Limits...
In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383.

Ufuk A, Somers G, Houston JB, Galetin A - Pharm. Res. (2015)

Bottom Line: Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs.Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

View Article: PubMed Central - PubMed

Affiliation: Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, The University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, UK.

ABSTRACT

Purpose: To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.

Methods: For all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.

Results: Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.

Conclusions: This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

No MeSH data available.


Related in: MedlinePlus

Cell-to-medium concentration ratio (Kp) of imipramine (a), formoterol (b) and clarithromycin (c), and total uptake rate of clarithromycin (d) over a range of concentrations in NR8383 cells. Data represent mean ± SD of 3 experiments except for the first two data points (0.1–0.5 μM) of imipramine (n = 1). Solid line represents the fit for total uptake at 37°C. Symbols represent the observed data in the absence ( imipramine;  formoterol;  clarithromycin) and presence () of 20 mM NH4Cl at 37°C. For Kp and uptake kinetic parameter estimation, the fitting was obtained using the two-site binding model (Eq. 2) and the conventional two-step model (Eq. 3).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4628094&req=5

Fig1: Cell-to-medium concentration ratio (Kp) of imipramine (a), formoterol (b) and clarithromycin (c), and total uptake rate of clarithromycin (d) over a range of concentrations in NR8383 cells. Data represent mean ± SD of 3 experiments except for the first two data points (0.1–0.5 μM) of imipramine (n = 1). Solid line represents the fit for total uptake at 37°C. Symbols represent the observed data in the absence ( imipramine; formoterol; clarithromycin) and presence () of 20 mM NH4Cl at 37°C. For Kp and uptake kinetic parameter estimation, the fitting was obtained using the two-site binding model (Eq. 2) and the conventional two-step model (Eq. 3).

Mentions: For a number of basic drugs, accumulation in the NR8383 was assessed further over a range of substrate concentrations. A concentration-dependent accumulation was observed for the drugs investigated, as illustrated in Fig. 1. The two-site binding model was fitted to the experimental data and the estimated uptake parameters Kp,U1,max, Kp,min and KU1 are shown in Table III. In contrast to the Kp value obtained at 5 μM, imipramine maximum Kp of 1166 was estimated by the modelling approach (corresponds to concentration <0.1 μM). The Kp,min of 141, 24 and 10 was estimated for imipramine, clarithromycin and formoterol, respectively. The analysis indicated that half of maximal Kp associated with saturable uptake would be reached at concentrations below 2 μM for these three drugs (Table III). The fold difference between Kp,max and Kp,min ranged between 3 and 8 for clarithromycin and imipramine, respectively.Fig. 1


In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383.

Ufuk A, Somers G, Houston JB, Galetin A - Pharm. Res. (2015)

Cell-to-medium concentration ratio (Kp) of imipramine (a), formoterol (b) and clarithromycin (c), and total uptake rate of clarithromycin (d) over a range of concentrations in NR8383 cells. Data represent mean ± SD of 3 experiments except for the first two data points (0.1–0.5 μM) of imipramine (n = 1). Solid line represents the fit for total uptake at 37°C. Symbols represent the observed data in the absence ( imipramine;  formoterol;  clarithromycin) and presence () of 20 mM NH4Cl at 37°C. For Kp and uptake kinetic parameter estimation, the fitting was obtained using the two-site binding model (Eq. 2) and the conventional two-step model (Eq. 3).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4628094&req=5

Fig1: Cell-to-medium concentration ratio (Kp) of imipramine (a), formoterol (b) and clarithromycin (c), and total uptake rate of clarithromycin (d) over a range of concentrations in NR8383 cells. Data represent mean ± SD of 3 experiments except for the first two data points (0.1–0.5 μM) of imipramine (n = 1). Solid line represents the fit for total uptake at 37°C. Symbols represent the observed data in the absence ( imipramine; formoterol; clarithromycin) and presence () of 20 mM NH4Cl at 37°C. For Kp and uptake kinetic parameter estimation, the fitting was obtained using the two-site binding model (Eq. 2) and the conventional two-step model (Eq. 3).
Mentions: For a number of basic drugs, accumulation in the NR8383 was assessed further over a range of substrate concentrations. A concentration-dependent accumulation was observed for the drugs investigated, as illustrated in Fig. 1. The two-site binding model was fitted to the experimental data and the estimated uptake parameters Kp,U1,max, Kp,min and KU1 are shown in Table III. In contrast to the Kp value obtained at 5 μM, imipramine maximum Kp of 1166 was estimated by the modelling approach (corresponds to concentration <0.1 μM). The Kp,min of 141, 24 and 10 was estimated for imipramine, clarithromycin and formoterol, respectively. The analysis indicated that half of maximal Kp associated with saturable uptake would be reached at concentrations below 2 μM for these three drugs (Table III). The fold difference between Kp,max and Kp,min ranged between 3 and 8 for clarithromycin and imipramine, respectively.Fig. 1

Bottom Line: Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs.Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

View Article: PubMed Central - PubMed

Affiliation: Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, The University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, UK.

ABSTRACT

Purpose: To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.

Methods: For all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.

Results: Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.

Conclusions: This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

No MeSH data available.


Related in: MedlinePlus