Volume of Distribution Vd

A high volume of distribution (Vd) can be advantageous for targets where efficacy correlates with the ability of compounds to penetrate tissues, for instance, high tumor concentrations for oncology targets and high brain uptake for CNS targets. In addition, for compounds with comparable clearance, a high Vd translates into longer half-life (t1/2) values and better durations of action. In contrast, high volumes may also result in pharmacology based toxicities due to potentially ubiquitously higher tissue exposures.

While volume of distribution is mostly determined via the conventional plasma pharmacokinetics, prediction of Vd has been attempted by a number of laboratories using tissue/ plasma drug concentration ratio, plasma protein binding, lipophilicity (e.g. logD), etc. (Poulin and Theil, 2002; Lombardo et al., 2002 and 2004).

A. Brain uptake

A number of imidazole-based small molecules were identified as antagonists for the melanocortin 4 (MC4) receptor, a CNS target responsible for body weight regulation and energy homeostasis (Vos et al., 2004 and Marsilje et al., 2004). Permeability studies in Caco-2 cells suggested that these molecules are not substrates for efflux pumps (e.g. Pgp). Uptake of these molecules into brain was

Figure 5. Correlation between the brain plasma ratios vs. volume of distributions at steady state (Vss) (R2 = 0.8) for a group of seven MC4 antagonists (♦ one-hour samples, ■ four-hour samples).

Figure 5. Correlation between the brain plasma ratios vs. volume of distributions at steady state (Vss) (R2 = 0.8) for a group of seven MC4 antagonists (♦ one-hour samples, ■ four-hour samples).

evaluated in rats. Compounds were dosed intravenously (IV) to rats (1mg/kg) and the PK parameters CLp, Vd, t1/2, and AUC were calculated. Oral studies at the doses of 10 mg/kg were also conducted and brain tissues were collected. Brain concentrations of drug molecules were measured and brain to plasma ratios were calculated. A good correlation (R2 = 0.8) between the brain to plasma ratios vs. Vd was observed (Figure 5) (Yin et al., 2004). This result led the DMPK team to use Vd values in screening compounds for selective brain uptake studies, which expedited compound advancement to oral efficacy evaluations.

B. RBC partition

PK parameters (e.g. CLp, t1/2, Vd, etc.) of drug molecules are conventionally derived from plasma exposure measurements. This practice can sometimes give rise to misinterpretation of the disposition profiles of drug molecules. ML-X was a lead compound in one of our discovery programs and served as an example of an incorrect conclusion being made from only the plasma PK parameters. ML-X possesses high plasma CLp (8.7 L/hr/kg) and Vd (8.2 L/kg) in rats. However, the rat liver microsomal CLhep was relatively low (~2 L/h/kg) and the renal excretion was insignificant, suggesting that the high CLp may be due to a) the extra-hepatic metabolism, b) plasma instability, or c) high red blood cell (RBC) uptake. ML-X was found to be stable in the rat plasma. The ML-X RBC/plasma partitioning experiment was then conducted and the RBC/plasma ratios of ML-X in rats, mice, and humans were determined to be 28, 9, and 3, respectively. In addition, the whole blood CL (0.13 L/hr/kg) and Vd (0.58 L/kg) of ML-X in rats were determined and shown to be much lower than the plasma CLp and Vd (Figure 6). The RBC binding of ML-X was determined to be tight, concentration dependent, but reversible. Further studies in the blood of xenograft mice suggested that MLX binds to the carbonic anhydrase present in the RBCs (Wong et al., 1994). ML-X bound to RBC can be displaced by its structural analog and by chlorthalidone (Johnston et al., 1981), a sulfonamide diuretic known to strongly bind to carbonic

Figure 6. Whole blood and plasma concentration-time profiles of ML-X in male Sprague-Dawley rats following 1 mg/kg IV bolus dose (■ whole blood concentrations, ▲ plasma concentrations).

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Figure 7. Plasma concentration-time profiles of ML106 in mice (♦), Sprague-Dawley rats (■), dogs (▲), and monkeys (•) following 1 mg/kg IV bolus dose (rats and mice) or 5 min infusion (dogs and monkeys).

Figure 7. Plasma concentration-time profiles of ML106 in mice (♦), Sprague-Dawley rats (■), dogs (▲), and monkeys (•) following 1 mg/kg IV bolus dose (rats and mice) or 5 min infusion (dogs and monkeys).

anhydrases in the cytosol of RBC (Lettieri and Portelli 1983; Xia et al., 2004). Nova screen results also indicate that ML-X binds to carbonic anhydrases I and II with the IC50 value of 0.63 |M and < 0.16 |M, respectively.

The above results suggest that the high plasma CL and Vd of ML-X was a reflection of high RBC/plasma partitioning and tight binding to carbonic anhydrases. The DMPK team decided to screen compounds from this discovery project using whole blood PK parameters.

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