In Vitro and In Vivo Pharmacokinetics of Aminoalkylated Diarylpropanes NP085 and NP102

Abstract

Malaria remains a great burden on humanity. Although significant advances have been made in the prevention and treatment of malaria, malaria control is now hindered by an increasing tolerance of the parasite to one or more drugs within artemisinin combination therapies; therefore, an urgent need exists for development of novel and improved therapies. The University of the Free State Chemistry Department previously synthesized an antimalarial compound, NP046. In vitro studies illustrated an enhanced efficacy against Plasmodium falciparum. However, NP046 showed low bioavailability. Efforts to enhance the bioavailability of NP046 have resulted in the synthesis of a number of aminoalkylated diarylpropanes, including NP085 and NP102. Pharmacokinetic studies were conducted in C57BL/6 mice, with 15 mg/kg NP085 or NP102 administered orally and the 5 mg/kg NP085 or NP102 administered intravenously. Blood samples were collected by means of tail bleeding at predetermined time intervals. Drug concentrations were determined using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and subsequently pharmacokinetic modeling was done for both compounds. NP085 and NP102 were incubated in vitro with human and mouse liver microsomes. Both compounds were also subjected to a parallel artificial membrane permeation assay. In vitro studies of NP085 and NP102 illustrated that both of the compounds are rapidly absorbed and undergo rapid hepatic metabolism. The maximum concentration of drug (C_max) obtained following oral administration of NP085 and NP102 was 0.2 ± 0.4 and 0.7 ± 0.3 μM, respectively; the elimination half-life of both compounds was 6.1 h. NP085 and NP102 showed bioavailability levels of 8% and 22%, respectively.

INTRODUCTION

Malaria is a mosquito-borne disease caused by a parasite. Humans are infected largely by four parasite species, of which the most common is Plasmodium vivax while Plasmodium falciparum elicits the most severe infections. The World Health Organization (WHO) currently recommends the use of artemisinin combination therapy (ACT) as the first-line treatment regimen for uncomplicated falciparum malaria (1). Overall, possibly through the use of ACTs coupled with prophylaxis and vector control, the worldwide incidence of malaria has decreased over the past decade such that in 2013 there were approximately 198 million cases worldwide, with 584,000 deaths (1). However, in spite of the apparent utility of combination therapies in suppressing the emergence of resistance (2), it is particularly alarming to note that malaria control is now hindered by the tolerance of the parasite to one or more drugs within the ACTs (3–5). Irrespective of the precise cause, an inability to control malaria with ACTs will have a catastrophic consequence. Therefore, an urgent need exists for treatment modalities that may overcome resistance, such as the development of novel more efficient antimalarial drugs and new drug combinations.

The Chemistry Department of the University of the Free State (UFS) produced a series of aminoalkylated chalcones in an attempt to increase the efficacy of chalcones against malaria (6). NP046, which resulted from this series, demonstrated enhanced in vitro efficacy against drug-sensitive and -resistant strains of P. falciparum (22 to 31.29 nM). Further, NP046 has been subjected to a comprehensive pharmacokinetic (PK) study in mice but showed a very low oral bioavailability of approximately 6% (7). Efforts to enhance the bioavailability of NP046 have resulted in the synthesis of a number of aminoalkylated diarylpropanes, including NP085 and NP102. The difference in the three structures resides in the p-substituent in the benzene ring: for NP046, a fluoro substituent, and for NP085 and NP102, an ethyl substituent and trifluoromethyl substituent, respectively. NP085 should be the most lipophilic, while NP046 should be the least lipophilic.

Similar to NP046 (7), both NP085 and NP102 illustrated activity against the intraerythrocytic stages of drug-sensitive and -resistant strains of P. falciparum (8.89 to 15 nM and 5.6 to 10.6 nM, respectively). In this study, the in vitro permeability and metabolic stability of the two aminoalkylated diarylpropanes were evaluated, as well as their in vivo pharmacokinetic properties in mice (Fig. 1).

FIG 1.

Structures of NP046 (a), NP085 (b), and NP102 (c).

MATERIALS AND METHODS

Ethical statement.

All studies and procedures were conducted with prior approval of the Ethics Committee of the University of Cape Town (approval number 011/022) in accordance with the National Code for animal use in research, education, diagnosis, and testing of drugs and related substances in South Africa (based on the NIH Guide for the Care and Use of Laboratory Animals [8]).

In vitro permeability and metabolism. (i) Materials.

The University of the Free State supplied hydrochloric salts of NP085 and NP102. Human liver microsomes and mouse liver microsomes were obtained from Xenotech (Kansas City, KS, USA). Carbamazepine, hexane, hexadecane, and NADPH were purchased from Sigma-Aldrich (Steinheim, Germany). Human plasma was obtained from the Western Cape Transfusion Service. All other reagents were of bioanalytical grade.

(ii) PAMPA.

A parallel artificial membrane permeation assay (PAMPA) was performed in triplicate in 96-well MultiScreen filter plates (0.4-μm-pore-size polycarbonate [PCTE] membrane; Millipore). Membrane filters were precoated with 5% hexadecane in hexane and allowed to dry prior to the assay. A membrane integrity marker, Lucifer yellow, was added to the apical wells of the precoated MultiScreen plate donor/drug solutions containing test compound. Phosphate buffer (pH 7.4) was added to the 96-well acceptor plate. Test compound was used to spike (1 μg/ml) the donor buffer at physiologically relevant pHs (pH 4, 6.5, and 8); the donor plate was slotted into the acceptor plate and incubated for 4 h at room temperature with gentle shaking (40 to 50 rpm). Following the incubation, samples from the acceptor wells and theoretical equilibrium wells were transferred to the analysis plate and matrix matched with blank donor buffer. Acetonitrile containing an internal standard (0.0236 μg/ml carbamazepine) was added to all samples; the samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Agilent Rapid Resolution high-performance liquid chromatograph [HPLC] and AB Sciex 4500 MS), and peak area ratios were used to calculate the apparent permeability (P_app). Membrane integrity was assessed by calculating the P_app of Lucifer yellow (acceptable values of <50 nm/s) using a Modulus microplate reader (excitation, 490 nm; emission, 510 to 570 nm).

(iii) Plasma protein binding and nonspecific liver microsomal binding.

Plasma protein binding was measured using an ultracentrifugation method. Briefly, the assay was performed in a 96-well microtiter plate with pooled human plasma, spiked with the test compound (5 μM). An aliquot was immediately removed and quenched using ice-cold acetonitrile containing an internal standard (0.0236 μg/ml carbamazepine) and placed in the freezer. This served as the total-concentration sample. After preincubation (37°C for 1 h) duplicate aliquots of the spiked plasma were transferred to ultracentrifugation tubes and ultracentrifuged for 4 h (42,000 rpm at 37°C) (Optima L-80XP; Beckman) (9). Analyte concentrations of all compounds and sample types were determined by means of LC-MS/MS (Agilent Rapid Resolution HPLC and AB Sciex 4000 QTRAP MS).

The ultracentrifuge method was also used to determine the nonspecific binding of NP085 and NP102 in mouse liver microsomes (MLM) and human liver microsomes (HLM). Stock solutions of compound (10 mM) were used to spike (1 μM) duplicate samples of microsomal solution (0.5 mg/ml) in phosphate buffer (pH 7.4). The same methodology as for plasma protein binding was utilized. Analyte concentrations of all compounds and sample types were determined by means of LC-MS/MS (Agilent Rapid Resolution HPLC and AB Sciex 4000 QTRAP MS).

Metabolic stability.

A metabolic stability assay was performed in duplicate in a 96-well microtiter plate. NP085 and NP102 (0.1 M) were incubated individually in mouse and pooled human liver microsomes (0.4 mg/ml) at 37°C in the presence and absence of the cofactor NADPH (1 mM). An aliquot was taken at 0, 5, 10, 30, and 60 min, and the reaction was quenched by the addition of 300 μl of ice-cold acetonitrile containing an internal standard (0.0236 μg/ml carbamazepine) (10). The test compounds in the supernatant were analyzed by means of LC-MS/MS (Agilent Rapid Resolution HPLC and AB Sciex 4000 QTRAP MS).

(i) Determination of intrinsic clearance.

Intrinsic clearance (CL_int) studies were performed for NP085 and NP102 using the substrate depletion approach (10, 11), where drug concentrations are determined after different times of incubations with microsomes. The fundamental basis of this approach lies in the derivation of the integrated Michaelis-Menten equation (12):

$$V_M\times dt=\frac{-K_{M\text{app}}+\left[S\right]}{\left[S\right]}\times d\left[S\right]$$ (1)

where V_M is the maximum rate of metabolism, K_Mapp is the apparent K_M, [S] is the concentration of substrate, and t is time. Over one half-life, t_1/2 (i.e., when [S] = 0.5[S]_{t = 0}), the following equation applies:

$$\frac{V_M\times t_{1\hspace{0.17em}/\hspace{0.17em}2}}{K_{M\text{app}}}=\ln \hspace{0.17em}2+\frac{0.5{\left[S\right]}_{t=0}}{K_{M\text{app}}}$$ (2)

A necessary assumption in this approach, which is included in the experimental design, is that the substrate concentration used is below the K_Mapp value such that:

$$\frac{0.5\left[S\right]}{K_{M\text{app}}}⪡\ln \hspace{0.17em}2$$ (3)

The in vitro t_1/2 is incorporated into the following equation:

$${\text{CL}}_{\mathrm{int}\hspace{0.17em}=\frac{\ln \hspace{0.17em}2\hspace{0.17em}\times \hspace{0.17em}\text{volume of incubation}}{t_{1/2}\hspace{0.17em}\times \hspace{0.17em}\text{protein or enzyme amount}}}$$ (4)

where the volume of incubation is expressed in microliters, the amount of protein or enzyme is in micrograms, and the half-life is in minutes.

(ii) In vitro-in vivo correlations.

The CL_int in the incubations was expressed as microliters/minute/milligram (microsomes) and was scaled to the apparent clearance for the whole liver (whole-liver CL_int). The following scaling factors were used for the microsomal protein yield per gram of liver (MPGL): human, 52.5 mg/g (13); mouse, 87.5 mg/g (14).

The in vivo hepatic clearance (CL_H) was estimated according to the “restrictive well-stirred liver” model (15). The in vivo hepatic clearance (CL_H) is expressed as:

$$In\hspace{0.17em}vivo\hspace{0.17em}{\text{CL}}_H=\frac{Q_H\times \left(f_{u\left(p\right)}\right)(B:P)\times ({\text{CL}}_{\mathrm{int}\hspace{0.17em}}÷f_{u\left(inc\right)})}{Q_H+\left(f_{u\left(p\right)}\right)(B:P)\times ({\text{CL}}_{\mathrm{int}\hspace{0.17em}}÷f_{u\left(inc\right)})}$$ (5)

where Q_H is the hepatic blood flow. The values used for hepatic blood flow (Q_H) were 21 ml/min/kg and 90 ml/min/kg for human and mouse, respectively (16). B:P is the blood-to-plasma ratio and is assumed to be 1, f_u(p) is the unbound drug fraction in the plasma, and f_u(inc) is the unbound drug fraction within the microsomes.

In vivo pharmacokinetics. (i) Materials.

Acetonitrile (LiChrosolv) and formic acid (proanalysis grade) were purchased from Merck (Darmstadt, Germany). The Department of Chemistry (UFS) supplied test compounds (NP085, NP102, and NP046 [internal standard]). Water used to prepare solutions was purified by Millipore Elix 10 reverse osmosis and a Milli-Q (Millipore, USA) Gradient A 10 polishing system.

(ii) Animals.

Healthy C57BL/6 mice weighing approximately 30 g were used. The mice were maintained at the animal facility of the University of Cape Town. Mice were housed in 27- by 21- by 18-cm cages, under controlled environmental conditions (26 ± 1°C and a 12-h light/dark cycle). Their diet consisted of standard laboratory food. Water was available ad libitum.

(iii) Drug administration. (a) Oral administration.

NP085 and NP102 were administered orally at a dose of 15 mg/kg (n = 3). Both NP085 and NP102 were dissolved in water. Drug administration was accomplished via oral gavage. The total volume per administration was 200 μl. Blood samples were collected via tail bleeding predose and at predetermined time intervals of 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose in 0.5-ml lithium heparin microvials to prevent blood coagulation. The samples were stored at −80°C.

(b) Intravenous administration.

NP085 and NP102 were administered intravenously (i.v.) at a dose of 5 mg/kg in water (n = 3). The total volume per administration was 80 μl. Blood samples were collected via tail bleeding at predetermined time intervals predose and at 0.083, 0.5, 1, 2, 4, 8, and 24 h postdose in 0.5-ml lithium heparin microvials to prevent blood coagulation. The samples were stored at −80°C.

(iv) Sample analysis.

The whole-blood concentrations of NP085 and NP102 were determined using a quantitative LC-MS/MS assay developed in the Division of Clinical Pharmacology, University of Cape Town. Sample preparation was achieved with a protein precipitation extraction method, using 20 μl of whole blood and 150 μl of acetonitrile containing an internal standard (NP046); 5 μl was injected onto the analytical column.

Chromatography was performed on a Synergi Hydro RP (2.5-μm particle size, 100-Å pore size, 50 by 2 mm) analytical column. The mobile phase consisted of 0.1% formic acid in water and acetonitrile and was delivered at 0.4 ml/min, using a gradient. An AB Sciex API 3200 mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, monitoring the transition of the protonated molecular ions at m/z 338.2 to the product ions at m/z 86.0 for NP085, the protonated molecular ions at m/z 378.1 to the product ions at m/z 86.2 for NP102, and the protonated molecular ions at m/z 328.2 to the product ions at m/z 242.9 for the internal standard (NP046). The ion source settings are summarized in Table 1. The calibration range was between 2 ng/ml and 6,250 ng/ml (0.01 μM to 18.55 μM; 0.01 μM to 16.58 μM) for both NP085 and NP102, respectively.

TABLE 1.

Ionization source settings for NP085, NP102, and NP046^a

Electrospray ionization settingb	Value
Nebulizer gas (AU)	40
Turbo gas (AU)	20
Curtain gas	20
CAD gas	5
Temp (°C)	500
IS (V)	5,500

^aNP046 is the internal standard.

^bAU, arbitrary unit; CAD, collision-activated dissociation; Temp, source temperature; IS, ion spray.

Statistical analysis.

The experimental data were evaluated in terms of drug concentration versus time. The following parameters were calculated from whole-blood drug levels obtained:

• Peak drug concentration (C_max) in micromolar units

• Time to peak concentration (T_max) in hours

• Elimination half-life (t_1/2) in hours

• Area under the concentration-time curve from time zero to infinity (AUC_0–∞) in minutes per micromole/liter

• Clearance (CL) in milliliters/minute/kilogram

• Volume of distribution (V) in liters/kilogram

• Bioavailability in percents

Results are reported as means ± standard errors of the means (SEM).

Noncompartmental analysis was used to calculate the PK parameters of NP085 and NP102 (PK Solutions, version 2.0; Summit Research Services, Montrose, CO, USA). The area under the plasma concentration curve up to the last time point (AUC_last) was calculated by using the trapezoidal rule. The area under the curve extrapolated to infinity (AUC_0–∞) was determined by adding C_last/k_ε to AUC_last, where C_last is the concentration of the last time point and k_ε is the elimination rate constant. The half-life is calculated as t_1/2 = ln(2)/k_ε. Apparent volume of distribution (V) is based on the trapezoidal AUC (area) and the elimination rate constant. Clearance was calculated as CL = (FD/AUC_0–∞), where FD is the fraction of the dose absorbed, and was normalized using the weight of each mouse.

RESULTS AND DISCUSSION

In vitro permeability and metabolism.

In an effort to evaluate the metabolic stability of NP085 and NP102, the in vitro clearance was investigated in systems comprising HLM and MLM. Studies were performed to determine the in vitro intrinsic clearance (CL_int) of the two compounds by measuring their depletion over time from an initial substrate concentration of 0.1 M. The metabolic in vitro half-life (t_1/2) was calculated using the slope of the linear regression from the natural logarithmic percentage of the substrate remaining versus incubation time. The in vitro half-life (t_1/2) of NP102, compared to that of NP085, was increased in HLM and MLM (Table 2). The in vitro half-life was used to determine the intrinsic clearance (CL_int) of the compounds. The results are given in Table 2. It is evident from the data that the intrinsic clearance as well as the estimated in vivo hepatic clearance in HLM and MLM is high for both aminoalkylated diarylpropanes. Further, NP085 and NP102 illustrated high in vitro permeability, with log P_app values of −4.7 and −3.6, respectively. In addition, NP085 and NP102 were highly bound to microsomes (98.6% and 98.3%, respectively) and moderately bound to plasma proteins (78.8% and 79%, respectively). The results for the three quality controls included during the determination of plasma protein binding were within accepted criteria: high, warfarin (99.1% protein bound); moderate, internal compound MMV390048 (85% protein bound); and low, caffeine (24.4% protein bound).

TABLE 2.

Calculated intrinsic clearance (CL_int) of NP085 and NP102 using the t_1/2 in HLM and MLM and estimated in vivo hepatic clearance (CL_H)

Compound	Degradation half-life (min)		In vitro CLint (μl/min/mg)		Estimated in vivo CLH (ml/min/kg)
	HLM	MLM	HLM	MLM	HLM	MLM
NP085	43	21	46.7	322.0	20.7	89.7
NP102	58	37	34.2	184.1	20.1	89.1

In vivo pharmacokinetics.

The LC-MS/MS method performed well during sample analysis. The calibration standards and quality-control standards were analyzed in duplicate in the study sample batch. A quadratic regression, with peak area ratio (drug/internal standard) against concentration with 1/concentration (1/x) weighting, was fitted to the calibration curves. The combined accuracy (% nom) statistics were between 93.8 and 109.8 for the standards and between 100.8 and 111.8 for the quality controls. The combined precision (coefficient of variation [CV], expressed as a percentage) statistics were below 15.

The pharmacokinetic parameters for the oral and i.v. groups were calculated for both NP085 and NP102, respectively. Results are presented in Table 3. The whole-blood profiles obtained for the i.v. group and oral group are graphically presented in Fig. 2 and Fig. 3.

TABLE 3.

Summary of the pharmacokinetic parameters of NP085 and NP102 following oral and i.v. administration in mice

Parameter	Value of the parameter by administration route (mean ± SEM)a
	NP085		NP102
	Oral	i.v.	Oral	i.v.
Cmax (μM)	0.16 ± 0.04		0.7 ± 0.3
Tmax (h)	0.5 ± 0		0.5 ± 0
Elimination half-life (h)	5.9 ± 3.7	6.1 ± 0.4	4.6 ± 0.9	6.1 ± 0.1
CL (ml/min/kg)		105.7 ± 17.4		60.1 ± 8.5
V (liters/kg)		56.7 ± 13.5		32.4 ± 7.6
AUC0-∞ (min · μmol/liter)	36 ± 9	145 ± 21	147 ± 40	226 ± 29
Bioavailability (%)	8 ± 2		22 ± 6

^aThe doses were 15 mg/kg and 5 mg/kg for oral and i.v. administration, respectively.

FIG 2.

Mean (SEM) whole-blood concentration-time profile of NP085 and NP102 following i.v. administration of 5 mg/kg to mice (n = 3).

FIG 3.

Mean (SEM) whole-blood concentration-time profile of NP085 and NP102 following a single oral administration of 15 mg/kg in mice (n = 3).

Following i.v. administration (Fig. 2) of NP085 and NP102 at a dose of 5 mg/kg, the mean elimination half-life was 6.1 h for both NP085 and NP102. The mean AUC_0–∞ values quantified were 145 min · μmol/liter (NP085) and 226 min · μmol/liter (NP102).

Following oral administration (Fig. 3), mean maximum whole-blood concentrations (C_maxs) of 0.16 ± 0.04 μM and 0.7 ± 0.3 μM were observed for NP085 and NP102, respectively. Thereafter the concentrations declined rapidly. The mean AUC_0–∞ values quantified were 36 min · μmol/liter (NP085) and 147 min · μmol/liter (NP102). The observed maximum peak whole-blood concentration was reached at approximately 0.5 h for both compounds. It is evident that the C_max value obtained for NP102, an analogue of NP046 in the current study, is increased compared to that in a previous study. In addition, the in vivo half-life values were also increased for both NP085 and NP102 compared to the half-life of 3.2 ± 0.35 h observed for NP046 (7).

Interestingly, based on allometric scaling of the intrinsic clearance to an estimated in vivo hepatic clearance (Table 2), we expected NP085 and NP102 to be rapidly cleared, with estimated CL_H values of 89.7 ml/min/kg and 89.1 ml/min/kg, respectively. The same was true for the clearance calculated during the pharmacokinetic studies in mice; NP085 had a clearance of 105 ml/min/kg while NP102 had a clearance of 60 ml/min/kg. It is evident that the in vitro and in vivo clearance displayed by NP085 was more rapid than that of NP102, which could possibly be due in part to oxidative cleavage of the ethyl group. Additionally, during the in vitro PAMPA, it is clear that the permeability of both compounds is high, with that of NP102 being slightly higher than that of NP085.

Conclusion.

The results of the in vitro absorption and metabolism and in vivo pharmacokinetics studies confirm the increase in absorption of both NP085 and NP102 compared to that of NP046. Further, the in vitro and in vivo clearance values correlate well. However, it is evident from the results obtained in the current study that both NP085 and NP102 are cleared rapidly; this has triggered structure-activity relationship (SAR) optimization in an attempt to block metabolic sites without compromising the antimalaria activity of future compounds within this series. Therefore, we presume that the increase in bioavailability of NP102 over that of NP085 is due to the increase in absorption, a lower volume of distribution, and delayed hepatic clearance of the drug.