Once the chemical series (pharmacophores with low micromolar in vitro IC50 potency) are selected from the HTS and there is confidence that a predictable SAR relationship can be established, the project is advanced to the HTL stage. Both in vitro ADME experiments and in vivo PK studies will be conducted for at least five compounds per series in parallel to identify DMPK issues and to select appropriate in vitro tools for screening new molecules. In vitro parameters such as projected hepatic clearance CLhep (e.g. derived from the measured intrinsic clearance CLint in microsomes), Caco-2 permeability (Papp), and competitive CYP inhibition (e.g. IC50 values) are collected. PK parameters such as CLp, Vss, CLr, 11/2, Tmax, Cmax, and AUC in rats (a commonly used preclinical PK species) are also determined. Equations 1 and 2 illustrate the calculation of intrinsic clearance (CLint) and predicted "hepatic" clearance (CLhep) using human liver microsomes without considering the unbound fraction:
CLint = (0.693 / in vitro t1/2) x (incubation volume/mg of microsome) x (20
mg microsome/gram of liver) x (45 gm of liver / kg body weight) (1)
where Qh is the human hepatic blood flow. The major contributing factors for the lower plasma exposure or shorter t1/2 (e.g. hepatic and extra-hepatic metabolism, renal and biliary excretion and volume of distribution) will be first determined. If the rat CLp values agree well with the rat CLhep values and the SAR of rat CLhep correlates well with the human CLhep, hepatic metabolism is then regarded as the primary route of elimination and the rat liver microsomal stability experiment will be implemented as the screening tool. If direct conjugations (e.g. glucuronidation and sulfation) are possible, due to the structural features of drug molecules, S9 or hepatocyte stability experiments are carried out instead (Lu, 2004). Other animal models (i.e. mouse, dog, guinea pig and monkey) will be evaluated if rat is not the suitable species for the human PK optimization. For example, if the dog microsomal clearance correlates well with both human microsomal and dog plasma clearance, the dog microsomal assay can then be used as a human clearance model in rank ordering compounds. Biliary excretion may be examined if CLp is less than hepatic blood flow but greater than the predicted CLhep (using microsomes, S9, or hepatocytes) and the renal clearance CLr is low (Qh > CLp > CLhep >> CLr). Extrahepatic metabolism will be investigated if CLp is greater than the Qh and the CLr is low. Caco-2 permeability data can be used to assist in the oral bioavailability assessment, and in some reported cases (Lohmann et al., 2002), brain penetration. An in vivo cassette dosing PK scheme is generally used as the screening tool when renal or biliary excretions are the major routes of clearance.
In general, extrahepatic clearance is evaluated when the CLp is greater than the hepatic blood flow (e.g. >3.3 L/hr/kg for rats). However, surprises do arise sometimes. For example, a series of arylalkyl primary amine compounds were evaluated as potential therapeutic agents in one of our discovery programs. Pharmacokinetic studies at the HTL stage revealed high CLp values for these primary amines in rodents. The in vitro CLhep values (measured in both microsomes and S9 fractions) were much lower than the in vivo CLp values. This poor in vitro/in vivo correlation (IVIVC) was observed in rats, dogs, and monkeys. The CLp value in rats was much higher than the rat liver blood flow (Qh) with less than 20% urinary excretion and less than 2% biliary excretion, suggesting the possibility of extrahepatic metabolic clearance. There was no obvious CYP-mediated metabolism when these alkyl amines were incubated with microsomes from various species. The blood to plasma ratio was close to unity, thus blood clearance was similar to that in plasma. Metabolism of these amines by monoamine oxidases (MAOs) was not observed. Schiff's base formation between these amines and endogenous sugars was also not observed, nor was there any significant in vivo lung first-pass effect. Surprisingly, an N-acetylated metabolite was identified in rat bile. This uncommon (though precedented) N-acetylated metabolite was subsequently observed in rat blood, plasma, urine, hepatocytes, as well as liver subcellular fractions when incubated with acetyl CoA indicating N-acetylation as a primary route of metabolism for the alkyl primary amines in this program. Acetylation may also be the general mechanism of clearance in the rat as N-acetylated ML106 (the lead compound) was detected in rat liver, kidney, and lung S9 incubations. Species specificity was observed with the degree of the N-acetylation much greater in rat S9, followed by monkey and human S9 fractions,
Rat Human Monkey Dog
Figure 4. Species specific N-acetylation of ML106 in rat, human, monkey, and dog liver S9 fractions.
and absence in the dog S9 (Figure 4) (Li et al., 2004). While the identification of the enzyme(s) responsible for the N-acetylation of these arylalkyl amines is in progress, the dog is a species known to lack N-acetyl transferases (NAT1 and NAT2), the enzymes that catalyze the N-acetylation of "aromatic" amines. The high clearance of these primary amines in rats precluded the potential to rank order compounds in this species. The absence of N-acetylation in dogs led the DMPK team to select monkeys as the appropriate animal surrogate for humans to screen new compounds.
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