WO2003102240A1

WO2003102240A1 - A method for identification of obese il-6 responsive patients

Info

Publication number: WO2003102240A1
Application number: PCT/SE2003/000903
Authority: WO
Inventors: John-Olov Jansson; Ingrid Wernstedt
Original assignee: Sahltech I Göteborg AB
Priority date: 2002-06-03
Filing date: 2003-06-03
Publication date: 2003-12-11
Also published as: SE0201689D0; AU2003238990A1

Abstract

This invention relates to a method for identification of an obese patient that is responsive to treatment with a substance that upon administration so said obese patient will lead to an increased level of an IL-6 receptor agonist for the treatment of obesity, wherein a sample from a person is analysed with a method that can detect a CC of GC genotype in position -174 from initiation of transcription of the IL-6 gene, and/or a AA or GA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or a genotype that is associated with said CC or GC genotype and/or said AA or GA genotype, and the presence of at least one of said genotypes indicates that said person is identified as said obese patient. Furthermore, the invention relates to a kit for identification of such an obese patient, as well as to pharmaceutical compositions and methods for treatment of such obese patients.

Description

A METHOD FOR IDENTIFICATION OF OBESE IL-6 RESPONSIVE

PATIENTS

Background of the invention

Obesity is a major risk factor for cardiovascular disease, which is the most common cause of death in the western societies. Evidence for genetic control of human body weight and composition is well established from adoption, twin and family studies¹. One of the best examples is the small intra-pair differences between the adaptation to overfeeding by monozygotic twins². The mapping of the human genome has revealed that in many genes, there are small variations of separate nucleotides, so- called single nucleotide polymorphisms (SNPs) . It is known that such hereditary variations in the genome may cause differences in phenotypes, including susceptibility to disease. The cytokine interleukin-6 (IL-6) is known to be important for the immune system, but is also a candidate mediator for the regulation of body weight and composition. The results of a recent study show that IL-6 knockout mice develop mature onset obesity indicating that IL- 6 plays an important role in the energy balance³ (see also International Patent Application WO 01/03725) . Moreover, IL-6 treatment decreased body fat mass in normal rats⁴.

Several SNPs are described in the region of the IL-6 gene, including the -174 G/C and the -597 G/A SNP, which are located within the promoter. These SNPs are strongly associated with each other¹⁴. Thus, a subject with -174 GG genotype often also has a -597 GG genotype.

Both -174 alleles are abundant in European popula- tions, but the -174 C may be slightly less common with a frequency of about 0.4^5,e. The physiological role of this polymorphism in humans is unclear. The G allele has been associated both with higher serum IL-6 levels^5,7"10, and with lower serum IL-6 levels¹¹'¹². Although the -174 IL-6 G/C SNP has been associated with susceptibility to e.g. myocardial infarction, longeviety and diabetes, the results have been contradictory. Some authors found that the C allele is associated with higher cardiovascular and general morbidity and mortality⁶'¹², while others have found the opposite^7"9. Some studies have indicated an association between the -174 G allele and insulin resis- tance⁷, while others have not found this¹⁰. In addition, it is unclear whether serum IL-6 levels correlate positively with insulin resistance as suggested by Bastard et al¹³, or if there is not a correlation, as reported in a large study on 228 healthy subjects by Fernandez-Real⁷ and by Villuendas¹⁰. These contradictions may raise doubts that there are any relevant associations between the -174 G/C polymorphism and metabolic parameters or regulation of body composition. In agreement with this assumption, Villuendas et al found no association between the -174 G/C SNP or the -597 G/A SNP and BMI in a very recent study¹⁰.

Obesity is a disease that probably has several different causes¹⁷. Therefore, it is likely that there is a substantial number of patients that are obese due to other causes than inappropriate IL-6 production. These patients will be obese despite the fact that they have sufficient endogenous IL-6-production. These patients are also unlikely to respond to IL-6 treatment. Of course it would be very advantageous to be able to distinguish in- dividuals being particularly susceptible for IL-6 treatment, but so far, there has been no way of determining a certain patient group being particularly suitable for IL- 6 treatment . Summary of the invention

The aim of the present invention is to provide a method for identifying individuals being responsive to the anti-obesity effect of IL-6 by determining the -174 and/or -597 IL-6 gene genotypes in obese subjects, and identify the persons with -174 CC/GC or -597 AA/GA genotypes and then treat them with IL-6 to decrease their obesity.

More precisely, the invention relates to a method for identification of an obese patient that is responsive to treatment with a substance that upon administration to said patient will lead to an increased level of an IL-6 receptor agonist for the treatment of obesity, wherein a sample from a person is analysed with a method that can detect

• a CC or GC genotype in position -174 from initiation of transcription of the IL-6 gene, and/or

• a AA or GA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or • a genotype that is associated with said CC or GC genotype and/or said AA or GA genotype, and the presence of at least one of said genotypes indicates that said person is identified as said obese patient.

The invention also relates to a kit for identifica- tion of an obese patient that is responsive to treatment with a substance that upon administration to said obese patient will lead to an increased level of an IL-6 receptor agonist for the treatment of obesity, comprising means for isolation of DNA from a sample from a person, means for amplification of at least the DNA sequence surrounding the biallelic -174 G/C polymorphism in position -174 from initiation of transcription of the IL-6 gene and/or the biallelic -597 G/A polymorphism in position - 597 from initiation of transcription of the IL-6 gene and/or an associated polymorphism, and means for analysing said DNA sequence to detect at least one of the genotypes selected from the group consisting of: • a CC genotype in position -174 from initiation of transcription of the IL-6 gene, and/or

• a GC genotype in position -174 from initiation of transcription of the IL-6 gene, and/or • a AA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or

• a GA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or

• a genotype that is associated with said CC, GC, AA and/or GA genotype.

In addition, the invention relates to the use of a substance that upon administration to an obese patient having

• a AA or GA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or

• a genotype that is associated with said CC or GC genotype and/or said AA or GA genotype, will lead to an increased level of an IL-6 receptor agonist for the production of a pharmaceutical composition for the treatment of obesity.

Furthermore, the invention relates to a method for treatment of obesity wherein a pharmaceutically effective amount of a substance, that upon administration to an obese patient having

• a genotype that is associated with said CC or GC genotype and/or said AA or GA genotype, will lead to an increased level of an IL-6 receptor agonist, is administered to said patient. Preferably, said genotype is the CC or GC genotype at the biallelic -174 G/C polymorphism in position -174 from initiation of transcription of the IL-6 gene and/or the AA or GA genotype at the biallelic -597 G/A polymorphism in position -597 from initiation of transcription of the IL-6 gene.

There are several advantages with being able to identify persons being responsive to IL-6-treatment by determining the -174 and -597 IL-6 gene genotypes in obese subjects, and identify the persons with -174 CC/GC and/or -597 AA/GA genotypes and then treat them with IL-6 to decrease their obesity: 1) It is economically beneficial to avoid to treat patients with the -174 GG and -597 GG genotypes, as they are likely to have appropriate endogenous production of IL-6 at appropriate site. Therefore, they are less likely to respond to IL-6 treatment. 2) All treatments are associated with side effects in some patients. Therefore, it is important to identify patients with -174 GG and -597 GG that are unlikely to respond to the anti-obesity effect of IL-6. If given IL-6 treatment, these patient would risk side effects and at the same time have small chance of positive fat reducing effect. 3) Specifically, IL-6 may induce hyperglycemia¹⁸ and should therefore not be given to patients that are unlikely to respond to the anti-obesity effect of IL- 6. As obesity is associated with type two diabetes¹⁷, an effective reduction of body fat in patients with - 174 GG and -597 GG genotypes may also reduce the risk of diabetes as a side effect of IL-6 treatment.

Detailed description of the invention

The inventors have found that obese persons that are responsive to the anti-obesity effect of interleukin-6 (IL-6) can be identified by detecting the presence of one or more of the following genotypes; -597 AA, -597 GA, -174 CC or -174 GC in the IL-6 promoter region of the IL- 6 gene . In the research work leading to the present invention the role of -174 G/C and -597 G/A SNP was examined by genotyping 30 subjects of different body mass index (BMI) . It was surprisingly found that obese individuals had reduced incidence of the -174 G and -597 G allele.

Further, the present inventors have shown that the C containing genotypes of the common -174 G/C polymorphism of the IL-6 promoter is associated with increased indices of body fat mass . The present inventors have thus found that persons with -174 GG genotype and/or -597 GG genotype are less likely to be obese. However, since obesity is a polygenic disease it is likely that there is a subgroup of obese patients that are obese despite having the -174 GG and/or -597 GG genotype and thus presumably having sufficient endogenous IL-6 production at appropriate site. These persons will be obese due to other causes, and would unlikely respond to IL-6 treatment.

The results thus indicate that the -174 CC and -174 GC genotypes, as well as the -597 AA and -597 GA genotypes are associated with insufficient production of endogenous IL-6 at a site important for its body fat reducing effect, whereas the -174 GG and -597 GG genotypes are not . The present inventors have thus surprisingly found that persons having the -174 GG genotype or the -597 GG genotype are not likely to be responsive to treatment with IL-6 or IL-6 analogues, while persons having the -174 CC/GC or -597 AA/GA genotypes are responsive to such treatment.

The invention thus relates to a method and a kit for identifying obese individuals having the -174 CC/GC or -597 AA/GA genotypes and also to pharmaceutical compositions and methods for treatment of such obese patients with IL-6 or IL-6 analogues. By a polymorphism is meant one of a number of alternative versions of a gene, resulting from a change in the base sequence of the DNA.

By a single nucleotide polymorphism (SNP) is meant a polymorphism, which consist of the change of only one nucleotide at a certain site.

The term "biallelic polymorphism" used herein relates to a certain site within the genome where only two alternative nucleotides exist in a population. The least common nucleotide has a prevalence of 1 % or more.

The position of a SNP is often calculated using the transcription start site as reference point . The first nucleotide of the transcription start is position number one (1) and the first nucleotide upstream from the start site is minus one (-1) . However, in some literature the nomenclature uses the translation start as reference point. The translation start is located approximately 65 nucleotides downstream from the transcription start in the IL-6 gene. Furthermore, the numbering may also be inconsistent due to individual variations of certain AT- repeat stretches within the IL-6 promoter. However, the DNA sequence that surround the specific SNP is unaltered.

By the term "position -174 from initiation of transcription" is thus meant the position in the nucleotide sequence which is located at a distance of 174 nucleotides in the 5' direction from the site where transcription starts, where the nucleotide immediately upstream (i.e. in the 5' direction) of the first transcribed nucleotide is defined as -1. To give an unambiguous definition of the -174 polymorphism site, the surrounding sequences are given, see below.

...cctagttgtgtcttgc (G/C) atgctaaaggacg... -174 G/C SNP

Accordingly, by the term "position -597 from initiation of transcription" is meant the position in the nucleotide sequence which is located at a distance of 597 nucleotides in the 5' direction from the site where transcription starts, where the nucleotide immediately upstream (i.e. in the 5' direction) of the first transcribed nucleotide is defined as -1. To give an unambiguous definition of the -597 polymorphism site, the surrounding sequences are given, see below.

...cacgaaatttgagg (G/A) tggccaggcagtcta... -597 G/A SNP

Also included in the scope of the invention are other polymorphisms associated with the -597 G/A and/or -174 G/C biallelic polymorphisms.

By "a genotype that is associated with the CC or GC genotype in position -174, and/or the AA or GA genotype in position -597 from initiation of transcription of the IL-6 gene" is meant a genotype having a polymorphism linked to, and inherited together with, the -174 G/C and/or the -597 A/G polymorphisms. Certain genotypes are inherited together and it is not easily determined which of them that exerts the possible biological effect.

Thus, by determining the nucleotide sequence at an associated site, it would be possible to indirectly determine the nucleotides in positions -174 and -597 from initiation of transcription of the IL-6 gene. Therefore, determination of the nucleotide sequence at an associated site is an alternative way to determine the IL-6 responsive genotype and thus an alternative embodiment of the invention.

A suitable sample according to the present invention may be a blood sample or any other available tissue e.g. a fat biopsy or a tissue sample.

Isolation of DNA from said suitable sample may be performed with any available commercial kit or any conventional method known in the art . For example, DNA may be isolated from blood samples. The first step of the isolation procedure is lysis of red blood cells by hypotonic solution, leaving the leukocytes intact. The leukocytes are collected by centrifugation and their cellular membranes are solubilised by the use of a lysis-solution. Contaminating cellular proteins are removed by salt precipitation and centrifugation. The DNA, which remains in the supernatant, is precipitated by isopropanol and then washed with 70 % ethanol .

The sample according to the invention may be analysed with any method that can detect the certain geno- types. Preferably, said sample according to the invention is analysed with a method using the sequencing with poly- merase chain reaction, the sequencing with polymerase chain reaction followed by another method or with dynamic allele-specific hybridization (DASH) . However, the geno- typing can be performed with any of the methods described in the literature with non published methods, or with novel methods invented in the future .

The principle of the DASH method is that the target sequence is amplified by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin-coated microtiter plate well, and the non- biotinylated strand is rinsed away with alkali. An oli- gonucleotide probe, specific for one allele, is hybridised to the target at low temperature. This forms a du- plex DNA region that interacts with double strand- specific intercalating dye. Upon excitation, the dye emits fluorescence proportional to the amount of probe- target duplex present. The sample is the steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the ^λmelting' temperature of the probe-target duplex. A single-base mismatch between the probe and the target results in a dramatic lowering of melting temperature that can easily be detected¹⁹.

The substance according to the invention may be an IL-6 receptor agonist. A preferred example of such an agonist is IL-6. It is also possible to use functionally equivalent analogues of IL-6. Further, it is possible to use a naturally occurring agonist, such as IL-6, as well as a synthetically produced agonist, such as an IL-6 mimetic. Examples of synthetically produced IL-6 receptor agonists are given in US 550 61 07 (Cunningham et al) , US 589 19 98 (Rocco et al) , and US 591 41 06 (Gennaro et al) . Said substance may also be a substance that upon administration will lead to the release of an endogenous occurring IL-6 receptor agonist, preferably IL-6.

The expression "IL-6 receptor agonist" used herein relates to all substances that bind to and activate the same receptor proteins as IL-6.

The expression "functionally equivalent analogue" used herein relates to any substance that is structurally similar to IL-6 and has essentially the same pharmacol- ogical and/or therapeutical effects.

The term "patient" used herein relates to any human or non-human mammal in need of being tested and/or treated with the methods, kit or pharmaceutical composition according to the invention. The term "treatment" used herein relates to both treatment in order to cure or alleviate a disease or a condition, and to treatment in order to prevent the development of a disease or a condition.

The pharmaceutical composition and the method ac- cording to the invention are suitable for treatment of different pathological disturbances of regulation of body adipose tissues. More precisely, the pharmaceutical composition and the method according to the invention are suitable for treatment of obesity and overweight by re- ducing adipose tissue mass.

Obesity includes visceral or general obesity that is due to genetic predisposition, a condition sometimes described as the thrifty genotype. Obesity caused by lifestyle and environment, such as lack of exercise, or diets with high caloric content or high fat content, can also be treated as described herein. The pharmaceutical composition and the method according to the invention could also be used to enhance the effects of exercise and/or diet .

The reduction in adipose tissue mass according to the invention preferably results in a weight reduction that is larger than 5% of body weight at the start of treatment .

The pharmaceutical composition or pharmaceutical composition or pharmaceutical preparation according to the invention may also comprise other substances, such as an inert vehicle, or pharmaceutical acceptable adjuvants, carriers, preservatives etc., which are well known to persons skilled in the art.

The substance according to the invention is preferably administered in a dose of 20 ng to 200 μg per kg body weight. Said substance can be administered subcutane- ously, intramuscularely, intravenously, intranasally or orally.

The expression "pharmaceutically active amount" of the substance relates to a dose of the substance that will lead to the desired pharmacological and/or therapeutic effect. The desired pharmacological and/or therapeutic effect is, as stated above, to cure or alleviate different pathological disturbances of regulation of body adipose tissues, leading to obesity, i.e. treatment of obesity and overweight by reducing adipose tissue mass.

Furthermore, it is possible to combine the treatment according to the invention with other conventional pharmacological treatments of obesity. The substance according to the invention may thus be administered in combina- tion with other conventional pharmaceuticals used to treat obesity.

The invention will now be further explained in the following examples . These examples are only intended to illustrate the invention and should in no way be consid- ered to limit the scope of the invention. Examples

Example 1

Materials and methods Blood samples from 30 male subjects, with a BMI range of 21 to 36, were retrieved from another obesity study. From these blood samples, DNA was isolated by a commercial kit. The sequence of interest was amplified by PCR and the genotyping were then performed by Dynamic Al- lele Specific Hybridization (DASH) .

Results

Lean subjects (BMI < 25) had allele frequencies of 0.62 and 0.38 for -174 G and C respectively, which is similar to what is described in literature⁵'⁶. However, overweight and obese subjects (BMI > 29) proved to have a different allele frequency, -174 G had a frequency of 0.40 and consequently -174 C had a frequency of 0.60. The proportions of subjects with CC, GC and GG were 0.30, 0.58 and 0.12 respectively. In line with this, the χ² analysis showed that the obese subjects in the present study had a significantly different distribution between the CC, CG and GG genotypes compared to the patients in the ECTIM study⁶ (P=0,03). In a similar way, we compared the allelic frequencies for bialleic G/A polymorphism at -597 from transcription initiation of the IL-6 gene. It has been reported previously that the -597 G/A and -174 G/C polymorphisms are in almost complete association¹⁴. In line with this, we saw similar differences in -597 G/A allele frequencies between obese and non obese subjects as those observed for -174 G/C polymorphism. In obese subjects, the -597 G and -597 A frequencies were 0.40 and 0.60 respectively. In non obese subjects, the -597 G and 597 A frequencies were 0.62 and 0.38 respectively. Example 2

In example 2, it was hypothesized that low IL-6 gene transcription, caused by the -174 C allele, could contribute to obesity in humans.

Methods

Study subjects

The study population was recruited by local advertisement at Huddinge hospital, Stockholm. 74 subjects (females, mean age of 38 years) were genotyped for the -174 IL-6 G/C SNP to investigate association with indices of body fat mass such as body mass index (BMI) and serum leptin levels. Additional blood chemistry was also measured e.g. IL-6, glucose, total cholesterol, low- density lipoprotein (LDL) , high-density lipoprotein (HDL) and triglycerides . The clinical and biochemical characteristics of the subjects are represented in table 1. The study was approved by the ethics committees of the University of Gothenburg and the Karolinska Institute.

Table 1 Clinical and biochemical characteristics of the study subj cts

Subjects:

Gender (M / F) 0 /74

-174 G/C IL-6 SNP (f_G/ f_c) 0.57 / 0.43

Age (years) 38.3 ± 1.24

BMI (kg/m≥) 23.9 ± 0.31

SBP (mmHg) 114 + 1.91

DBP (mmHg) 72.2 + 1.23

Serum leptin (ng/ml) 12.3 ± 0.81 Serum IL-6 (pg/ml)

B-Glucose (mM) 4.86 ± 0.07

Total cholesterol (mM) 5.00 + 0.13

LDL (mM) 3.31 + 0.12

HDL (mM) 1.50 ± 0.04

Triglycerides (mM) 1.07 ± 0.08

(SBP = systolic blood pressure, DBP = systolic blood pressure, data are expressed as mean ± SEM) . Biochemical analyses

Glucose, cholesterol, triglycerides and HDL were analysed by Huddinge University Hospital ' s routine chemistry laboratory. LDL was not measured directly, but was calculated from the following equation: LDL = Total cholesterol - HDL - triglycerides/5. Serum leptin levels were analysed by Human Leptin radioimmunoassay kit (Linco, St. Charles, MO).

DNA geno typing

The genotyping was performed by PCR-RFLP as described⁷ using primers 5 ' -TGACTTCAGCTTTACTCTTTGT-3 ' and 5' -CTGATTGGAAACCTTATTAAG-3 ' and subsequent digestion of the PCR-product with SfaNI restriction enzyme.

Statistical methods

Data were analyzed with the SPSS program (version 11.0.0; SPSS, Chicago, IL) . To compare the variables between the three different genotypes, we used the Kruskal- Wallis one-way (K-W 1-way) ANOVA due to non-normally distributed data. Chi square analysis (χ²-analysis) was used for the investigation of presence of Hardy-Weinberg equilibrium and for overall comparison between overweight (BMI > 25 kg/m²) and lean (BMI < 25 kg/m²) subjects. Odds ratios for genotypes were calculated by logistic regression. We calculated the population-attributable risk as described before²⁰. The population-attributable risk = (X-D/X, where X = (1-f)² + 2f (l-f)rι + f²r₂, f is the frequency of the risk allele (f_c = 0.47) and ri and r₂ are the estimated genotype risk ratios of the GC (1.179; 95% confidence interval 1.023-1.359) and CC (1.229; 95% confidence interval 1.055-1.432) genotypes relative to GG. The confidence intervals for the risk ratios were used to calculate the confidence interval for the population- attributable risk. To investigate the effects of gender and -174 G/C genotype on serum leptin levels, we used two-way (2-way) ANOVA. Data of serum leptin levels were transformed with Blom's method in order to obtain normally distributed residuals in the 2-way ANOVA²². Data in text or tables are given as mean ± standard error of the mean (SEM) . P-values of < 0.05 were considered as sig- nificant.

Results

The frequencies of the -174 C and G alleles in the study population were 0.43 and 0.57 respectively. Seven- teen subjects (23.0 %) had the CC genotype, 30 subjects

(40.5 %) the GC genotype and 27 (36.5 %) had the GG genotype. The genotypes were in Hardy-Weinberg equilibrium (p = 0.57) . The relation between -174 G/C genotype and BMI and serum leptin levels are shown in Table 2. Subjects with the CC and GC genotypes had a mean BMI 5.3 % and 9.2 % higher, respectively, than the mean BMI of the GG genotypes (p = 0.03). Compared to the GG genotype, serum leptin levels tended to be higher in subjects with CC and GC genotypes (66.8 % and 26.4 % higher respectively), but the difference did not reach significance (p = 0.09) . Total cholesterol, LDL, HDL, triglycerides and b-glucose did not differ between the genotypes (data not shown) .

Table 2 The age, BMI and serum leptin levels in the study population according to the -174 G/C genotype

■174 IL-6 genotype

CC GC GG p-value (K-W)

Subjects (nr / %) 17 / 23.0 30 / 40.5 27 / 36.5 Age (years) 40.5 ± 2.49 36.9 ± 1.94 38.3 ± 2.15 0.53 BMI (kg/m*) 24.0 + 0.62 24.9 ± 0.49 22.8 ± 0.46 0.03 Serum leptin (ng/ml) 16.1 + 2.42 12.2 + 1.11 9.65 ± 0.75 0.09

The p-values are for comparison of the three genotypes (K-W 1-way ANOVA) . Data are shown as mean ± SEM. Conclusions

The present findings show, surprisingly, that the allelic frequencies of G at -174 and at -597 of the promoter of the IL-6 gene are lower in obese persons than the frequency reported in non-obese persons. Further, it was shown that the -174 C genotype of the IL-6 gene promoter is associated with increased susceptibility to develop overweight. The -174 G/C and the -597 G/A polymorphisms are strongly connected with each other with -174 C and the -597 A genotypes being associated in the same haplotypes¹ . Therefore, it can be concluded that the -597 A genotype of the IL-6 gene promoter is associated with increased susceptibility to develop overweight.

The present inventors have previously found that that IL-6 can prevent obesity³'⁴. The present results indicate that obese subjects with -174 CC/GC and -597 AA/GA genotypes, i e, carriers of the -174 C and the -597 A alleles, have impaired ability to produce sufficient amounts of IL-6 at sites where high levels of IL-6 are of importance for prevention of obesity. This is surprising, given that previous studies have not shown any consistent relation between -174 G/C or -597 G/A SNPs and various metabolic functions, that in turn are related to obesity

6-8,12,15,15

References

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2. Bouchard, C. et al . The response to long-term overfeeding in identical twins . N Engl J Med 322, 1477-82. (1990) .

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5. Fishman, D. et al . The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 102, 1369-76. (1998) .

6. Georges, J. L. et al . Interleukin-6 gene polymorphisms and susceptibility to myocardial infarction: the ECTIM study. J Mol Med 79, 300- 305. (2001) . 7. Fernandez-Real , J. M. et al . Interleukin-6 gene polymorphism and insulin sensitivity. Diabetes 49, 517-20. (2000) .

8. Burzotta, F. et al . Relation of the -174 G/C polymorphism of interleukin-6 to interleukin-6 plasma levels and to length of hospitalization after surgical coronary revascularization. Am J Cardiol 88, 1125-8. (2001).

9. Olivieri, F. et al . The -174 C/G locus affects in vitro/in vivo IL-6 production during aging. Exp Gerontol 37, 309-14. (2002).

10. Villuendas, G. , San Millan, J. L. , Sancho, J. & Escobar-Morreale, H. F. The -597 G-->A and -174 G-->C polymorphisms in the promoter of the IL-6 gene are associated with hyperandrogenism. J Clin Endocrinol Metab 87, 1134-41. (2002).

11. Brull, D. J. et al . Interleukin-6 gene -174g>c and -572g>c promoter polymorphisms are strong predictors of plasma interleukin-6 levels after coronary artery bypass surgery. Arterioscler Thromb Vase Biol 21, 1458-63. (2001).

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14. Jordanides, N. , Eskdale, J. , Stuart, R. & Gallagher, G. Allele associations reveal four prominent haplotypes at the human interleukin-6 (IL- 6) locus. Genes Immun 1, 451-5. (2000). 15. Fernandez-Real, J. M. , Broch, M. , Vendrell, J. ,

Richart, C. & Ricart, W. Interleukin-6 gene polymorphism and lipid abnormalities in healthy subjects. J Clin Endocrinol Metab 85, 1334-9. (2000) . 16. Rauramaa, R. et al . Stromelysin-1 and interleukin-6 gene promoter polymorphisms are determinants of asymptomatic carotid artery atherosclerosis. Arterioscler Thromb Vase Biol 20, 2657-62. (2000) . 17. Barsh, G. S., Farooqi, I. S. & O'Rahilly, S. Genetics of body-weight regulation. Nature 404, 644-51. (2000) . 18. Tsigos, C. et al . Dose-dependent effects of re- combinant human interleukin-6 on glucose regula- tion. J Clin Endocrinol Metab 82, 4167-70.

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Claims

1. A method for identification of an obese patient that is responsive to treatment with a substance that upon administration to said patient will lead to an in- creased level of an IL-6 receptor agonist for the treatment of obesity, wherein a sample from a person is analysed with a method that can detect

• a CC or GC genotype in position -174 from initiation of transcription of the IL-6 gene, and/or • a AA or GA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or

• a genotype that is associated with said CC or GC genotype and/or said AA or GA genotype, and the presence of at least one of said genotypes indicates that said person is identified as said obese patient.

2. A method according to claim 1, wherein said genotype being detected is a CC genotype in position -174 from initiation of transcription of the IL-6 gene.

3. A method according to claim 1, wherein said geno- type being detected is a GC genotype in position -174 from initiation of transcription of the IL-6 gene.

4. A method according to claim 1, wherein said genotype being detected is a AA genotype in position -597 from initiation of transcription of the IL-6 gene.

5. A method according to claim 1, wherein said genotype being detected is a GA genotype in position -597 from initiation of transcription of the IL-6 gene.

6. A method according to any one of the claims 1-5, wherein said sample is a blood sample.

7. A method according to any one of the claims 1-5, wherein said sample is a fat biopsy.

8. A method according to any one of the claims 1-5, wherein said sample is a tissue sample.

9. A method according to any one of the claims 1-8, wherein said sample is analysed with dynamic allele- specific hybridisation (DASH) .

10. A method according to any one of the claims 1-8, wherein said sample is analysed with a method using the sequencing with polymerase chain reaction

11. A method according to any one of the claims 1-8, wherein said sample is analysed with a method using the sequencing with polymerase chain reaction followed by another method.

12. A method according to any one of the claims 1-

11, wherein said substance is an IL-6 receptor agonist.

13. A method according to any one of the claims 1-

12, wherein said substance is IL-6 or a functionally equivalent analogue thereof.

14. A kit for identification of an obese patient that is responsive to treatment with a substance that upon administration to said obese patient will lead to an increased level of an IL-6 receptor agonist for the treatment of obesity, comprising means for isolation of DNA from a sample from a person, means for amplification of at least the DNA sequence surrounding the biallelic - 174 G/C polymorphism in position -174 from initiation of transcription of the IL-6 gene and/or the biallelic -597 G/A polymorphism in position -597 from initiation of transcription of the IL-6 gene and/or an associated polymorphism, and means for analysing said DNA sequence to detect at least one of the genotypes selected from the group consisting of:

• a CC genotype in position -174 from initiation of transcription of the IL-6 gene, and/or

• a GC genotype in position -174 from initiation of transcription of the IL-6 gene, and/or

• a AA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or

• a GA genotype in position -597 from initiation of transcription of the IL-6 gene, and/or • a genotype that is associated with said CC, GC, AA and/or GA genotype .

15. Use of a substance that upon administration to an obese patient having

• a genotype that is associated with said CC or GC genotype and/or said AA or GA genotype, will lead to an increased level of an IL-6 receptor ago- nist for the production of a pharmaceutical composition for the treatment of obesity.

16. Use according to claim 15, wherein said obese patient has a CC genotype in position -174 from initiation of transcription of the IL-6 gene.

17. Use according to -claim 15, wherein said obese patient has a GC genotype in position -174 from initiation of transcription of the IL-6 gene.

18. Use according to claim 15, wherein said obese patient has a AA genotype in position -597 from initia- tion of transcription of the IL-6 gene.

19. Use according to claim 15, wherein said obese patient has a GA genotype in position -597 from initiation of transcription of the IL-6 gene.

20. Use according to any one of the claims 15-19, wherein said substance is an IL-6 receptor agonist.

21. Use according to any one of the claims 15-20, wherein said substance is IL-6 or a functionally equivalent analogue thereof .

22. A method for treatment of obesity wherein a pharmaceutically effective amount of a substance, that upon administration to an obese patient having

• a genotype that is associated with said CC or GC genotype and/or said AA or GA genotype, will lead to an increased level of an IL-6 receptor agonist, is administered to said patient.

23. A method according to claim 22, wherein said obese patient has a CC genotype in position -174 from initiation of transcription of the IL-6 gene.

24. A method according to claim 22, wherein said obese patient has a GC genotype in position -174 from initiation of transcription of the IL-6 gene.

25. A method according to claim 22, wherein said obese patient has a AA genotype in position -597 from initiation of transcription of the IL-6 gene.

26. A method according to claim 22, wherein said obese patient has a GA genotype in position -597 from initiation of transcription of the IL-6 gene.

27. A method according to any one of the claims 22-

26, wherein said substance is an IL-6 receptor agonist.

28. A method according to any one of the claims 22-

27, wherein said substance is IL-6 or a functionally equivalent analogue thereof.

INTERNATIONAL SEARCH REPORT International application No.

PCT/SE 03/00903

C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

Clin. Invest., Vol. 102, no. 7, October 1998, 1-28 Daniel Fishman et al: "The Effect of Novel Polymorphisms in the Interleukin-6 (IL-6) Gene on IL-6 Transcription and Plasma IL-6 Levels, and an Association with Systemic-Onset Juvenile Chronic Arthritis", page 1369 - page 1376, page 1370, left column, lines 38-41; page 1374, figure 4

Form PCT/ISA/210 (continuation of second sheet) (July 1998)