Characterization of in vivo metabolites of WR319691, a novel ...

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Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-011-0047-8

ORIGINAL PAPER

Characterization of in vivo metabolites of WR319691, a novel compound with activity against Plasmodium falciparum Erin Milner • Jason Sousa • Brandon Pybus • Victor Melendez • Sean Gardner • Kristina Grauer • Jay Moon • Dustin Carroll • Jennifer Auschwitz • Montip Gettayacamin • Patricia Lee • Susan Leed William McCalmont • Suzanne Norval • Anchalee Tungtaeng • Qiang Zeng • Michael Kozar • Kevin D. Read • Qigui Li • Geoffrey Dow



Received: 19 April 2011 / Accepted: 6 June 2011 ! Springer-Verlag France 2011

Abstract WR319691 has been shown to exhibit reasonable Plasmodium falciparum potency in vitro and exhibits reduced permeability across MDCK cell monolayers, which as part of our screening cascade led to further in vivo analysis. Single-dose pharmacokinetics was evaluated after an IV dose of 5 mg/kg in mice. Maximum bound and unbound brain levels of WR319691 were 97 and 0.05 ng/g versus approximately 1,600 and 3.2 ng/g for mefloquine. The half-life of WR319691 in plasma was approximately 13 h versus 23 h for mefloquine. The pharmacokinetics of several N-dealkylated metabolites was also evaluated. Five of six of these metabolites were detected and maximum total and free brain levels were all lower after an IV dose of

This manuscript was reviewed by the Walter Reed Army Institute of Research and the U.S. Army Medical Research and Materiel Command, and there is no objection to its publication or dissemination. The opinions expressed herein are those of the authors and do not necessarily reflect the views or opinions of the Department of the Army and the Department of Defense. All animal experiments were conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhere to the principles stated in the Guide for the Care and Use of Laboratory Animals (National Academy Press, 1996). The MDCK permeability assays were performed under contract by Absorption Systems (Exton PA).

Electronic supplementary material The online version of this article (doi:10.1007/s13318-011-0047-8) contains supplementary material, which is available to authorized users. E. Milner (&) ! J. Sousa ! B. Pybus ! V. Melendez ! S. Gardner ! K. Grauer ! J. Moon ! D. Carroll ! J. Auschwitz ! P. Lee ! S. Leed ! W. McCalmont ! Q. Zeng ! M. Kozar ! Q. Li ! G. Dow Department of Medicinal Chemistry, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910-7500, USA e-mail: [email protected]

5 mg/kg WR319691 compared to mefloquine at the same dose. These data provide proof of concept that it is feasible to substantially lower the brain levels of a 4-position modified quinoline methanol in vivo without substantially decreasing potency against P. falciparum in vitro. Keywords Mefloquine ! Malaria ! N-dealkylated metabolites ! Brain levels 1 Introduction Intermittent preventive treatment (IPTx) of malaria involves periodic administration of a full treatment level dose of an antimalarial drug to infants (IPTi), in pregnancy (IPTp) or, putatively to travelers (IPTt) in order to prevent malaria and morbidity. IPTx differs from treatment because individuals are asymptomatic and prophylactic because the doses given are higher and not administered in a continuous regimen. Mefloquine (MQ) is indicated for prophylaxis during pregnancy and is more effective than other regimens for IPTx due to its long half-life, but the high incidence of central nervous system (CNS) adverse events at treatment level doses will likely limit its widespread application for administration to asymptomatic individuals (Dow et al. 2008; Briand et al. 2009; Cairns et al. 2010; CDC 2008; Gosling et al. 2009; McGready 2010). A MQ-like compound that is better tolerated and suitable for single dose M. Gettayacamin ! A. Tungtaeng United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand S. Norval ! K. D. Read Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, UK

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permeability assay which we showed previously to be reasonably predictive of maximum brain levels in vivo (Milner et al. 2010b). Potential late leads were selected on the basis of exhibiting IC90 s \ 100 ng/ml against at least one strain of Pf together with an apparent permeability \ 7.4 9 1-E-6 cm/s in the A–B direction across MDCK monolayers. The latter threshold was established as being indicative of lower CNS concentrations of mefloquine in our earlier work. Once the preliminary in vitro benchmarks were achieved, our general intent was to declare a compound a late lead if the absolute maximum bound and unbound brain concentrations were five fold lower than mefloquine at the minimum curative dose after oral administration in mice. The rationale for this CNS safety benchmark was outlined in our earlier work (Dow et al. 2011). During the synthesis of WR319670 (structure in Fig. 1), a byproduct dimer was isolated, characterized, and submitted to preliminary in vitro studies. Interestingly,

administration would offer a significant improvement over the available alternatives. The putative CNS targets of mefloquine are not known, but there are likely many (Dow et al. 2006). A reduction in partitioning into the central nervous system, if it could be engineered into the quinoline methanol scaffold without affecting the desirable properties of MQ, such as its halflife and intrinsic potency, may provide a significant improvement in the therapeutic index without compromising utility. In recent studies we constructed a library of next generation quinoline methanols in order to determine if the MQ scaffold could provide sufficient chemical space to maintain Pf potency while reducing CNS accumulation (Milner et al. 2010a). We employed a primary in vitro testing cascade involving in vitro susceptibility screening against two drug-resistant strains of P. falciparum (mefloquine resistant D6 and multiple drug resistant C235) and a MDCK

OH HO

N

N H

CF 3 N

CF3

CF3

N H

OH HO

N

N

CF3

CF3

HO OH

H2N

CF3

N

N H N

O

HO

N H

N

CF3

CF3 WR319707 m/z:367.11

Fig. 1 Predicted N-dealkylation metabolites of WR319691

123

O N

CF3

CF3 WR308245 m/z:338.09

m/z:366.08

m/z:323.04

OH

O

NH 2

HN

CF3

H 2N

HO CF 3

N

OH N H

CF 3

N CF3

WR318973 m/z:381.13

WR319670 m/z:381.13

CF 3 N

CF3

CF 3

CF3

OH

CF3

HO

NH

CF3

CF3

N

CF3

OH HO

N

CF3 m/z:323.04

CF3

CF3

CF 3 N

N

OH N

N H

CF 3

CF3

CF3

O HO

N

OH

N

N H

CF 3 N

CF3

WR319691 m/z:688.17

OH

OH HO

N

N CF3 WR160972 m/z:309.02

CF 3 N CF3 WR308314 m/z:324.07

Eur J Drug Metab Pharmacokinet

WR319691 exhibited favorable in vitro Pf potency and substantially reduced permeability across MDCK cell monolayers relative to mefloquine (Table 1). This observation led to the profiling of this compound as a potential lead. As part of this process it was important to evaluate

efficacy after oral dosing in vivo since low in vitro permeability might reflect low in vivo permeability. In parallel we considered it important to evaluate pharmacokinetic parameters after intravenous (IV) dosing to aid interpretation in the event of an efficacy failure via the oral route,

Table 1 In vitro data Structure

WR no.

HN

SYBR green D6 IC90 (ng/ml)

SYBR green C235 IC90 (ng/ml)

Half life human liver microsomes

Half life mouse liver microsomes

MDCK A–B Papp (10-6 cm/s)

142490

15

42

[60

[60

9.4

319691

32

80

[60

[60

0.14

308245

45

58

[60

[60

318973

[500

[500

[60

[60

8.3

319670

388

[500

[60

[60

4.9

319707

[500

[500

ND

ND

3.1

308314

[500

[500

ND

ND

HO

N

CF3

CF 3

OH HO

N

N H

CF3 N

N

CF 3

CF3

CF 3

HO

N H

N

14

CF3

CF3 HO

NH

N H N

CF3

CF3 OH N

H2N

CF 3 N CF3

HO

NH 2

N H N

CF3

CF 3 OH

ND

H2N CF3 N CF3

D6 is chloroquine sensitive but a mefloquine resistant strain of African origin. C235 is a multiple drug resistant strain of Thai origin. Papp in A–B direction in 9 10-6 cm/s ND no data

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Eur J Drug Metab Pharmacokinet

and also to assess exposure in the CNS. Finally, the metabolism of the WR319691, via three N-dealkylation pathways, can be predicted, a priori, based on the compound’s structure (Fig. 1). The predicted metabolites were synthesized and several putative metabolites exhibit antimalarial activity in their own right. Determining which of these pathways predominates in vivo was considered critical to determining which future analogs of WR319691 should be made, to either inhibit or promote metabolism to specific active intermediates as appropriate.

2.3 Method development—WR319691 and metabolites Stock compound was infused on mass spectrometric detectors to determine optimal mass or mass transitions for analysis. Liquid chromatography gradients were developed to provide adequate pre-concentration and/or separation of the compound prior to mass spectrometric detection. All methods remained valid in the various matrices used in these studies. All chromatography was performed using a Waters XTerra MS C18 column (50 mm 9 2.1 mm id; 3.5 lm particle size).

2 Materials and methods 2.4 Analyte determination in plasma, brain, whole blood, and liver samples

2.1 Liver microsomal methods Sample stocks at 10 or 20 mM (depending on solubility) in DMSO were diluted to a final concentration of 1 lM into a mixture containing, 0.5 mg/mL of pre-warmed pooled human or mouse liver microsomes (BD Gentest), 1.3 mM NADP (Sigma), 3.3 mM MgCl2 (Sigma), and 0.1 M pH 7.4 PBS using a TECAN Genesis robotic liquid handler. The reaction was started with the addition of 1 U/mL glucose-6-phosphate dehydrogenase (G6PD). The mixture was incubated on a shaking platform at 37"C, and aliquots were taken and quenched with the addition of an equal volume of cold acetonitrile at 0, 10, 20, 30, and 60 min. Samples were centrifuged at 3,700 rpm for 10 min at 20"C to remove debris. Sample quantification was carried out by LC/MS, and metabolic half-life was calculated by log plots of the total ion chromatograph area remaining. 2.2 Dosing of animals and collection of samples Groups of four male 5- to 6- week-old FVB mice were dosed with 5 mg/kg compound IV. Whole blood, plasma, liver and brain samples were obtained from the animals at 5 min, 1, 6, 24, 48, 96 and 120 h. Brains and livers were collected and frozen at -80"C per established procedures. Whole blood was collected by cardiac puncture and plasma was separated from whole blood. The indicated whole blood and plasma volumes were collected. Actual dosing volumes, dosing times and sample times are presented in Table 2.

Table 2 In vivo efficacy data

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Plasma, whole blood, and liver standard curves were prepared via serial dilutions from the high concentration value (generally 500 ng/mL) through a series of 10–11 lower concentration values (see Standard Curve). The serial dilution included the preparation of 4–5 QC samples for each of a low range point and a high range point (generally 10 ng/mL and 100 ng/mL, respectively). Once the standard curve dilutions had been prepared, a 100 lL aliquot was removed and extracted with 200 lL acetonitrile. The extracted samples were centrifuged at 10,000 rpm for 10 min and the supernatant was removed for analysis by LC–MS/MS. Plasma, whole blood, and liver PK samples were extracted in the same manner as the standard curve and QC samples, where 100 lL aliquots were extracted with 200 lL acetonitrile, centrifuged, and the supernatant analyzed by LC–MS/MS. Sample drug concentrations were interpolated directly from the standard curve. Blank brain homogenate was prepared by adding 5 mL of water for each gram of brain, then homogenizing the mixture using an ultrasonication probe. The homogenate was then used to prepare standard curve and QC solutions. Brain standard curves were prepared via serial dilutions from the high concentration value (generally 500 ng/mL) through a series of 10–11 lower concentration values (see Standard Curve). The serial dilution included the preparation of 4–5 QC samples for each of a low range point and a high range point (generally 10 ng/mL and 100 ng/mL, respectively). Once the standard curve dilutions have been

Compound

Dose

Vehicle

No. of surviving mice (days of euthanasia)

Vehicle



ECEL-HECT

0 (6, 6, 6, 6, 6)

Mefloquine

40 mg/kg 91

ECEL-HECT

1 (17, 17, 17, 17)

WR319691

40 mg/kg 91

ECEL-HECT

0 (10, 10, 10, 10, 10)

Eur J Drug Metab Pharmacokinet

prepared, a 100 lL aliquot was removed and extracted with 200 lL acetonitrile. The extracted samples were centrifuged at 10,000 rpm for 10 min and the supernatant was removed for analysis by LC–MS/MS. Brain samples were homogenized and extracted in the same manner as the standard curve and QC samples, where 100 lL aliquots were extracted with 200 lL acetonitrile, centrifuged, and the supernatant analyzed by LC–MS/MS. Sample drug concentrations were first interpolated from the standard curve, then multiplied by a factor of six to account for the drug levels present in the brain prior to dilution with water for homogenization. 2.5 Pharmacokinetic parameter determination Drug concentrations were generated for the test drug. A measured plasma drug concentration versus time curve was produced, in graphic and tabular form, for each subject on both linear/linear and log/linear scales, for the parent compound. Mean plasma drug concentration versus time curves were also presented separately. For the determination of PK parameters of WR319691 in plasma and other tissues after systemic application, a non-compartmental analysis was performed using WinNonlin (version 5.2; Pharsight Corp., Mountain View, CA). The area under the curve (AUC) was determined by the linear trapezoidal rule with extrapolation to infinity based on the concentration of the last time point divided by the terminal rate constant. The elimination half-life (t1/2), maximum plasma concentration (Cmax), and time to Cmax (Tmax) of WR319691 and its metabolites were also calculated. The log–linear trapezoidal rule was used to estimate the respective AUCinf values, and the AUCinf ratios of the metabolite to the parent compound WR319691 were calculated to determine exposure to any metabolite compared with the parent drug. Mean clearance rate (CL) was determined by dividing the dose by the AUCinf for intravenous injection for plasma samples. The mean CL adjusted with absolute bioavailability (F) was determined for other tissue samples. Mean residence time (MRT) was determined by dividing the area under the first moment curve (AUMC) by AUC. The volume of the central compartment (Vz) and volume of the tissue compartment (Vz/F) were calculated as the product of CL and MRT. 2.6 Determination of mouse brain tissue binding The methodology employed was a modification of that reported previously (Summerfield et al. 2007). In brief, a 96 well equilibrium dialysis apparatus was used to determine the free fraction in the brain for each test compound (HT Dialysis LLC, Gales Ferry, CT). Membranes (12–14kDA cut-off) were conditioned in deionised water for 60 min,

followed by conditioning in 80:20 deionised water:ethanol for 20 min, and then rinsed in artificial cerebrospinal fluid (CSF) before use. Control mouse brain was removed from the freezer and allowed to thaw on the day of experiment. Thawed brain tissue was homogenized with artificial CSF to a final composition of 1:2 brain:artificial CSF using a Covaris S2 (K Biosciences, Hoddesdon, UK). Diluted brain homogenate was then spiked with test compound (10 lg/g), and 150 lL aliquots (n = 6 replicate determinations/compound) loaded into the 96-well equilibrium dialysis plate. Dialysis versus artificial CSF (150 lL) was carried out for 5 h in a temperature controlled incubator at ca. 37"C (Barworld scientific Ltd, UK) using an orbital microplate shaker at 125 revolutions/min (Barworld scientific Ltd, UK). At the end of the incubation period, aliquots of brain homogenate or artificial CSF were transferred to micronic tubes (Micronic B!V., the Netherlands) and the composition in each tube balanced with control fluid, such that the volume of artificial CSF to brain is the same. Sample extraction was performed by the addition of 400 lL of acetonitrile containing an appropriate internal standard. Samples were allowed to mix for 1 min and then centrifuged at 3,000 rpm in 96-well blocks for 15 min (Allegra X12-R, Beckman Coulter, USA). All samples were analyzed by means of UPLC/MS/MS on a Quattro Premier XE Mass Spectrometer (Waters Corporation, USA). The unbound fraction was then determined for each compound as the ratio of the peak area in artificial CSF to that in brain, with correction for dilution factor according to Eq.1 (Kalvass and Maurer 2002), Undiluted fu ¼ !!

1=D " " 1=fu;apparent # 1 þ 1=D

ð1Þ

where D is the dilution factor in brain homogenate and fu,apparent is the measured free fraction of diluted brain homogenate.

3 Results and discussion Our overall objective involves a lead candidate with reduced partitioning into the CNS without affecting the desirable properties of mefloquine (MQ), such as its halflife and intrinsic potency. WR319691 demonstrated excellent in vitro potency and favorable permeability across MDCK cell monolayers, but lacked in vivo potency, had a short half-life, and much lower brain levels than mefloquine. All of the predicted metabolites were synthesized and utilized as standards. Each of the metabolites was detected in the brain at lower exposure levels than mefloquine. WR319691 delayed mortality, but did not cure malariainfected mice as does mefloquine. While the compound

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methanols which have demonstrably less microsomal stability (e.g. WR308278) (Milner et al. 2010b). It is likely that WR319691 also exhibits lower relative bioavailability than MQ, which would need to be addressed in a new series of analogs. The blood and tissue concentrations for mefloquine (MQ, WR142490) and WR319691 for a single intravenous dose of 5 mg/kg are presented in Figs. 2 and 3. As expected based on the MDCK permeability data, maximum bound and unbound brain levels of WR319691 were 16 and 64-fold lower than mefloquine. Also, the distribution of drug varied for WR319691 compared to MQ. For WR319691, the concentration of drug followed the trend of liver [ whole blood [ plasma [ brain, while MQ is corresponds to liver [ brain [ whole blood [ plasma. All putative metabolites were detected with the exception of WR319707 (Tables 3, 4, 5). Maximum bound and unbound brain levels of all metabolites measured were lower than the parent compound with the exception of WR308245. The potent metabolite WR308245 accumulated in the brain at lower levels than mefloquine despite higher in vitro MDCK permeability (Table 6). This may be because the major pathways of metabolism, based on maximum liver levels, trend towards the less permeable and less potent metabolites WR318973 and WR319670. If this were not the case, it might be necessary to selectively block N-dealkylation to the more active and brain-penetrant species WR308245, in a new series of analogs. Based on these observations, the future of WR319691 as an antimalarial is probably limited because of poor efficacy after oral dosing. However it does provide precedent that it is possible to dramatically lower the brain levels of a 4-position modified quinoline methanol, without altering intrinsic in vitro potency. To address this, one would need to increase bioavailability by lowering the molecular weight of the compound. Whether this could be done without simultaneously increasing brain levels remains to be seen. Lowering LogP in this manner could also increase metabolic stability and might improve the PK profile of a new analog. Since pathways of metabolism towards less brain-penetrating compounds predominate, blocking of these pathways in an attempt to increase metabolic stability

was metabolically stable when incubated with liver microsomes it exhibited a shorter half-life than mefloquine after intravenous dosing. It is logical to suppose that a shorter half-life might contribute to the lack of in vivo efficacy relative to mefloquine, although exposure data after PO dosing would be needed to confirm this. Moreover, it cannot completely explain the efficacy outcome since we have observed superior efficacy in other quinoline

Tissue Concentration [ng/g] Plasma/Blood Concentration [ng/ml]

Average blood/tissue concentrations for WR142490(5 mg/kg IV) WR142490 [Brain] WR142490 [Plasma] WR142490 [Liver] WR142490 [Whole Blood]

100000 10000 1000 100 10 1 0.01

0.1

1

10

100

1000

Time (h)

Fig. 2 Plasma, blood and tissue concentration–time curves for Mefloquine (WR142490)

Tissue Concentration [ng/g] Plasma/Blood Concentration [ng/ml]

Average blood/tissue concentrations for WR319691 (5 mg/kg IV) WR319691 [Brain] WR319691 [Plasma] WR319691 [Liver] WR319691 [Whole Blood]

100000 10000 1000 100 10 1 0.1 0.01

0.1

1

10

100

1000

Time (h)

Fig. 3 Plasma, blood and tissue concentration–time curves for WR319691

Table 3 Maximum concentrations of metabolites in brain, plasma, and liver after administration of 5 mg/kg WR319691 IV in mice

Metabolite

Cmax (ng/g) WR160972

1

WR318973

22

WR319670 WR319707 WR308245

123

Brain

4.1 0 85

Plasma Tmax (h)

Cmax (ng/g)

6

344

Liver Tmax (h)

Cmax (ng/g)

Tmax (h)

6

339

6

120

7.4

6

1,180

1

24 NA

4 0

6 NA

498 830

1 6

6

0

NA

112

1

Eur J Drug Metab Pharmacokinet Table 4 Pharmacokinetic parameters for WR319691 in different tissues after a 5 mg/kg dose IV PK parameters

WR319691 (plasma)

Cmax (ng/ml)

WR319691 (brain)*

WR319691 (liver)

95 ± 9

27,637 ± 942

2,473 ± 151

Tmax (h)

WR319691 (whole blood) 5,286 ± 507

0.08 ± 0

0.08 ± 0

0.08 ± 0

AUC0–120 (ng h/ml)

21,281 ± 816

1,924 ± 311

4,93,506 ± 27,766

32,895 ± 1,493

AUCinf (ng h/ml)

21,298 ± 814

2,055 ± 343

4,93,842 ± 27,823

33,044 ± 1,601

12.81 ± 1.55

36.99 ± 8.60

15.98 ± 1.05

15.59 ± 0.97

235.02 ± 9.03

2,484.80 ± 418.19

10.15 ± 0.61

151.59 ± 7.53

Vz or Vz/F (ml/kg)

4,345 ± 569

1,31,151 ± 30,012

MRT b (h)

15.34 ± 1.31

t1/2-elimination (h) CL or CL/F (ml/h/kg)

50.18 ± 14.49

0.08 ± 0

234 ± 12

3,404 ± 161

15.15 ± 0.40

11.33 ± 0.94

* signifies emphasis on brain levels Table 5 Pharmacokinetic parameters for mefloquine in different tissues after a 5 mg/kg dose IV PK parameters

WR142490 (plasma)

WR142490 (brain)*

WR142490 (liver)

WR142490 (whole blood)

Cmax (ng/ml)

709 ± 138

Tmax (h)

0.08 ± 0

6.00 ± 9.00

0.08 ± 0

AUC0–168 (ng h/ml)

14,623 ± 928

73,929 ± 7,162

3,60,132 ± 67,652

29,582 ± 3,222

AUCinf (ng h/ml)

14,832 ± 819

75,865 ± 7,757

3,71,923 ± 74,251

29,995 ± 3,466

27.71 ± 7.79

32.75 ± 4.63

26.38 ± 9.58 168.47 ± 20.50

t1/2-elimination (h)

23.87 ± 7.55

1,672 ± 281

19,434 ± 3,528

1,722 ± 153 0.08 ± 0

CL or CL/F (ml/h/kg)

337.88 ± 18.40

66.46 ± 7.25

13.80 ± 2.37

Vz or Vz/F (ml/kg)

11,732 ± 3,976

2,610 ± 620

661.66 ± 179.79

6,293 ± 1,965

34.57 ± 6.26

41.31 ± 5.71

34.95 ± 2.10

32.95 ± 6.12

MRT b (h)

* signifies emphasis on brain levels Table 6 Brain binding, maximum bound and unbound brain concentrations of mefloquine (5 mg/kg IV), WR319691 (5 mg/kg IV), and three metabolites of WR319691 (after administration of 5 mg/kg WR319691 IV) in mice

Compound

Mouse brain binding (% bound)

Whole brain maximum concentration (ng/g)

Free brain maximum concentration (ng/g)

Mefloquine

99.8

1,600

3.2

WR319691

99.95

97

WR318973

99.53

22

WR319670

99.56

WR308245

98.78

may have the inadvertent effect of increasing brain levels of WR308245-like species. Acknowledgments We gratefully acknowledge substantial financial support from Medicines for Malaria Venture, Military Infectious Diseases Research Program (MIDRP), and the United States Department of Defense.

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