All animal studies had the approval of the Institutional Animal Ethics Committee of Advinus Therapeutics Ltd. (an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility) and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments CHIR 99021 on Animals (Government of India). Animals were acclimatized in study rooms for at least three days prior to dosing. Hamsters and mice were housed in polypropylene cages (3 per cage, marked for identification), rats were housed singly and dogs were housed in individual pens maintained in controlled environmental conditions

(22 ± 3 °C; 40–70% Relative Humidity; 10–15 fresh air change cycles/h) with 12 h light and dark cycles. All animals were bred in-house except hamsters which were obtained from the Central Drugs Research Institute, Lucknow,

India. Hamsters, mouse and rats were given Ssniff® Rodent pellet food (ssniff Spezialdiäten GmbH, Germany) ad S3I-201 manufacturer libitum and dogs were given Pedigree® standard dog chow (manufactured by Effem India Private Limited, India) 300 g once a day. Good quality water passed through activated charcoal filter and exposed to UV rays was provided ad libitum throughout the study to all animals. In hamsters and mice, blood samples were collected through retro-orbital plexus using a sparse sampling design. In rats and dogs, a serial sampling design was used

with blood samples withdrawn through jugular vein in rats and cephalic vein in dogs. In rats, surgery was performed 48 h before study conduct and no surgery was performed in dogs. The IV solution vehicle comprised 20% (v/v) N-methyl-2-pyrrolidinone Florfenicol (NMP) and 40% (v/v) polyethylene glycol 400 (PEG-400) in 100 mM citrate buffer pH 3. The PO vehicle comprised 7% (v/v) Tween® 80 and 3% (v/v) ethanol in water for hamster and mouse studies. Oral solutions in rat and dog used the same vehicle as IV. Suspension formulations comprised 0.08% (v/v) Tween® 80 in 0.5% (w/v) sodium carboxymethyl cellulose (medium viscosity). The IV dose volume was 1 mL/kg for hamsters, rats and dog and 2 mL/kg for mice. The oral dose volume was 10 mL/kg for hamsters and mice, 5–10 mL/kg for rats and 2–5 mL/kg for dogs. Formulations were prepared on the day of dosing. Rats were anesthetized using 1 mL/kg body weight of a mixture of ketamine (40 mg/mL) and xylazine (4 mg/mL). The depth of anesthesia was assessed by sensory and motor responses. Rats were placed in supine position and a 2 cm ventral cervical skin incision was made on the right side. Tissues were cleaned to visualize jugular vein following which a sterile PE-50 cannula was inserted into the vein and secured in place with a suture. The cannula was exteriorized through the scapulae.