USE OF AN INHIBITOR OF NTSR1 ACTIVATION OR EXPRESSION FOR PREVENTING WEIGHT LOSS, MUSCLE LOSS, AND PROTEIN BLOOD LEVEL DECREASE IN SUBJECTS IN NEED THEREOF

Abstract

Cachexia is a potentially lethal syndrome afflicting mammals, frequently complicates the treatment of infection, inflammation and cancer. It is characterized by involuntary weight loss, including muscle loss and decrease in protein blood level content. The inventors now show in 2 animal models (mice fed with normal diet and mice fed with high fat diet) that neutralisation of the long fragment of neurotensin with an inhibitor of NTSR1 activation or expression prevents weight loss, muscle loss and protein blood level decrease. Accordingly, the present invention relates to use of an inhibitor of NTSR1 activation or expression for preventing weight loss, muscle loss, and protein blood level decrease in subjects in need thereof.

Claims

1. A method of preventing weight loss, muscle loss and/or protein blood level decrease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of NTSR1 activation or expression.

2. The method of claim 1 wherein the subject is underweight.

3. The method of claim 1 wherein the subject suffers from a wasting disorder selected from the group consisting of anorexia cachexia, anorexia of the aged, anorexia nervosa, cachexia associated with cancer, cachexia associated with AIDS, cachexia associated with heart failure, cachexia associated with cystic fibrosis, cachexia associated with rheumatoid arthritis, cachexia associated with kidney disease, cachexia associated with chronic obstructive pulmonary disease (COPD), cachexia associated with ALS, cachexia associated with renal failure or cachexia associated, and other disorders associated with aberrant appetite, fat mass, energy balance, and/or involuntary weight loss.

4. The method of claim 1 wherein the subject suffers from cachexia.

5. The method of claim 4 wherein the subject suffers from cancer.

6. The method of claim 1 wherein the inhibitor of NTSR1 activation or expression is an antibody.

7. The method of claim 6 wherein the antibody is humanized or human antibody.

8. The method of claim 6 wherein the antibody contains the heavy chain CDRs of the heavy chain variable region of NTSp27.7.4 (SEQ ID NO:3) as represented by SEQ ID NO:7-9 or FLp26-8.2 (SEQ ID NO:4), as represented by SEQ ID NO:13-15.

9. The method of claim 6 wherein the antibody of the present invention comprises the light chain CDRs of the light chain variable region of NTSp27.7.4 (SEQ ID NO:5) as represented by SEQ ID NO:10-12 or FLp26-8.2 (SEQ ID NO:6) as represented by SEQ ID NO:16-18.

10. The method of claim 6 wherein the antibody comprises the heavy chain CDRs of the heavy chain variable region of NTSp27.7.4 (SEQ ID NO:3) and the light chain CDRs of the light chain variable region of NTSp27.7.4 (SEQ ID NO:4).

11. The method of claim 6 wherein the antibody comprises the heavy chain CDRs of the heavy chain variable region of FLp26-8.2 (SEQ ID NO:5) and the light chain CDRs of the light chain variable region of FLp26-8.2 (SEQ ID NO:6).

12. The method of claim 8 wherein the antibody binds to an epitope comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid residues from amino acid residues 123 to 137 of SEQ ID NO: 2 or of SEQ ID NO: 20.

13. The method of claim 1 wherein a competing antibody cross-competes for binding to the epitope comprising amino acid residues from amino acid residues 123 to 137 of SEQ ID NO: 2 or of SEQ ID NO: 20 with the monoclonal antibody comprising a heavy chain comprising the following CDRs: i) the H-CDR1 of NTSp27.7.4 as set forth in SEQ ID NO: 7, ii) the H-CDR2 of NTSp27.7.4 as set forth in SEQ ID NO: 8 and iii) the H-CDR3 of NTSp27.7.4 as set forth in SEQ ID NO: 9 and a light chain comprising the following CDRs: i) the L-CDR1 of NTSp27.7.4 as set forth in SEQ ID NO 10, ii) the L-CDR2 of NTSp27.7.4 as set forth in SEQ ID NO: 11 and iii) the L-CDR3 of NTSp27.7.4 as set forth in SEQ ID NO: 12.

14. The method of claim 1 wherein a competing antibody cross-competes for binding to the epitope comprising amino acid residues from amino acid residues 123 to 137 of SEQ ID NO: 2 or of SEQ ID NO: 20 with the monoclonal antibody comprising a heavy chain comprising the following CDRs: i) the H-CDR1 FLp26-8.2 as set forth in SEQ ID NO: 13, ii) the H-CDR2 FLp26-8.2 as set forth in SEQ ID NO: 14 and iii) the H-CDR3 FLp26-8.2 as set forth in SEQ ID NO:15 and light chain comprising the following CDRs: i) the L-CDR1 FLp26-8.2 as set forth in SEQ ID NO:16, ii) the L-CDR2 FLp26-8.2 as set forth in SEQ ID NO:17 and iii) the L-CDR3 FLp26-8.2 as set forth in SEQ ID NO:18.

Description

FIGURES

[0054] FIG. 1. LF-NTS mAb treatments induced a gain of weight under regular diet. (A) Weight of mice fed with chow treated or not once a weeks with PBS, or 5 mg/kg i.v. LF-NTS mAb n=10 In a two-way ANOVA statistical analysis, was P<0.05 from day 145 to day 247. Inset Graph displaying the data from day 1 to 29. (B) Percentage of weight gained from day 1 by the mouse over time. In t test was p<0.05 from day 150 to day 248.

[0055] FIG. 2. LF-NTS mAb treatment induced an increase in blood albumin level. Ratio albumin/total protein from blood taking on fasted mice for 6 h at day 72 (n=6) comparison between PBS and LF-NTS mAb treated animals. In t test*p<0.05

[0056] FIG. 3. LF-NTS mAb treatment induced an increase mouse physical activity. Mouse activity recorded from individual mouse during 48 h (ADDENFI, Les Cordeliers, Paris, France) at day 63 (A) and day 125 (B). At D300, activity with recorded with an actimeter (Immetronic, Z, France), horizontal activity (C) and rearing (D) were recorded. Recording performed on PBS or LF-NTS mAb treated animals at day 63 (n=6), d125 (n=8), and d300 (n=8) was split in three time period. Int test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0057] FIG. 4. LF-NTS mAb treatment induced an increase the weight of the muscle. At the sacrifice the tibialis (A) and gastrocnemius (B) were dissected and weight (n=9).

[0058] FIG. 5. LF-NTS mAb treatment induced an increase the weight of the major organs. At the sacrifice the Liver (A), spleen (B), pancreas (C) and Inguinal white adipose tissue (D) were dissected and weight (n=9).

[0059] FIG. 6. LF-NTS mAb treatments induced a gain of weight under high fat diet. (A) Weight of mice fed with 60% high fat diet (HFD) treated or not once a weeks with PBS or 5 mg/kg i.v. LF-NTS mAb n=5. In a two-way ANOVA statistical analysis *p<0.05 (B) Percentage of weight gained from day 1 by the mouse over time. In t test p<0.05 at day 75 and 100;

[0060] FIG. 7. LF-NTS mAb treatments induced an increase in food intake. The weight of HFD eaten per cage of 5 animals was calculated. The graph represents the weight of food eaten per week and per animal treated or not with LF-NTS mAb.

[0061] FIG. 8. LF-NTS mAb treatment induced an increase in blood protein and albumin level. Level of albumin or protein from blood taking on fasted mice for 6 h at day 110 (n=5) fed with HFD, and treated or not with LF-NTS mAb. In t test *p<0.05

[0062] FIG. 9. LF-NTS mAb treatment induced an increase mouse physical activity. (A-B) Mouse activity recorded from individual mouse during 48 h (ADDENFI, Les Cordeliers, Paris, france). Recording was split in three time period, the recording was performed at day 53 and day 120 (n=5) of HFD on animals treated with PBS or LF-NTS mAb. (C) Rearing was recorded with an actimeter (Immetronic, France) over a period of 48 h at day 110 on another set of mice with an average weight of 50 g. Int test *p<0.05, **p<0.01, ***p<0.001,

[0063] FIG. 10. LF-NTS mAb treatment induced an increase of the muscle fibers surface. At day 130 after HFD, (A) calculation of gastrocnemius muscle fibers surface performed on 200 fibers (n=7) (B) calculation of tibialis muscle fibers surface performed on 150 fibers (n=6). (C) and (D) Distribution of the muscle fibre size of gastrocnemius or tibialis, respectively, on animal treated or not with LF-NTS mAb. In t test ****p<0.0001.

[0064] FIG. 11. LF-NTS mAb prevent the cachexia induced by cancer. (A) LF-NTS mAb treatment decrease the tumor growth rate of mouse lung carcinoma sub-clone (LLC1-A1) n=10 In 2way Anova *p<0.05. (B) Calculation of the percentage of weight (body without tumor) as compared to the weight at day 1 (day of grafting). In t test *p<0.05. (C) Weight (g) of the carcass (animals without organs, with skin, bones muscles, and head). In t test one tail *p<0.05.

[0065] FIG. 12. LF-NTS mAb treatments prevent the emptying of epididymal white adipose tissues. (A) LF-NTS mAb treatment reduced the weight loss of epididymal white adipose tissues induced by cachexia n=9. In t test *p<0.05 **p<0.01. (B) Size of epididymal white adipocytes from mice nonbearing tumor, mice bearing tumor treated with PBS or LF NTS mAb. In t test *p<0.05 ***p<0.001. (C) Frequency distribution (percentage) of the epididymal white adipocytes from nonbearing tumor mice, or mice bearing tumor treated with PBS or with LF NTS mAb. (n=7)

[0066] FIG. 13. LF-NTS mAb treatments prevent the emptying of retroperitoneal white adipose tissues. (A) LF-NTS mAb treatment reduced the weight loss of retroperitoneal white adipose tissues induced by cachexia n=9. In t test *p<0.05 **p<0.01. (B) Size of retroperitoneal white adipocytes from nonbearing tumor mice, tumor bearing mice treated with PBS or LF NTS mAb, Int test *p<0.05 **p<0.01. (C) Frequency distribution (percentage) of the retroperitoneal white adipocyte from nonbearing tumor mice, tumor bearing mice treated with PBS or LF NTS mAb (n=6).

EXAMPLE

Material & Methods

[0067] Animals

[0068] Male C57BL/6j mice were purchased at 4 or 3 weeks old from (Janvier™) after a week of acclimation mice were separated in two groups and treated once a week i.v. with PBS or 5 mg/kg for 247 days. Mice were fed by regular chow, LASQCdiet ROD-16R LAS vendi or by high fat diet (HFD) 14.6% protein, 58.8% fat, and 26;7% carbohydrate from SAFE (ref 260 HF). All the procedures were in accordance with the “Guide of the Care and Use of laboratory Animals”. Institutional Review Board approval was obtained by «Le Comité d'Ethique en l'Expérimentation Animale Charles Darwin # B751201».

[0069] Plasma Biochemical Analysis.

[0070] Total protein and albumin were determined using a benchtop biochemistry analyzer according to the manufacturers' protocol (Randox Laboratories Ltd, Roissy en France, France).

[0071] Activity

[0072] Global activity was measured on an activity platform to measure the (ADDENFI, Les Cordeliers, Paris, France). Mice were individually housed in a modular chamber (45×35 cm) surrounded by 50 cm-high walls. In this system, the floor plate recover with litter is resting on piezoelectric pressure sensors, providing continuous analog signal generated by the subtle changes in floor-plate pressure due to animal movement. Signals were analysed by Alab suite fida software.

[0073] Rearing was measured in an actimeter (Immetronic, France) composed of eight cages (19×11×14 cm) under low illumination (<5 lx). One mouse was placed in each box and its displacements were measured by photocell beams located across the long axis and above the floor. Rearing activity was recorded during 36 h and expressed in counts/12 h as the total number of interruption of the photocell beams.

[0074] Muscular Fibers and Adipocytes Surface Calculation

[0075] After dissection, the muscle and adipocytes were fixed with 4% paraformaldehyde, and embedded in paraffin wax. Standard haematoxylin and eosin staining was performed. Surface calculation was performed using Image J software

[0076] Tumors

[0077] Eight-week-old male athymic NMRI-Foxn1nu/nu mice (Janvier™) and C57BL/6j (Janvier™) were used. Mice were injected in the flanks with 0.25×10.sup.6 of mouse lung carcinoma cells, LLC1 subclone A1, and allowed to grow until tumors reached a volume of 35 to 40 mm.sup.3 (tumor volume were calculated using ellipsoid formula). Animals were then randomized. Two groups of 10 mice were formed and treated by intra-orbital injections with LF NTS mAb (5 mg/kg once a week), or vehicle (PBS).

[0078] Results

[0079] Effect of LF-NTS mAb on Mice Fed with Normal Diet.

[0080] Effect of LF-NTS mAb treatment was evaluated on C57BL/6j mice treated once a week with 5 mg/kg, or PBS for 247 days, and under normal diet. The first injection was performed when the mice were 5 weeks old. As seen on FIG. 1A the mice treated with NTS mAb put on weight along the treatment as compared to control mice. The percentage of weight gained was calculated from day 1. The inset graph shows that the gain of weight start just after the first injection and was significant at day 24. The FIG. 1B shows that the percentage of gained weight is increasing with time. The average increase rate was of 22%. No difference in food intake was observed (Data not shown). Basic blood parameters, glucose cholesterol, and triglyceride levels were not modified by the treatment (Data not shown). The weight of feces collected during 48 h was equivalent (Data not shown). The ratio albumin to protein was significant 17% higher in LF-NTS mAb treated mice as compared to control (FIG. 2). Mice treated with LF-NTS mAb are more active than PBS treated mice, this increase and persist with time. At day 63 and 125 the global activity was measured with the ADDENFI cage (FIGS. 3A and 3B) At day 300 the actimeter measurement revealed that rearing was preferentially increased (FIGS. 3C and 3D). At the sacrifice, the weight of muscle, tibialis and gastrocnemius (FIGS. 4A and 4B), as well as the major organs, liver, pancreas, spleen, and inguinal white adipose tissue (Iwat) was also increased with the LF-NTS mAb (FIGS. 5A to 5D)

[0081] Effect of LF-NTS mAb on Mice Fed with High Fat Diet.

[0082] Similar experiment was performed on C57BL/6j mice treated once a week with 5 mg/kg, or PBS for 120 days, and under high fat diet. After weaning the mice were on chow for a week and at 4 week old mice the food was switch to HFD. The first injection was performed two days before the diet switch. As seen previously the mice treated with NTS mAb put on weight along with the treatment as compared to control mice (FIGS. 6A & 6B), the percentage of gained weight is increasing with time the average increase rate was of 19%. Food intake was evaluated every 10 days, mice treated LF-NTS mAb ate 30 to 50% more than the control mice (FIG. 7). Blood sample taken after 110 day of diet and treatment showed that protein and albumin were 34 and 18% higher respectively when mice were treated with antibody (FIG. 8). Finally, the mice under NTS-LF mAb were also more active as shown in FIG. 9, in particular the exploratory behavior induced by novelty, rearing was strongly increased in mice treated with mAb (FIGS. 9A to 9C). This more active behavior affects muscle fibers size of the gastrocnemius and the tibialis. The sire of the fibers was 34 and 110% higher in the gastrocnemius and the tibialis, respectively (FIGS. 10A and 10B). The distribution of the fiber sizes shown in FIGS. 10C and 10D show that globally all the muscle fibers have grown; the small fibers have almost disappeared in the animals treated with the antibody.

[0083] Effect of LF-NTS mAb on Tumor Bearing Mice

[0084] We tested the LF-NTS antibody on tumor growth of LLC1-A1 sub-clone. We observed a diminution of the tumor growth rate (FIG. 11A). Eighteen days after grafting the animals were sacrificed and weighed. The tumor bearing animals lost 7.67±3.4% of their weight as compared to the weight before grafting. This weight lost was not observed in the mice-bearing tumor and treated with LF-NTS mAb, or mice with no tumor (FIG. 11B). The weight of the carcass was also diminished when mice were treated with PBS, as compared to mice treated with LF NTS mAb, or mice not bearing tumors (FIG. 11C). The LLC1-A1 cells grafted in mice induced cachexia and the LF NTS mAb inhibited the weight lost.

[0085] Epididymal (EWAT) and the retro-peritoneal (RPWAT) white adipose tissues were weighted after sacrifice. In animals bearing tumors, both EWAT and RPWAT were lighter as compared to animals nonbearing tumors (FIGS. 12A & 13A), indicating that this model is able to induce cachexia. When animals are treated with LF-NTS mAb the EWAT and RPWAT were heavier (FIGS. 12A & 13A). We calculated the size of the adipocytes of EWAT and RPWAT. We observed that the adipocytes from animals bearing tumors are small with the highest frequency at 600 to 800 μm.sup.2, and 400 to 600 μm.sup.2 for EWAT and RPWAT, respectively (FIGS. 12B and 13C & 13B and 13C). Whereas, in animals bearing tumors, treated with LF NTS mAb, the size of EWAT was equivalent to the EWAT from nonbearing tumors animals, with a highest frequency of 1300 μm.sup.2 (FIGS. 12B and 12C). LF-NTS mAb prevent partially the emptying of the adipocytes of the RPWAT. The highest frequency was around 900 μm.sup.2 and 1500 μm.sup.2 for animals treated with LF NTS mAb and animals without tumors, respectively (FIGS. 13B and 13C). These observations on adipocytes confirm the hypothesis as LF NTS mAb counteract cachexia induced by cancer.

REFERENCES

[0086] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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