[0001] Active immunisation against GIP
[0002]BACKGROUND OF THE INVENTION
[0003]This invention relates to a composition for active immunisation, the composition comprising an immunogen, the immunogen comprising human GIP (SEQ ID NO: 1), or an analogue thereof, coupled by a crosslinker to an antigenicity-conferring carrier protein material; and an immunologically acceptable vehicle; use of said immunogen for the manufacture of a medicament for the alleviation of obesity and / or insulin resistance; and a method of moderating blood glucose excursions and / or alleviating obesity and / or insulin resistance, the method comprising administering said composition to a host animal.
[0004]By the term "active immunisation" is meant the induction of GlP-specific neutralising antibodies. This is in contrast to "passive immunization" which is intended to mean the administration of GIP-specific neutralising antibodies. In the present context, passive immunization might involve: generating N-terminally reactive GIP antiserum in, for example, rabbits by repeated immunisation with an immunogen, for example, human GIP(I-11) coupled to ovalbumin using glutaraldehyde; and then administering the GIP antiserum to the target population, hi contrast, active immunisation involves administering the immunogen to the target population, in which a desired therapeutic effect is intended, thereby causing the target animal to induce the formation of GIP- specific neutralising antibodies within the target animal.
[0005]RELATED BACKGROUND ART
[0006]Obesity has long been identified as a major global health problem and a significant cause of preventable death (Mokdad et al. 2004). As such, obesity is a major risk factor for chronic diseases including, but not limited to, type 2 diabetes, cardiovascular disease, hypertension, dyslipidaemia and certain forms of cancer. Existing medical drug strategies to achieve and maintain weight loss have limitations and are associated with various unpleasant side-effects (Lean & Finer, 2006). Thus, new therapeutic approaches to prevent or even reverse the human obesity epidemic are currently sought.
By chronic administration is meant administration at intervals over a treatment period having a duration of greater than 90 days. By sun-chronic is meant administration at intervals over a treatment period having a duration of less than 90 days, for example in the range of 5 to 89 days.
[0007]Gastric inhibitory polypeptide (GIP) is a 42 gastrointestinal hormone secreted from enteroendocrine K-cells, located predominantly in the jejunum and duodenum, following ingestion of carbohydrate, protein and particularly fat (Brown 1994). Although for some time it was believed to be purely an incretin hormone potentiating pancreatic beta cell insulin release, several studies have indicated that GIP has other effects at extrapancreatic sites (Vella and Rizza, 2004). For example, functional GIP receptors are present on adipocytes (Yip et al. 1998) and there is a potent and protracted secretion of GIP following fat ingestion (Ross and Dupre, 1978). Furthermore, GIP has various anabolic effects in adipocytes, including stimulation of glucose uptake, lipoprotein lipase activity, fatty acid synthesis and fatty acid incorporation into adipose tissue (Eckel at el. 1979, Oben et al. 1991, Kim et al. 2007).
[0008]Early studies have already established a link between hyperphagia, high fat diets, K-cell hyperplasia, increased cellular GIP and elevated circulating GIP (Flatt PR et al. 1984; Flatt PR et al. 1983; Bailey CJ et al. 1986). Moreover, recent advances in the understanding of GIP physiology have shown GIP-R signalling as a key link between consumption of Westernised energy-rich high fat diets and obesity-diabetes (Miyawaki et al. 2002, McClean et al. 2007, Hansotia et al., 2007). Thus, both normal and obese- diabetic (ob/ob) mice with genetic knockout of the GIP receptor (GIP-R) are protected from diet-induced obesity (Miyawaki et al., 2002, Hansotia et al., 2007). Our own previous studies in ob/ob and diet-induced obese mice have shown that sub-chronic daily administration of the specific and stable GIP receptor antagonist, PrO3GIP, (Gault et al., 2002), can prevent or reverse many of the established metabolic abnormalities associated with obesity-diabetes (Gault et al., 2005; 2007, McClean et al., 2007, Irwin et al., 2007). Furthermore, targeted chemical knock-out of GIP producing K-cells reduces obesity and insulin resistance in high-fat fed mice (Althage et al. 2008). Hence, there is now accumulating evidence to suggest a possible role for disruption or ablation of GIP signalling in the alleviation of obesity and insulin resistance.
The induction of GIP neutralising antibodies is another particularly attractive possibility of disrupting GIP-R signalling. We have recently shown that active immunisation against GIP in ob/ob mice resulted in improved glucose tolerance and circulating glucose levels (Irwin N et al. January 2009), these observations were largely confirmed by another independent research team (Fulurija A et al. 2008). Importantly, these studies were not associated with adverse reactions, in keeping observations that genetic GIP-R knock-out and chronically treated Pro GIP mice do not exhibit notable pathologies (Miyawaki K et al. 2002; Gault VA et al. 2002; Gault VA et al. 2005; Gault et al. 2007; Hansotia Tet al. 2007; Irwin N et al. 2007). We have now extended this by actively immunising insulin resistant high fat fed mice against PrO3GIP. This high fat fed mouse model for dietary-induced obesity-diabetes more closely resembles the human obesity epidemic that is also fuelled by increase caloric intake, thus bringing this research a step closer to clinical realisation. The approach of PrO3GIP immunisation may have dual therapeutic utility. Firstly, antibodies generated against the C-terminus of PrO3GIP will neutralize circulating native GIP, since native GIP differs from PrO3GIP by only 1 amino acid in the N-terminus. Secondly, albumin-bound PrO3GIP may function as a long-acting GIP-receptor antagonist in its own right enhancing the overall blockade of GIP signalling.
[0009]WO06045796 provides a composition comprising a virus-like particle (VLP) and at least one antigen, wherein said antigen is a GIP protein or a GIP fragment linked to the VLP, respectively. WO06045796 also provides a method for producing the aforesaid composition. The compositions of WO06045796 are stated to be useful in the production of vaccines, in particular, for the prevention and/or treatment of obesity, in particular, by inducing efficient immune responses, in particular antibody responses.
[0010]Ebert and Creutzfeldt, 1982 disclose the use of a specific GIP antiserum to experimentally induce a GIP deficiency. However, Ebert and Creutzfeldt, 1982, neither disclose nor suggest how this GIP antiserum is prepared. In addition, Ebert and Creutzfeldt, 1982 neither disclose nor suggest active immunisation with an immunogen comprising human GIP (SEQ ID NO: 1) or an analogue thereof, coupled by way of a crosslinker to an antigenicity-conferring carrier protein material. Moreover, Ebert and Creutzfeldt, 1982 neither disclose nor suggest alleviating diabesity and / or insulin resistance and / or moderating blood glucose excursions.
It is an object of the present invention to at least partly inhibit GIP signalling by the induction of GIP-specific neutralising antibodies. This method is particularly attractive, given that the blockade of GIP might be long-lasting.
[0011]None of the prior art either discloses or suggests immunisation methods using immunogens comprising human GIP (SEQ ID NO: 1), or an analogue thereof, coupled by way of a crosslinker, to an antigenicity-conferring carrier protein material for the manufacture of a medicament for the alleviation of obesity and / or insulin resistance.
[0012]In the present study we have actively immunised against GIP to evaluate the role of endogenous circulating GIP in obesity-diabetes as manifested in ob/ob mice. Ob/ob mice have well characterised K-cell hyperplasia, increased cellular GIP and elevated circulating GIP concentrations (Flatt et al. 1984, Bailey et al. 1986), and thus represent an excellent challenge for evaluation of the sub-chronic effects of active GIP immunisation, hi the present study we have actively immunised and against PrO3GIP to evaluate the role of active PrO3GIP immunisation in high-fat fed mice.
[0013]SUMMARY OF THE INVENTION
[0014]According to a first aspect of the invention, there is provided a composition for active immunisation, the composition comprising an immunogen, the immunogen comprising human GIP (SEQ ID NO: 1), or an analogue thereof, coupled by a crosslinker to an antigenicity-conferring carrier protein material; and an immunologically acceptable vehicle. Optionally, the immunogen comprises SEQ ID NO: 1 or SEQ ID NO: 2 coupled by the crosslinker to the antigenicity-conferring carrier protein material.
[0015]Optionally, the analogue of human GIP comprises at least 12 amino acid residues from the N-terminal end of SEQ ID NO: 1. Further optionally, the analogue of human GIP comprises at least 12 amino acid residues from the N-terminal end of SEQ ID NO: 1 and comprises one or more amino acid substitutions or modifications selected from the group consisting of: an amino acid substitution or modification at position 1, an amino acid substitution or modification at position 2, and an amino acid substitution or modification at position 3. Alternatively, the analogue of human GIP comprises SEQ
ID NO: 1 and comprises one or more amino acid substitutions or modifications selected from the group consisting of: an amino acid substitution or modification at position 1, an amino acid substitution or modification at position 2, and an amino acid substitution or modification at position 3.
[0016]Further optionally, the analogue of human GIP comprises either at least 12 amino acid residues from the N-terminal end of SEQ ID NO: 1 or SEQ ID NO: 1 and further comprises:
[0017](a) N-terminal glycation and an amino acid substitution or modification at one or more of positions 1, 2, and 3
[0018](b) amino acid substitution or modification at each of positions 1, 2, and 3;
[0019](c) amino acid substitution or modification at two of positions 1, 2, and 3, wherein each amino acid substitution or modification is selected from the group consisting of: (i) N-terminal glycation;
[0020](ii) N-terminal alkylation;
[0021](iii) N-terminal acetylation;
[0022](iv) N-terminal acylation;
[0023](v) the addition of an N-terminal isopropyl group; (vi) the addition of an N-terminal pyroglutamic acid;
[0024](vii) substitution at position 1 by a D-amino acid;
[0025](viii) substitution at position 1 by an L-amino acid;
[0026](ix) substitution at position 2 by a D-amino acid;
[0027](x) substitution at position 2 by an L-amino acid; (xi) substitution at position 2 by amino isobutyric acid or sarcosine;
[0028](xii) substitution at position 3 by a D-amino acid;
[0029](xiii) substitution at position 3 by an L-amino acid;
[0030](xiv) substitution at position 3 by amino isobutyric acid or sarcosine;
[0031](xv) conversion of the Ala2-Glu3 bond to a ψ[CH2NH] bond; (xvi) conversion of the Ala2-Glu3 bond to a stable isostere bond; and
[0032](xvii) substitution by beta-alanine, an omega-amino acid, 3 -amino propionic acid, 4-amino butyric acid, ornithine, citrulline, homoarginine, t-butylalanine, t-butylglycine, N-methylisoleucine,
phenylglycine, cyclohexylalanine, norleucine, cysteic acid and methionine sulfoxide; and (d) amino acid substitution or modification at one of positions 1, 2, and 3, wherein the amino acid substitution or modification is selected from the group consisting of:
[0033](i) N-terminal glycation; (ii) N-terminal alkylation; (iii) N-terminal acetylation; (iv) N-terminal acylation; (v) the addition of an N-terminal isopropyl group;
[0034](vi) the addition of an N-terminal pyroglutamic acid; (vii) substitution at position 1 by a D-amino acid; (viii) substitution at position 1 by an L-amino acid; (ix) substitution at position 2 by an D-amino acid; (x) substitution at position 2 by an L-amino acid
[0035](xi) substitution at position 2 by amino isobutyric acid or sarcosine; (xii) substitution at position 3 by a D-amino acid; (xiii) substitution at position 3 by an L-amino acid; (xiv) substitution at position 3 by amino isobutyric acid or sarcosine; (xv) conversion of the Ala2-Glu3 bond to a ψ[CH2NH] bond;
[0036](xvi) conversion of the Ala2-Glu3 bond to a stable isostere bond; and (xvii) substitution by beta-alanine, an omega-amino acid, 3 -amino propionic acid, 4-amino butyric acid, ornithine, citrulline, homoarginine, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, cysteic acid and methionine sulfoxide.
[0037]Preferably, the carrier protein material is selected from a protein, a protein fragment, a synthetic polypeptide or a semi-synthetic polypeptide. Suitable protein carrier materials will elicit an immunogenic response when administered to a host animal as part of the immunogen of the present invention. Appropriate carrier protein materials commonly contain poly(amino acid) segments and include polypeptides, proteins and glycoproteins. Illustrative examples of useful carrier protein materials are bovine serum albumin (BSA), ovalbumin, such as egg ovalbumin, bovine gamma globulin, bovine
thyroglobulin (BTG), keyhole limpet haemocyanin (KLH) etc. Alternatively, synthetic poly(amino acids) having a sufficient number of available amino groups, such as lysine, may be employed, as may other synthetic or natural polymeric materials bearing reactive functional groups. Ovalbumin is a suitable carrier protein material.
[0038]Preferably, the human GIP (SEQ ID NO: 1), or the analogue thereof, is modified by crosslinker addition at an epsilon amino group of at least one lysine residue of the human GIP, or the analogue thereof. More preferably, the lysine residue is chosen from the group consisting of Lyslό, Lys30, Lys32, Lys33, and Lys37.
[0039]Alternatively, the human GIP (SEQ ID NO: 1), or the analogue thereof, is modified by a lysine substitution and by crosslinker addition at an epsilon amino group of the substituted lysine.
[0040]Preferably, the crosslinker is derived from an aldehyde, optionally, a di-aldehyde, including, but not limited to, glutaraldehyde.
[0041]According to a second aspect of the invention, there is provided use of an immunogen comprising human GIP (SEQ ID NO: 1), or an analogue thereof, coupled by a crosslinker to an antigenicity-conferring carrier protein material for the manufacture of a medicament for the alleviation of diabesity and / or insulin resistance. By diabesity, we mean that, since type 2 diabetes is obesity dependent, and obesity is the main aetiogical cause of type 2 diabetes, we use the term 'diabesity' or "obesity-diabetes" to refer to diabetes as a result of obesity and high caloric intake.
[0042]According to a third aspect of the invention, there is provided use of an immunogen comprising human GIP (SEQ ID NO: 1), or an analogue thereof, coupled by a crosslinker to an antigenicity-conferring carrier protein material for the manufacture of a medicament for the moderation of blood glucose excursions.
[0043]According to a fourth aspect of the invention, there is provided a method of alleviating diabesity and / or insulin resistance, the method comprising administering a composition of the first aspect of the invention to a host animal (a member of the target population).
According to a fifth aspect of the invention, there is provided a method of moderating blood glucose excursions, the method comprising administering a composition of the first aspect of the invention to a host animal (a member of the target population).
[0044]According to a sixth aspect of the invention, there is provided a method of treating diabesity, the method comprising administering a composition of the first aspect of the invention to a host animal (a member of the target population). By "diabesity" is meant diabetes caused by excessive adiposity; the condition of having both diabetes and obesity.
[0045]In the methods and uses of the invention, the immunogens useful in the first aspect of the invention are administered to host animals (a member of the target population), such as humans, to elicit production of specific antibodies to human GIP within the host animal.
[0046]In the methods and uses of the invention, the composition of the first aspect of the invention is administered at intervals, optionally at regular intervals. For example, the regular interval is fortnightly. Alternatively, the regular interval is every 3 or 4 weeks. In the methods and uses of the invention, the composition of the first aspect of the invention is sub-chronically administered at intervals, optionally at regular intervals, over a treatment period having a duration of less than 90 days, for example in the range of 5 to 89 days. In the methods and uses of the invention, the composition of the first aspect of the invention is chronically administered at intervals, optionally at regular intervals, over a treatment period having a duration of greater than 90 days.
[0047]BRIEF DESCRIPTION OF THE FIGURES
[0048]Figure 1. Sub-chronic (administration for up to 90 days) effects of active immunisation with human GIP(I -42) on body weight, food intake and non- fasting glucose and insulin in ob/ob mice. A 1 :1 ratio of incomplete Freund's adjuvant and either the immunogen or ovalbumin (control) were administered subcutaneously once every 14 days for 56 days as indicated by the horizontal black bar. Parameters were measured for 5 days
prior to and for the 56 days of the treatment period. Values are mean ± S.E.M. for 8-10 mice. *P<0.05 compared to control.
[0049]Figure 2. Sub-chronic effects of active immunisation with human GIP(I -42) on glucose tolerance in ob/ob mice. Tests were conducted after 56 days active immunisation. Glucose (18 mmol/kg body weight, ip) was administered at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 min with identical baseline subtraction are also shown. There were no clear differences in glucose-stimulated insulin release in the GIP immunised mice. Values are mean ± S.E.M. for 8-10 mice. *P<0.05 compared to control.
[0050]Figure 3. Sub-chronic effects of active immunisation with human GIP(l-42) on insulin sensitivity in ob/ob mice. Tests were conducted after 56 days active immunisation. Insulin (50 U/kg body weight) was administered at the time indicated by the arrow. Panel A depicts whole plasma glucose values whilst panel B shows percentage plasma glucose change from basal. Values are mean ± S.E.M. for 8-10 mice. *P<0.05 compared to control.
[0051]Figure 4. Chronic (administration for more than 90 days) effects of active PrO3GIP immunisation on (A) body weight and (B) energy intake in high-fat fed mice and in lean controls. Complexed PrO3GIP peptide or ovalbumin alone (control) were administered subcutaneously once every 14 days for 112 days (day 0 is the day that complexed PrO3GIP peptide or ovalbumin alone (control) were first administered). Immunised and non-immunised control high fat mice were then placed on a high fat diet on day 35, as indicated by the black horizontal bar. Lean controls were retained on the standard rodent maintenance diet. Values are mean ± S.E.M. for 8-10 mice. **P<0.01 and ***P<0.001 compared to lean control.
[0052]Figure 5. Chronic effects of active PrO3GIP immunisation on non-fasting glucose in high-fat fed mice. Complexed PrO3GIP peptide or ovalbumin alone (control) were administered subcutaneously once every 14 days for 112 days (day 0 is the day that complexed PrO3GIP peptide or ovalbumin alone (control) were first administered). Immunised and non-immunised control high fat mice were then placed on a high fat diet on day 35, as indicated by the black horizontal bar. Lean controls were retained on
the standard rodent maintenance diet. Values are mean ± S.E.M. for 8-10 mice. *P<0.05, **P<0.01 and ***P<0.001 compared to lean control. ΔΔPO.01 and ΔΔΔP<0.001 compared to non-immunised high-fat control.
[0053]Figure 6. Chronic effects of active immunisation against PrO3GIP on glucose tolerance in high-fat fed mice and in lean controls. Tests were conducted after fortnightly administration subcutaneously once every 14 days for 112 days of active PrO3GIP immunisation. Lean controls were retained on the standard rodent maintenance diet. Immunised and non-immunised control high fat mice were placed on a high fat diet on day 35. Glucose (18 mmol/kg body weight, i.p.) was administered at the time indicated by the arrow (A). Plasma glucose AUC values for 0-60 min are also shown (B). Values are mean ± S.E.M. for 8-10 mice. *P<0.05, **P<0.01 and ***P<0.001 compared to lean control. ΔΔP<0.01 compared to non-immunised high- fat control.
[0054]Figure 7. Chronic effects of active immunisation against PrO3GIP on glycaemic response to GIP in high-fat fed mice and in lean controls. Tests were conducted after fortnightly administration subcutaneously once every 14 days for 112 days of active PrO3GIP immunisation. Lean controls were retained on the standard rodent maintenance diet. Immunised and non-immunised control high fat mice were placed on a high fat diet on day 35. Glucose (18 mmol/kg body weight, i.p.) in combination with GIP (25 nmol/kg body weight) was administered at the time indicated by the arrow (A). Plasma glucose AUC values for 0-60 min are also shown (B). Values are mean ± S.E.M. for 8-10 mice. *P<0.05 and **P<0.01 compared to lean control. ΔPO.05 and ΔΔP<0.01 compared to non- immunised high-fat control.
[0055]Figure 8. Chronic effects of active immunisation against PrO3GIP on insulin sensitivity in high-fat fed mice and in lean controls. Tests were conducted after fortnightly administration subcutaneously once every 14 days for 112 days of active PrO3GIP immunisation. Lean controls were retained on the standard rodent maintenance diet. Immunised and non- immunised control high fat mice were placed on a high fat diet on day 35. Insulin (20 U/kg body weight, i.p.) was administered at the time indicated by the arrow (A). Plasma glucose AUC values for 0-60 min are also shown (B). Values are mean ± S.E.M. for 8-10 mice. *P<0.05 compared to control. *P<0.05 and **P<0.01 compared to lean control. ΔP<0.05 compared to non-immunised high-fat control.
DETAILED DESCRIPTION OF THE INVENTION
[0056]Although human GIP provides defined structural epitopes, human GIP is not, in itself, immunogenic and needs to be conjugated to carrier protein materials, in order to elicit an immunogenic response when administered to a host animal. Appropriate carrier protein materials commonly contain poly (amino acid) segments and include polypeptides, proteins and glycoproteins. Illustrative examples of useful carrier protein materials are ovalbumin, bovine serum albumin (BSA), egg ovalbumin, bovine gamma globulin, bovine thyroglobulin (BTG), keyhole limpet haemocyanin (KLH) etc. Alternatively, synthetic poly(amino acids) having a sufficient number of available amino groups, such as lysine, may be employed.
[0057]MATERIALS AND METHODS
[0058]Preparation of Immunogen used in Example 1
[0059]Human GIP(I -42) obtained from Sigma Genosys was confirmed as being pure by HPLC. Molecular weight of GIP is 4983 Da.
[0060]Carrier protein material, for example, ovalbumin (18 mg) was added to human GIP (4 mg in 1 mL PBS) at the correct molar ratio (about 1 mole of GIP per 50 amino acids of carrier) and stirred for 2 hours at room temperature. An equal volume of freshly prepared glutaraldehyde was added dropwise to the GIP/carrier protein solution and incubated with constant agitation for 1 hour at room temperature. The peptide solution was incubated for 1 hour following addition of 1 mLl M glycine in PBS (at a final concentration of 200 mmol/L) to quench unreacted aldehyde. The immunogen was separated from free peptide by dialysis with 4 changes of PBS overnight at 40C.
[0061]A 4mg batch of human GIP-ovalbumin was dialysed overnight against PBS at 40C. The dialysate was aliquoted and stored at -200C prior to injection. Each mouse received 80 μg immunogen (80 μg human GIP-ovalbumin) per injection.
[0062]Preparation of Immunogen (Complexed PrO3GIP peptide) used in Example 2
Pro3 GIP(I -42) was obtained from Sigma Genosys and was confirmed as being pure by HPLC. Pro3 GIP(I -42) comprises human GIP (1-42) in which GIu (Glutamic acid) at position 3 has been replaced with Pro (Proline). Molecular weight of PrO3GIP 4951.
[0063]Pro3 GIP(I -42) was coupled to ovalbumin as outlined above in relation to human
[0064]GIP(I -42) using the cross-linking glutaraldehyde method. A 4 mg batch of Pro3 GIP(I- 42) -ovalbumin (complexed PrO3GIP peptide) was dialysed overnight against PBS at 40C. The dialysate was aliquoted and stored at -20°C prior to injection. Each mouse received 80 μg immunogen (80 μg Pro3GIP-ovalbumin or "complexed PrO3GIP peptide") per inj ection.
[0065]Animals used in Example 1
[0066]Equal numbers of male and female obese diabetic (ob/ob) mice originally derived from the colony maintained at Aston University, UK (Bailey et al., 1982) were used at 7-9 weeks of age for active immunisation studies. 7-9 weeks old mice were chosen because, with the ob/ob model, the onset of diabetes occurs at 4-5 weeks. Day 0 is the first day GIP-ovalbumin was administered subcutaneously (the mice were 7-9 weeks old on day 0). Animals were age-matched, divided into groups and housed individually in an air-conditioned room at 22 ± 2°C with a 12 h light: 12 h dark cycle (08:00 - 20:00 h). Drinking water and a standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK) were freely available before commencement of experiment. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.
[0067]Animals used in Example 2
[0068]Male National Institutes of Health (NIH) Swiss mice obtained from Harlan Limited were used at 12-15 weeks of age for active PrO3GIP immunisation studies. Day 0 is the first day GIP-ovalbumin was administered subcutaneously (the mice were 12-15 weeks old on day 0). Animals were divided into groups and housed individually in an air- conditioned room at 22 ± 2°C with a 12 h light: 12 h dark cycle (08:00 - 20:00 h).
[0069]Drinking water and a standard rodent maintenance diet (10% fat, 30% protein and 60% carbohydrate, Trouw Nutrition, Cheshire, UK) were freely available before commencement of experiment. At day 35, the high-fat mice were then placed on a high fat diet (45% fat, 20% protein and 35% carbohydrate; percent of total energy of
26.15kj/g; Special Diets Service, Essex, UK), the aim being to induce obesity and glucose intolerance, whilst the lean controls were retained on the standard rodent maintenance diet. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.
[0070]Experimental protocol for active immunisation - Example 1
[0071]Groups of ob/ob mice (n=8-10) were then injected subcutaneously with a 1/1 mixture of incomplete Freund's adjuvant and 80 μg immunogen comprising human full length GIP coupled by a crosslinker (glutaraldehyde) to ovalbumin (an antigenicity-conferring carrier protein material). Four further subcutaneous booster injections were performed with the same quantity of immunogen, mixed 1/1 with incomplete Freund's adjuvant, after 2, 4, 6 and 8 weeks. Control mice received an equivalent amount of ovalbumin in Freund's adjuvant. Food intake, body weight, non-fasting plasma glucose and insulin concentrations were monitored at 4-6 day intervals. Intraperitoneal glucose tolerance (18 mmol/kg body weight) and insulin sensitivity (50 U/kg body weight) tests were performed at 56 days in non- fasted animals. All acute experiments commenced at 10:00 h.
[0072]Experimental protocols for active immunisation — Example 2 Groups of mice (n=10) were injected subcutaneously with a 1/1 mixture of incomplete Freund's adjuvant and 80 μg complexed PrO3GIP peptide comprising human full length Pro GIP coupled by a crosslinker (glutaraldehyde) to ovalbumin (an antigenicity- conferring carrier protein material). Eight further subcutaneous booster injections were performed with the same quantity of antigen, mixed 1/1 with incomplete Freund's adjuvant after 2, 4, 6, 8, 10, 12, 14 and 16 weeks. Control non-immunised mice received an equivalent amount of ovalbumin in Freund's adjuvant. Food intake, body weight, non-fasting plasma glucose and insulin concentrations were monitored at 4-6 day intervals. At week 5 (day 35), PrO3GIP immunised and control non- immunised mice were transferred to a high fat diet (45% fat, 35% carbohydrate and 20% protein). A third group of lean control mice were maintained on standard rodent maintenance diet throughout the 16 weeks. Intraperitoneal glucose tolerance (18 mmol/kg body weight) and insulin sensitivity (20 U/kg body weight) tests were performed at the end of the study period in non- fasted animals. Furthermore, the glycaemic response to glucose (18 mmol/kg) in combination with native GIP (25 nmol/kg body weight) was examined at
the end of the study period in non-fasted animals. All acute experiments commenced at 10:00 h.
[0073]Biochemical analysis Blood samples taken from the cut tip of the tail vein of conscious mice at the times indicated in the Figures were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 s at 13,000 g. The resulting plasma was then aliquoted into fresh Eppendorf tubes and stored at -200C prior to analysis. Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, 1971) using a Beckman Glucose Analyzer II (Beckman Instruments, Gal way, Ireland). In the case of insulin sensitivity testing, blood glucose was measured for convenience using the Ascensia Contour® Blood Glucose Meter (Bayer AG, Leverkusen, Germany).
[0074]Statistics Results are expressed as mean ± S.E.M.. Data were compared using ANOVA, followed by a Student-Newman-Keuls post hoc test. Areas under the curve (AUC) analyses were calculated using the trapezoidal rule with baseline subtraction (Burington, 1973). P < 0.05 was considered to be statistically significant.
[0076]Active immunisation using an immunogen comprising human GIP(I -42)- glutaraldehyde-ovalbumin resulted in progressively lowered circulating plasma glucose and insulin levels that were significantly (P<0.05) decreased compared to control levels on day 56 and 30, respectively (Figure 1). These changes in glycaemic status were not accompanied by changes in body weight or food intake (Figure 1) over the 56-day period of observation.
[0077]Active immunisation with an immunogen comprising human GIP(I -42)-glutaraldehyde- ovalbumin for 56 days had a tendency to reduce plasma glucose levels at all time points during glucose tolerance testing (Figure 2). Furthermore, the overall glycaemic excursion (plasma glucose AUC) was significantly decreased (P<0.05) by active immunisation with an immunogen comprising human GIP(I -42)-glutaraldehyde- ovalbumin, compared to controls. These observations of glucose homeostasis were independent of any changes in glucose-stimulated insulin release (Figure 2).
As shown in Figure 3, the hypoglycaemic action of insulin was similar in both groups. Thus, despite significantly lower basal glucose concentrations (Figure 3A), the relative reduction of glucose following exogenous insulin administration was similar in mice actively immunised with an immunogen comprising human GIP(I -42)-glutaraldehyde- ovalbumin, compared to controls (Figure 3B).
[0078]Recent research suggests that ablation of GIP receptor signalling can reverse or prevent many of the metabolic abnormalities associated with dietary and genetically induced obesity-diabetes .
[0079]The present study was designed to assess the sub-chronic effects of active immunisation against GIP using an immunogen comprising human GIP(I -42)-glutaraldehyde- ovalbumin. Active immunisation using this immunogen for 56 days in young ob/ob mice resulted in significantly (p<0.05) reduced circulating plasma glucose and insulin concentrations on day 56 and 30, respectively, compared to control mice. The glycaemic response to intraperitoneal glucose was correspondingly improved (p<0.05) in mice immunised with an immunogen comprising human GIP(I -42)-glutaraldehyde- ovalbumin. These changes were independent of any effects on food intake, body weight or glucose-stimulated insulin secretion. Furthermore, insulin sensitivity was similar in mice immunised with an immunogen comprising human GIP(I -42)-glutaraldehyde- ovalbumin and respective controls.
[0080]Overall, the results reveal beneficial effect of blockade of GIP receptor action in obesity-diabetes with active immunisation using an immunogen of the present invention.
[0082]Active immunisation against GIP, with a composition of the first aspect of the present invention, was associated with improved metabolic control in ob/ob mice. This is consistent with the capture of GIP by antibodies and reducing of markedly elevated biologically active concentrations of circulating GIP. Since GIP promotes marked hyperinsulinaemia and insulin resistance (Bailey et al. 1982), this modulates basal hyperglycaemia. Thus, despite the prevalence of gross obesity, attenuation of GIP
signalling by active immunisation modestly but significantly decreased the hyperglycaemia and hyperinsulinaemia in ob/ob mice. Furthermore, the biological effects of active immunisation using this immunogen were not associated with changes of energy intake, in agreement with previous studies (Miyawaki et al. 2002; Gault et al. 2005, Irwin et al. 2007). This confirms a clear dissociation of the antidiabetic actions of sustained GIP receptor ablation from other possible benefits that might occur from reduced caloric intake and/or alleviation of obesity.
[0083]Since GIP is a major incretin, a safety concern regarding therapy based on the current rationale is disturbance of glucose homeostasis. However, assessment of glucose tolerance at the end of the study period actually revealed a clear-cut beneficial effect of prolonged active immunisation using a composition of the first aspect of the present invention on glucose concentrations and overall glycaemic excursion in ob/ob mice. Furthermore, glucose-stimulated insulin levels were unaltered compared to controls. There was no apparent enhancement of insulin sensitivity in mice immunised with a composition of the first aspect of the present invention. However, starting glucose concentrations were substantially lower and counter-regulatory mechanisms may come into play to prevent lowering of glucose concentrations beyond 6 mmol/1. Despite this, there is clear evidence that active immunisation with a composition of the first aspect of the present invention had obvious beneficial effects on glucose homeostasis in ob/ob mice. Thus, the only concern associated with the current therapeutic regime would be the induction of adverse reactions. Whilst we were unable to assess this fully, all mice displayed normal behavioural patterns with no effect on food intake. Furthermore, induction of long-term genetic or chemically-induced GIP deficiency is without detrimental effect (Irwin et al 2007; Hansotia et al 2007).
[0084]In conclusion, this study has shown that active immunisation with a composition of the first aspect of the present invention was associated with an improvement of glucose tolerance and circulating glucose levels as previously noted in ob/ob mice with either genetic or chemical ablation of GIP receptor signalling (Miyawaki et al. 2002, Gault et al. 2005, Irwin et al. 2007). Further detailed studies are needed to extend these observations and fully elucidate the possible use of immunisation with a composition of the first aspect of the present invention as a potential therapy for obesity-diabetes.
EXAMPLE 2
[0085]Chronic effects of active PrO3GIP immunisation on body weight and energy intake in high-fat fed mice. Active immunisation against PrO3GIP did not result in significant changes in body weight compared to non-immunised controls (Figure 4A). Furthermore, shortly after administration of high-fat diet (day 35), both high-fat fed groups had significantly increased body weights compared to lean controls throughout the reminder of the study period (Figure 4A). In addition, whilst there was a tendency for increased energy intake in high-fat fed mice, there was no significant change in energy intake between the three groups of mice (Figure 4B).
[0086]Chronic effects of active PrO3GIP immunisation on non-fasting glucose levels in high- fat fed mice. Active immunisation against PrO3GIP resulted in a marked decrease in non-fasting glucose levels compared to non-immunised high-fat control mice, with significant reductions (PO.05-PO.01) on days 98 and 116 (Figure 5). Furthermore, on day 116 non-fasting glucose concentrations in Pro GIP immunised mice were not significantly different from lean control mice (Figure 5).
[0087]Chronic effects of active PrO3GIP immuni sation on glucose tolerance in high-fat fed mice. Active immunisation against PrO3GIP resulted in a significant (PO.01) decrease in glucose levels at 15, 30 and 60 minutes post glucose injection when compared to non-immunised high-fat control mice (Figure 6A). However, glucose levels in PrO3GIP immunised mice were still significantly elevated at 30 and 60 minutes post injection compared to lean controls (Figure 6A). This was corroborated in the 0-60 min AUC values with PrO3GIP immunised mice having a significantly (PO.01) reduced overall glycaemic excursion compared to non-immunised high-fat control mice, but significantly (PO.05) elevated compared to lean controls (Figure 6B).
[0088]Chronic effects of active PrO3GIP immunisation on glycaemic response to native GIP in high-fat fed mice. Administration of GIP significantly (PO.05 - PO.01) reduced glucose levels in lean control and non-immunised high-fat control mice at 15, 30 and 60 min post- injection compared to PrO3GIP immunised mice (Figure 7A). This was supported by the 0-60 min AUC values with PrO3GIP immunised mice having a significantly (PO.05) elevated overall glycaemic excursion compared to non-
immunised high-fat control mice and lean controls (Figure 7B). This demonstrates the presence of specific GIP neutralising antibodies.
[0089]Chronic effects of active PrO3GIP immunisation on insulin sensitivity in high-fat fed mice. Active immunisation against Pro3GIP resulted in a significantly reduced (P<0.05) glucose levels 30 minutes post insulin injection when compared to non- immunised high-fat control mice (Figure 8A). However, the 0-60 min AUC values revealed PrO3GIP immunised mice to have a significantly (PO.05) elevated overall glycaemic excursion compared to lean controls, with no significant difference compared to non-immunised high-fat control mice (Figure 8B).
[0091]The present study has illustrated the potential benefit of active immunisation against
[0092]PrO3GIP for the treatment of obesity-diabetes or "diabesity". Whilst, high-fat fed PrO3GIP immunised mice did not display any reductions in body weight, there was a significant restoration of glycaemic control. Indeed, by the end of the study PrO3GIP immunised mice had similar non-fasting glucose levels to normal lean control mice. This is consistent with view that GIP promotes marked hyperinsulinaemia and insulin resistance and subsequent elevated glucose levels, as observed with genetic or chemical ablation of GIP receptor function in mice with diet-induced obesity or double mutation of leptin encoding ob gene (Miyawaki K et al. 2002; Gault VA et al. 2005). Thus, the present observation is consistent with the ongoing capture of GIP by circulating antibodies, thereby reducing markedly elevated glucose levels. As expected, administration of native GIP in combination with intraperitoneal glucose had no effect on glucose homeostasis in PrO3GIP immunised mice. This observation evidences the biological effectiveness of the GIP-specific neutralising antibodies generated.
[0093]The biological effects of active PrO3GIP immunisation was not associated with any changes of energy intake indicating a clear dissociation of the antidiabetic actions from possible benefits that might occur from reduced caloric intake and/or alleviation of obesity. Since GIP is a major incretin, an issue regarding therapy based on the current rationale is a possible deficit in insulin secretion due to neutralisation of the incretin effect. However, assessment of glucose tolerance at the end of the study period actively immunised mice clearly revealed beneficial effects of prolonged active PrO3GIP
immunisation on glucose concentrations and overall glycaemic excursion when compared to high-fat controls. There were still mild impairments of glucose tolerance in PrO3GIP immunised mice when compared to control mice. This could reflect length of time on high-fat diet, age of mice, duration of treatment regime or the relatively small number of animals tested.
[0094]In the present study, there was no apparent enhancement of insulin sensitivity in PrO3GIP immunised mice. However, starting glucose concentrations were substantially lower and counter-regulatory mechanisms may come into play to prevent lowering of glucose concentrations beyond 6 mmol/1. Despite this, there were still a mild impairment of insulin sensitivity in PrO3GIP immunised mice when compared to lean control mice, that may be attributable to factors outlined above. Furthermore, these observations are essentially comparable to a previous study highlighting the beneficial effects of active immunisation against GIP in an animal model of genetically-induced obesity-diabetes (Irwin N et al. 2009).
[0095]In conclusion, this study has shown that GIP is an important hormone affecting glucose homeostasis in high-fat fed mice. Furthermore, active immunisation against PrO3GIP was associated with an improvement of glucose tolerance and circulating glucose levels as previously noted in ob/ob mice with either genetic or chemical ablation of GIP receptor signalling (Miyawaki K et al. 2002; Gault VA et al. 2005). Further detailed studies are needed to fully elucidate the possible use of immunisation against PrO3GIP as a potential therapy for obesity-diabetes.
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