Potency and plasma protein binding of drugs in vitro—a potentially misleading pair for predicting in vivo efficacious concentrations in humans

Abstract

In drug discovery or preclinical stages of development, potency parameters such as IC 50 , K i , or K d in vitro have been routinely used to predict the parameters of efficacious exposure (AUC, C min , etc.) in humans. However, to our knowledge, the fundamental assumption that the potency in vitro is correlated with the efficacious concentration in vivo in humans has not been investigated extensively. Thus, the present review examined this assumption by comparing a wide range of published pharmacokinetic (PK) and potency data. If the drug potency in vitro and its in vivo effectiveness in humans are well correlated, the steady-state average unbound concentrations in humans [ C u_ss.avg = f u ·F·Dose/( CL ·τ) = f u ·AUCss/τ] after treatment with approved dosage regimens should be higher than, or at least comparable to, the potency parameters assessed in vitro . We reviewed the ratios of C u_ss.avg /potency in vitro for a total of 54 drug entities (13 major therapeutic classes) using the dosage, PK, and in vitro potency reported in the published literature. For 54 drugs, the C u_ss.avg / in vitro potency ratios were < 1 for 38 (69%) and < 0.1 for 22 (34%) drugs. When the ratios were plotted against f u (unbound fraction), “ratio < 1” was predominant for drugs with high protein binding (90% of drugs with f u ≤ 5%; i.e., 28 of 31 drugs). Thus, predicting the in vivo efficacious unbound concentrations in humans using only in vitro potency data and f u should be avoided, especially for molecules with high protein binding.

Human Plasma In‐Cell Western Assays—An In vitro Predictor for In vivo Pharmacology in Oncology Drug Discovery

“…As shown in Figure 2C, large discrepancies between in vivo potencies derived from these two methods are observed for highly protein‐bound compounds (human PPB >90%, or f u < 0.1), for which the traditional method tends to underestimate compound activities. This is consistent with the findings of others (Yim, 2019). Consequently, the human plasma ICW assay described in this article represents a novel yet simple method to evaluate in vivo compound potencies more accurately than traditional methods, in anticipation of in vivo pharmacology studies.…”

“…When comparing potencies obtained from the assay described in this protocol to those obtained by simply adjusting f u , i.e., IC 50, human plasma versus IC 50, PPB adjusted , this is an overall agreement between the two methods for compounds that bind less to plasma proteins (PPB <90%, or f u > 0.1). However, for compounds that bind more extensively to plasma proteins (PPB >90%, or f u < 0.1), IC 50, human plasma values are consistently 3-5-fold less than those of IC 50, PPB adjusted , indicating that their in vivo activities may be underestimated by the PPB adjustment technique (Yim, 2019).…”

“…Traditionally, this has been achieved by determining the in vitro potency and percent of human plasma protein binding (PPB), then adjusting the former with the latter to correct for PPB when estimating a dose for in vivo studies. However, this approach can yield inaccurate and misleading findings (Yim, 2019), especially when used to assess test agents that bind extensively to plasma proteins (Fig. 2C and Table 1).…”

“…Combining f u with in vitro cellular potencies, such as the half maximal inhibitory concentration (IC 50 ) against the target of interest, is typically used in a drug discovery program to estimate in vivo activity (IC 50, PPB adjusted = IC 50 /f u ). However, a recent review comparing steady-state average unbound drug concentrations -computed from f u and other pharmacokinetic parameters following treatment with therapeutic doses of approved drugs-to their in vitro potencies revealed that the majority of drugs examined (38/58, 69%) generated a ratio <1, with high protein binding agents (f u < 5%) displaying the most extreme discrepancy (Yim, 2019). This suggests that PPB or f u adjustment may not accurately predict in vivo potencies, especially for high protein binding molecules.…”

Review article: time to revisit Child‐Pugh score as the basis for predicting drug clearance in hepatic impairment

“…This factor will mainly affect the systemic clearance of low clearance or low extraction ratio drugs. 88 It is also important to note that differences in protein binding obtained from in vitro experiments and in vivo situations can be observed, 89,90 and this can contribute to the poor predictive performance for some drugs in HI populations.…”