B

Figure 5.4. (a) Caco-2 cell permeability (A to B) for a list of compounds within a research program. Permeability values were obtained using Caco-2 cells cultured for ~21 days followed by transport studies at 200 |M. (b) Bi-directional Caco-2 cell permeability and PAMPA permeability values for some randomly selected compounds within the program. PAMPA study was performed at 100 |M

Compound number

Figure 5.4. (a) Caco-2 cell permeability (A to B) for a list of compounds within a research program. Permeability values were obtained using Caco-2 cells cultured for ~21 days followed by transport studies at 200 |M. (b) Bi-directional Caco-2 cell permeability and PAMPA permeability values for some randomly selected compounds within the program. PAMPA study was performed at 100 |M

absorption characteristics of these compounds. A randomly selected set of compounds with Caco-2 cell permeability values lower than 100nm/s was evaluated for their PAMPA permeability as well as their bidirectional permeability in Caco-2 cell model. The results from these two permeability models are shown in Fig. 5.4b. The combination assay demonstrated that the low A to B Caco-2 cell permeability of many compounds was due to the fact that they are substrates of P-gp (i.e., B to A permeability is much higher than A to B permeability) rather than due to low intrinsic permeability of the compound. The high PAMPA permeability coupled with low Caco-2 cell permeability would indicate an active efflux process (in Caco-2 cells) and not necessarily low intrinsic permeability of the test compound. It can be expected that these compounds may have more than adequate intestinal permeability if intestinal P-gp is saturated due to high drug concentration in the gut. In fact, many of these compounds were shown to exhibit good oral bioavailability in preclinical animal models. Despite being a substrate of P-gp, these compounds were well absorbed in vivo, and the high PAMPA permeability value was reflective of in vivo absorption.

A combination of PAMPA and unidirectional (A to B) Caco-2 cell permeability models can synergistically provide invaluable permeability/absorption assessment of test compounds. If both PAMPA and A to B Caco-2 cell permeability are low, then the compound can be deprioritized to have low intrinsic permeability and thus likely to have poor absorption in humans. However, if A to B Caco-2 cell permeability is low but PAMPA is high, then the compound is likely to be a substrate of P-gp and not necessarily a poorly permeable compound in vivo. At a relevant oral dose level, P-gp can be saturated to allow adequate absorption to occur in humans. A parallel combination assessment of PAMPA and A to B Caco-2 cell permeability followed by more detailed bidirectional Caco-2 cell assay might be a prudent path for research programs.

Drug absorption primarily occurs in the small intestine where the pH may vary from acidic to neutral and slightly basic (Dressman et al., 1990; Russell et al., 1993). In the upper small intestine where pH is likely to be more acidic, weakly acidic drugs exist primarily as unionized form and the passive transcellular pathway becomes the dominant permeation route. On the contrary, weakly basic drugs will be mostly in the form of ionized species, and consequently the passive tran-scellular route plays a minor role. Therefore, the apical pH used in the permeability assay becomes critical. Both PAMPA and Caco-2 cell permeability assays are typically performed at a single donor pH 6.5 to maintain adequate throughput (i.e., maximum number of compounds evaluated in minimum time frame). Single donor pH is perfectly appropriate for neutral and zwitterionic compounds where a change in pH is not expected to affect the ionization status. However, a majority of new drug candidates are either weak bases or weak acids. Therefore, the permeability of drug candidates with ionizable groups depends significantly on the experimental pH used. Table 5.4 lists a handful of drugs (acids and bases) along with their permeability values in the PAMPA model under two different experimental pH conditions. As expected, acidic drugs (e.g., ibuprofen, ketoprofen, piroxicam, etc.)

Table 5.4. Effect of pH on PAMPA permeability

PAMPA (nm /s)

Percentage of FA

pH 5.0

pH 7.4

ACIDS

Glipzide

562 i 27

14a i 1

95

Ibuprofen

756 i 69

5a i 1

100

Ketoprofen

567 i 49

9a i 1

90

Naproxen

500 i 85

17a i 2

100

Piroxicam

621 ± 42

21a ±3

100

BASES

Timolol

34a i 2

595 i 17

90

Hydralazine

2a i 2

177 i 44

90

Metoprolol

40a i 8

512 i 124

95

aPermeability value is inconsistently low despite complete absorption in humans aPermeability value is inconsistently low despite complete absorption in humans showed significantly higher permeability when studied at pH 5.0 than 7.4, and the basic drugs (e.g., metoprolol, timolol, etc.) had much higher permeability at pH 7.4 than 5.0. All drugs listed are almost completely absorbed in humans (>90%). But the permeability of these drugs when inappropriate pH was used is comparable to drugs that are not absorbed at all. Thus, it appears that an appropriate donor pH should be used depending on the physicochemical properties of test compounds. The degree of pH dependency is often very difficult to estimate because the pKa of a large number of drug candidates cannot be measured experimentally due to resources limitation. To minimize the occurrence of false negatives (i.e., classified as a poorly permeable when it is not), it becomes necessary to run the assay at two different pH condition (low of pH ~5 and high pH ~7.4) simulating the dynamic pH environment in the intestine to capture true intrinsic permeability. Because of a higher throughput potential, the PAMPA model can be used in the first tier pH dependent permeability study for similar chemotypes. Once an optimum pH is identified, all subsequent studies (including cell-based, tissue-based studies) can be carried out at that pH. However, it should be recognized that the permeability studies performed at multiple pH would negatively impact the throughput and cost involved.

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