9th Euro-Global Summit on Toxicology and Applied Pharmacology Paris. France June 22-24. 2017 Comparative repeated toxicity study in rats: enoxaparin biosimilar product versus reference product. Zina Kobbi Pharm D. PhD Faculty of Pharmacy of Monastir. university of Monastir. Tunisia Principal consultant of “AREMA”

1- Introduction: Tunisian context/ Biosimilars Content 1- Introduction: Tunisian context/ Biosimilars 2- Method 2-1-Products: Enoxaparine (biosimilar vs reference) 2-2-Lab Animal: Wistar Rats 2-3-Strategy: repeated toxicity 2-4-Tests: comparative assays 3- Results 4- conclusion and perspectives

1- Introduction Tunisia is amoung first African countries to developp active pharmaceutical industries. Since 1990/ 36 units (human). Two pharma industries developping biological products and have arround 10 products under developpment.

1- Introduction Expiry of many patents of biologicals Explosion of biosimilars/ biosimilar pharmaceutical industries. Requirements and approval of biosimilars by the competent authorities not yet harmonized : a real dilemma. « enoxaparin » Europe (EMA) and USA (FDA) : different requirements.

Big challenge! 1- Introduction Our study : set up the first preclinical study in Tunisia according international standards. part of enoxaparin biosimilar evaluation Big challenge! Comparative repeated toxicity study in rats of Enoxaprine (biosimilar versus innovator). Preparation (3 years): setting up protocol , order of materials and reagents, animals selection and preparation, pilot studies …

2- Method 2-1-Products Enoxparin test drug product. a similar biological medicinal drug products, at 4000 IU anti-Xa/0.4 ml : Enoxa; manufactured by les Laboratoires Médis (Tunisia). Enoxparin reference drug product, at 4000 IU anti-Xa/0.4 ml: Lovenox;  manufactured by Sanofi-Aventis (France). Control product: sodium chloride solution at 0.9 %.

2- Method 2-2-Animals White Wistar rats, Forty (40) rats of each sex, six to seven-week-old 14 days quarantine/acclimation period Randomly assigned One (1) control group Two (2) treatment groups

Animals and facilities : 2- Method 2-3-Strategy Animals and facilities : Pasteur institute of Tunis ( different services) National veterinary university of Sidi Thabet (Tunis)  Laboratory of biochimestry at Child hospital of Tunis Biosimilar (Enoxa) was subject to a comparative 2 and 4 weeks repeated toxicity study in vivo with reference product (Lovenox) To detect differences in toxicological response To assess similarity in the safety profile

2- Method 2-3-Strategy

2- Method 2-4-Tests Animal observation: Hematology: Biochemistry: Once daily for morbidity and twice daily for mortality (Montgomery )/ Body weight Hematology: On day 14 and day 28 Parameters measured by MINDRAY, auto Hematology analyzer, BC-2800Vet Biochemistry: On day 14 and day 28 Parameters measured by roche cobas 6000

Organ-to-body weight ratios 2- Method 2-4-Tests Terminal procedure. organ weight: Necropsy (all rats) Organs trimmed and weighed: the liver, brain, lung, kidney and heart. Organ-to-body weight ratios Anatomo-histopathology : The organs from all groups Fixed in 10 % formalin solution, embedded in paraffin, sliced (2 to 4 µm) and stained with hematoxylin-eosin for microscopic analysis

2- Method Statistical analysis On: body weight, hematology and biochemistry parameters, organ-to-body weight ratios. Method: XLSTAT, Kruskal-Wallis test and bonferroni correction.

3- results Food consumption and body weight: Clinical observation: No major effect of the drug product or drug dosage Body weight depends mainly on animal sex/ fairly constant in female rats and increased in male rats. Clinical observation: At dose of 100 mg/kg/jour Mortality 25 % (4) rats under biosimilar/ reference (3 days)

3- results Clinical observation: At dose of 100 mg/kg/jour Severe toxic effects (fig): fig a and b  nodule/ hemorrhagic cyst ; fig c and d: hematoma/ bluish coloration ; fig e genital swelling ; fig f : diarrhea

3- results Clinical observation: At dose of 40 mg/kg/jour One rat died under biosimilar At dose of 20 mg/kg/jour Adverse effects/ Bluish coloration

Product (dose mg/kg/j) No significant statistical difference 3- results Hematology: 14 days (male group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 WBC (109 /L) 15.4 ±3.54 14.9 ±2.76 11.72 ±3.53 12.97 ±4.75 12.17 ± 1.24 Lymphocytes (109/L) 9.2 ± 2 8.32 ± 2.35 7.07 ± 2.02 8.1 ± 2.86 Lymphocytes (%) 59.60±2.92 55.25±6.70 60.28±1.13 62.45±3.54 Monocyte (109 /L) 0.53±0.19 0.60±0.20 0.40±0.14 0.45±0.19 Monocyte (%) 3.65±0.41 3.88±0.55 3.68±0.22 3.53±0.34 Granylocytes (109/L) 5.68±1.45 5.98±0.66 4.25±1.38 4.43±1.80 Granylocytes (%) 36.75±3.27 40.88±6.91 36.05±1.01 34.03±3.47 RBC (1012 /L) 10.21±0.43 9.67±0.39 9.66±0.56 9.19±0.85 9.78±0.59 Hemoglobin (g/dL) 16.75±0.70 16.08±0.50 15.38±1.05 15.53±1.14 Hematocrit (%) 65.25±2.47 62.35±2.18 62.20±4.15 62.13±5.19 65.43±0.95 MCV ( fL) 64.00±0.83 64.60±3.11 64.43±0.98 67.70±0.85 67.15±4.31 MCH (pg) 16.38±0.13 16.60±0.57 15.88±0.46 16.85±0.34 16.60±0.94 MCHC (g/dL) 25.60±0.36 25.73±0.43 24.68±0.46 24.93±0.30 24.80±0.36 RDW (%)   11.43±0.45 11.63±1.73 11.65±0.91 11.73±0.87 13.20±1.06 PLT (109 /L) 846.75±23.3 808.67±82.6 755.00±37 760.00±38.3 875.67±76.04 MPV (fL) 6.23±0.13 6.58±0.26 6.43±0.05 6.68±0.38 6.60±0.22 PDW 14.88±0.10 15.25±0.44 15.05±0.13 15.38±0.19 15.30±0.14 pct (%) 0.53±0.01 0.56±0.09 0.48±0.02 0.53±0.08 0.58±0.04 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference. No significant statistical difference

Product (dose mg/kg/j) No significant statistical difference 3- results Hematology: 14 days (female group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 WBC (109 /L) 12.67 ± 2.1 9.47 ± 1.2 9.82 ± 2.45 8.77 ± 2.23 9 ± 1.12 Lymphocytes (109/L) 7.35 ± 2.1 4.85 ± 0.75 5.15 ± 1.4 5.25 ± 1.43 4.72± 0.95 Lymphocytes (%) 58.00±4.48 51.08±4.20 52.03±7.42 59.45±2.57 52.20±5.67 Monocyte (109 /L) 0.38±0.05 0.40±0.00 0.28±0.05 0.30±0.00 Monocyte (%) 3.35±0.40 4.25±0.62 4.10±0.42 3.68±0.44 3.60±0.34 Granylocytes (109/L) 4.95±1.23 4.23±0.68 4.30±1.42 3.25±0.81 3.98±0.56 Granylocytes (%) 38.65±4.48 44.68±4.25 43.88±7.70 36.88±2.29 44.20±5.43 RBC (1012 /L) 9.59±0.74 8.62±0.67 9.11±0.31 8.51±0.86 9.08±0.38 Hemoglobin (g/dL) 16.15±0.89 15.08±1.35 15.55±0.71 14.60±1.92 15.45±0.10 Hematocrit (%) 64.63±3.56 58.03±5.02 62.98±2.98 59.58±7.94 61.30±0.84 MCV ( fL) 67.50±1.63 67.38±1.86 69.23±3.83 69.88±3.37 67.65±2.81 MCH (pg) 16.80±0.40 17.45±0.59 17.05±0.90 17.08±0.78 16.98±0.75 MCHC (g/dL) 24.95±0.13 25.93±0.19 24.68±0.21 24.45±0.17 25.15±0.24 RDW (%) 9.60±0.33 11.00±2.62 11.98±2.17 11.78±1.91 13.20±1.06 PLT (109 /L) 800.25±65 811.67±56.8 768.33±27.7 779.0±161.2 950.67±50.65 MPV (fL) 6.35±0.21 6.40±0.22 6.25±0.06 6.18±0.31 6.40±0.14 PDW 15.13±0.13 15.23±0.17 15.13±0.10 15.10±0.26 15.28±0.13 pct (%) 0.51±0.05 0.56±0.10 0.50±0.10 0.62±0.03 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference. No significant statistical difference

Product (dose mg/kg/j) 3- results Hematology: 28 days (male group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 WBC (109 /L) 11.42± 1.92 10.85± 1.55 12.95± 3.44 10.125± 1.33 17.55± 7 Lymphocytes (109/L) 7.42 ± 1.36 6.5 ± 0.9 7.12 ± 0.97 5.9 ± 0.92 11.5 ± 5.24 Lymphocytes (%) 65.15±6.49 59.90±4.14 56.30±7.30 58.35±1.81 64.75±4.03 Monocyte (109 /L) 0.35±0.06 0.68±0.10 0.88±0.36 0.80±0.22 0.75±0.35 Monocyte (%) 3.10±0.47 6.50±0.39* 6.83±1.32 * 7.58±1.62 * 4.05±0.49 Granylocytes (109/L) 3.65±1.05 3.68±0.78 4.95±2.21 3.43±0.33 5.30±1.41 Granylocytes (%) 31.75±6.08 33.60±4.34 36.88±6.67 34.08±1.96 31.20±4.53 RBC (1012 /L) 9.93±0.70 8.51±0.54 9.08±0.33 9.16±0.34 8.71±1.35 Hemoglobin (g/dL) 16.53±1.11 14.30±0.88 15.73±0.49 15.40±0.63 14.83±2.10 Hematocrit (%) 64.35±4.05 38.68±2.88 43.00±1.53 42.20±1.88 57.97±8.28 MCV ( fL) 64.93±1.67 45.50±0.48 47.40±0.49 46.13±0.82 66.73±1.43 MCH (pg) 16.63±0.46 16.75±0.13 17.28±0.22 16.78±0.13 17.03±0.32 MCHC (g/dL) 25.63±0.15 36.95±0.54 36.53±0.21 36.45±0.70 25.53±0.06 RDW (%) 11.65±1.19 10.70±0.74 12.75±2.04 10.90±0.58 12.83±2.59 PLT (109 /L) 831.25±64.2 397.50±39.9 412.50±39.3 450.50±51.9 539.50±84.15 MPV (fL) 6.10±0.14 5.33±0.22 5.45±0.13 5.33±0.19 6.57±0.40 PDW 14.90±0.08 14.35±0.19 14.48±0.17 14.38±0.17 15.33±0.25 pct (%) 0.51±0.05 0.21±0.03 0.22±0.02 0.24±0.04 0.64±0.03 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference. Monocyte decreased for rats receiving test and reference drug products when compared to control

Product (dose mg/kg/j) 3- results Hematology: 28 days (female group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 WBC (109 /L) 10.15 ± 0.61 9.02 ± 1.86 9.15 ± 2.55 9.52 ± 1.66 10.65 ± 1.91 Lymphocytes (109/L) 6.45± 0.38 4.55 ± 1.12 5.47 ± 1.14 4.97 ± 0.92 6.02± 1.24 Lymphocytes (%) 63.18±1.58 50.03±5.40 60.20±4.34 52.58±7.74 56.60±4.45 Monocyte (109 /L) 0.28±0.05 0.53±0.15 0.58±0.22 0.65±0.25 0.55±0.21 Monocyte (%) 3.35±0.42 5.93±1.21 6.40±0.67 6.88±1.89 5.05±1.35 Granylocytes (109/L) 3.43±0.30 3.95±0.86 3.10±1.22 3.90±1.14 4.08±0.72 Granylocytes (%) 33.48±1.96 44.05±6.56 33.40±3.86 40.55±6.80 38.35±3.16 RBC (1012 /L) 8.92±0.21 8.81±0.10 8.64±0.37 8.32±0.63 8.56±0.55 Hemoglobin (g/dL) 14.88±0.41 15.23±0.31 15.25±0.82 14.18±1.46 15.33±0.81 Hematocrit (%) 58.85±1.64 41.60±0.79 41.95±2.11 38.78±4.04 51.70±9.46 MCV ( fL) 66.03±0.61 47.30±0.68 48.63±1.03 46.60±1.48 60.73±12.08 MCH (pg) 16.60±0.08 17.20±0.23 17.58±0.40 16.98±0.54 17.88±0.46 MCHC (g/dL) 25.23±0.21 36.55±0.58 36.33±0.15 36.50±0.26 30.40±6.18 RDW (%) 9.55±0.34 10.38±0.59 10.45±0.41 10.38±0.29 12.63±2.08 PLT (109 /L) 934±78.97 365±32.28* 380±83.54 417.5±63.94 691.75±31.94 MPV (fL) 6.33±0.13 5.33±0.13 5.43±0.34 5.45±0.21 5.73±0.43 PDW 15.13±0.10 14.43±0.10 14.53±0.32 14.48±0.29 14.75±0.31 pct (%) 0.59±0.06 0.19±0.01 0.20±0.04 0.23±0.04 0.41±0.21 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference. Platelet decreased for rats receiving test and reference drug products when compared to control.

Changes only in Platelet and monocytes. 3- results Hematology: Changes only in Platelet and monocytes. Changes only between test and reference drug products when compared to control. No statistically significant differences in all parameters between rats of both sexes treated with test and reference products.

Product (dose mg/kg/j) No significant statistical difference 3- results Biochemistry: 14 days (male group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 ALP (U/L) 104.25±17.06 128.75±19.29 105.00±17.61 143.50±39.82 131.75±30.54 ALT (U/L) 62.05±6.96 62.18±22.01 67.55±12.82 73.73±13.04 62.40±13.90 AST (U/L) 178.98±15.75 141.25±23.46 137.25±26.82 138.13±31.54 154.53±34.90 BUN (mg/dL) 7.43±0.96 6.83±1.72 7.73±1.06 7.00±0.74 6.98±0.53 Triglyceride (mg/dL) 1.96±0.52 1.40±0.22 1.37±0.60 0.59±0.16 0.90±0.53 total protein (/dL) 72.23±1.36 72.90±6.01 67.40±6.00 77.55±12.85 69.83±6.21 Creatinine (mg/dL) 0.26±0.02 0.29±0.06 0.23±0.06 0.20±0.13 Total cholesterol (mmol/L) 1.45±0.13 1.11±0.53 1.21±0.22 1.61±0.13 1.33±0.21 Total bilirubin (U/L) 0.89±0.41 0.99±0.08 0.70±0.21 0.67±0.19 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference.   No significant statistical difference

Product (dose mg/kg/j) No significant statistical difference 3- results Biochemistry: 14 days (female group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 ALP (U/L) 61.33±24.85 55.75±13.96 75.25±14.24 46.50±25.25 54.25±17.61 ALT (U/L) 62.97±1.25 43.50±6.93 54.18±10.89 51.33±12.01 62.70±17.84 AST (U/L) 158.30±22.63 151.18±42.87 106.95±13.21 276.85±29.11 133.20±30.17 BUN (mg/dL) 8.73±2.75 6.35±0.67 6.95±1.99 9.90±1.99 6.83±1.24 Triglyceride (mg/dL) 1.08±0.40 0.91±0.24 0.59±0.22 1.12±0.21 0.50±0.12 total protein (/dL) 75.17±1.86 73.15±2.79 71.60±1.62 66.88±5.46 78.38±3.69 Creatinine (mg/dL) 0.22±0.15 0.36±0.04 0.27±0.03 0.45±0.09 0.28±0.03 Total cholesterol (mmol/L) 1.25±0.11 1.01±0.27 1.12±0.51 1.61±0.46 1.29±0.19 Total bilirubin (U/L) 1.05±0.45 0.85±0.34 0.69±0.21 0.98±0.47 0.80±0.47 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference. No significant statistical difference

Product (dose mg/kg/j) No significant statistical difference 3- results Biochemistry: 28 days (male group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 ALP (U/L) 129.00±2.83 122.00±10.80 168.25±42.74 170.00±51.08 194.67±45.08 ALT (U/L) 64.08±45.76 47.65±8.38 64.93±8.05 71.38±7.42 82.57±17.05 AST (U/L) 128.90±67.61 98.53±14.43 120.95±41.18 145.60±69.45 132.90±49.36 BUN (mg/dL) 6.98±1.33 5.53±0.63 6.40±0.88 6.05±0.79 7.43±0.83 Triglyceride (mg/dL) 2.51±1.97 1.78±1.13 3.80±1.23 0.90±0.19 1.15±0.27 total protein (/dL) 79.05±2.71 75.18±8.21 82.60±6.83 86.18±3.81 82.53±9.79 Creatinine (mg/dL) 0.25±0.05 0.22±0.02 0.25±0.03 0.21±0.03 0.15±0.11 Total cholesterol (mmol/L) 1.90±0.28 1.62±0.38 1.98±0.25 2.24±0.75 1.97±0.47 Total bilirubin (U/L) 0.74±0.17 0.77±0.47 0.89±0.17 0.59±0.37 0.97±0.17 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference. No significant statistical difference

Product (dose mg/kg/j) No significant statistical difference 3- results Biochemistry: 28 days (Female group): Parameter (units of measure) Product (dose mg/kg/j) Control Reference 3.5 Test 3.5 Reference 20 Test 20 ALP (U/L) 79.33±17.62 87.75±24.43 71.00±21.37 82.75±11.79 89.25±27.73 ALT (U/L) 71.30±11.75 66.20±6.01 68.73±20.79 82.23±9.68 70.13±18.62 AST (U/L) 114.40±11.53 92.00±10.49 118.03±51.65 108.00±19.88 103.67±13.20 BUN (mg/dL) 7.60±1.25 5.60±0.98 5.80±0.73 6.03±1.39 6.48±0.63 Triglyceride (mg/dL) 2.63±0.68 1.83±0.65 3.02±1.57 0.73±0.13 0.93±0.30 total protein (/dL) 87.87±5.35 82.88±2.13 87.45±5.80 81.28±3.62 78.93±4.18 Creatinine (mg/dL) 0.20±0.13 0.30±0.02 0.29±0.03 0.28±0.02 0.23±0.03 Total cholesterol (mmol/L) 2.06±0.17 1.39±0.24 2.10±0.47 1.55±0.30 1.34±0.27 Total bilirubin (U/L) 0.54±0.26 1.22±0.19 1.20±0.81 1.14±0.25 0.73±0.35 * Significant difference when test or reference compared with control. ** Significant difference when test compared with reference. No significant statistical difference

3- results Biochemistry: Alanine aminotransferase increased after 28 days of injections in rats of both test and reference drug products and with doses 20mg/kg/day when compared to control and to doses 3.5 mg/kg/day. No statistically significant differences were observed in all parameters between rats of both sexes treated with test and reference products.

Product (dose mg/kg/j) No significant statistical difference 3- results Organ weights: 14 days groups Product (dose mg/kg/j) Organ (%) Control Reference 3.5 Test 3.5 Reference 20 Test 20 Males   Kidney 0.85±0.06 0.53±0.37 0.89±0.11 0.80±0.08 0.78±0.09 Lungs 0.76±0.08 0.91±0.08 0.88±0.12 0.82±0.32 0.68±0.46 Heart 0.32±0.03 0.30±0.20 0.40±0.06 0.32±0.01 0.32±0.04 Brain 0.47±0.10 0.43±0.30 0.63±0.07 0.58±0.04 0.59±0.04 Liver 3.24±0.27 2.40±1.74 3.92±0.59 3.09±0.22 3.02±0.17 Females 0.84±0.04 0.90±0.06 0.85±0.10 0.71±0.47 0.74±0.15 0.60±0.43 0.88±0.15 0.82±0.19 0.57±0.14 0.42±0.06 0.42±0.04 0.41±0.05 0.30±0.21 0.77±0.14 0.93±0.07 0.83±0.06 0.86±0.09 2.94±0.39 3.12±0.75 3.41±0.20 3.43±0.25 3.12±0.09 No significant statistical difference

Product (dose mg/kg/j) No significant statistical difference 3- results Organ wheights: 28 days groups Product (dose mg/kg/j) Organ (%) Control Reference 3.5 Test 3.5 Reference 20 Test 20 Males   Kidney 0.94±0.10 0.85±0.12 0.79±0.08 0.98±0.05 0.95±0.11 Lungs 0.79±0.10 0.92±0.06 0.80±0.09 0.81±0.09 0.78±0.16 Heart 0.40±0.06 0.38±0.03 0.34±0.03 0.35±0.03 0.36±0.04 Brain 0.57±0.09 0.50±0.07 0.48±0.06 0.55±0.03 0.55±0.06 Liver 4.84±0.47 3.82±0.24 4.86±0.57 5.05±0.52 5.73±0.65 Females 0.99±0.11 0.92±0.04 0.82±0.03 0.96±0.04 0.90±0.05 0.85±0.32 0.50±0.04 0.52±0.05 0.80±0.18 0.70±0.12 0.39±0.06 0.42±0.05 0.39±0.04 0.38±0.02 0.39±0.05 0.66±0.13 0.82±0.05 0.71±0.05 0.74±0.08 0.72±0.04 5.37±0.83 4.47±0.43 3.82±0.19 4.43±0.28 4.59±0.53 No significant statistical difference

3- results Histopathology: Female rats (28 days). Scalebar:100 mm (40 x magnification). Liver. lung. heart. kidney. and brain from control product (A. D. G. J. and M. respectively); from test product at 20mg/ kg/day (B. E. H. K. and N. respectively). and from reference product at 20 mg/kg/day (C. F. I. L. and O. respectively). Comparable features No remarkable changes

3- results Histopathology: Histopathology of lung from a control rat showing interstitial alveolar hemorrhage. Scale bar: 100 mm (40 x magnification) Change discarded as observed also in control groups and not due to product but caused by CO2 used in anesthesia.

3- conclusion Results from different doses and analysis performed on rats of different groups treated with biosimilar and reference (innovator) product are similar. No adverse effects were observed at doses of 3.5mg/kg/day and doses of 5mg/kg/day and few adverse effects were observed at doses 20 mg/kg/day and mortality at doses 100 mg/kg/day and 40mg/kg/day.

3- conclusion Taken together, these findings suggest that the similar biological version of enoxaparin drug product “Enoxa” and reference product “Lovenox” have comparable safety profile in rats. The similar biological product is considered suitable for human trial to continue clinical investigation of biosimilarity, using appropriate clinical studies to confirm its safety and efficacy.

Perspectives Local tunisian /African /middle east countries, could develop complicated biosimilar products. First demonstration of the ability to acess bio-safety of biosimilar products Assesment of comparative toxicity profile is the starting point and could enable performing additional human trial to confirm safety and efficacy of these products Harmonization of worldwide biosimilar guidelines could help strategic decision for pharmaceutical companies and commercial facilities that provide animal-testing services.

This work was published in the journal: Regulatory Toxicology and Pharmacology 84 (2017) 9-17 Comparative subcutaneous repeated toxicity study of enoxaparin products in rats Kobbi Zina. Kraiem Hazar. Benlasfar Zakaria. Marouani Ammar.Massoud Taieb. Boubaker Samir. Bouhaouala-Zahar Balkiss and Fenina Nadia (Faculty of pharmacy of Monastir and Pasteur Institute of Tunis )

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