FIELD OF INVENTION

The present invention relates to efficacy studies of the test compound Etanercept in preventing the arthritic pathology developing in the Tg197 humanized mouse model of arthritis.

OBJECT OF INVENTION

The object of this study was to assess the prophylactic effect afforded by recombinant Etanercept (TNFR:Fc) on the development of pathology in the Tgl97, human TNF transgenic murine model of Rheumatoid Arthritis (Keffer et al., 1991). Transgenic mice were separated into 2 groups (G1 & G2) of 8 mice each, as well as an additional group of 16 mice (G3) and received twice weekly intraperitoneal administration of 10|il of test compounds per gram of body weight ,. Treatment was initiated prophylactically, before the onset of arthritis (end of third week of age), and continued for 7 weeks. One additional group of four transgenic mice was sacrificed just prior to the initial dose administration to be used as control for disease onset, individual body weights and arthritic scores were recorded weekly. At 10 weeks of age, mice from all experimental groups were sacrificed and mouse sera and ankle joints were collected. The sera were stored at -80°C to be forwarded for further analysis, whereas the ankle joints were fixed in formalin, decalcified in 30% formic acid and then embedded in paraffin. Haematoxylin/eosin-stained paraffin sections of ankle joints from all experimental animals were assessed histopathologically in a blinded fashion.

SUMMARY OF PRESENT INVENTION

The results of this study show that the test compound Etanercept which was used at 30 mg/kg (Group 3) was very significantly effective in preventing the arthritic pathology developing in the Tgl97 mice compared to mice treated with formulation buffer alone (Group 2). A marked improvement was evident in Group 3 in comparison to Group 2 in all scores measured, namely statistically very significant arthritis inhibition of -60% in histopathology and -80% with in-life clinical measurements. The positive control Enbrel, which was also used at 30mg/kg in Group 1, afforded equivalent very significant inhibition of - 70% with respect to in-life arthritic scores and -60% with respect to histopathological analysis, compared to buffer-treated Group 2. In addition, treatment with the test material Etanercept appeared significantly more efficient in inhibiting clinical oedema in arthritic in-life scores, compared to positive control Enbrel treatment by week 10, although this improvement was not statistically significant in the corresponding histology scores.

None of the treatments was statistically effective in improving the initial clinical and histology scores witnessed by the 3 week control Tgl97 mice. Although the mean histology and arthritic scores at the end of study in the group treated with the test compound
Etanercept, but not the positive control Enbrel, indicated an improvement towards 25% compared to the 3 week-old control group, this effect did not reach statistical significance.

In conclusion, the test material Etanercept proved to be effective in treating the progressive arthritis of the Tgl97 humanized mouse model of arthritis.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Body weight curve: The effect of the treatments on body weight gain of experimental Tgl97 mice. By week 10 mean body weights of the bi-weekly treated groups were as follows: (Gl) Positive control-Enbrel 30mg/kg = 20.9g, (G2) Vehicle/buffer =16.3g, (G3) Test material-Etanercept 30 mg/kg = 21.8g. Bars indicate standard error of the mean.

Figure 2. Arthritic Evaluation: The effect of the treatments on arthritic scores of experimental Tgl97 mice. By week 10 the mean disease severity scores of the bi-weekly treated groups were as follows: (Gl) Positive control-Enbrel 30mg/kg = 0.45, (G2) Vehicle/buffer =1.49, (G3) Test material-Etanercept 30 mg/kg = 0.23. The control mice at week 3 had a score of 0.38. Bars indicate standard error of the mean.

Figure 3. Histopathological evaluation: The effect of the treatments on the histological score of experimental Tgl97 mice. By week 10, the mean histopathological scores of the bi¬weekly treated groups were as follows: (Gl) Positive control-Enbrel 30mg/kg = 1.69, (G2) Vehicle/buffer =3.94, (G3) Test material-Etanercept 30 mg/kg =1.43. The control mice at week 3 had a score of 2.19. Bars indicate standard error of the mean.

Figure 4. Arthritic Score (AS) and Histopathology score (HS) of ankle joints of Tg 197-end of study

DETAILED DESCRIPTION OF THE INVENTION

Drugs: Dilutions of the test compound recombinant Etanercept were received on December 10"^ 2009, together with formulation buffer. In total 36 X 0.55ml vials containing 25mg/ml Etanercept and a polypropylene container with 100ml of formulation buffer were stored at 4°C upon arrival. Enbrel ® was purchased as positive control on the 8"^ of December 2009 ( LOT : E04637, expiry date: 02/2011) and was also stored at 4°C. Dilutions from the appropriate vials were prepared immediately prior to administration according to the company's instructions. Hence, for Ig mouse body weight=IO|a.l injection volume of the appropriate concentration was calculated for the number of mice per group.

Animals: Male Tgl97 mice (maintained in a CBAxC57BL/6 genetic background) were crossed with (CBAXC57BL/6) F1 females. Their heterozygous Tgl97 transgenic offspring were used in this study. Animals were housed and maintained at the facilities of the animal house unit of BSRC "Al. Fleming".

Breeding:
Breeders: Animal house unit, BSRC "Al. Fleming", Athens, Greece.

Housing:
Type of housing: Ventilated cages (Techniplast, UK)
Bedding: Sterilised wood chips
Room No.: Room 8
Animals per cage: 4
Room temperature: 20-23°C
Humidity: 40-70%
Air cycle: Room: 8-12 times/hr
Lighting (light-dark cycle): 10 hour cycle, 5:00 am-7:00 pm light

Feed and drinking water:
Diet: Standard diet
Manufacturer: Altromin (Germany)
Feed supply: Ad libitum
Drinking water: Chlorinated Tap water
Supply of drinking water: Ad libitum
Water dispenser: Plastic, drilled bottle

Animal experiments were performed according to the protocol itemized below: In the beginning of the study, four Tgl97 littermates of the experimental animals were sacrificed and ankle joints were harvested and processed accordingly. Heterozygous Tgl97 mice were divided into 3 groups, weight and gender -matched of 8 mice (4 male and 4 female) each for G1 and G2 and 16 mice (8 male and 8 female) for G3. Each group received twice weekly intraperitoneal injections as follows:

Group 1 received lOpl per gram body weight of Enbrel® (30 mg/kg Etanercept)
Group 2 received 10|xl per gram body weight of formulation buffer
Group 3 received lOpl per gram body weight of test Etanercept (30mg/kg)

The weight of each mouse was recorded and the average weight of each group was calculated weekly, in order to estimate the volume of the tested compounds (10|j1 per gram) to be administered intraperitoneally to mice belonging to the same group.

The first weight measurements and injections were performed on the 16'*' of December 2009. An additional male mouse was included in G2, treated with formulation buffer, as a precaution for possible losses from ensuing morbidity towards the end of the study. Injections two times a week commenced when the animals were 3 weeks old and continued for 7 weeks. Each experimental week began with scoring of macroscopic changes in joint morphology (in-life arthritic scores) on each Wednesday followed by the first weekly injection on Thursday and the second weekly injection on the following Monday.

The end of the in-life stage of the study was carried out on the of February 2010, whereupon all mice were sacrificed at 10 weeks of age. The final dosing (last 9 Week) was on Monday February 1®', 2010 from 11:00 to 11:30am. The sacrifice occurred in numerical order (mouse 1, mouse 2, mouse 3, etc) from 11:00am to 12:30pm preceded by a final measurement score the day before (11:00-11:30) on Wednesday February 3'rd 2010. The sampling time from the last injection to the sacrifice ranged from 72 to 74 hours.

Sera were collected by cardiac puncture and stored at -80°C. Ankle joints were harvested, fixed in formalin, decalcified and embedded in paraffin blocks. Ankle joint paraffin sections were stained with haematoxylin/eosin and a series of sections were prepared to evaluate microscopically histopathological scores.

3.1 Arthritic Scores and Body Weights.

The effect of the tested compounds was monitored by weekly assessment of clinical scores and body weight measurements. Arthritic scores were evaluated weekly macroscopically on ankle joint morphological and functional changes based on the following scoring scale:

Arthritis scoring system:

0 = no arthritis (normal appearance, mouse can support upside its weight, whole body flexibility/ evasiveness normal, grip strength maximum)
0,5= onset of arthritis (mild joint swelling above paw, all other parameters as above) 1,0 = mild arthritis (joint distortion by swelling, inflamed paw, all other parameters as above) 1,5 = mild to moderate arthritis ( joint-paw swelling, distortion + last finger inward deformation, brief support upside its weight borderline yes/no, whole body flexibility reduced, less grip strength)
2,0 = moderate arthritis (severe joint, paw and finger swelling, joint -leg deformation, no support upside its weight falls off, no whole body flexibility, no grip strength, climbing/feeding affected)
2,5= moderate to heavy arthritis (as above 2 + finger deformation in paws, mouse movement impaired)
3,0 = heavy arthritis (ankylosis detected on flexion and severely impaired movement, mouse moribund).

The addition of an extra 0,25 score signifies a tendency towards the next more severe phenotype, i.e. when one, but not all the criteria from the next scale of severity are present. For example, "1,75" means "1,5" with severe swellmg but no joint deformation and some strength on flexion. Arthritic score (AS) and body weight measurements with group average scores are shown in TABLE 1 and TABLE 2:



M32 was found dead on week 7 and was unsuitable for tissue analysis due to cannibalism and decay. The effect of the tested compounds on body weight gain of experimental Tgl97 mice is shown in FIGURE 1. The effect of the tested compounds on arthritic scores of experimental Tgl97 mice is shown in FIGURE 2.

Histopathological scoring system

Specimens were processed and scored as described previously (Douni et al., 2004). The histopathological score of ankle joints was evaluated microscopically using haematoxylin/eosin staining of paraffin sections in a blinded fashion in a scale of 0-4 as follows:

0 = no detectable pathology
1= hyperplasia of the synovial membrane and presence of polymorphonuclear infiltrates 2= pannus and fibrous tissue formation and focal subchondrial bone erosion 3= cartilage destruction and bone erosion 4= extensive cartilage destruction and bone erosion

Half marks were given when some but not all of the features from the next higher score were present. Hence, a score of "2.5" means pannus and fibrous tissue formation and focal subchondrial bone erosion (score 2), with more bone erosion spread outside and around subchondrial foci, but not as broad and with cartilage destruction, as to justify a score "3".

Histopathological score of ankle joints of Tgl97 mice

To monitor disease onset, 4 littermates (coded as Conl-Con4) of the experimental Tgl97 mice used in the present study were sacrificed at 3 weeks of age, which was the treatment starting point. The histopathological analysis showed ankle joint morphology with evidence of arthritis onset, as well as more established arthritic pathology (AS 0.4, HS 2.2). Ankle joints from mouse M32 which died during treatment period on week 7 were not collected since they were not suitable for analysis. At 10 weeks of age, experimental mice were sacrificed and total histopathological analysis of the ankle joints was performed and is presented in tables 4 and 5 as well as in FIGURE 3.


The effect of the tested compounds on histology scores of experimental Tgl97 mice is shown in FIGURE 3, as well as a comparative graph of histology and arthritic scores shown in FIGURE 4.

Statistical analysis on arthritic and histopathology scores

Arthritic and histopathology scores were statistically evaluated at the end of study i.e. at 10 weeks of age. Normality distribution (Gaussian) and variances were checked prior to analysis of variance for multiple groups and pair-wise testing using Tukey's multiple comparison test for statistical significance. This analysis was erroneous due to unequal variances and /or failure in the normality test for a group. The non-parametric Kruskal-Wallis multiple comparison test with Dunn's pair-wise testing when significant differences were observed was performed to confirm significance, shown in (*) in Table 6. Data were also analysed with two-tailed, unpaired Student's t-test, (with Welch's correction where appropriate) for greater power of analysis also shown in Table 6 .

Conclusions

The results of this study show that the test compound Etanercept which was used at 30 mg/kg (Group 3) was very significantly effective in preventing the arthritic pathology developing in the Tgl97 mice compared to mice treated with formulation buffer alone (Group 2). A marked improvement was evident in Group 3 in comparison to Group 2 in all scores measured, namely statistically very significant arthritis inhibition of -60% in histopathology and -80% with in-life clinical measurements. The positive control Enbrel, which was also used at 30mg/kg in Group 1, afforded equivalent very significant inhibition of ~ 70% with respect to in-life arthritic scores and -60% with respect to histopathological analysis, compared to buffer-treated Group 2. hi addition, treatment with the test material Etanercept appeared significantly more efficient in inhibiting clinical oedema in arthritic in-life scores, compared to positive control Enbrel treatment by week 10, although this improvement was not statistically significant in the corresponding histology scores.

None of the treatments was statistically effective in improving the initial clinical and histology scores witnessed by the 3 week control Tgl97 mice. Although the mean histology and arthritic scores at the end of study in the group treated with the test compound Etanercept, but not the positive control Enbrel, indicated an improvement towards 25% compared to the 3 week-old control group, this effect did not reach statistical significance.

In conclusion, the test material Etanercept proved to be effective as the commercially available Enbrel, in treating the progressive arthritis of the Tg197 humanized mouse model of arthritis.

WE CLAIM

1. A method to assess the prophylactic effect afforded by recombinant biomolecule on the development of pathology in the Tg197, human TNF transgenic murine model of Rheumatoid Arthritis.

2. The recombinant biomolecule of claim 1, wherein the biomolecule is a biosimilar protein.

3. The recombinant biomolecule of claim 1, wherein the biomolecule is a biosimilar fusion protein.
4. The recombinant biomolecule of claim 1, wherein the biomolecule is a biosimilar Etanercept, recombinant soluble Tumor Necrosis Factor Alfa receptor (TNFR)- Human IgG Fc fusion protein.

5. Method according to claim 1, wherein the biosimilar Etanercept used at a concentration of about 20 to 40 mg/Kg, preferably 25 to 35 mg/Kg of body weight.

6. Method according to claim 1, concentration of biosimilar Etanercept used is effective in preventing the arthritic pathology developing in the Tgl97 mice.

7. Method according to claim 6, concentration of biosimilar Etanercept used is effective in preventing the arthritic pathology developing in the Tgl97 mice, whereas statistical score of arthritis inhibition ranges between 60-80% in histopathology.

8. Method according to claim 6, concentration of biosimilar Etanercept used is effective in preventing the arthritic pathology developing in the Tgl97 mice, whereas statistical score of arthritis inhibition ranges between 70-90% with in-life clinical measurements.

9. Method according to claim 1, concentration of biosimilar Etanercept used is equally effective in inhibiting clinical oedema in arthritis, in-life scores, compared to positive control treatment by week between 6 to 14, preferably by week between 8 to 12.

10. The test material recombinant biomolecule is at least equally effective as the positive control treatment, in treating the progressive arthritis of the Tgl97 humanized mouse model of arthritis.

11. The recombinant biomolecule of claim 10, wherein the biomolecule is a biosimilar protein.

12. The recombinant biomolecule of claim 10, wherein the biomolecule is a biosimilar fusion protein.

13. The recombinant biomolecule of claim 10, wherein the biomolecule is a biosimilar Etanercept, recombinant soluble Tumor Necrosis Factor Alfa receptor (TNFR)- Human IgG Fc fusion protein.