Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/2264—Obesity-gene products, e.g. leptin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/204—IL-6
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics

Definitions

• the present invention relates to a new medicinal product and a new method for treatment of pathological disturbances of regulation of body fat tissue mass and/or obesity associated disorders.
• the 16 kDa protein leptm is almost only produced white adipocytes from which leptm is then released to circulation. Leptm production by fat and plasma leptm levels is highly correlated with adipose tissue mass
• Obese ( ob) mice which lack leptm show many of the abnormalities seen m starved animals, including hyper- phagia, decreased body temperature, decreased energy ex- penditure, decreased immune function, and infertility. Leptin replacement corrects all of these abnormalities implying that ob mice live m a state of "perceived starvation" due to lack of leptin and that the biological response m the presence of food leads to obesity.
• the leptin receptor is normally expressed at high levels m hypothalamic neurons and m other cell types, including T cells and vascular endothelial cells.
• Ob-R The leptin receptor
• DMH dorsomedial hypothalamic nucleus
• PVN paravent ⁇ cular nucleus
• VN ventrome- dial hypothalamic nucleus
• LH lateral hypothalamic nucleus
• Each of these nuclei express one or more neuropeptides and neurotransmitters such as neuropeptide Y (NPY) and mela- nocyte-stimulating hormone alpha ( -MSH) , that regulate food intake and/or body weight, probably by actions downstream of leptin (Friedman JM and Halaas JL, 1998; Flier JS and Maratos-Flier E, 1998) .
• neuropeptide Y neuropeptide Y
• -MSH mela- nocyte-stimulating hormone alpha
• leptm m The role of leptm m the pathogenesis of obesity may be inferred by measurement of plasma leptin.
• An increase m plasma leptin suggests that obesity is the re- suit of resistance to leptin.
• a low or normal plasma concentration of leptin suggests that obesity is due to decreased production of leptin.
• This interpretation is similar to that used m studies of msulm and the pathogenesis of type I and type II diabetes.
• mutations of leptin and its receptor are rare m human obesity, but most obese individuals still have higher levels of leptin than do non-obese individuals, an indication of leptin resistance that might be receptor-independent (Flier JS, 1997) .
• genes involved m development of obesity have recently been found and most of them seem to act down- stream of leptin at the hypothalamic level.
• Other genes that are involved m development of obesity encode neuro- peptides, e.g. leukocyte adhesion receptors, which are important cell -cell adhesion molecules the mflamma- tory and immune systems (Dong ZM et al . , 1997), and neu- rocytokmes like ciliary neurotrophic factor, whose receptor subumts share sequence similarity with the leptin receptor (Gloaguen I et al . . , 1997) .
• the identification of anti-obesity mechanisms that act independently or to- gether with the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance .
• Leptin has immuno- regulatory activi ty Exogenous leptin up-regulates both phagocytosis and the macrophage production of promflammatory cytokmes such as tumor necrosis factor (TNF- ) and mterleukm-6 (Loffreda S et al . , 1998) . It has been suggested that the up-regulation of inflammatory immune responses by leptin may contribute to several of the major complications of obesity such as increased incidence of infection, diabetes and cardiovascular disease (Loffreda S et al . , 1998; McCarty MF, 1999) . This hypothesis is attractive since it would implicate a common pathogenic mechanism (lack of leptin action) for both obesity and some of its major complications. However, an alternative possibility is that regulatory mechanisms usually connected to e.g. immune functions also are of importance for the regulation of body fat .
• the cytokmes act as hormonal regulators of the immune system and m the body's reactions during trauma and inflammation.
• the cytokme mterleukm-6 (IL-6) is known to be important the development of B-lymphocytes and m the change of plasma protein production of the liver during trauma and inflammation, the so-called acute phase response. In line with this, IL-6 levels are markedly increased during acute phase response. It has been shown that IL-6-type cytokme receptors share functional specificity with the long form of the leptin receptor (Baumann H et al., 1996) .
• the role of the cytokmes including IL-6 m healthy animals and humans is not well known and they are suggested to have little effect, partly because circulating levels often are low m the absence of illness (Hirano T, 1998) .
• Interleukm-6 exerts its biological effects through the ligand-specifIC IL-6 receptor, which belongs to the cytokme receptor superfamily.
• the multisubunit IL-6 receptor complex consists of the IL-6R ⁇ subunit which binds to IL-6 and the membrane associated glycopro- tem gpl30 which s a signal transducer.
• the IL-6R subunit can be activated by ligand binding m both its membrane bound and its soluble form.
• IL-6 induces heterodime ⁇ zation between IL- ⁇ R ⁇ and gpl30, which m turn leads to homodime ⁇ zation of gpl30 to a second gpl30 molecule (see e.g.
• IL-6/lL-6R ⁇ complexes can be potent activators of gpl30, including cells that lack membrane bound IL-6R . Since gpl30 can be activated by several other ligand-receptor complexes, these effects may not reflect the physiological role of IL-6 (see e.g. Schirmacher, P., et al .
• IL-6 plays a role for different parts of the immune response (see e.g. Hirano, T. (1998) , supra) . It is well known that production of IL-6 as well as the circulating levels of this cytokme is enhanced during so-called acute phase reaction (APR) . Moreover, IL-6 is considered a key mediator of APR, especially after infection with gram positive bacteria (see e.g. Kopf, M., et al . (1994), Impaired immune and acute-phase responses m mterleukm- 6-def ⁇ c ⁇ ent mice. Nature 368, 339-342) . The APR is characterized by changes m the composition of the proteins released into plasma from the liver. APR is seen m pathological conditions with an inflammatory component such as trauma, infections, autoimmune disease, and tumors. These conditions are also associated with catabo- lism, i.e. decreased growth and increased degradation of tissues belonging to the fat free mass m the body.
• Aging is associated with several somatic changes including increased body fat mass m general and visceral fat mass m particular (see e.g. Rudman, D., et al . , (1990) , Effects of human growth hormone m men over 60 years old. N. Engl . J. Med. 323, 1-6; Flier, J. S. and Foster D. W. (1998) supra) .
• the proportion of the popula- tion that have disturbances of blood fats such as pathologically elevated serum triglyce ⁇ des also increase with age and is higher middle aged than m young adult persons (Brown, M. S., and Goldstein, J. L. (1983) Disorders of lipid metabolism, Harrison's principle of internal medicine, 10th Ed, 547-559.
• mice with complete IL-6 deficiency due to targeted dis- ruption of the IL-6 gene have few reports of differences between mice with complete IL-6 deficiency due to targeted dis- ruption of the IL-6 gene, and normal wild type mice m the absence of provocations (see e.g. Hirano, T. (1998), supra) . It is known that these mice develop normally to adulthood and they are fertile (see e.g. Kopf, M. , et al , supra, and Poll, V., et al . , (1994) . Interleukm-6 deficient mice have been reported to be protected from bone loss caused by estrogen depletion. EMBO J. 13, 1189- 1196) . It has also been reported that IL-6 mice might have a defective fever response (see e.g. Hirano, T.
• IL-6 is released during acute phase reaction. Therefore, it is not surprising that IL-6 production is enhanced by gram-positive as well as by gram-negative bacteria. The latter seem to release IL-6 via production of an antigen called lipopolysaccha ⁇ de (LPS) (see e.g. Kopf, M. , et al . (1994), supra).
• LPS lipopolysaccha ⁇ de
• the pro- duction of IL-6 is enhanced by tumor necrosis factor- ⁇ , TNF- ⁇ , a cytokme that is thought to play a role for the induction of type 2 diabetes, an illness associated with visceral obesity and cardiovascular disease. TNF- ⁇ production is enhanced from adipocytes that have accumulated fat (see e.g. Hotamisligil G. S.
• IL-6 is a major mediator of APR, a condition associ- ated with wasting and decreased appetite. However, it is still by no means certain that IL-6 also causes these an- orectic and wasting effects. In fact, there are data indicating that this is not the case, although lipopolysac- charides (LPS) were reported to induce weight loss mice and that this effect can be significantly prevented by treatment with ant ⁇ -IL-6 monoclonal antibodies. However, m the same study the ant ⁇ -IL-6 antibodies did not prevent the hypertnglyceridemia induced by LPS, possibly suggesting that IL-6 is less important for changes m fat metabolism during APR (Strassman, G. et al . (1993), The role of mterleukm-6 m lipopolysaccha ⁇ de- induced weight loss, hyperglycemia and flbrmogenproduction. Cytokme 5, 285-290) .
• alcohol can suppress the concentration of circulating IL-6 (see e.g. Akerman, P. A., et al . (1993), Long-term ethanol consumption al- ters the hepatic response to the regenerative effects of tumor necrosis factor-alpha. Hepatology 17, 1066-1073). It is also well known that ethanol can cause visceral obesity as well as deranged blood fats including enhanced serum triglyceride levels (Brown, M. S., and Goldstein, J. L. (1983), supra) .
• TNF- ⁇ is a stimulator of IL-6 production.
• This effect of TNF- ⁇ is exerted via the type 1 (p55) receptor, since it has been shown that IL-6 levels are decreased m mice with TNF receptor 1, but not TNF receptor 2, gene knock out (Yamada, Y., et al . (1998), Analysis of liver regeneration m mice lacking type 1 or type 2 tumour necrosis factor receptor: re- quirement for type 1 but not type 2 receptor.
• mice lacking the TNF- ⁇ ligand have not been reported to be obese (Uysal, K. T., et al (1997), Protection from obesity induced msulm resistance m mice lacking TNF- ⁇ , Nature 389,610-614), and there was no obesity mice deficient m the both of the two receptors, type 1 ( ⁇ 55) and type 2 (p75) , that are thought to mediate the biological effects of TNF- ⁇ . Actually, mice deficient the type 2 (p75) receptor ga less weight when given high fat diet, suggesting that TNF- ⁇ might even stimulate obesity via this receptor type (Schreyer, S. A.
• TNF- ⁇ often enhances msulm resistance, a symptom often associated with obesity (see Flier, J. S. and Foster D. W. (1998) , supra) .
• IL-6 has been reported to increase circulating triglyce ⁇ des by release of triglyce ⁇ des from the liver (Nonogagi K, et al . (1995), Interleukm-6 stimulates hepatic t ⁇ glyceride secretion m rats, Endocrinology 136, 2143-2149) .
• the object of the present invention is to provide new medicinal products and methods for treatment of obe- sity and/or obesity associated disorders.
• the invention relates to the use of a substance that upon administration to a patient will lead to an increased level of an mterleukm-6 (IL-6) receptor agonist for the production of a medicinal product for the treat - ment of obesity and/or obesity associated disorders.
• IL-6 mterleukm-6
• the invention relates to a method for treatment of obesity and/or obesity associated disorders wherein a pharmaceutically effective amount of a substance that upon administration to a patient will lead to an increased level of an mterleukm-6 (IL-6) receptor agonist is administered to said patient.
• IL-6 mterleukm-6
• the metabolic syndrome (also called syndrome X) comprises obesity (m particular abdominal obesity) , disturbances of blood fats (e g triglyce ⁇ des) , and diabetes type II.
• the invention thus relates to medicinal products comprising a substance that upon administration to a patient will lead to an increased level of an mterleukm-6 (IL-6) receptor agonist.
• Said substance may be an IL-6 receptor agonist .
• a preferred example of such an agonist is IL-6.
• IL-6 IL-6
• 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, from different cells, such as endothe- lial cells, or organs, such as
• IL-6 receptor agonist used herein relates to all substances that bind to and activate the same receptor proteins as IL-6.
• patient used herein relates to any human or non-human mammal m need of treatment with the medicinal product or method according to the invention.
• treatment used herein relates to both treatment m order to cure or alleviate a disease or a condition, and to treatment m order to prevent the development of a disease or a condition. The treatment may either be performed m an acute or m a chronic way.
• the invention is suitable for treatment of high levels of triglyce ⁇ des .
• high levels of triglyce ⁇ des relates to amounts of this compound that are higher than for a normal, healthy person.
• the medicinal product and the method according to the invention are suitable for treatment of different pathological disturbances of regulation of body fat tissues, leading to obesity and/or obesity associated disor- ders.
• One example is visceral or general obesity that is due to genetic predisposition, a condition sometimes described as the thrifty genotype.
• Another example is diet- mduced obesity, a condition that often is resistant to leptin treatment .
• the medicinal product and the method according to the invention are e.g. suitable for treatment of cardiovascular disease, since obesity and obesity associated disorders are associated with an increased risk of cardiovascular disease.
• the medicinal product and the method according to the invention are also suitable for treatment of persons that have been exposed to high doses of glucocorticoid hormone, e.g.
• glucocorticoids due to tumours producing such hormones, due to treatment with glucocorticoids against certain dis- eases, or due to abuse of glucocorticoids. It is known that high levels of glucocorticoids cause visceral obesity and disturbed blood fats. It has been shown that glucocorticoids under certain circumstances can decrease IL-6 production.
• Other patients which may be treated with the medicinal product or the method according to the invention are persons with obesity, obesity associated disorders, and/or low endogenous production of IL-6 during normal state, i.e., m the absence of APR.
• persons with obesity and/or obesity associated disorders m combination with msensitivity to IL-6 may be treated with the medicinal product and the method according to the invention.
• the IL-6 msensitivity could e.g.
• IL-6R ⁇ low levels of the receptor protein IL-6R ⁇ on the cell surface or low levels of the glycoprotem gpl30 which normally mediates the effects of IL-6.
• the IL-6 produced by the patients themselves may not be sufficient to inhibit development of obesity and/or obesity associated disorders.
• Another example of a group of patient which may be treated according to the invention are patients suffering from normal agmg.
• the production of IL-6 m important tissues could be insufficient although the circulating levels often are increased.
• a possible IL-6 insufficiency m agmg may also be due in part to msensitivity to IL-6.
• GH growth hormone
• IL-6 m combination with soluble IL-6R ⁇ may exert unspecific effects, including even on cells that do not have membrane bound IL-6R ⁇ (see e.g. Peters, M. (1997), supra).
• the medicinal product 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 the art .
• the medicinal product according to the invention may be formulated for enteral (e.g. oral or per oral) or par- enteral administration.
• the invention also relates to use of a substance that upon administration to a patient will lead to an increased level of an mterleukm-6 (IL-6) receptor agonist for a medicinal product for treatment of the above speci- fied conditions.
• IL-6 mterleukm-6
• the invention relates to a method for treatment of pathological disturbances of fat metabolism wherein a pharmaceutically effective amount of a substance that upon administration to a patient will lead to an increased level of an mterleukm-6 (IL-6) receptor agonist is administered to said patient.
• a pharmaceutically effective amount of a substance that upon administration to a patient will lead to an increased level of an mterleukm-6 (IL-6) receptor agonist is administered to said patient.
• said substance is administered together with a factor that will intensify the effect of said mterleukm-6 (IL-6) receptor agonist. Since these effects of IL-6 on fat metabolism were first seen m the work leading to the present invention after removal of endogenous IL-6, it seems appropriate to use IL-6 according to the invention m doses that previously have been used to substitute for IL-6 deficiency.
• Such a dose would be about 1 mg/kg body weight given as a subcutaneous injection to mice (se e.g. e.g. Cressman, D. E., et al . , (1996), Liver failure and defective hepa- tocyte regeneration m mterleukm-6-deflcient mice. Science 274, 1379-1383) .
• mice se e.g. e.g. Cressman, D. E., et al . , (1996), Liver failure and defective hepa- tocyte regeneration m mterleukm-6-deflcient mice. Science 274, 1379-1383) .
• the dose of IL-6 m humans could be quite different. The dose may be higher m older individuals, since it has been shown that IL-6 levels m- crease with age. The dose may be lower than those doses that would result IL-6 levels found during APR, to avoid side effects similar to the symptoms of APR.
• Fig 1 A shows the effect of mterleukm-6 gene knock out m male mice on mean body weight at different ages.
• Fig 1 B shows the physical appearance of IL-6 knock out male mice at 9-10 months of age.
• the photo shows repre- sentative body shapes of IL-6 -/- and IL-6 +/+ male mice.
• the computerized tomography (CT) shows transverse sections of the abdomen of representative IL-6 -/- and IL-6
• Fig 2 A, B and C illustrates the effects of mter- leukm-6 gene knock out on mean body weight at different ages m female mice (Fig 2 A) and the effect of mterleukm-6 gene knock out on mean body mass index (Fig 2 B) (BMI, body weight/ (crown-rump length) 2 ) and mean visceral transversal width (mm) (Fig 2 C) were also investigated m 9 month-old female mice.
• Fig 3 Shows the measured daily food intake during three consecutive days m 11 month-old female IL-6 +/+ and IL-6 -/- mice.
• Fig 4 A and B illustrates the effects of mterleu- km- 6 gene knock out m female mice on serum triglyceride levels (Fig 4 A) and serum leptin levels (Fig 4 B) .
• Fig 5 shows the possible sources of IL-6 that could be of importance for body composition and leptin sensitivity.
• Fig 6 shows the effect of vehicle and leptin administration on food intake 15 month-old wild-type and IL- 6 knockout (IL-6 _/ ) male mice.
• Thick black bars represent leptin treatment period. Vehicle or leptin was injected mtrape ⁇ toneally twice daily. Values are indicated as mean ⁇ SEM.
• Fig 7 shows the effect of vehicle and leptin administration on body weight m 15 month-old wild-type and IL- 6 knockout (IL-6 ) male mice.
• Thick black bars represent leptin treatment period.
• Vehicle or leptin was injected mtrape ⁇ toneally twice daily. Values are mdi- cated as mean ⁇ SEM.
• Fig 8 shows relative weights of different fat depots (% fat weight/body weight) m IL-6 + + and
• IL-6 _ ⁇ mice Three mtra-abdommal fat pads (gonadal, retroperitoneal and mesenteric) and the femoral fat pad (a subcutaneous fad pad on the outer thigh) were dissected and weighed 18-month-old male (A) and female (B) IL-6+/+ and IL-6-/- mice. There were 4-10 mice each group. * P ⁇ 0.05, ** P ⁇ 0.01 and *** P ⁇ 0.001, vs. corresponding IL-6+/+ mice.
• Fig 9 shows comparison of the effect of IL-6 treatment m IL-6 + + and IL-6 ⁇ _ mice.
• the mice were treated with gradually increasing doses of IL-6 (40 ng/day, days 0-4; 80 ng/day, days 5-12; 160 ng/day, days 13-18).
• Figures 11 B and C compare values at day 0 before initiation of IL-6 treatment with day 18 after IL-6 treatment m IL-6 +/ + and IL-6 " _ mice.
• the total abdominal area was calculated from the CT scans (B) .
• the mtraperitoneal area containing fat was measured separately by calculating the darker areas with attenuation similar to subcutaneous fat (C) . Both the total mtraperitoneal and mtraperitoneal fat areas were calculated blindly by two different people, with no connection to the study. There were 5 mice in each group. All animals were 12 -month-old at the start of the treatment. * P ⁇ 0.05, ** P ⁇ 0.01 and *** P ⁇ 0.001, vs. corresponding control mice. # P ⁇ 0.05, vs. the corresponding group before initiation of IL-6 treatment.
• mice The IL-6 knock out mice (i.e. IL-6 -/- mice) and the corresponding controls used m these examples were kindly provided by Dr. Manfred Kopf at Basel Institute of Immu- nology, Basle, Switzerland (see e.g. Kopf, M. (1994), supra) .
• the IL-6 -/- mice were back crossed 7-8 times with C57B1/6 mice to ga a strain of mice genetically consisting of more than 95 % C57B1/6.
• mice As controls to the IL-6 -/- mice, wild type C57B1/6 mice (i.e. IL-6 +/+ mice) (Bomholtgard Breeding & Research Centre A/S) were used m examples 1-4. These mice were kept at standardized conditions with standard low fat chow and water freely available. Food intake was measured keeping two female mice per cage. The amount of chow was recorded once per day. In example 5 age-matched normal C57BL/6 male mice from B&K Universal AB (Sollen- tuna, Sweden) were used as wild-type controls. All male mice were housed separately (due to aggressiveness) m standard cages under standardised environmental conditions, i.e.
• mice with IL-6 gene knock-out were generated as described by Kopf et ai (12) .
• the IL-6 _ geno- type was moved onto C57BL/6 background by eight successive back crosses.
• the resulting strain of mice consists genetically of more than 99.5% C57BL/6.
• Normal C57BL/6 mice from B&K Universal (Sollentuna, Sweden) were used as wild-type controls for the IL-6 _ ⁇ mice.
• the animals were maintained under standardized environmental conditions, i.e. 24-26°C, 50-60% relative humidity, artificial lighting at 05.00-19.00 h, with water and pelleted food ad li bi tum . All procedures regarding the mice were conducted accordance with protocols approved by the institutions (Goteborg and Lund) and the local ethical committees on animal care .
• the body weight of the IL-6 -/- mice and wild type control female mice were recorded regularly.
• the crown-rump length and the transversal abdominal diameter were measured m anesthetized animals by dual x-ray absorptiometry (DEXA) using the Norland pDEXA Sabre (Fort Atkinson, WI , USA) .
• Body mass index was then calculated for each mouse as body weight/crown-rump length 2 .
• Visceral and subcutaneous obesity was also evaluated by computerized tomography (CT) at a level 5 mm cranially of the junction between the L6 and SI vertebras .
• CT computerized tomography
• body weight was measured using a weighing scale (A S D Instruments, EK-200G) .
• Food consumption was measured daily by weighing the food left over 24 h after the previous fillup.
• Basal food intake was measured during pre-treatment with saline injections before onset of the leptin treatment.
• Body weight and food intake was measured for 3 days after the end of leptin treatment.
• Plasma leptin was determined with a recently described radioimmunoassay (Ahren, B. et al . (1997) Regulation of plasma leptin m mice: Influence of age, high-fat diet and fasting, Am. J. Physiol. 273, R113-R120; Lmco Research, St Charles, Mo, USA) .
• the method uses a poly- clonal rabbit antibody raised against recombmant mouse leptin, 125 I -labeled tracer prepared with recombmant mouse leptin and mouse leptin as standard. Rabbit anti- rabbit IgG was used for separation of bound and free leptin.
• tail blood samples were collected from young (4 months) and old (12 months) wild-type and IL-6 1 male mice. Differences between IL-6 -/- and IL-6 +/+ control mice were determined by Student's t-test. When more than two groups were compared, statistics were calculated by one-way ANOVA followed by Student-Newman-Keuls multiple range test .
• IL-6 -/- knock-out male mice were not heavier than their wild type littermates at 2-5 months of age. However, the body weight of 9 months old IL-6 -/- male mice was higher than that of the corresponding wild type animals, as evident from Fig. 1 A. The physical appearance of male mice at 9-10 months of age clearly showed that the IL-6 -/- mouse was considerably fatter than a wild type control of the same age, as shown Fig. 1 B) . Computerized tomography (CT) of the abdomen clearly indicated that both visceral (mtraabdommal) and subcutane- ous fat mass were markedly increased m the IL-6 -/- mice compared to the wild type control, as evident from Fig. 1 C.
• CT Computerized tomography
• IL-6 knock-out mice were studied at different ages m female mice.
• the body weight did not differ between wild type and knock-out female mice between two and five months of age, but between seven and nine months of age the body weight was significantly higher m IL-6 -/- than m wild type +/+ mice, as seen m Fig. 2 A.
• the body mass index of 9- 10 months old IL-6 knock-out female mice was higher than that of the corresponding wild type females, which is illustrated m Fig. 2B.
• the transversal abdominal diameter, as measured by DEXA, was also larger m IL-6 knock-out female mice than wild type controls at 9-10 months of age (Fig. 2C) .
• Serum triglyceride and cholesterol levels of 11 months old female IL-6 -/- mice were compared to wild type IL-6 +/+ controls. As can be seen m Fig. 4 A the serum triglyceride was considerably higher m the IL-6 - /- mice. Also the circulating levels of leptin were mark- edly higher, i.e., about three times, compared to those of wild type mice, as seen m Fig. 4 B.
• Example 5 15-month-old IL-6 and wild-type males received mtraperitoneal dp) injections of leptin at doses of 120 ⁇ g/day or 240 ⁇ g/day or vehicle twice daily (at 08:30 and 17:00) for 3 consecutive days.
• Human leptin was obtained from PeproTech (Rocky Hill, NJ, USA) and dissolved m sterile PBS, 0.1% BSA. In order to get the animals used to injections, mice were given saline injections twice daily before the start of the leptin treatment.
• CNTF ciliary neurotrophic factor
• the receptor subunits for ciliary neurotrophic factor (CNTF) have been shown to share sequence similarities with Ob-Rb, Gloaguen I et al . , 1997) and IL-6 receptors.
• CNTF can reverse obesity m various animal models, including db mice lacking leptin receptors (Gloaguen I et al . , 1997) . All three of these systems, leptin, IL-6 and CNTF, signals through the JAK-STAT pathway to regulate gene expression (Flier JS , 1997; Hirano T, 1998; Gloaguen I et al . , 1997) .
• Cross-reactivity between the three systems at the receptor or post -receptor level may serve as an explanation for the link between regulation of body weight by leptin and IL-6 (as well as CNTF) .
• IL-6 mice are preferred for studying infection and mflam- mation.
• the IL-6 mice m this study were back-crossed for 8 generations to a 99.5% pure C57BL/6 background, which may be of importance for the development of the obese phenotype. If so, this raises the question whether the obese phenotype is exclusive for IL-6 _ ⁇ mice with a C57BL/6 background or if it also would be seen m other mice strains deficient for IL-6.
• the weight gam m the IL-6 ⁇ _ mice could be secondary to the development of leptin resistance indicated by this study. If this is the case, one could expect the IL- 6 /_ mice to have a higher level of basal food intake compared to wild-type mice.
• the plasma leptin levels of the IL-6 '/ mice were expected to be higher than m the wild-type mice.
• leptin resistance m the IL-6 ⁇ /_ mice is associated with increased plasma leptin levels. For instance, elevation of plasma leptin is seen m most obese humans with leptin resistance (Flier JS and Foster DW, Williams textbook of endo- crmology 9 th edition) .
• Other measurements of plasma leptin levels m female mice have shown increased levels m the IL-6 " _ mice compared to wild-type mice (Wallenius V and Jansson JO, unpublished results) .
• the increase m body fat caused by IL-6 deficiency was confirmed by fat dissections 18- month-old male (shown Fig. 8 A) and female (shown m Fig. 8 B) mice.
• Four different fat pads were dissected from these mice.
• the male and female IL-6 ⁇ /_ and IL-6 + + mice were first weighed and then three mtra-abdominal fat pads (gonadal, retroperitoneal and mesenteric) and the femoral fat pad (subcutaneous pad m the groin of the thigh) were dissected and weighed. All investigated fat pads, except the male mesenteric fat pad (Fig.
• Example 7 In this example female IL-6 " _ and IL-6 + + mice were treated with IL-6 to see if it was possible to reverse some of the phenotypical changes observed m the IL-6 mice.
• Figure 9 A shows that 18 days of IL-6 treatment reduced body weight to a larger extent m IL-6 mice than m IL-6 + + mice. Quantification of several CT scans performed before the start of IL-6 treatment showed that the mtraperitoneal area was significantly higher m the IL- 6 _/ ⁇ mice compared to the IL-6 + + (Fig. 9 B) . After 18 days of IL-6 treatment the total abdominal area had decreased significantly m the IL-6 _ mice while there was no such effect m the IL-6 + + mice (Fig. 9 B) .
• Intraperitoneal ar- eas were also measured, and they had a similar attenuation on the CT scans as subcutaneous fat. This quantification, excluding non-fat tissues, indicated an even larger increase m the fat content m IL-6 "/" mice compared to the IL-6 + + mice (Fig. 9 C) . There was a signifi- cant decrease the mtraperitoneal areas with fat-like attenuation after IL-6 treatment to the IL-6 _ " mice (Fig. 9 C) . Before IL-6 treatment, leptin levels were almost three times higher m the IL-6 _ ⁇ mice compared to the IL- 6 + + mice. IL-6 replacement for 18 days to the IL-6 _ ⁇ mice caused a significant decrease m leptin levels compared to before treatment .
• CTs computerized tomographies
• pQCT Stratec peripheral quantitative computerized tomography
• XCT Research M software version 5.4B; Norland Medical Systems Inc., Fort Atkinson, WI
• the section was made at the same point all mice, i.e. 5 mm proxi- mally of the cri sta illiaca .

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Animal Behavior & Ethology (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Obesity (AREA)
• Gastroenterology & Hepatology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Immunology (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Epidemiology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Diabetes (AREA)
• Hematology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Child & Adolescent Psychology (AREA)
• Genetics & Genomics (AREA)
• Endocrinology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26663620

Family Applications (1)

Country Status (4)

Families Citing this family (4)

Family Cites Families (2)

• 1999
  • 1999-12-03 SE SE9904424A patent/SE9904424D0/xx unknown
• 2000
  • 2000-07-13 EP EP00950167A patent/EP1194159A1/de not_active Withdrawn
  • 2000-07-13 AU AU63303/00A patent/AU6330300A/en not_active Abandoned
  • 2000-07-13 WO PCT/SE2000/001491 patent/WO2001003725A1/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events