the physiological loss of muscle mass and strength in elderly resulting in impaired physical capacity. In addition, circulating ghrelin levels are 20-35% lower in elderly than in young subjects and a reduced GH-response to exogenous ghrelin administration has been observed in older subjects suggesting a decline in ghrelin sensitivity with increasing age ((Chapman IM. Endocrinology of anorexia of ageing. Best Pract Res Clin Endocrin Metab 2004;18:437-52). Hypoghrelinemia and ghrelin resistance may play a role in the loss of appetite and the loss of body weight seen in frail elderly.

The orexigenic and anabolic effects of the binding member of the present invention will be beneficial in frail elderly subjects, stimulating appetite and food intake, reversing or limiting body weight loss and loss of muscle mass, reducing weakness and improving physical capacity.

Further indications

Throughout the application, including the present section, it will be understood that whenever the binding members according to the present invention are mentioned as useful for a specific indication, the term "binding member" encompasses the binding member as such as well as a salt, hydrate or any other derivative thereof, optionally in the form of a pharmaceutical composition as described herein.

The present invention relates to use of one or more of the binding members according to the present invention in the manufacture of a medicament for the treatment of an individual in need thereof. Preferably, said individual is suffering from, or at risk of suffering from, a pathological condition treatable with the binding members of the present invention. The present invention also relates to a method of treatment of a individual in need thereof, comprising administering to said individual one or more of the binding members according to the present invention. Thus, the binding members of the present invention may be used to treat any individual capable of receiving benefit from said treatment.

In one embodiment the binding member may be used for prevention and treatment of cachexia/malnutrition or maintenance of metabolic homeostasis in patients. The foremost sign of cachexia is weight loss, not only of fatty tissue but also of muscle tissue and even bone. This non-fatty tissue is also known as "lean body mass." In addition, there is loss of appetite (anorexia), weakness (asthenia), and a drop in hemoglobin level (anemia).

Treatment of cachexia is not simply a matter of eating more. Even if the person wants to eat, even if he or she tries to eat, even if the person is given nutrients through a stomach tube or intravenously, the condition will normally not be reversed.

Recent research has revealed that the condition is now regarded as part of the body's reaction to the presence of the underlying disease. Recent research also indicates that, in some cases, tumors themselves produce substances that induce cachexia.

Thus, in one aspect, the invention relates to the use of a binding member disclosed herein for the preparation of a medicament for

a) stimulation of appetite, and/or b) stimulation of food intake, and/or c) stimulation of weight gain, and/or d) increasing body fat mass, and/or e) increasing lean body mass.

In particular the binding member may be used in the prevention and treatment of cachexia or malnutrition in individuals suffering from: - Cancer - AIDS - Cardiac failure - Pulmonary insufficiency due to for instance COPD, pulmonary fibrosis, cystic fibrosis or D-i -antitrypsin deficiency - Renal failure due to for instance glomerulonephritis, tubulointerstitial nephropathy, hereditary nephropathy (e.g. polycystic kidney disease), hypertension, diabetes mellitus, vascular disease, obstructive uropathy - Liver failure due to for instance viral hepatitis, toxic hepatitis, fibrosis, cirrhosis, primary biliary cirrhosis, hereditary liver diseases - Autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus - Severe chronic infections such as tuberculosis - Patients undergoing gastrectomy - frailty - Organ transplantation patients Patients recovering from major surgery - Patients recovering from trauma, such as especially trauma requiring surgical intervention - Premature children and children born small for gestastional age

It is also preferred that the binding member according to the invention may be administered prophylactically for preventing a cachectic state. Thus, in addition to the treatment of cachexia and malnutrition, the binding members according to the present invention may be useful in prevention or treatment of a catabolic state, such as a catabolic state resulting from: - Critical illness Severe infections such as sepsis - Severe burns - Major trauma - Major surgery such as gastrointestinal surgery, cardiothoracic surgery, transplantation - Treatment with catabolic agents such as glucocorticoids Due to the positive effect of the binding members on quality of life (QOL), for example as is caused by improved appetite and/or body weight and/or nutritional status, the binding members according to the invention are suitable for prevention and treatment of frailty in elderly persons. In another embodiment the binding member according to the invention is useful in the prevention or treatment of heart failure. In particular the heart failure may be due to ischaemic heart disease, cardiomyopathy, hypertension, valvular dysfunction or heart failure due to pulmonary disease such as chronic bronchitis, pulmonary hypertension, emphysema or heart failure caused by genetic defects.

Furthermore, the binding member according to the invention is useful in prevention or treatment of tissue ischaemia. For example the ischaemia may be cardiac ischaemia due to for instance coronary artery disease, or cerebral ischaemia due to thrombo-embolism Furthermore, the binding members according to the invention are suitable for prevention and/or treatment of bone and cartilage related diseases. In particular the binding members are useful in the prevention or treatment of osteoporosis.

Also, the binding members according to the invention are suitable in the treatment of bone fractures, by acceleration of fracture healing and recovery following major fractures.

The binding members of the present invention also improve the nutritional condition of the individuals being treated due to an increase of gastric motility and gastric emptying. Accordingly, the binding member of the present invention may be used in the treatment or prevention of delayed gastric emptying/gastroparesis, such as in patients with diabetes mellitus, patients with renal failure patients with liver failure, patients with idiopathic gastroparesis, critically ill patients, patients undergoing anaestesia and patients undergoing surgery.

Furthermore, the binding members may be used for prevention and treatment of postoperative ileus.

In a further embodiment the binding members are useful in the prevention and treatment of inflammatory diseases, such as inflammatory bowel diseases.

In a further embodiment the binding member according to the invention is useful in the treatment of malignant diseases, such as breast cancer and thyroid cancer. In a further embodiment the binding member according to the invention is useful in the treatment of hyperthyroidism, thus, binding members according to the invention may be used for preventing weight gain in individuals being converted from a hyperthyroidic state to euthyroid state. The binding members according to the invention are also useful in the treatment of sleeping disorders, and accordingly, the binding members may be used for treating sleeping disorders.

In yet another embodiment, the invention relates to use of a binding member in the treatment of a lipodystrophic syndrome, or for the manufacture of a medicament for the prevention or treatment of a lipodystrophic syndrome. Lipodystrophic syndromes encompass a heterogeneous group of rare disorders characterized by partial or generalized loss of adipose tissue depots. Some patients may have only cosmetic problems while others may also have severe metabolic complications such as dyslipidemia, hepatic steatosis, and severe insulin resistance. These disorders can either be inherited (familial or genetic lipodystrophies) or can occur secondary to various types of illnesses or drugs (acquired lipodystrophies).

Pharmaceutical compositions further comprising a secretagogue compound

In one preferred embodiment of the present invention, the pharmaceutical compositions of the present invention described herein further comprise a secretagogue compound, such as ghrelin or any of the secretagogue compounds described in the above section on ghrelin-like compounds.

Without being bound by theory, it is envisaged that a secretagogue such as ghrelin may be used as a substance to increase the anabolic factor IGF-1 , and that as a result leads to increased body weight and/or prevention of loss of body weight and body fat. It is furthermore believed, without being bound by theory, that the binding member prolongs the half-life of a secretagogue, such as ghrelin or a ghrelin-like compound, within the individual and/or increases the action of a secretagogue, such as ghrelin or an analogue thereof, thus prolonging the actions (such as appetite promotion) of said secretagogue. Thus, the antibody acts to potentiate the effects of said secretagogue. This potentiation is presumably increased when a secretagogue, such as ghrelin is administered to the individual, which is particularly desirable in instances of ghrelin deficiency. Further combinations

It is further envisaged that the pharmaceutical compositions of the present invention can be administered in combination with other compounds and/or pharmaceutical compositions. By the term "in combination" is meant herein that said composition may be co-formulated with other compounds in the same composition, and/or that said other compound(s) and/or pharmaceutical composition(s) are administered before, during (including concurrently with) and/or after administration of the compositions of the present invention.

Suitable compounds and/or pharmaceutical compositions for use in combination with the pharmaceutical compositions of the present invention comprise, but are not restricted to, other body weight and body fat inducing factors. Exemplarily mentioned factors are melanin-concentrating hormone (MCH), MCH receptors agonists, especially MCH receptor 1 agonists, neuropeptide Y (NPY), NPY receptor 1 agonists, NPY receptor 5 agonists, and NPY receptor 2 antagonists including peptide YY (PYY) and PYY (3-36), alpha-melanocyte stimulating hormone (alpha- MSH, alpha-melanocortin), melanocortin-3 receptor (MC3R) antagonists, melanocortin-4 receptor (MC4R) antagonists, agouti-related peptide (Agrp), Agrp- agonists, cocaine- and amphetamine-regulated transcript (CART) antagonists, orexin receptor 1 and receptor 2 agonists, growth hormone (GH), GH receptor agonists, insulin-like growth factor-1 (IGF-1), and IGF-1 receptor 1 agonists.

In another preferred embodiment, the compositions of the present invention may be used in combination with one or more other stomach-derived factor. This other stomach-derived factor may include any hormone, acylated or nonacylated peptide, amino acid derivative, nucleotide, fatty acid derivative, carbohydrate or other substance derived or secreted from the stomach, and may preferably (but not exclusively) be selected from the following list: Pacreastatin, gastrin, histamine, resistine, prostaglandins such as prostaglandin E2, intrinsic factor.

In addition to "stomach derived factors", a secretagogue, such as ghrelin can also be used in combination with any synthetic low or high molecular weight agonist acting on the the same receptor as a "stomach derived factor" . Combination therapy

Administration of the pharmaceutical composition of the present invention may be used in combination with any suitable therapy. By treatment "in combination" with another treatment regime is meant that said another treatment regime may be carried out before, during (including concurrently with) and after treatment of an individual with the compositions of the present invention.

In one embodiment of the present invention, said therapy is an anti-cancer therapy, including chemotherapy, radiotherapy and surgical treatment. In particular it is used in combination with chemotherapy and radiotherapy. Thus in one embodiment the present invention relates to a method of treating cancer comprising administering an effective amount of radiotherapy and an effective amount of pharmaceutical composition of the present invention.

As anorexia nervosa is psychological based appetite regulating treatment may not be sufficient. Thus the pharmaceutical composition may be administrated in combination with other pharmaceutical compositions, such as pysocofamaca/ and anti depressives. In another embodiment, the compositions of the present invention may be administered with psychotherapy or similar treatments.

The treatment of a gastrectomized individual may comprise the administration of the pharmaceutical compositions in combination with a stomach derived factor, either by formulation of a pharmaceutical composition comprising both active molecules or by separate administration of two pharmaceutical compostions.

Individual in need

According to the invention, any suitable individual who may draw benefit from the compositions of the present invention may be treated with said compositions. Preferably, said individual is suffering from a disorder of appetite regulation.

In addition a person in need of gaining weight for athletic performance or for cosmetic reasons may also be considered as an individual in need treatment.

Furthermore, the invention may relate to stimulation of appetite and increasing the rate of weight gain in animals, particularly young animals or weanlings, and more particularly young animals of species that are utilized for meat, such as sheep, pigs, cows, horses, poultry and game animals.

Form of pharmaceutical compositions Whilst it is possible for the compounds or salts of the present invention to be administered as the raw chemical, it is preferred to present them in the form of a pharmaceutical composition. Accordingly, the pharmaceutical composition according to the present invention preferably further comprises a pharmaceutically acceptable carrier or a diluent.

The compositions of the present invention may preferably be delivered to an individual in any way so as to achieve a beneficial effect, preferably by stimulating appetite and/or preventing malnutrition, and/or improving the individual's sense of well-being or quality of life. In one preferred embodiment, a composition according to the present invention is administered via an oral, nasal, pulmonary, transdermal or parenteral route. More preferably, the composition is administered via the oral or pulmonary route. Other drug-administration methods, which are effective to deliver the drug to a target site or to introduce the drug into the bloodstream, are also contemplated.

The compounds according to the invention may be administered with at least one other compound. The compounds may be administered simultaneously, either as separate compositions or combined in a unit dosage form, or administered sequentially.

In a preferred embodiment, the pharmaceutical composition is not immunogenic when administered to a individual for therapeutic purposes, unless that purpose is to induce an immune response.

Preferably, the composition comprises a binding member or pharmaceutically acceptable salt thereof and pharmaceutically acceptable carriers, vehicles and/or excipients and/or transport molecules.

Transport molecules act by having incorporated into or anchored to it the compound according to the invention. Any suitable transport molecules known to the skilled person may be used. Examples of transport molecules may be liposomes, micelles, and/or microspheres.

A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4, 235,871 ,

4,501 ,728 and 4,837,028, all of which are incorporated herein by reference.

Micelles are formed by surfactants (molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups) in aqueous solution. As the concentration of a solid surfactant increases, its monolayers adsorbed at the air/water or glass/water interfaces become so tightly packed that further occupancy requires excessive compression of the surfactant molecules already in the two monolayers. Further increments in the amount of dissolved surfactant beyond that concentration cause amounts equivalent to the new molecules to aggregate into micelles. This process begins at a characteristic concentration called "critical micelle concentration".

The shape of micelles formed in dilute surfactant solutions is approximately spherical. The polar head groups of the surfactant molecules are arranged in an outer spherical shell whereas their hydrocarbon chains are oriented toward the center, forming a spherical core for the micelle. The hydrocarbon chains are randomly coiled and entangled and the micellar interior has a nonpolar, liquid-like character. In the micelles of polyoxyethylated nonionic detergents, the polyoxyethlene moieties are oriented outward and permeated by water. This arrangement is energetically favorable since the hydrophilic head groups are in contact with water and the hydrocarbon moieties are removed from the aqueous medium and partly shielded from contact with water by the polar head groups. The hydrocarbon tails of the surfactant molecules, located in the interior of the micelle, interact with one another by weak van der Waals forces.

The size of a micelle or its aggregation number is governed largely by geometric factors. The radius of the hydrocarbon core cannot exceed the length of the extended hydrocarbon chain of the surfactant molecule. Therefore, increasing the chain length or ascending homologous series increases the aggregation number of spherical micelles. If the surfactant concentration is increased beyond a few percent and if electrolytes are added (in the case of ionic surfactants) or the temperature is raised (in the case of nonionic surfactants), the micelles increase in size. Under these conditions, the micelles are too large to remain spherical and become ellipsoidal, cylindrical or finally lamellar in shape.

Common surfactants well known to one of skill in the art can be used in the micelles of the present invention. Suitable surfactants include sodium laureate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PLURONIC F-127 (Wyandotte Chemicals Corp.). Preferred surfactants are nonionic polyoxyethylene and polyoxypropylene detergents compatible with IV injection such as, TWEEN-80, PLURONIC F-68, n-octyl-.beta.-D-glucopyranoside, and the like. In addition, phospholipids, such as those described for use in the production of liposomes, may also be used for micelle formation.

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon an individual without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.

The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The pharmaceutical composition of the present invention can include pharmaceutically acceptable salts of the compounds therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide).

Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydriodic, phosphoric, sulpfuric and nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, ethylenediaminetetraacetic (EDTA), p-aminobenzoic, glutamic, benzenesulfonic and ptoluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutical acceptable salts listed in J. Pharm. Sci. 1977,66,2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium and magnesium salts and the like.

Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium and tetramethylammonium salts and the like.

Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Also included within the scope of compounds or pharmaceutical acceptable acid addition salts thereof in the context of the present invention are any hydrates (hydrated forms) thereof.

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water.

The pharmaceutical compositions formed by combining the compounds of the invention and the pharmaceutical acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The compositions may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

In a preferred embodiment of the invention the composition comprises the binding member or a salt thereof as a lyophilisate and the composition further comprises a solvent. In another embodiment the composition is a solution of the binding member or a salt thereof. Preferably, the solvent may be any suitable solvents, such as described herein, and preferably the solvent is saline or a physiological buffer like phosphate buffer.

The invention also relates to a method for preparing a pharmaceutical composition or pharmaceutical composition comprising an compound of the invention, comprising admixing at least one binding member as defined above with a physiologically acceptable carrier.

In a still further aspect, the invention relates to a pharmaceutical composition comprising, as an active ingredient, a compound as defined above or a pharmaceutical acceptable salt thereof together with a pharmaceutical acceptable carrier.

Accordingly, the composition may further include the transport molecules as described above.

In a further aspect of the invention the present compounds may be administered in combination with further pharmacologically active substances known to increase body weight, e. g. melanin-concentating hormone (MCH), MCH receptors agonists, especially MCH receptor 1 agonists, neuropeptide Y (NPY), NPY receptor 1 agonists and NPY receptor 5 agonists, NPY receptor 2 antagonists including pepetide YY (PYY) and PYY (3-36), alpha-melanocyte stimulating hormone (alpha- MSH, alpha-melanocortin), melanocortin-3 receptor (MC3R) antagonists, melanocortin-4 receptor (MC4R) antagonists, agouti-related peptide (Agrp), Agrp- agonists, cocaine- and amphetamine-regulated transcript (CART) antagonists, orexin receptor 1 and receptor 2 agonists, growth hormone (GH), GH receptor agonists, and insulin-like growth factor-1 (IGF-1), IGF-1 receptor 1 agonists or other pharmacologically active material. Moreover, the further active substance may comprise other stomach derived factors including, but not restricted to, one or more of gastrin, pancreostatin, histamine, resistine, prostaglandins such as prostaglandin

E2, intrinsic factor.

In another embodiment the secretagogue is administered in combination with a NSAID, such as indomethacin, and COX1 inhibitors or COX2 inhibitors. Another combination may be with erythropoietin/EPO. Another combination may be with one or more of leptin, agonists of the renin-angiotensin system, opioid receptor agonists or peroxisome proliferator-activated receptor gamma agonists. In another preferred embodiment, the secretagogue may be administered in combination with a growth hormone, preferably hGH. The combination may be in the form of kit-in-part systems, wherein the combined active substances may be used for simultaneous, sequential or separate administration.

Compositions for parenteral administration

The compounds of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. Aqueous solutions should be suitably buffered if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.

Solutions of a binding member or pharmaceutically acceptable salt thereof, (and for example antigenic epitopes and protease inhibitors) can be prepared in water or saline, and optionally mixed with a nontoxic surfactant. Compositions for intravenous or intra-arterial administration may include sterile aqueous solutions that may also contain buffers, liposomes, diluents and other suitable additives.

Oils useful in parenteral compositions include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such compositions include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral compositions include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral compositions include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides; (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-.beta.-aminopropionates, and 2-alkyl- imidazoline quaternary ammonium salts, and (e) mixtures thereof.

The parenteral compositions typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such compositions will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral compositions can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions comprising the active ingredient that are adapted for administration by encapsulation in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.

Sterile injectable solutions are prepared by incorporating a secretagogue, such as ghrelin or an analogue or pharmaceutically acceptable salt thereof in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.

Compositions for administration as suppositories

The compounds of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.

The active compound may be formulated into a suppository comprising, for example, about 0.5% to about 50% of a compound of the invention, disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%].

Compositions for aerosol, nasal or inhalation delivery

It is contemplated that the compounds of the present invention may be formulated for administration to the respiratory tract and including intranasal administration, and for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The compositions may be provided in a single or multidose form. In the latter case of a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomizing spray pump. A suitable formulation for nasal administration is described in EP 1 466 610.

For inhalation, the compounds can be formulated as using methods known to those skilled in the art, for example an aerosol, dry powder or solubolized such as in microdroblets, preferably in a device intended for such delivery (such as commercially available from Aradigm, Alkerme or Nektar).

Compositions administered by aerosols may be prepared, for example, as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, employing fluorocarbons, and/or employing other solubilizing or dispersing agents in accordance with methods known in the art.

Pharmaceutically acceptable salts Pharmaceutically acceptable salts of the instant compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases. Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent.

The compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral

(including subcutaneous) route, in an effective amount.

Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.

Administration - dosing regimes

The pharmaceutical composition may be prepared so it is suitable for one or more particular administration methods. Furthermore, the method of treatment described herein may involve different administration methods.

The pharmaceutical composition comprising said binding member may be administrated to an individual in need there of by any suitable method.

The binding members of the invention can for example be administered parenterally by injection or by gradual infusion over time. Thus, binding members of the invention may be administered parenterally, such as intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be delivered by peristaltic means.

In a preferred embodiment the pharmaceutical composition is administered in a concentration equivalent to from 0.1 unit to 1 unit per kg bodyweight, such as from 0.5 unit to 0.5 unit per kg bodyweight, such as from 1.0 unit to 0.1 unit per kg bodyweight, such as from 1.0 unit to 50 unit per kg bodyweight, such as from 1.0 unit to 10 unit per kg bodyweight.

In a preferred embodiment the pharmaceutical composition is administered in a concentration equivalent to from 0.1-1 mg binding member per kg bodyweight, such as from 0.5 to 5 mg per kg bodyweight, such as from 1.0 mg to 10 mg per kg bodyweight, such as from 5 to 50, such as from 5 to 250 mg per kg bodyweight.

In a preferred embodiment the pharmaceutical composition is administered in a concentration equivalent to from 0.1-1 mg per kg bodyweight per day, such as from 0.5 to 5 mg per kg bodyweight per day, such as from 1.0 mg to 10 mg per kg bodyweight per day, such as from 5 to 50 mg per kg bodyweight per day

The administration route must ensure that the non-degraded, bioactive form of the binding member will be the dominating form in the circulation, which will reach ghrelin and any secondary binding ligands, for example a GHS receptor. Thus, in order to obtain the maximum effect of the pharmaceutical composition it is preferably administered from one to three times daily, each administration being within 45 minutes of a meal, such as within 30 minutes of a meal, such as within 25 minutes of a meal, such as within 20 minutes of a meal, such as within 15 minutes of a meal, such as within 10 minutes of a meal, such as within 5 minutes of a meal. More preferred the pharmaceutical composition is administered prior to each main meal, such as administered three times daily.

The pharmaceutical preparations described herein are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. When desired, compositions can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. In one aspect of the present invention, a suitable dose of the compositions described herein is administered in pharmaceutically effective amounts to an individual in need of such treatment. Herein, "pharmaceutically effective amounts", is defined as an administration involving a total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound, alone or in combination with other agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an " effective amount" or an amount necessary to achieve an "effective level" in the individual patient.

The dosage requirements will vary with the particular drug composition employed, the route of administration and the particular subject being treated. Ideally, a patient to be treated by the present method will receive a pharmaceutically effective amount of the compound in the maximum tolerated dose, generally no higher than that required before drug resistance develops Suitable dosing regimens are preferably determined taking into account factors well known in the art including type of subject being dosed; age, weight, sex and medical condition of the subject; the route of administration; the renal and hepatic function of the subject; the desired effect; and the particular compound employed.

Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

A preferred dosage of a composition employed according to the invention is in a concentration equivalent to from about 0.1 mg to about 10 mg ghrelin per kg bodyweight, which is preferably administered daily. More preferably, the pharmaceutical composition comprises a unit dosage form of from about 5 to about 250 mg of ghrelin or an analogue thereof, more preferably from about 20 mg to about 200 mg, more preferably from about 20 mg to about 100 mg. The secretagogues of the present invention may be administered admixed with a pharmaceutically acceptable carrier or diluent.

It should be noted that the normal ghrelin response which occurs before a meal is a short-lived surge in plasma concentrations of ghrelin and that due to the relative short half life of the peptide an i.v. injection of a secretagogue in combination with the binding members of the present invention will ensure that a similar short-lived peak on ghrelin concentrations can be obtained. The administration route must ensure that the non-degraded, bioactive form of the secretagogue will be the dominating form in the circulation, which will reach the ghrelin receptors and stimulate these. Thus, in order to obtain the maximum effect of the pharmaceutical composition it is preferably administered from one to three times daily, each administration being within 90 minutes of a meal, such as within 85 minutes of a meal, such as within 80 minutes of a meal, such as within 75 minutes of a meal, such as within 70 minutes of a meal, such as within 65 minutes of a meal, such as within 60 minutes of a meal, such as within 55 minutes of a meal, such as within 50 minutes of a meal, such as within 45 minutes of a meal, such as within 40 minutes of a meal, such as within 35 minutes of a meal, such as within 30 minutes of a meal, such as within 25 minutes of a meal, such as within 20 minutes of a meal, such as within 15 minutes of a meal, such as within 10 minutes of a meal, such as within 5 minutes of a meal. More preferred the pharmaceutical composition is administered prior to each main meal, such as administered three times daily.

For the present invention the dosage will vary depending on the compound employed and the mode of administration. Dosage levels will vary between about 0.01 μg/kg body weight to 10 mg/kg body weight daily, preferably between about 0.01 μg/kg body weight to 1 mg/kg body weight, more preferably between 0.1 to 10 μg /kg body weight. For all methods of use disclosed herein for the compounds, the daily oral dosage regimen will preferably be from about 0.01 μg to about 80 mg/kg of total body weight. The daily parenteral dosage regimen about 0.01 μg to about 80 mg/kg of total body weight. The daily topical dosage regimen will preferably be from 0.01 μg to 150 mg, administered one to four, preferably two or three times daily. The daily inhalation dosage regimen will preferably be from about 0.01 μg /kg to about 1 mg/kg per day. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Furthermore, since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on interindividual differences in pharmacokinetics, drug distribution, and metabolism. The "effective level" can be defined, for example, as the blood or tissue level desired in the patient that corresponds to a concentration of one or more compounds according to the invention.

In one preferred embodiment of the present invention, the compositions of the present invention are administered in a dose of from about 0.1 mg/kg/day binding member or analog thereof to about 10 mg/kg/day. In another preferred embodiment, the compositions are administered in a dose of from about 5 to about 250 mg of the binding member.

The pharmaceutical composition may also be a kit-in-part further including an antibiotic agent, such as antibiotics selected from β-lactams, cephalosporins, penicilins and aminoglycosides, and/or include an immunostimulating agent, such as cytokines, interferons, growth factors, for example GCSF or GM-CSF. The kit-in-part may be used for simultaneous, sequential or separate administration.

Isolated nucleic acid molecule/vector/host cell

In one aspect the invention relates to an isolated nucleic acid molecule encoding at least a part of the binding member as defined above. In one embodiment the nucleic acid molecule encodes a light chain and another nucleic acid encodes a heavy chain. The two nucleic acid molecules may be separate or they may be fused into one nucleic acid molecule, optionally spaced apart by a linker sequence. In particular in relation to antibody fragments the nucleic acid molecule may encode the whole binding member, however dependant on the design of the binding member this may also be relevant for some larger binding members.

In one embodiment the nucleic acid molecule encodes an affibody sequence or an antibody-affibody chimera sequence. In a further aspect of the invention the nucleic acids encodes two nucleic acid molecules, separate or fused. In particular more than one antibody-affibody chimera sequence may be included, for example two, three or four or more than four antibody-affibody chimera sequences may be included.

The nucleic acid molecule preferably is a DNA sequence, more preferably a DNA sequence comprising, in its upstream end, regulatory elements promoting the expression of the binding member once the nucleic acid molecule is arranged in a host cell.

Accordingly, in one embodiment the invention relates to a polynucleotide selected from the group consisting of i) polynucleotide encoding a fragment of a polypeptide, where in said fragment a) is capable of recognising an antigen also being recognised by the binding member and/or. b) is capable of binding selectively to an antigen, wherein said antigen is also bound selectively by the binding member and/or c) has a substantially similar or higher binding affinity to an antigen bound selectively by the binding member ii) a polynucleotide, the complementary strand of which hybridize under stringent conditions, with a polynucleotide as defined in i), iii) a polynucleotide comprising a nucleotide sequence which is degenerate to the nucleotide sequence of a polynucleotide as defined in i) and the complementary strand of such a polynucleotide.

The invention further relates to a vector comprising the nucleic acid molecule as defined above, either one vector per nucleic acid, or two or more nucleic acids in the same vector. The vector preferably comprises a nucleotide sequence which regulates the expression of the binding member encoded by the nucleic acid molecule.

In yet another aspect the invention relates to a host cell comprising the nucleic acid molecule as defined above. Such a host cell may be a prokaryotic cell, for example E. coli, or a eukaryotic cell as for example a yeast strain or a mammalian cell line.

Also, the invention relates to a cell line engineered to express the binding member as defined above, this cell line for example being, Escherichia coli, Saccharomyces cerevisiea, Pichia pastoris, or a hybridoma of a murine lymphocyte and an immortalised cell line. Said cell line may be any suitable cell line, however the cell line P3 is preferred. In another embodiment a CHO cell line is preferred.

Therapeutic methods

The binding members according to the present invention are particular useful in therapeutic methods due to their high affinity and specificity. Accordingly, the binding members can be used immunotherapeutically towards a disease or disorder associated with a secretagogue, in particular ghrelin, more particularly human ghrelin.

The term "immunotherapeutically" or "immunotherapy" as used herein in conjunction with the binding members of the invention denotes both prophylactic as well as therapeutic administration. Thus the pharmaceutical pharmaceutical composition comprising the binding members can be administered to patients in order to prevent the development of a disease of appetite regulation, in order to minimise the severity of a disorder in appetite regulation, or to patients already suffering from a disorder in appetite regulation. Furthermore the therapeutic method may prevent reoccurrence of disorders of appetite regulation.

The dosage ranges for the administration of the binding members of the invention are those large enough to produce the desired effect in which the symptoms of the disease are ameliorated. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

A therapeutically effective amount of an binding member of this invention is typically an amount of antibody such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 unit per milliliter (ml) to about 100 unit/ml, preferably from about 1 unit/ml to about 5 unit/ml, and usually about 5 unit/ml, in one or more dose administrations daily, for one or several days.

Use of an isolated binding member for the production of a pharmaceutical compositions

In another embodiment of the present invention, the isolated binding members of the present invention are used for the production of any of the pharmaceutical compositions desrcibed herein, for the treatment of an individual in need thereof. By "treatment" is also meant prophylaxis and aftercare, and/or lessening of disease symptoms and/or possible disease prevention and/or cure. Furthermore are provided in the scope of the present invention methods of treatment of an individual in need thereof, comprising administering to said individual an effective amount of one or more of the pharmaceutical compositions described herein. Said individual is preferablys suffering, or at risk of, one or more of the health problems described earlier herein, such as a disorder of appetite regulation, and/ or ghrelin deficiency. Said method may be for the treatment of loss of body fat mass or loss of bone mass of a gastrectomized individual and/or comprise improving the sense of well-being and the quality of life in an individual. In another embodiment, the treatment stimulates appetite and prevents malnutrition of the individual. Said method may involve one or more of the combination treatments as disclosed herein

Diagnostic methods

The present invention also contemplates various assay methods for determining the presence, and preferably the amount of a secretagogue, preferably ghrelin, typically in a biological sample.

Accordingly, the present invention relates to a method of detecting or diagnosing a disease or disorder of appetite regulation in an individual comprising

providing a biological sample from said individual,

adding at least one type of the binding members of the present invention, capable of specifically binding a secretagogue, such as ghrelin or a ghrelin-like compound, to said biological sample

detection of binding members bound to said biological sample, thereby detecting or diagnosing the disease or disorder.

The bound binding member may be detected either directly or indirectly and thereby the amount of a secretagogue, such as ghrelin or ghrelin-like compound, in the sample is measured.

Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures in which a binding reagent of this invention can be used to form a binding reaction product whose amount relates to the amount of the ligand in a sample. Thus, while exemplary assay methods are described herein, the invention is not so limited.

Various heterogenous and homogeneous protocols, either competitive or noncompetitive, can be employed in performing an assay method of this invention.

Binding conditions are those that maintain the ligand-binding activity of the receptor. Those conditions include a temperature range of about 4 to 50 degrees Centigrade, a pH value range of about 5 to 9 and an ionic strength varying from about that of distilled water to that of about one molar sodium chloride.

The detecting step can be directed, as is well known in the immunological arts, to either the complex or the binding reagent (the receptor component of the complex). Thus, a secondary binding reagent such as an antibody specific for the receptor may be utilized.

Alternatively, the complex may be detectable by virtue of having used a labeled receptor molecule, thereby making the complex labeled. Detection in this case comprises detecting the label present in the complex.

A further diagnostic method may utilize the multivalency of a binding member composition of one embodiment of this invention to cross-link ligand, thereby forming an aggregation of multiple ligands and polypeptides, producing a precipitable aggregate. This embodiment is comparable to the well known methods of immune precipitation. This embodiment comprises the steps of admixing a sample with a binding member composition of this invention to form a binding admixture under binding conditions, followed by a separation step to isolate the formed binding complexes. Typically, isolation is accomplished by centrifugation or filtration to remove the aggregate from the admixture. The presence of binding complexes indicates the presence of the preselected ligand to be detected.

Thus, a further embodiment of the invention relates to a kit comprising at least on binding member capable of binding a secretagogue, such as ghrelin, specificallly, said molecule being labelled.

The present invention also describes a diagnostic system, preferably in kit form (describe below), for assaying for the presence of ghrelin, in a biological sample where it is desirable to detect the presence, and preferably the amount, of ghrelin in a sample according to the diagnostic methods described herein.

The diagnostic system includes, in an amount sufficient to perform at least one assay, a binding member composition according to the present invention, preferably as a separately packaged reagent, and more preferably also instruction for use.

The biological sample can be a tissue, tissue extract, fluid sample or body fluid sample, such as blood, plasma or serum.

"Packaged" refers to the use of a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a binding member of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated labelled binding member preparation, or it can be a microtiter plate well to which microgram quantities of a contemplated binding member has been operatively affixed, i.e., linked so as to be capable of binding a ligand.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.

A diagnostic system of the present invention preferably also includes a label or indicating means capable of signalling the formation of a binding reaction complex containing a binding member complexed with the preselected ligand.

Any label or indicating means can be linked to or incorporated in an expressed polypeptide, or phage particle that is used in a diagnostic method. Such labels are themselves well-known in clinical diagnostic chemistry.

The labelling means can be a fluorescent labelling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labelling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante

(FITC), 5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diammobenzidine. An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labelling agents and are used illustratively herein. An exemplary radiolabelling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴ I, ¹²⁵ 1, ¹²⁸ 1, ¹³² 1 and ⁵¹ Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is ¹²⁵ 1. Another group of useful labelling means are those elements such as ¹¹ C, ,¹⁸ F, ¹⁵ O and ¹³ N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such ¹¹¹ indium of ³ H.

The linking of labels, i.e., labelling of, polypeptides and proteins or phage is well known in the art. For instance, proteins can be labelled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a molecular entity capable of selectively binding a binding member species of the present invention or a complex containing such a species, but is not itself a binding member of the present invention. Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the binding member species when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labelled. However, when the diagnostic system includes a specific binding agent that is not labelled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labelled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of a preselected ligand in a fluid sample. "ELISA" refers to an Enzyme-Linked Immunosorbent Assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample and is readily applicable to the present methods.

Thus, in some embodiments, a binding member of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption methods are described herein.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from

Abbott Laboratories of North Chicago, III.; polyvinyl chloride, polystyrene, cross- linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride. The binding member species, labelled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

Methods for production of secretagogues for use in combination with the binding members of the present invention

Secretagogues such as ghrelin-like compounds can be produced using techniques well known in the art. For example, a polypeptide region of a secretagogue can be chemically or biochemically synthesized and modified. Techniques for chemical synthesis of polypeptides are well known in the art. (See e. g., Vincent in Peptide and Protein Drug Delivery, New York, N. Y., Dekker, 1990.) Examples of techniques for biochemical synthesis involving the introduction of a nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2 d Edition, Cold Spring Harbor Laboratory Press, 1989.

Pharmaceutical compositions containing a compound of the present invention may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications.

According to the present invention ghrelin may be utilized as the well-known acylated 28 amino acid peptide and may be produced by chemical synthesis or recombinant techniques. Techniques for producing peptides and linking an octanoyl ester to the serine no. 3 are well within the technical person's skill. Alternatively any of the analogues mentioned in the documents referred to above may be utilized. Preferred compounds are the compounds designated as NN 703 [5-Amino-5- methylex-2-enoic acid N-methyl-N-((1 R)-1-(methyl-((1 R)-1-(methylcarbamoyl-2- phenylethylcarbomoyl)-2-(naphtalen-2-yl)ethyl)amide] and MK677 [sometimes also designated MK0677, cf. Drug Discovery Today, vol. 4, No.11 , November 1999, 497- 506] or NNC 26-1291 , or NNC 26-1187 are growth hormone secretagogues of a non-peptidyl described in WO 99/58501 and WO 00/26252, respectively, all of which documents are incorporated herein by way of reference.

Claims

80Claims

1. A pharmaceutical composition comprising an isolated binding member, said isolated binding member comprising at least one binding domain capable of specifically binding to a secretagogue compound with a dissociation constant K_d for said secretagogue compound that is less that 1x 10^"4 M.

2. The pharmaceutical composition of claim 1 , wherein said at least one binding domain is capable of specifically binding ghrelin.

3. The pharmaceutical composition of any of the preceding claims, wherein the isolated binding member is a pure isolated binding member.

4. The pharmaceutical composition of any of the preceding claims, wherein the binding member is selected from antibodies or other immunologically active molecules.

5. The pharmaceutical composition of claim 4, wherein the antibodies are selected from monoclonal antibodies, polyclonal antibodies or a mixture of monoclonal antibodies.

6. The pharmaceutical composition according to claim 5, wherein the active fragment of antibodies are selected from Fab, Fab', F(ab)₂ and Fv.

7. The pharmaceutical composition any of claims 1-4, wherein the binding member is an affibody or a mixture of affibodies.

8. The pharmaceutical composition of any of claims 1-4, wherein the binding member is an antibody affibody chimera.

9. The pharmaceutical composition according to any of claims 1-4, wherein the the immunologically active molecule is a mixture of antibody affibody chimeras

10. The pharmaceutical composition according to any of the preceding claims, comprising at least two different binding members. 81

11. The pharmaceutical composition according to any of the preceding claims, wherein said binding member binds to a epitope on said secretagogue, said epitope being residues 1-15 or 2-16 of SEQ ID NO: 1.

12. The pharmaceutical composition according to any of the preceding claims, wherein said binding member binds to a epitope on said secretagogue, said epitope being residues 16-25 of SEQ ID NO: 1.

13. The pharmaceutical composition according to any of the preceding claims, wherein said binding member binds to a epitope on said secretagogue, said epitope being residues 17-25 of SEQ ID NO: 1.

14. The pharmaceutical composition according to any of the preceding claims, wherein said binding member binds to a epitope on said secretagogue, said epitope being residues 16-23 of SEQ ID NO: 1.

15. The pharmaceutical composition according to any of the preceding claims, wherein said binding member binds to a epitope on said secretagogue, said epitope being residues 18-25 of SEQ ID NO: 1.

16. The pharmaceutical composition of any of the preceding claims, wherein the binding member is capable of prolonging the plasma half-life of a secretagogue compound within an individual.

17. The pharmaceutical composition of claim 16, wherein said prolonging of the plasma half-life is caused by binding of the binding member to the secretagogue compound.

18. The composition according to any of the previous claims, wherein the binding member comprises a plurality of binding sites.

19. The composition according to claim 18, wherein the binding member comprises 2 binding sites. 82

20. The pharmaceutical composition according to any of the previous claims, further comprising a secretagogue compound.

21. The pharmaceutical composition according to claim 20, wherein the secretagogue is ghrelin or a pharmaceutically acceptable salt thereof.

22. The pharmaceutical composition according to any of the preceding claims, wherein the secretagogue is a ghrelin-like compound or a pharmaceutically acceptable salt thereof wherein the ghrelin-like compound comprises a structure defined by formula I

Z¹ - (X¹)_m - (X²) - (X³)_n- Z², wherein Z¹ is an optionally present protecting group each X¹ is independently selected from an amino acid, wherein said amino acid is selected from naturally occurring and synthetic amino acids, X² is any amino acid selected from naturally occurring and synthetic occurring amino acids, said amino acid being modified with a bulky hydrophobic group, preferably an acyl group, or a fatty acid, each X³ is independently selected from an amino acid, wherein said amino acid is selected from naturally occurring and synthetic amino acids, wherein one or more of X¹ and X³ optionally may be modified by a bulky hydrophobic group, preferably an acyl group, or a fatty acid, Z² is an optionally present protecting group, m is an integer in the range of from 1-10 n is 0 or an integer in the range of from 1-35. 83

23. The pharmaceutical composition according to claim 22, wherein m is an integer in the range of from 1-9, such as of from 1-8, such as of from 1-7, such as of from 1-6, such as of from 1-5, such as of from 1-4, such as of from 1-3, such as of from 1-2, such as 2,

24. The pharmaceutical composition according to any of claims 22-23, wherein X² is selected from the group of modified Ser, modified Cys and modified Lys, such as wherein X² is modified Ser.

25. The pharmaceutical composition according to any of claims 22-24, wherein the ghrelin-like compound is selected from a compound of formula II Z¹ - Gly- (X¹)_m.ι - (X²) - (X³)_n- Z², formula III Z¹ - Gly- Ser- (X²) - (X³)_n- Z², and formula IV Z¹ - Gly - (X²) - (X³)_n- Z².

26. The pharmaceutical composition according to claim 25, wherein the ghrelin-like compound is having formula III.

27. The pharmaceutical composition according to any of claims 22-26, wherein (X³)_n comprises a sequence selected from one or more of the sequences shown below:

Phe Leu Ser Pro Glu His Gin Phe Leu Ser Pro Glu His Phe Leu Ser Pro Glu

Phe Leu Ser Pro Phe Leu Ser 84 Phe Leu Phe

28. The pharmaceutical composition according to any of claims 22-27, wherein n is an integer in the range of from 1-25, such as of from 1-24, such as from 1-15, such as of from 1-10, such as of from 10-25, such as of from 10-24, such as of from 15-25, such as of from 15-24.

29. The pharmaceutical composition according to any of claims 22-28, wherein the acyl group is a C1-C35 acyl group.

30. The pharmaceutical composition according to any of claims 22-29, further comprising a pharmaceutically acceptable carrier or a diluent.

31. The pharmaceutical composition according to any of the preceding claims, formulated for parenteral administration.

32. The pharmaceutical composition according to any of the preceding claims, formulated for nasal administration.

33. The pharmaceutical composition according to any of the preceding claims, formulated for subcutaneous administration.

34. The pharmaceutical composition according to any of the preceding claims, formulated for pulmonal administration.

35. A method of detecting a disorder in appetite regulation in an individual comprising providing a biological sample from said individual, - adding at least one binding member as defined in any of claims 1- 7 to said biological sample 85 - detection of binding members bound to said biological sample, thereby detecting or diagnosing the disease or disorder.

36. A kit for detecting a disorder in appetite regulation in an individual, said kit comprising at least one binding member as defined in any of claims 1- 19, said at least one binding member being labelled.

37. Use of an isolated binding member for the production of any of the pharmaceutical compositions described in claims 1-36, for the treatment of an individual in need thereof.

38. Use of claim 37, wherein said individual is suffering from a disorder of appetite regulation.

39. Use of any of claims 37-38, wherein said disorder of appetite regulation is caused by, or associated with one or more of cachexia, a neoplastic disorder such as cancer, or AIDS-related cachexia

40. Use of any of claims 37-39, wherein said individual is suffering from one a pathological disorder associated with ghrelin deficiency.

41. Use of any of claims 37-40, for the treatment of loss of body fat mass and/or loss of bone mass, in a gastrectomized individual.

42. Use of any of claims 37-41 , wherein the treatment comprises improving the sense of well-being and the quality of life in the individual.

43. Use according to any of claims 37-42, wherein the treatment stimulates appetite and prevents malnutrition of the individual.

44. Use according to any of claims 37-43, wherein the composition is administered in a dose of from about 0.1 mg/kg/day to about 10 mg/kg/day 86

45. Use according to any of claims 37-44, wherein the composition is administered in unit dosage form comprising from about 5 to about 250 mg of the binding member as defined in any of the claims 1-33.

46. Use according to any of claims 37-45, wherein said pharmaceutical composition is administered in combination with a stomach-derived factor.

47. Use according to claim 46, wherein said stomach-derived factor is ghrelin or a ghrelin-like compound.

48. A method of treatment of an individual in need thereof, comprising administering to said individual an effective amount of one or more of the pharmaceutical compositions defined in claims 1-34.

49. The method according to claim 48, wherein said individual is suffering from a disorder of appetite regulation.

50. The method according to claim 48, wherein said disorder of appetite regulation is caused by, or associated with one or more of cachexia, cancer cachexia or AIDS-related cachexia.

51. The method according to claim 48, wherein said individual is suffering from a pathological disorder associated with ghrelin deficiency.

52. The method according to claim 48, for the treatment of loss of body fat mass or loss of bone mass of a gastrectomized individual.

53. The method according to any of claims 48-52, wherein the treatment comprises improving the sense of well-being and the quality of life in the individual.

54. The method according to any of claims 48-53, wherein the treatment stimulates appetite and prevents malnutrition of the individual.

55. The method according to any of claims 48-54, wherein the composition is administered in a dose of from about 0.1 mg/kg/day to about 10 mg/kg/day 87

56. The method according to any of claims 48-55 where the composition is administered in unit dosage form comprising from about 5 to about 250 mg of the binding member as defined in any of the claims 1-33.

57. The method according to claim 56 wherein said pharmaceutical composition is administered in combination with a stomach-derived factor.

58. The method according to any of claims 48-57, wherein said stomach-derived factor is ghrelin or a ghrelin-like compound.

59. An isolated nucleic acid molecule encoding at least a part of the binding member as defined in any of the claims 1-19.

60. A vector comprising the nucleic acid molecule as defined in claim 59.

61. The vector according to claim 60, comprising a nucleotide sequence witch regulates the expression of the binding member encoded by the nucleic acid molecule.

62. A host cell comprising the nucleic acid as defined in any of claims 59-61.

63. A cell line engineered to express the binding member as defined in any of the claims 1-19.