The pharmacodynamic effects of tigilanol tiglate have been investigated in several in vitro and in vivo mice model studies; no pharmacodynamic studies were performed in dogs or on mast cell tumour cells. In these non-clinical pharmacology studies, it was demonstrated that tigilanol tiglate activates the protein kinase C (PKC) signalling cascade. In addition, necrosis is induced in cells that are in direct contact with tigilanol tiglate.

A single intratumoral injection of tigilanol tiglate was shown to elicit a rapid and localised inflammatory response, via activation of PKC, loss of integrity of the tumour vasculature and induction of tumour cell death. These processes led to haemorrhagic necrosis and destruction of the tumour mass.

In dogs treated with tigilanol tiglate, treatment results in an acute inflammatory response with swelling and erythema extending to the tumour margins and immediate surrounds. This acute inflammatory response generally resolves within 48 to 96 hours. Necrotic destruction of the tumour is seen within 4 to 7 days of treatment, but sometimes takes longer. In dogs, this is characterised by blackening, shrinkage and ‘softening’ of the tumour and by a leakage of a thick discharge composed of the tumour remnants and dried blood. The necrotic tumour mass will begin to fall away through the ischaemic surface forming a wound with a pocket or crater-like defect. Healthy granulation tissue then rapidly fills the newly-created wound bed, with full wound closure occurring typically within 4 to 6 weeks.

The efficacy and safety of the veterinary medicinal product was evaluated in a multi-centre, clinical study using 123 client owned dogs with a single mast cell tumour that measured up to 10 cm3 at the time of initial treatment. Only four dogs had tumours with a volume of >8 cm3 , so it is not recommended to use STELFONTA in tumours with a volume greater than 8 cm3.

Dogs aged 1 year or older were included in the study if they were diagnosed with a subcutaneous MCT located at or distal to the elbow or the hock, or with a cutaneous MCT, at WHO stage Ia or IIIa without regional lymph node involvement, or clinical signs of systemic disease. Dogs included had a measurable tumour less than 10 cm3 that was not excoriated or abraded, and which was not a recurrence following surgery, radiation therapy or systemic therapy.

The following concomitant medication was given. Prednisone or prednisolone was initiated 2 days prior to study treatment at a dose of 0.5 mg/kg orally twice daily for 7 days (2 days before, on the day of treatment, and 4 days post treatment), then 0.5 mg/kg once daily for an additional 3 days. Famotidine (0.5 mg/kg orally twice daily) and diphenhydramine (2 mg/kg orally twice daily) were initiated on the day of study treatment and continued for 7 days. Treatment with the veterinary medicinal product was given once on treatment day and again 4 weeks later if any residual tumour was detected. Tumour response was measured via RECIST scores: complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD).

Four weeks after first treatment, 60/80 (75%) achieved a complete response (CR), and another four weeks later CR was observed in 8/18 (44.4%) of remaining dogs that were treated twice. Therefore, overall 68/78 (87.2%) of dogs achieved a CR result after one to two doses of the veterinary medicinal product. Of the treated dogs with CR, which were available for follow up 8 and 12 weeks after the last injection, 59/59 (100%) and 55/57 (96%), respectively, remained disease free at the site of the treated tumour.

Efficacy of the product in high grade tumours (as determined by cytological grading) was only evaluated in a limited number of cases. Ten out of 13 tumours in the study that were categorised as either "high grade" or "suspected high grade" received STELFONTA. Of these, 5 achieved a complete response after 1 or 2 treatments, four of which were still tumour free after 84 days after their final treatment. From the 5 complete response cases, 3 were confirmed being "high grade", and 2 were of "suspected high grade".

In this multi-centre clinical study, 98% of dogs treated with the veterinary medicinal product developed a wound at the site of the treated tumour (an intended reaction to treatment). 56.5% of these wounds were fully healed at 28 days post treatment. By 42 days post treatment, 76.5% of wounds were fully healed. By 84 days post treatment, 96.5% of wounds were fully healed.

Pharmacokinetic parameters of tigilanol tiglate were evaluated in a study monitoring systemic plasma levels of 10 dogs following intratumoral injection into 5 cutaneous and 5 subcutaneous MCTs with the recommended treatment dose. A dose of 0.5 mg/cm3 (= 0.5 ml/cm3 ) tumour volume was used in animals with tumour volumes ranging from 0.1 to 6.8 cm3 , resulting in dose rates ranging from 0.002 to 0.145 mg/kg bodyweight (mean 0.071 mg/kg bodyweight).

Due to varying dose rates and limitations in sampling timepoints, a reliable determination of Cmax and AUC values could not be obtained, but measurements indicated a mean Cmax of 5.86 ng/ml (range: 0.36-11.1 ng/ml) and a mean AUClast of 14.59 h*ng/ml (range: 1.62-28.92 h*ng/ml). Large interindividual variability has been observed when determining half-life following intratumoral injection ranging from 1.24-10.8 hours. Tigilanol tiglate appears to exhibit flip-flop kinetics (sustained release rate) since a considerable shorter half-life of 0.54 hours was determined after intravenous infusion of 0.075 mg/kg in 12 dogs.

In vitro metabolite screening in canine liver microsomes demonstrated a half-life of tigilanol tiglate in hepatocytes of 21.8 minutes and a total of thirteen metabolites. Metabolic products were more polar and oxygenated than the parent compound. Studies have shown some functional group substitutions of this nature resulting in reduced in vitro biological activity (≥60X reduction of activity on PKC compared with parent compound).

The route of excretion of tigilanol tiglate or its metabolites has not been determined. Analysis of urine, faeces and saliva samples from dogs treated with the veterinary medicinal product show the appearance of tigilanol tiglate in isolated samples with no trend or consistency at levels of 11-44 ng/g (ml).