Kinase experiment:

Dipeptidyl peptidase IV activity is determined using the fluorogenic substrate Gly-Pro-4-methoxy-2-naphthylamide. Hydrolysis of L-Ala-4-methoxy-2-naphthylamide in 60 mM phosphate buffer pH 7.4 is chosen for the determination of aminopeptidase M activity. The intramolecularly quenched fluorogenic substrate Lys (2,4 dinitrophenyl)-Pro-Pro-NH-CH2-CH,-NH,-aminobenzoyl-2HCl is used to assay aminopeptidase P activity. Prolyl oligopeptidase (PO) is measured with N-benzyloxycarbonyl-Gly-Pro-7-amino-4-methylcoumarin. One unit of enzymatic activity is defined as the amount of enzyme catalyzing the formation of 1 μmol of assay product per minute under the conditions used[1].

Animal experiment:

Rabbits[1] Male New Zealand white rabbits (3-4 kg) are used. EXPERIMENT 1 The rabbits (n=1 for each condition) receive a single iv. injection of 10 mg Prodipine or saline. Blood samples are taken 5 min before injection and 30 min, 1 hr, 2 hr, and 24 hr after the treatment. EXPERIMENT 2 The rabbits (n=1 for each condition) receive a single i.v. bolus injection of saline or are treated with 1, 5, or 10 mg Prodipine (also in a single i.v. bolus) at time 0. The animals are kept under observation for 40 days. At several time points (-5 min, 4 hr, and at days 1, 2, 3, 6, 12, 21, and 40), blood samples are taken and body weight determined. Rabbits are killed with pentobarbital after termination of the observation period.

Prodipine, a diphenyl-phosphonate derivative. The IC50s of Prodipine for purified and plasma Dipeptidyl peptidase IV (DPP IV) from the rabbit are 4.5 μM and 30 μM, respectively.

Prodipine, a diphenyl-phosphonate derivative selected from a group of slow-binding irreversible inhibitors of this enzyme. The IC50 values of Prodipine for purified and plasma DPP IV from the rabbit are 4.5 μM and 30 μM, respectively[1].

Male rabbits weighing 3-4 kg receive a single intravenous injection with 10 mg Prodipine or saline. After 1 hr, plasma DPP IV activity has decreased to less than 20% of the preinjection value and remaines unchanged during a 24 hr observation period. A profound and long-lasting inhibition of plasma DPP IV activity is observed in the treated animals (1, 5 or 10 mg). It takes 5 to 8 days to reach half of the pretreatment DPP IV activity and generally more than 20 days for a complete recovery[1].

[1]. De Meester I, et al. In vivo inhibition of dipeptidyl peptidase IV activity by pro-pro-diphenyl-phosphonate (Prodipine). Biochem Pharmacol. 1997 Jul 1;54(1):173-9.