SB1518

Metabolism and Disposition of Pacritinib (SB1518), an Orally Active Janus Kinase 2 Inhibitor in Preclinical Species and Humans

Ramesh Jayaraman1,2, Mohammed Khalid Pasha1,3, Anthony Williams1,4, Kee Cee Goh1,5 and Kantharaj Ethirajulu1,*

1S*BIO Pte Ltd, 1 Science Park Road, # 05-09, the Capricorn, Singapore Science Park II, Singapore 117528; 2TheraIndx LifeSciences Pvt. Ltd.,# 449, 10th Cross, 4th Phase, Peenya Industrial Area, Bangalore 560058, India; 3Department of Genetics, Osmania University, Hyderabad, Andhra Pradesh, India; 4Institute of Chemical and Engineering Sciences: Organic Chemistry 11 Biopolis Way, Helios, #03-08, Singapore 138667; 512 Windrest Way, Point Cook, VIC 3030, Australia

Abstract: The ADME of Pacritinib (SB1518), an orally active JAK 2 inhibitor, was investigated in vitro and in vivo in preclinical species and humans. Pacritinib showed ~ 5 fold higher affinity to human plasma proteins relative to mouse in vitro. It was metabolized by human CYP3A4 in vitro, and did not significantly induce CYP3A and 1A2 in human hepatocytes. In vitro metabolism studies with
mouse and human liver microsomes showed the presence of four major metabolites of Pacritinib -M1 (oxidation), M2 (dealkylation), M3 (oxidation), M4 (reduction). The in vitro and in vivo metabolic patterns observed in mice and humans were in good agreement. Qualitatively and quantitatively, none of the metabolites formed in vivo was > 10% of Pacritinib in mouse, dog and humans. Pacritinib showed systemic clearance of 8.0, 1.6, 1.6 l/h/kg, volume of distribution of 14.2, 7.9, 8.5 l/kg, t1/2 of 5.6, 6.0, 4.6 h, and oral bioavailability of 39, 10, and 24% in mouse, rat and dog, respectively. In radiolabeled mass balance and QWBA studies in mice, ~ 91% of the dose was recovered in feces, suggesting biliary clearance, and maximum radioactivity was seen in the gastrointestinal tract followed by the kidney, heart and low activity in the brain. The relatively high exposures of Pacritinib in humans might be attributed to its very high plasma protein binding, low metabolic and/or biliary clearance.
Keywords: ADME, CYP450, JAK2, metabolism, pharmacokinetics, pacritinib/SB1518, QWBA.

INTRODUCTION
Janus Kinases (JAKs) play a critical part in signal transduction systems that regulate the processes of hematopoiesis, immune system development and inflammation [1]. Dysregulated expression of JAK2 has been implicated in the development of myeloproliferative neoplasms and lymphomas [2]. Pacritinib (SB1518) (11-(2-Pyrrolidin-1-yl- ethoxy)-14, 19-dioxa-5, 7, 26-triaza-tetracyclo [19.3.1.1
(2,6).1(8,12)] heptacosa 1(25), 2(26), 3, 5, 8, 10, 12(27), 16,
21, 23-decaene) is a potent inhibitor of JAK2 and FLT3 [3], that showed promising efficacy in preclinical models of myelofibrosis and lymphoma [4]. Pacritinib is now being evaluated in late stage clinical trials in myelofibrosis and lymphoma patients [5-7]. The aim of this manuscript is to assess the predictability of pharmacokinetics, drug-drug interactions, and the metabolites of pacritinib in humans based on preclinical ADME and PK, and human in vitro ADME.

*Address correspondence to this author at the S*BIO Pte Ltd, 250 North Bridge Road, #28-00 Raffles City Tower, Singapore 179101;
Tel: +6596304929 Fax: +6564788768;
E-mail: [email protected]
MATERIALS AND METHODS
Chemicals and Reagents
Pacritinib (SB1518) (11-(2-Pyrrolidin-1-yl-ethoxy)- 14, 19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]
heptacosa 1(25), 2(26), 3, 5, 8, 10, 12(27), 16, 21, 23-
decaene);the HCl or citrate salts were used in all the in vitro and in vivo studies), and its metabolites SB2403, SB2442, SB2537 and SB1777 (Fig. 1) were synthesized in SBIO Pte Ltd., Singapore. All other reagents were of research or analytical grade. Nicotinamide adenine dinucleotide phosphate reduced (NADPH) was bought from the Sisco laboratories (India). Human liver microsomes (Lot number 0710619) were purchased from the Xenotech (U.S.A). Mouse liver microsomes were prepared, based on an SOP, in house.

Relative Plasma Protein Binding (RPPB)
The RPPB was estimated based on a method reported by Collins and Klecker [8], in triplicates, at Pacritinib concentrations of 1, 10 and 100 µg/ml between human and mouse plasma. The dialysis was performed in a Microequilibrium dialyzer with a chamber volume of 0.5 ml. Before use, the membrane was rinsed with Milli Q water

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O O

-
O
H

Pacritinib, MW 473, [M] SB2442 ,MW 489, [M3]

O

O

O

SB2537, MW 487, [M1] SB1777, MW 475, [M4]

SB2403, MW 376, [M2]
Fig. (1). Chemical structure of Pacritinib (11-(2-Pyrrolidin-1-yl-ethoxy)-14, 19-dioxa-5, 7, 26-triaza-tetracyclo [19.3.1.1(2, 6).1(8,12)]
heptacosa 1(25), 2(26), 3, 5, 8, 10, 12(27), 16, 21, 23-decaene) and the authentic reference standards for its metabolites. The asterisk shown in the structure of Pacritinib indicates the carbon that was radiolabelled for the mass balance and QWBA studies.

and soaked overnight in PBS. Stock solutions of 0.1, 1 and 5 mg/ml of Pacritinib were prepared in DMSO, and spiked into human and mouse plasma to final conc of 1, 10 and 100 µg/ml. The spiked samples were vortexed and 0.25 ml of human plasma was transferred into one of the dialyzer chambers. The other chamber was filled with 0.25 ml of mouse plasma containing compound. The dialysis assembly was placed in a water bath at 37oC and dialysis was done for 24 h. Following dialysis, 50 µl of dialyzed plasma samples from both the cells was transferred into Eppendorf tubes, and extracted with 1.25 ml of tertiarymethylbutylether [MTBE] for 30 min. After extraction, the samples were centrifuged at 4oC for 10 min at 13000 rpm, and 1500 µl of supernatant was transferred into 1.5 ml Eppendorf tubes, dried in a Turbovap system at 35oC for 30 min. The dried samples were dissolved in 0.1 ml of methanol/milliQ water (60:40), mixed and centrifuged, and 90 µl was analyzed by the LC- MS/MS method described below. The RPPB was estimated using the equation
RPPB human/mouse =
In Vitro CYP450 Isoform Typing
The cDNA expressed human CYP450 enzyme preparations co-expressed with human NADPH CYP450 reductase (BactosomesTM) were from the Cyprotex Ltd., UK, and stored at -80oC prior to use. BactosomesTM (final P450 conc CYP1A2 and CYP2C19 100 pmol/ml, CYP2C9 and CYP3A4 25 pmol/ml, CYP2D6 50 pmol/ml), 0.1 M phosphate buffer pH 7.4, Pacritinib 5 µM, DMSO conc 0.25% were pre-incubated at 37oC before the addition of NADPH (final conc 1 mM) to start the reaction. Control BactosomesTM (lacking CYP450 enzymes) were also incubated to check non-enzymatic degradation. The final reaction volume was
25 µl. CYP450 specific substrates were used as positive controls. Each compound was incubated for 0, 5, 15, 30, and 45 min with each isoform. The reactions were quenched by the addition of 50 µl of methanol containing internal standard at the different time points. The plates were centrifuged at 2500 rpm for 20 min at 4oC and the supernatants were analyzed using Cyprotex generic LC-MS/MS conditions. The peak area ratio was plotted against time on a log-linear scale, the first order elimination rate constant was estimated

from the slope, from which the t1/2 was estimated using the equation t1/2 = 0.693/Kel.

In Vitro Human CYP450 Induction
Human hepatocytes were supplied from the UK human tissue bank. The freshly isolated hepatocytes were added to a 24 well plate [final seeding density 0.15 x106 cells/cm2] in Williams E media. The cells were incubated at 37oC, 95% humidity, 5% CO2 for 48 h before the addition of test compound and controls. Dexamethasone (50 µM), rifampicin (10 µM) for CYP3A4 and omeprazole (50 µM) for CYP1A2 were used as control inducers. Solutions of the control inducers and Pacritinib (0.1, 1 and 10 µM) were prepared in culture media [final DMSO conc 0.1%]. Negative controls were included that had 0.1% DMSO in the control medium. Following 48 h of culture, the media was replaced with prewarmed media containing the control inducers or negative controls. The experiment was done in triplicates. The cells were further incubated for 72 h in the presence of control inducers, Pacritinib and negative controls. Media were changed every 24 h with fresh test compound or negative control. Probe substrate solutions, ethoxyresorufin (20 µM) and midazolam (20 µM), for CYP1A2 and CYP3A4, respectively, were prepared in prewarmed media. Following 72 h incubation with Pacritinib or control inducers, media were replaced with appropriate probe substrates. The hepatocytes were incubated with the probe substrates midazolam for 30 and 60 min for ethoxyresorufin. After incubation, the supernatants were removed and mixed with an equal volume of methanol (containing internal standard for CYP3A4 only). Standard curves were prepared in culture media for the metabolites, 1-hydroxymidazolam (0.001 to
1 µM) and resorufin (0.001 to 2.5 µM). Standards were
added to a plate containing an equal volume of methanol (with internal standard). The samples and standards were centrifuged at 2500 rpm for 20 min at 4oC, and the supernatant was analyzed using LC-MS/MS for CYP3A4 and fluorescence for CYP1A (Ex λ 535 nm; Em λ 595 nm). Metabolites of the probe substrates were quantitated using calibration curves. The statistical significance of Pacritinib’s induction potential, compared to negative controls, was evaluated using a one-way ANOVA with Dunnett’s post- test. The statistical significance of the control inducers relative to the negative controls was assessed using an unpaired, one-tailed t- test. A p value of < 0.05 was deemed significant in both the tests.
In Vitro Metabolite Profiling using Liver Microsomes
Preliminary experiments with Pacritinib incubated with liver microsomes from mouse, rat, dog and humans showed the formation of several metabolites out of which the major metabolites were M1, M2, M3 and M4 (Table 1). Based on the relative abundance and the proposed chemical structures based on the mass spectral data, the reference standards of SB2537 (M1), SB2403 (M2), SB2442 (M3) and SB1777
(M4) were synthesized and used to confirm these metabolites in human and mouse microsomal incubations and PK studies. The reaction mixture of 500 µl contained potassium phosphate buffer (0.1 mM, pH 7.4), Pacritinib (50 µM), and
HLM (1 mg/ml) or MLM (2 mg/ml). The reaction was started by the addition of NADPH (2 mM) and incubated at 37oC for 2 h. The reaction was terminated and extracted with MTBE (2.5 ml), dried and reconstituted in 100 µl of 60:40 methanol/water for the analysis by LC-MS/MS.
Identification of Metabolites in Mouse and Human Plasma
Mouse PK study: BALB/c nude mice (female, aged 10–12 weeks; weighing 16–21 g) were used in the study. Food and water were given ad libitum. Pacritinib was dosed at 50 mg/kg, by gavage (dose volume of 10 ml/kg) in 0.5% methylcellulose and 0.1% Tween 80. At 10 min (0.17 h) , 30
min (0.5 h), and 1, 2, 4, 6, 8, and 24 h post dose, mice (n=3/time point) were terminated by an overdose of CO2, and blood was collected by cardiac puncture and placed in tubes containing K3EDTA. Plasma was obtained by centrifuging the blood samples at 800 g for 10 min and storing at - 60 to - 80°C until analysis. Plasma samples were pooled into two sets. The first set was formed by pooling 50 µl plasma from the time points 0.17 h, 0.5, 1 and 2 h, and the 2nd set was formed by pooling 50 µl plasma from time points 4, 6, 8 and 24 h. The pooled plasma sets of 200 µl each were extracted using 2.5 ml of MTBE, dried and the samples were reconstituted in 100 µl of methanol/water (60:40) for LC-MS analysis.
Human PK studies: All clinical studies were performed as per the Helsinki declaration. Human plasma samples for the identification and quantitation of Pacritinib and metabolites were from patients in Phase 1 studies SB1518-2007-001 and SB1518-2008-003. SB1518-2007-001 was an open label Phase 1/2 study in advanced myeloid malignancies. In the Phase 1 portion cohorts of 3 to 6 patients received one of a series of escalating doses of Pacritinib ranging from 100 to 600 mg per day. There were 8 cohorts: cohort 1 (100 mg),
cohort 2 (150 mg), cohorts 3 (200 mg) and 6 (200 mg
SB1518 citrate), cohort 4 (300 mg), cohort 5 (400 mg),
cohort 7 (600 mg) and cohort 8 (500 mg).Pacritinib capsules were taken for 25 days followed by 3 days off drug and then orally once daily in continuous 28-day cycles. Pacritinib was taken in the morning before breakfast each day. Plasma samples for full PK assessments were taken just prior to dosing (time 0) and at 0.5, 1, 2, 3, 4, 5, 6, 8, and 24±2 hours after dosing on Days 1 (dose 1) and 15 (±3) of the first cycle of treatment (Cycle 1).
SB1518-2008-003 was an open label Phase 1/2 study in patients with myelofibrosis. In the Phase 1, portion cohorts of 3 to 6 patients received one of a series of escalating doses of Pacritinib ranging from 100 to 600 mg per day. The oral PK of pacritinib was assessed when dosed as a single agent, once daily, in a 28-day cycle. There were 5 cohorts: cohort 1 (100 mg), cohort 2 (200 mg), cohort 3 (400mg), cohort 4 (600 mg) and cohort 5 (500 mg). Pacritinib capsules were administered orally once daily in the morning before breakfast for 28 consecutive days. Plasma samples for full PK assessments were taken just prior to dosing (time 0) and at 0.5, 1, 2, 3, 4, 5, 6, 8, and 24±2 hours after dosing on Days
1 (dose 1) and 15 (±3) of the first cycle of treatment (Cycle 1),
Day 1 (time points were pre-dose, 0.5 h, 1, 2, 3, 4, 5, 6, 8 and

Table 1. Summary of metabolites of Pacritinib formed on incubation in human and mouse liver microsomes and in plasma based on LC/MS/MS analysis.

Compound Name Metabolite Structure MH+ ion

Pacritinib (SB1518)

Parent (M)

473

SB2537

M1
Oxidation

487

SB2403

M2
Dealkylation

376

SB2442

M3
Oxidation

489

SB1777

M4
Reduction

475

24 h post dose) and 15 PK (time points were pre-dose, 0.5 h, 1, 2, 3, 4, 5, 6, 8 and 24 h post dose) samples were from patient 701 (cohort 7, 600 mg, p.o., u.i.d.) from clinical study protocol SB1518-2007-001, and from patient 101 (cohort 1, 100 mg, p.o., u.i.d) from clinical study protocol SB1518- 2008-003. Plasma samples from patient 101 were used for the qualitative metabolite analysis. The first set was made by pooling 100 µl plasma from time points 0.5 h, 1, 2, 3, 4 h, and the 2nd set was made by pooling 100 µl plasma from time points 5, 6, 8 and 24 h). The pre-dose plasma sample was used as blank. The pooled sample sets, 500 µ l each, were extracted using 2.5 ml of MTBE. The supernatants were dried under a stream of Nitrogen and reconstituted in 100 µl methanol/water (60:40) for LC-MS analysis.
LC-MS/MS Analysis
LC-MS and LC-MS/MS Scans of Metabolite Reference Standards
LC-MS and LC-MS/MS analysis was done for the reference standards of SB2403, SB2442, SB2537, SB1777 and Pacritinib. The scans were performed under positive ionization mode using the method mentioned below. The LC-MS scans of reference standards were used to identify the retention time of the individual metabolites. Likewise, LC- MS/MS scans were run in order to identify the fragmentation pattern of individual metabolites. Both, the retention time and the fragmentation pattern of the reference standards were mapped with the in vitro and in vivo metabolites.

Metabolite Identification in Microsomal Incubations and Plasma by LC-MS/MS
All the samples were analyzed by HPLC-UV-MS/MS. HPLC: Agilent 1100 HPLC (Agilent Technologies, U.S.A.) coupled to a UV dual wavelength (λ1-254 nm and λ2 - 290 nm) absorbance detector 2487 (Waters, U.S.A.), and an Agilent column (C18, 5 µm, 150 x 4.6 mm i.d.). The mobile phases were: solvent A was 0.1 % formic acid (aqueous) and solvent B was 0.1% formic acid in acetonitrile (v/v). A step gradient, at a flow rate of 1.0 ml/min, was used: the initial mobile phase was maintained at 100% A for 3 min, followed by reduction to 80% A by 3.1 min, 45% A by 20 min, and brought to 100 % A by 25 min and maintained up to 30 min. The eluent from the HPLC was introduced via electrospray ionization directly into a QTRAP 3200 mass spectrometer (Applied Biosystems, Foster city, CA). The mass spectrometer was maintained with positive polarity, with the following parameter settings: de-clustering potential 70 V; entrance potential 9 V; collision energy 47 V; curtain gas 10; ion spray voltage 5500; temperature 500oC; ion source gas 1 60; ion source gas 2 65; collision gas medium and the interface heater on. Data were acquired in full scan-ion mode and enhanced product ion (EPI) mode for the selected ions.
Pharmacokinetic Studies
All the animal studies were done using protocols approved by the Institutional Animal Care and Use Committee at the Biological Resource Centre, Singapore.

Pharmacokinetics in Mice
BALB/c Nude Mice (Female, Aged 10–12 Weeks; Weighing 18–21 g), Obtained from Biological Resource Centre.
(BRC, Biopolis, Singapore), were used in the study. Food and water were given ad libitum. Pacritinib was dosed intravenously at a dose of 10 mg/kg a (1 mg/ml solution in 10% Dimethylacetamide [DMA] in saline) at a dose volume of 10 ml/kg. The oral dose, 50 mg/kg, was given by gavage (dose volume of 10 ml/kg) in 0.5% methylcellulose and 0.1% Tween 80. At 5 or 10 min, 30 min, and 1, 2, 4, 8, and 24 h post dose, mice (n=2/time point) were terminated by an overdose of CO2, and blood was collected by cardiac puncture and placed in tubes containing K3EDTA. Plasma was obtained by centrifuging the blood samples at 800 g for 10 min and stored at 60 to 80°C until analysis.

Pharmacokinetics in Rats
Wistar rats (male, aged 6–8 weeks; weighing 270–325 g), procured from the Biological Resource Centre (BRC, Biopolis, Singapore), were used in the study. Three rats were used for the intravenous and oral studies each, in a parallel design. A day before the study, the jugular vein was cannulated in all the rats. Animals were fasted overnight before dosing and feed was given 4 h post dose. Water was given ad libitum. Pacritinib (2 mg/kg) was dosed intravenously (bolus through the jugular vein), at a dose volume of 1.0 ml/kg in DMA: saline (10 %:90 % v/v). The oral dose (10 mg/kg) was dosed (gavage at a dose volume of 4 ml/kg) as a suspension in 0.5% methylcellulose and 0.1% Tween 80. Serial blood
samples (0.2 ml per sample) were drawn from each rat at 10 and 30 min and at 1, 2, 4, 8, and 24 h post i.v. dose and 15
and 30 min and at 1, 2, 4, 8, and 24 h post oral dose, and placed in tubes containing K3EDTA as the anticoagulant. Plasma was obtained by centrifuging the blood samples at 800 g for 10 min and storing at 60 to 80°C until analysis.

Pharmacokinetics in Dogs
The study was performed at Absorption Systems LP, PA, USA. Two male non-naïve Beagle dogs (one each for the i.v. and p.o. study) aged 1-2 years, weighing 8-11 kg, were used. The animals were healthy at the beginning of the study, and were acclimatized for at least 7 days. Food was withheld from the animals for a minimum of 12 h before and during the study, and were fed 4 h post dose. Water was given ad libitum. Pacritinib was dosed at 1 mg/kg i.v. as a solution in DMA: saline (10 %:90 % v/v) at a dose volume of 1 ml/kg, and 5 mg/kg p.o. in 0.5% methylcellulose and 0.1% Tween 80 in water at a dose volume of 2.5 ml/kg. Post dose, blood samples were drawn from the foreleg vein, at 2 min, 5, 15,
30 min, 1, 2, 4, 6, 8 and 24 h, and placed into chilled polypropylene tubes with K3EDTA. The blood samples were centrifuged at 3000 rpm for 15 min; plasma was separated and stored at -80oC until analysis.
Oral Pharmacokinetics of Pacritinib and exposures of SB2403 and SB2537
Human plasma samples used for the quantitation of pacritinib and its metabolites SB2403 and SB2537 were from patients in Phase 1 studies SB1518-2007-001 and SB1518-2008-003. The study design for the PK studies are mentioned in the section “Identification of metabolites in mice and human plasma”. Day 1 (time points were pre-dose,
0.5 h, 1, 2, 3, 4, 5, 6, 8 and 24 h post dose) and 15 PK (time points were pre-dose, 0.5 h, 1, 2, 3, 4, 5, 6, 8 and 24 h post dose) samples were from patient 701 (cohort 7, 600 mg, p.o., u.i.d.) from study SB1518-2007-001, and from patient 101 (cohort 1, 100 mg, p.o., u.i.d) from study SB1518-2008-003.

Sample Analysis
Mouse, rat and dog: To 50 µl of plasma, 10 µl of Carbamazepine (125 ng/ml in 50% methanol in water) internal standard was added and extracted with 1.25 ml of MTBE for 30 min on a vortexer. Following extraction, the samples were centrifuged (Eppendorf 5415R) at 1200 g for 10 min at 4°C. The supernatants were evaporated to dryness at 35°C in a turbovap for 30 min, reconstituted in mobile phase (70% methanol/30% water), and analysed by LC- MS/MS using an Alliance HT2795 HPLC system (Waters, Milford, MA) connected to a Quattro Micro Mass Spectrometer (Waters). The mobile phase consisted of methanol and 0.1% formic acid in water (70:30, v/v). The samples were separated on a column (Zorbax Eclipse C8 2X50 mm, 5 µm (Agilent)), maintained at 40°C, at a 0.3 ml/min flow rate with a 10 min run. The MS parameters for Pacritinib-MRM were: m/z 473 to m/z 96.8 (electrospray ionization positive); cone voltage, 40 CV; collision energy,
35 eV. The corresponding parameters for carbamazepine (internal standard) were m/z 237 to m/z 194 (electrospray ionization positive), 35 V, and 20 eV. The assay was linear

between 2.0 and 1000 ng/ml, accurate and precise with an LLOQ = 2.0 ng/ml
Humans: Pacritinib concentrations were quantified using a validated LC-MS/MS method [9]. The concentrations of SB2403 and SB2537 were quantified using a non-GLP method using authentic standards of SB2403 and SB2537. The assay was linear over the concentration range 1.0 –
500.0 ng of SB2403/ml and 1.0 – 500.0 of SB2537/ml of plasma. The LLOQ for both the metabolites was 1.0 ng/ml.

Pharmacokinetic Analysis
Non-compartmental (NCA) PK parameters were estimated using WinNonlin (version 5.2, Pharsight). The AUC was computed using the linear up-log down method. The following NCA parameters were estimated in mice, rats and dogs: Systemic Clearance (CL), Vss (Volume of distribution at steady state), kel, t1/2, AUC0-t, AUC0-∞, Cmax, AUC0-24h, and Tmax. The absolute oral bioavailability was estimated using the equation:
F(%) = Mean AUC0-, oral x Dose i.v. x100
procedure, and identified by individual animal number, tail marking, and study number. On the day before the study, group 1 animals were transferred to a metabolism cage for the duration of the excreta collections. Food and water were provided ad libitum.
Study Design
There were two groups in the study. Group 1 had 3 mice and group 2 had 7 mice. Both the groups were administered a single oral dose, by gavage, of 100 mg/kg (~ 100 µCi/kg) pacritinib at a dose volume of 10 ml/kg. The actual amount administered was determined by weighing the dose syringe before and after dose administration. After the dose was administered, and as the gavage tube was removed, a dose wipe was collected into a plastic container, and was retained at room temperature prior to analysis by liquid scintillation counting to determine residual radioactivity. The dose site wipes collected for Group 1 animals were pooled prior to radioanalysis. The radioactivity recovered was subtracted from the administered amount to give the actual radioactive dose administered. Urine and feces were collected from

Mean AUC

0-, i.v
Dose

oral
Group 1 animals pre-dose and at 0-12, 12-24, 24-48, 48-72,
72-96, 96-120, 120-144, and 144-168 hours post dose.

For the human study, Cmax, AUC0-24h, Tmax, and t1/2 were estimated for Pacritinib and its metabolites in the patients mentioned above. The AUC0-24h and Cmax for SB2403 and SB2537 were estimated as a percentage of the corresponding Cmax and AUC0-24h of pacritinib on days 1 and 15, respectively.

Radiolabeled Mass Balance Study, Pharmacokinetics and QWBA
Labeling of Pacritinib with 14C
Pacritinib was labeled at the benzylic position as shown in Fig. 1. The specific activity of the preparation was 52.9 mCi/mmol and the radiochemical purity was 99.7%.
Dosing Formulation
Fresh vehicle, 0.5% methylcellulose (4000 cps) and 0.1% Tween 80® in deionized water, was prepared for use on study once and was stored refrigerated at 2 to 8°C. The dosing formulation was prepared by mixing the appropriate amounts of radiolabeled 14C-pacrtitinib and non-radiolabeled pacritinib citrate salt with the required volume of vehicle, mixed with magnetic stirring bars to achieve a target concentration of 10 mg/ml (10 μCi/ml). The dosing formulation was prepared on the day prior to dosing, stirred overnight, and stored at room temperature. The dosing formulation was analyzed for radioactivity and radiochemical purity on the day of dosing. The concentrations of radioactivity in the formulation (based on the mean of the predose and postdose values) were as follows: Radioactivity Concentration: 26848236 dpm/g (12.1μCi/g), Pacritinib Concentration: 9.91478 mg/g, and the Specific Activity of Pacritinib: 1.2198 μCi/mg.
Animals
Male BALB/cAnNCrl mice (age 13 weeks, body weight range ~ 22-24 g) (Charles River Laboratories, Stone Ridge, NY) were used in the study. Animals were individually housed in suspended, stainless steel, wire-mesh type caging. Animals were assigned to cages using a simple randomization
Samples were pooled and collected into a plastic container on dry ice and maintained under these conditions or at -10 to
-30°C until processed for radioanalysis. The cage was rinsed with deionized water following each excreta collection at 24, 48, 72, 96, 120, and 144 h post dose. Following the final excreta collection at 168 h post dose, the cage was rinsed with 1% trisodium phosphate solution and wiped using gauze pad. The cage rinse and wipe sample were collected into separate plastic containers at room temperature and maintained under this condition until processed for radioanalysis. At the terminal time point of 168 h post dose, group 1 animals were euthanized by CO2 inhalation and euthanasia was confirmed via cervical dislocation. The carcasses were stored at -10 to -30°C until processed for radioanalysis. Blood samples (approximately 0.45 to 1 ml) were collected from one group 2 animal per time point via cardiac puncture following CO2 inhalation at 1, 4, 8, 24, 48, 72, and 168 h post dose into tubes containing K3EDTA on wet ice. An aliquot (approximately 0.25 ml) of whole blood was collected and stored on wet ice until processed for radioanalysis. The remaining blood was centrifuged at 4°C to obtain plasma and the samples were stored on wet ice until processed for radioanalysis.
QWBA Analysis
At each terminal time point of 1, 4, 8, 24, 48, 72, and 168 h post dose, one group 2 animal per time point was deeply anesthetized with CO2 and was immersed in a hexane/dry ice bath for 15 min. Following immersion in the hexane/dry ice bath, each carcass was drained, blotted dry, placed in an appropriately labeled plastic bag, and was placed on dry ice for approximately 2 to 14.5 h to complete the freezing process. Each carcass was removed from dry ice and stored frozen at -10 to -30°C until QWBA analysis. The frozen mouse carcasses were embedded in a 2% carboxymethylcellulose matrix and mounted on a microtome stage (Leica CM3600 Cryomacrocut and Vibratome 9800 microtomes, Nussloch, Germany) maintained at approximately –20°C. Sections that

were approximately 40 μm thick were taken in the sagittal plane, and captured on adhesive tape (Scotch Tape No. 8210, 3M Ltd., St. Paul, MN, USA). Sections were allowed to dry by sublimation in the cryomicrotome at ~20°C for at least 48
h. A set of sections were mounted on cardboard backing, covered with plastic wrapping, and exposed along with 14C- spiked calibration standards to 14C-sensitive imaging plates (Fuji Biomedical, Stamford, CT). The imaging plates and sections were enclosed in exposure cassettes and allowed to expose at room temperature for at least four days in a copper- lined lead safe. Selected sections were exposed to phosphor image screens, and tissue radioactivity concentrations were quantified from the whole-body autoradiograms using a validated image analysis system (Typhoon 9410™ image acquisition system [GE Healthcare/Molecular Dynamics, Sunnyvale, CA, USA] and MCID™ image analysis software [v. 7.0, Interfocus Imaging, Inc., Linton, Cambridge, UK]). Quantification, relative to the calibration standards, was performed by image densitometry using MCID image analysis software (Interfocus Imaging, Inc., Linton, Cambridge, UK) and a standard curve constructed from the integrated response (MDC/mm2) and the nominal concentrations of the 14C-blood calibration standards. The concentrations of radioactivity were expressed as μCi/g and were converted to μg equivalents of pacritinib per gram sample (μg equiv/g) using the specific activity of administered 14C-pacritinib (1.2198 μCi/mg). The lower limit of quantitation was 0.579 μg equiv/g.

RESULTS
Pacritinib showed approximately a 5 fold higher affinity to mouse plasma at 1 µg/ml and the affinity decreased with increasing concentration (Table 2). Pacritinib was mainly

Table 3. Metabolism of Pacritinib by recombinant human CYP450 isoforms in vitro.

CYP450
Isoform
t1/2(min) Mean Pacritinib Remaining at 45 min (% Relative to 0 min)
1A2 > 45 67.3
2C19 > 45 94.4
2C9 > 45 133.0
2D6 > 45 71.5
3A4 17.4 52.7

Table 4. In vitro induction of human CYP3A4 and CYP1A2 by Pacritinib following incubation in hepatocytes. The values shown are fold induction relative to negative controls.

Compound (µM) Fold Induction (mean ±SD)
CYP3A4 CYP1A2
Pacritinib (0.1) 1.4 ± 0.035* 1.02 ± 0.0377*
Pacritinib (1.0) 1.67 ± 0.102** 1.06 ±0.0204*
Pacritinib (10.0) 0.018 ± 0.019** 1.21 ± 0.0252**
Rifampicin (10.0) 20.9 ± 0.498*** NA
Dexamethasone (50.0) 8.88 ± 0.702*** NA
Omeprazole (50.0) NA 14.8 ± 0.0839**

metabolized by CYP3A4 with a t1/2 of 17.4 min and was not
*p < 0.05 : significantly different from 0.1% DMSO control
**p<0.01: significantly different from 0.1% DMSO control

significantly metabolized by the other CYP450s [t1/2> 45 min] (Table 3). Pacritinib did not significantly induce CYP3A4 and CYP1A2 (Table 4). The lower induction seen at 10 µM pacritinib could be due the precipitation of pacritinib at concentrations > 5 µM [3]. The level of induction by Pacritinib was < 40% of the corresponding positive controls for both CYP3A4 and 1A2.
Metabolite profiling studies in vitro following incubation of Pacritinib with human and mouse liver microsomal fractions indicated the formation of metabolites. Table 1 shows the metabolites of Pacritinib [MH+ with m/z 473] identified in HLM and MLM. The HPLC-UV, EMS-TIC, EMS-MS and EPI-MS/MS spectra for the reference standard

Table 2. The relative plasma protein binding of Pacritinib to human and mouse plasma determined by equilibrium dialysis.

Plasma Concentration of Pacritinib (µg/ml) RPPBhuman/mouse (Mean ±SD)
1.0 5.1 ± 0.28
10.0 1.9 ± 0.08
100.0 1.6 ± 0.04
***p<0.0001: significantly different from 0.1% DMSO control NA: Not Applicable

metabolites SB2403, SB2442, SB2537 and SB1777 are shown in supplementary Figs. (1, 2, 3, 4 and 5). Incubation of Pacritinib in HLM showed the formation of many metabolites (Fig. 2). The TIC-EMS showed MH+ ions of m/z 513 (MH+ +40, UIM), 543 (MH+ +70, UIM), 489 (MH+
+16, UIM), 473 (MH+ M, parent), 489 (MH+ +16, M3), 525
(MH+ +52, UIM), 442 (MH+ -31, UIM), 376 (MH+ -97, M2,
dealkylation metabolite) and 487 (MH+ +14, M1, oxidation metabolite), respectively. The metabolites corresponding to m/z 513, 543, 489, 525 and 442 are unknown and their ion intensities were <10% relative to the Pacritinib MH+ ion. The metabolic profile in MLM was similar to that of in humans in terms of the formation of M1, M2 and M3 (Fig. 3). The TIC-EMS showed, in addition to M1, M2 and M3, MH+ ions of m/z 513 (MH+ +40, UIM), 543 (MH+ +70, UIM), 489
(MH+ +16, UIM), 527 (MH+ +54, UIM), 541 (MH+ +68,
UIM), 525 (MH+ +52, UIM), 440 (MH+ -33, UIM), 442
(MH+ -31, UIM), and 523 (MH+ +50, UIM). The identities of M1, M2, and M3 in HLM and MLM were confirmed based on comparing the MS/MS spectrum of their peaks and retention times in the microsomal incubations with the MS/MS spectrum and retention times of their corresponding reference standards. Based on the confirmation of the

27 6.2
28

276 .2

276.3

28
Fig. (2). Metabolism of Pacritinib (50 µM) in HLM (a) HPLC-UV profile of pacritinib and its metabolites, (b) TIC-EMS of pacritinib and its metabolites, (c) XIC of MH+ of Pacritinib, (d) MS spectrum of Pacritinib, (e) XIC of MH+ of SB2537, (f) MS spectrum of SB2537, (g) XIC of MH+ of SB2403, (h) MS spectrum of SB2403, (i) XIC of MH+ of SB2442, and (j) MS spectrum of SB2442.

276.2 28
29
HPLC-UV profile
TIC-EMS of SB1518
incubation in MLM
276 .3

2

2
Fig. (3). Metabolism of pacritinib (50 µM) in MLM (a) HPLC-UV profile of pacritinib and its metabolites, (b) TIC-EMS of pacritinib and its metabolites, (c) XIC of MH+ of Pacritinib, (d) MS spectrum of Pacritinib, (e) XIC of MH+ of SB2537, (f) MS spectrum of SB2537, (g) XIC of MH+ of SB2403, (h) MS spectrum of SB2403, (i) XIC of MH+ of SB2442, and (j) MS spectrum of SB2442.

O O

O O O
O
OH
N N
H
N N N N
H O

M1, SB2537 (m/z 487)
M2, SB2403 (m/z 376)

O
N O
N N
H
M, SB1518 (m/z 473)

O O

O O
N O N O
-O
N N N N
H H

M4, SB1777(m/z 475)

Fig. (4). Schematic representation of molecular ionic structures of pacritinib and its metabolites.
M3, SB2442 (m/z 489)

metabolites in microsomal incubations, a metabolic pathway for Pacritinib was proposed (Fig. 4).
Analysis of pooled plasma from the oral PK study in mice showed the formation of M1, M2, M3 and M4 (reduction metabolite) (Fig. 5). Although the HPLC-UV profile did not show prominent peaks the TIC-EMS showed peaks at Rt 10.2, 12.74, 15.04, 16.0, 18.70, 19.81, and 21.88
min corresponding to MH+ ions of m/z 531 (MH+ +58, UIM), 473 (MH+, M), 369 (MH+ -104, UIM), 525 (MH+ +52,
UIM), 487 (MH+ + 14, M1), 409 (MH+, -64, UIM) and 409
(MH+ -64, UIM), respectively. The peak at 12.74 min corresponded to the parent compound. The XIC showed the formation of M1 and the TIC-EPI spectrum showed the formation of M2, M3 and M4.
The HPLC-UV profile of pooled human plasma showed parent compound at the Rt of 12.58 min, and the peaks of M1 (oxidation metabolite), M2 (dealkylation metabolite), and M3(oxidation metabolite) with low intensities (Fig. 6). However, the TIC-EMS showed prominent peaks at the Rt of 12.60, 13.25, 16.01, 16.42, 17.58, 18.48 and 18.73 min
corresponding to MH+ ions of m/z 416 (MH+ -57, UIM), 473 (MH+, M), 489 (MH+ +16, M3), 525 (MH+ +52, UIM), 440
(MH+ -33, UIM), 442 (MH+ -31, UIM), 376 (MH+ -97, M2)
and 487 (MH++14, M1), respectively. The XIC of pooled human plasma showed an extra peak M4 corresponding to mass ion with m/z 475 (MH++2, M4) at an Rt of 13.72 min. The formation of M1(oxidation metabolite), M2 (dealkylation metabolite), M3(oxidation metabolite) and M4(reduction metabolite) in both mouse and human plasma was confirmed
by the comparison with the reference standards’ LC-MS and LC-MS/MS profiles (supplementary Figs. 1, 2, 3, 4 and 5).
The PK profiles of Pacritinib, SB2403 (corresponding to the M2 dealkylation metabolite) and SB2537 (corresponding to the M1 oxidation metabolite) in human subjects are shown in Fig. 7 and the NCA PK parameters are shown in Tables 5 and 6. Subject 101 (100 mg): The PK profiles of the metabolites SB2403 and SB2537 were similar to pacritinib on days 1 and 15. The AUC0-24h and Cmax of SB2403 were 0. 4% and 0.3 % of that of pacritinib on day 1, and AUC0-24h and Cmax were 0.6 and 0.5 % of that of pacritinib on day 15. The AUC0-24h and Cmax of SB2537 were 9 % of that of pacritinib on day 1, and AUC0-24h and Cmax were 9 % of that of pacritinib on day 15. Subject 701 (600 mg): The PK profiles of the metabolites SB2403 and SB2537 were similar to pacritinib on days 1 and 15. The AUC0-24h and Cmax of SB2403 were 0.14% and 0.32% of that of pacritinib on day 1, and AUC0-6h and Cmax were 0.3 and 0.4 % of that of pacritinib on day 15. The AUC0-24h and Cmax of SB2537 were 5% of that of pacritinib on day 1, and AUC0-6h and Cmax were 5 % of that of pacritinib on day 15.
The results of the PK studies in mice, rats and dogs are summarized in Table 7 and Fig. 8. Mice: Pacritinib showed a biexponential decline, post IV dose, with a moderate mean terminal half-life of 5.6 h, high Cl (8 l/h/kg) and a high Vss (~14 l/kg). Post oral dosing, the absorption was rapid (tmax =
1.25 h) followed by a biexponential decline with a terminal t1/2 of 2.2 h, with a moderate oral bioavailability of 39%. Rat: Pacritinib displayed a biexponential decline, post an IV dose,

22.9

10.20

Fig. (5). contd….
Fig. (5). Pacritinib and its metabolites in pooled mice plasma: (a) HPLC-UV profile of pacritinib and its metabolites, (b) TIC-EMS of pacritinib and its metabolites, (c) XIC of MH+ of Pacritinib, (d) MS spectrum of Pacritinib, (e) XIC of MH+ of SB2537, (f) MS spectrum of SB2537, (g) EPI-TIC of SB2403, (h) MS/MS spectrum of SB2403, (i) EPI-TIC of SB2442, (j) MS/MS spectrum of SB2442, (k) EPI-TIC of SB1777, (l) MS/MS spectrum of SB1777.

W aters DWD from Sample 7 (SB1518_human_Cycle1_day15_inviv o_set_1) of DataSET 2.wiff Max.
5.0 12.58 a
4.5
4.0 HPLC-UV profile
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Time, mi n
TIC of +EMS: from Sample 7 (SB1518_human_Cycle1_day15_invivo_set_1) of DataSET 2.wiff (Turbo Spray) Max. 7.1e
7.0e8 12.60 b
6.0e8 TIC-EMS of SB1518 M
5.0e8 and its metabolites M1
4.0e8 in pooled human M2
plasma M3
3.0e8

2.0e8
18.73
1.0e8 16.01 16.42 18.48
13.25 17.58
0.0
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
XIC of +EMS: 473.0 to 473.5 amu from Sample 7 (SB1518_human_Cycle1_day15_invivo_set_1) of Data Time, mi n urbo Spray) Max. 1.0e
SET2.wiff (T
12.60 c
1.00e7
9.00e6 XIC of MH+ of
8.00e6
7.00e6 SB1518
6.00e6
5.00e6
4.00e6
3.00e6
2.00e6
1.00e6

0.00
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Time, min

+EMS: 12.590 min from Sample 7 (SB1518_human_Cycle1_day15_invivo_set_1) of DataSET2.wiff (Turbo Spray), Centroided Max. 2.9e
2.9e7 316.2 d
2.5e7 MS spectrum of
2.0e7 SB1518 290.3
276.3
1.5e7
260.2
1.0e7 473.2
115.1 244.2 258.3 287.3 306.3 332.2
5.0e6 128.2 143.2 170.2 220.3 246.2 274.2 300.2 328.2 495.2
118.2 151.1 161.2 186.3 205.3 229.3 250.9 281.2 279.4 320.3 334.3 356.2 385.3 404.2
455.3 475.4
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580
m/z, amu
XIC of +EMS: 487.0 to 487.5 amu from Sample 7 (SB1518_human_Cycle1_day15_invivo_set_1) of DataSET2.wiff (Turbo Spray) Max. 3.8e
2.8e5 18.74 e
XIC of MH+ of
2.5e5

2.0e5 SB 2537
1.5e5

1.0e5

23.
5.0e4
15.59 16.42 17.63 21.66 22.78
0.0
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Time, min

+EMS: 18.743 min from Sample 7 (SB1518_human_Cycle1_day15_invivo_set_1) of DataSET2.wiff (Turbo Spray), Centroided Max. 6.3e
6.0e6 398.2 f
509.2

5.5e6 MS spectrum of
5.0e6
4.5e6 SB2537 376.3
4.0e6
3.5e6 290.3 358.4 399.2
3.0e6
2.5e6
2.0e6 112.1 292.3 316.3 328.4 340.4
1.5e6
1.0e6 371.4
5.0e5 110.1 260.2 276.3 302.3 314.4 326.3 343.3 378.4 424.3 437.2 511.4
115.1 144.1 169.2 184.2 191.1 220.2 244.2 381.5 451.1 470.4 487.1 563.4 569.5
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580
m/z, amu

Fig. (6). contd….

XIC of +EMS: 376.0 to 376.5 amu from Sample 7 ( SB1518_hum an_Cycle1_day15_invivo_s et_1) of D ata SET2.wiff ( Turbo Spray) Max. 4 .2e
4.0e6 + g
18.74

3.5 e6 XIC of MH of
3.0 e6 SB2403
2.5 e6

2.0 e6

⦁ e6
1.0 e6 12.61
5.0 e5 16 .00
18.44
0.0
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
T ime, min

+EM S: 18.441 m in from Sam ple 7 (SB1518_human_Cycle1_day15_invivo_set_ 1) of D ata SET2.wiff (T urbo Spray), Centroided Max. 1 .8e
1.8 e6 288.2 h
⦁ e6
1.4 e6 MS spectrum of
276.2
1.2 e6 SB2403
1.0 e6
8.0 e5 290.2
6.0 e5 260.2
4.0 e5 277.3 286.2 304.3
328.2
2.0 e5 115.1 220.1 245.2 261.2 30 0.2 302.2 340.2 376.1 398.2 420.2
155 .2159.1 169 .1 186.2 230.0 269.2 279.6 358.3 435.9 465.4 475.6 483.1 519.8 526.4 551.3 577.5 5
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580
m/z, am u
XIC of +EMS: 489.0 to 489.5 amu from Sample 7 (SB1518_hum an_Cycle1_day15_invivo_s et_1) of D ataSET2.wiff ( Turbo Spray) Max. 1.5e
13.25 i
1.4e6
1.2e6 XIC of MH+ of
1.0e6 SB2442
8.0e5

6.0e5

4.0e5

2.0e5
11.89
0.0
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Time, min

+EMS: 13.244 m in from Sample 7 (SB1518_human_Cycle1_day15_invivo_set_1) of D ataSET2.wiff (Turbo Spray), Centroided Max. 1.7e
1.6e6 j
290.2 489.2
1.4e6 MS spectrum of 288.3
1.2e6 SB2442
1.0e6 276.3 424.2
8.0e5
6.0e5 306.3 316.3
262.2 304.2
4.0e5 302.2 511.2
2.0e5 115.1 234.2 260.2 274.2 300.2 332.2
128.1 155.1 184.2 218.2 233.2 264.2 272.2 298.2 346.3 383.3 402.2 426.4 465.2 473.3 491.3 512.2 571.1
553.2 59
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580
m/z, amu
XIC of +EMS: 475.0 to 475.5 amu from Sample 7 (SB1518_hum an_Cycle1_day15_invivo_s et_1) of D ataSET2.wiff ( Turbo Spray) Max. 8.9e
8.9e5 12.61 k
8.0e5 XIC of MH+ of
7.0e5
6.0e5 SB1777
5.0e5
4.0e5 M4
3.0e5
2.0e5
1.0e5 13.72
18.48
0.0
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
T ime, min

+EMS: 13.721 m in from Sample 7 (SB1518_human_Cycle1_day15_invivo_set_1) of D ataSET2.wiff (T urbo Spray), Centroided Max. 4.2e
4.0e5 l
316.2

3.5e5 MS spectrum of
3.0e5 SB1777
2.5e5

2.0e5

1.5e5
1.0e5 290.2 318.3 385.3
475.3
291.1
5.0e4 260.2 276.2 301.2 497.2
115.1 128.1 158.9 181.2 207.1 220.1 233.1 284.3 332.2 356.2 376.2 387.3 424.1 436.4 465.4 477.4 509.4 527.0 553.2 569.3 595.
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580
m/z, amu
Fig. (6). Pacritinib and its metabolites in pooled human plasma: (a) HPLC-UV profile of pacritinib and its metabolites, (b) TIC-EMS of pacritinib and its metabolites, (c) XIC of MH+ of Pacritinib, (d) MS spectrum of Pacritinib, (e) XIC of MH+ of SB2537, (f) MS spectrum of SB2537, (g) EPI-TIC of SB2403, (h) MS/MS spectrum of SB2403, (i) EPI-TIC of SB2442, (j) MS/MS spectrum of SB2442, (k) EPI-TIC of SB1777, (l) MS/MS spectrum of SB1777.

with a moderate mean terminal t1/2 of 6 h, a moderate Cl (1.6 l/h/kg) and a high Vss (~ 8 l/kg). Post an oral dose, the absorption was moderately fast (tmax = 4h), t1/2 was ~ 6 h, and the oral bioavailability was low (10%). Dog: Pacritinib showed a multiexponential decline post an IV dose with a high Cl (1.6 l/h/kg), high Vss (8.5 l/kg) and a moderate t1/2 of
~ 5 h. Post an oral dose, the absorption was moderately fast (tmax= 4h), the t1/2 was 4.4 h and the oral bioavailability was low (24%).
In the radiolabeled PK and mass balance study, radioactivity in plasma and blood peaked at 4 h post dose and was below limit of quantitation at 24 h post dose (Fig. 9). After the oral administration of 14C-pacritinib the recovery of dosed radioactivity over the 168-h study period was 92.97% (Table 9). The excretion pattern of radioactivity in urine and feces showed that the majority of the radioactive dose was recovered in the feces with a recovery of 90.98%. The urine

contained 1.49% of the total radioactivity. The majority of the dosed radioactivity recovered in the feces was obtained within 12 h (Table 8). The results from the QWBA study are summarized in Table 9 and supplementary Fig. 6. Pacritinib- derived radioactivity was widely distributed to tissues of mice through 8 h post dose, and decreased rapidly in most tissues from 24 to 168 h post dose. Most tissues showed maximal concentration at 1 h. The tissues with the highest concentrations (> 25,000 ng-eq/g) of pacritinib-derived radioactivity found at the maximal concentration were small intestine, cecum, large intestine, stomach, liver, kidney cortex, adrenal gland cortex, spleen, kidney medulla, and the bulbo-urethral gland. The lowest tissue concentrations observed at their maximal concentration were observed in the tissues of the central nervous system, and eye lens. Concentrations of radioactivity in most tissues declined rapidly over the course of the study and most tissue

10 10

Concentration (ug/mL)
Concentration (ug/mL)
1 1

0.1 0.1

0.01 0.01

0.001

0 4 8 12 16 20 24
Time (h)
0.001

0 4 8 12 16 20 24
Time (h)

C D
10 10

Concentration (ug/mL)
1 1

Concentration (ug/mL)
0.1 0.1

0.01 0.01

0.001

0 4 8 12 16 20 24
Time (h)
0.001

0 1 2 3 4 5 6
Time (h)

Fig. (7). Concentration-time profiles (semi logarithmic plot) of pacritinib and metabolites following oral doses, u.i.d., of pacritinib in a) subject 101, 100 mg, day 1, b) subject 101, 100 mg, day 15, c) subject 701, 600 mg, day 1, and d) subject 701, 600 mg, day 15. Dark filled squares: pacritinib; open squares: SB2403; filled circles: SB2537.

Table 5. NCA pharmacokinetic parameters of Pacritinib, SB2403, and SB2537 in subject 101 (100 mg), from phase 1 study SB1518- 2008-003, Cycle 1.

Parameter Day 1
Pacritinib SB2403 SB2537 SB2403
(% of Pacritinib) SB2537
(% of Pacritinib)
AUC0-24h(µg.h/ml) 29 0.10 3 0.4 9
Cmax(µg/ml) 2 0.006 0.15 0.3 9
Tmax (h) 4 24 6 NA NA
t1/2(h) 25 NE 28 NA NA
Day 15
AUC0-24h(µg.h/ml) 42 0.27 4.0 0.6 9
Cmax(µg/ml) 2 0.01 0.2 0.5 9
Tmax (h) 2 24 3 NA NA
t1/2(h) 26 NE 23 NA NA
NE: Not Estimated; NA: Not Applicable

Table 6. NCA pharmacokinetic parameters of SB1518, SB2403, and SB2537 in subject 701 (600 mg), from phase 1 study SB1518- 2007-001, Cycle 1.

Parameter Day 1

Pacritinib
SB2403
SB2537 SB2403
(% of Pacritinib) SB2537
(% of Pacritinib)
AUC0-24h(µg.h/ml) 110 0.15 6 0.1 5
Cmax(µg/ml) 7.2 0.02 0.36 0.3 5
Tmax (h) 2 2 2 NA NA
t1/2(h) 14.9 12.6 14.9 NA NA
Day 15*
AUC0-24h(µg.h/ml) 48 0.14 2.3 0.3 5
Cmax(µg/ml) 8.5 0.03 0.5 0.4 5
Tmax (h) 4 0 5 NA NA
t1/2(h) 6.3 6.5 NE NA NA
* tlast was 6 h ; AUC0-6h; NE: Not Estimated; NA: Not Applicable

Table 7. Noncompartmental pharmacokinetic parameters of Pacritinib estimated in mice, rats, and dogs.

Parameter Mouse (n=2) Rat (n=3) Dog (n=1)
Intravenous
Dose (mg/kg) 10 2 1
CL(l/h/kg) 8.0 1.6 ± 0.3 1.6
Vss(l/kg) 14.2 7.9 ±1.7 8.5
t1/2(h) 5.6 6 ± 2 4.6
AUC0-t(ng.h/ml) 1231 1243± 233 632
AUC0-∞(ng.h/ml) 1239 1302 ± 259 645
Oral
Dose (mg/kg) 50 10 5
tmax (h) 1.25 4 ± 0 4
Cmax(ng/ml) 1108 114 ± 25 89
t1/2(h) 2.2 5.7 ± 1.3 4.4
AUC0-t(ng.h/ml) 2438 599 ± 111 735
AUC0-∞(ng.h/ml) 2439 649 ± 100 761
CL/F(l/h/kg) 17.6 15.6 ± 2.3 6.6
V/F(l/kg) 57 13 ± 5 42
F (%) 39 10 ± 2 24

10000 1000

1000

Mean conc (ng/ml)
Mean conc (ng/ml)
100
100

10
10

1

0.1

0 4 8 12 16 20 24
time (h)

1
0 4 8 12 16 20 24
time (h)

1000

Concentration (ng/mL)
100

10

1
0 4 8 12 16 20 24
time(h)
Fig. (8). a) Mean concentration-time profiles (semi logarithmic plot) of pacritinib in mice after single intravenous (10 mg/kg; open circles) and oral (50 mg/kg; filled squares) doses. Each point represents the mean from two mice, b) Mean concentration-time profiles (semi logarithmic plot) of pacritinib in rats after single intravenous (2 mg/kg; open circles) and oral (10 mg/kg; filled squares) doses. Each point represents the mean ± S.E.M. from three rats, c) concentration-time profiles (semi logarithmic plot) of pacritinib in dogs after single intravenous (1 mg/kg; open circles) and oral (5 mg/kg; filled squares) doses.
100000 blood
plasma

ng-eg/g
10000

1000

100

0 2 4 6 8
time(h)

Fig. (9). Time course of pacritinib related radioactivity in mice.

Table 8. Cumulative recovery of radioactivity from mice in urine, feces and cage following a single oral dose of 14C-pacritinib.

Time Period (h) % Recovery
Urine Faeces Cage Rinse Cage Wipe Carcass
Predose 0 0
0.3 ND NA
0-12 1.19 84 ND NA
12-24 0.24 5.25 ND NA
24-48 0.04 1.26 0.06 ND NA
48-72 0.01 0.1 0 ND NA
72-96 0.01 0.04 0 ND NA
96-120 0.00 0.03 0 ND NA
120-144 0.00 0.02 0 ND NA
144-168 0.00 0.02 0 ND 0.14
subtotal 1.49 90.98 0.36 0.00 0.14
ND: Not done; NA: Not applicable

Table 9. Concentrations (μg equiv/g) of radioactivity in tissues of male BALB/cAnNCrl mice administered a single.

Tissue Type Tissue 1 h 4 h 8 h 24 h 48 h 72 h 168 h
Vascular/ Lymphatic Blood (cardiac) 3.451 0.770 BQL BQL BQL BQL BQL
Bone Marrow 11.517 9.166 1.337 BQL BQL BQL BQL
Lymph Node 11.603 7.119 1.113 BQL BQL BQL BQL
Spleen 29.621 11.631 2.004 BQL BQL BQL BQL
Thymus 5.808 4.832 1.337 BQL BQL BQL BQL
Excretory/ Metabolic Bile (in gall bladder) 693.496 418.232 2848.804 10.302 0.775 0.767 BQL
Kidney (cortex) 42.139 15.070 3.407 BQL BQL BQL BQL
Kidney (medulla) 28.470 5.616 2.444 BQL BQL BQL BQL
Liver 81.109 19.059 10.848 2.894 2.431 2.313 1.335
Urinary Bladder 21.493 11.843 4.419 BQL BQL BQL BQL
Urinary Bladder (contents) 37.717 53.063 17.409 0.730 BQL BQL BQL
Central Nervous System Brain (cerebrum) 1.131 BQL BQL BQL BQL BQL BQL
Brain (cerebellum) 0.792 BQL BQL BQL BQL BQL BQL
Brain (medulla) 0.836 BQL BQL BQL BQL BQL BQL
Spinal Cord 1.054 BQL BQL BQL BQL BQL BQL
Endocrine Adrenal Gland (cortex) 21.016 32.175 6.388 BQL BQL BQL BQL
Adrenal Gland (medulla) 22.695 11.729 3.008 BQL BQL BQL BQL
Pituitary Gland 4.230 5.730 1.120 BQL BQL BQL BQL
Thyroid 18.501 2.099 BQL BQL BQL BQL BQL
Secretory Harderian Gland 19.721 11.313 2.262 BQL BQL BQL BQL
Pancreas 21.841 7.292 1.413 BQL BQL BQL BQL
Salivary Gland 15.799 6.587 1.080 BQL BQL BQL BQL

Table 9. contd….

Tissue Type Tissue 1 h 4 h 8 h 24 h 48 h 72 h 168 h
Fatty Adipose (brown) 10.937 2.119 0.861 BQL BQL BQL BQL
Adipose (white) 2.771 1.055 BQL BQL BQL BQL BQL
Dermal Skin (non-pigmented) 3.381 1.806 BQL BQL BQL BQL BQL
Reproductive Bulbo-Urethral Gland 25.566 13.289 9.808 BQL BQL BQL BQL
Epididymis 15.099 1.976 1.005 BQL BQL BQL BQL
Prostate Gland 6.804 3.428 1.315 BQL BQL BQL BQL
Seminal Vesicles 4.623 3.549 1.858 BQL BQL BQL BQL
Testis 0.886 1.635 0.827 BQL BQL BQL BQL
Skeletal/Muscular Bone BQL 0.704 BQL BQL BQL BQL BQL
Heart (myocardium) 9.391 2.032 BQL BQL BQL BQL BQL
Skeletal Muscle 3.454 1.789 BQL BQL BQL BQL BQL
Respiratory Lung 23.924 6.032 1.049 BQL BQL BQL BQL
Alimentary Canal Cecum 10.959 78.834 245.696 BQL BQL BQL
Cecum (contents) 178.680 1821.769 3175.767 4.007 BQL BQL BQL
Large Intestine 9.868 13.177 229.445 0.963 BQL BQL BQL
Alimentary Canal Large Intestine (contents) BQL 1.331 *13580.619 11.748 0.897 0.928 1.386
Small Intestine 2 469.675 215.988 179.658 14.365 4.30 BQL BQL
Small Intestine (contents) *9517.684 3588.686 1466.205 7.043 1.004 1.016 BQ L
Stomach (gastric mucosa) 161.635 6.813 2.690 BQL BQL BQL BQL
Stomach (contents) 3351.976 1357.410 173.639 BQL BQL BQL BQL
Ocular Eye (lens) BQL BQL BQL BQL BQL BQL BQL
Eye (uveal tract) 7.453 2.399 0.946 BQL BQL BQL BQL
oral dose of 14C-pacritinib at 100 mg/kg (100 μCi/kg) BQL = Value is below the LLOQ
LLOQ = 0.00070600 μCi/g / 0.0012198 μCi/μg = 0.579 μg equivalent / g tissue ULOQ = 7.41040495 μCi/g / 0.0012198 μCi/μg = 6075.098 μg equivalent / g tissue
* = value is above the ULOQ

concentrations were BLQ at 24 hours post dose, except in the liver, large intestine, and small intestine. Elimination was nearly complete at 168 h, but radioactivity remained measurable in the liver.
DISCUSSION
Pacritinib is a small molecule macrocycle JAK2 inhibitor that is being evaluated in late stage clinical trials for efficacy in Myelofibrosis. Pacritinib conforms to the Lipinski rules for an orally bioavailable compound [3]. It is dibasic (pKa1 = 9.92, pKa2= 3.32) and moderately lipophilic (LogD7.4 = 1.76).
Pacritinib exhibits ~5 fold and ~3 fold higher protein binding in human plasma (99.88%) relative to mouse (99.41%) and dog (99.63%), respectively [3]. The limits on analytical sensitivity for determining the unbound fraction for highly bound drugs present difficulties for accurate estimation of PPB, and the relative PPB method has been proposed to address this issue [8]. Pacritinib showed fivefold higher affinity to human plasma relative to mouse plasma at
1000 ng/ml, consistent with results from the classic equilibrium dialysis [3].
Pacritinib appeared to be primarily metabolized by CYP3A4, which can be explained based on the fact that many macrocycle drugs such as erythromycin, cyclosporine, rapamycin, tacrolimus and taxol are known substrates for CYP3A4 [10]. Pacritinib did not significantly induce human CYP3A4 or CYP1A2. A less than 40% induction relative to the positive control means that the test compound may not show induction in vivo [11]. Pacritinib did not significantly inhibit any of the 5 major CYP450 enzymes [3]. Thus pacritinib did not show the potential to cause drug-drug interactions, inhibition or inhibition of the major CYP450 enzymes in vivo, which needs verification in clinical studies. Indeed, the exposure of pacritinib did not decrease more than two fold in patients upon repeated dosing, suggesting that CYP3A4 may not have been induced significantly [9].
In in vitro microsomal metabolism studies in MLM and HLM, Pacritinib was biotransformed to several metabolites

of which the majority were oxidation products and a dealkylated metabolite. The identity of the major metabolites M1(oxidation metabolite), M2 (dealkylation metabolite), M3 (oxidation metabolite) and M4(reduction metabolite) were confirmed with authentic reference standards in microsomes and in mouse and human plasma. Because the steady state exposures of the major metabolites M1 and M2 were < 10% of Pacritinib in humans at 100 and 600 mg, they are not required to be assessed for toxicity individually [12]. This also indicates that metabolic clearance may not be the primary clearance mechanism in humans, and inhibition of CYP3A4 may not influence the PK of Pacritinib in terms of clearance and oral bioavailability.
In nonclinical PK studies, Pacritinib showed high (>100% LBF), moderate (~ 50 % LBF) and high (86% LBF) plasma clearance, relative to liver blood flow [13], in mice, rat and dog, respectively. The plasma Cl can exceed the LBF in a species, like in mouse, if the fu*Clint, u significantly exceeds the LBF and has a CB/CP >1[14], or if it is cleared by other routes like the kidneys, in addition to metabolic clearance. Pacritinib was highly distributed in mouse, rat and dog (Vss > 2 l/kg), which may be attributed its basic nature (pKa=9.9) and lipophilicity (logD = 1.74). Drugs that are basic and lipophilic show high Vss [15]. The absolute oral bioavailability of pacritinib was moderate in mouse, and low in dog and rat. Prediction of Cl/F and V/F in humans, by allometric scaling, was not possible because of poor linear relationships between Cl/F and V/F with body weights of nonclinical species (data not shown). This could be due to differences in oral bioavailability, which have been shown to confound predictions of oral PK parameters [16]. The observed exposures in humans at the starting dose of 100 mg far exceeded (100-200 fold) the predicted exposures based on dog and mice PK data from toxicology studies (data not shown). This could be because of vertical allometry [16], where the predicted and observed values for Cl/F and V/F differ by > 1000%. Tang and Meyersohn have suggested that drugs that tend to show vertical allometry are those which show low metabolic clearance, a fu, human/fu,rat ratio of ≥ 5 and a cLogP of > 2 [16]. Pacritinib seems to fit the criteria in terms of low metabolic clearance and high cLogP of 4.1[3]. Although fu values from rat are not available for Pacritinib, it may well show a fu, human/fu, rat ratio of ≥ 5, since it shows a 5 fold higher affinity to human plasma than to mouse.
Although the exposures were high in humans at the starting
dose of 100 mg, relative to the 1/3rd NOAEL exposures in dog, no significant adverse events were observed [9]. This may be explained by the 3 fold higher PPB to human plasma relative to the dog [3].
Pharmacologically active concentrations were achieved in mice at 50 mg/kg, which formed the basis for designing dose and dose regimen in the efficacy experiments, suggesting a PKPD basis for the observed efficacy in pharmacology models of myelofibrosis and leukemia [4]. Pharmacologically active concentrations were achieved in human patients even at the starting dose of 100 mg which explains the observed efficacy at higher doses [9].
Based on the oral bioavailability and the pharmacology model, in addition to the formation of metabolites, the mouse and dog were chosen for sub-chronic and chronic toxicology
studies. Mass balance studies in mice indicated that biliary clearance is a major route of elimination for Pacritinib, which suggests that the same may be the case in humans. In the QWBA study, pacritinib related radioactivity was distributed across tissues, which was consistent with its high Vss in mice. Radioactivity was observed in bone marrow, the site of hematopoiesis and the location of JAK2, the target of pacritinib, consistent with the observed pharmacological activity in mice. Radioactivity levels in the heart and brain were not high, relative to blood, suggesting that Pacritinib may not show neuro- and cardiac toxicity in mice and was later confirmed in chronic toxicology studies in mice and dogs. No major neuro- or cardiac toxicities were observed in human patients upon chronic dosing of Pacritinib [9]. Radioactivity was detected at significant levels up to 7 days post dose in bile, liver, and contents of small intestine and large intestine suggesting that Pacritinib was eliminated by biliary clearance in mice. Based on this, and the fact that metabolites formed were <10% of pacritinib’ s AUC in mice, it is plausible that pacritinib may have a longer terminal t1/2 than what was estimated in the IVPO study in mice (t1/2 of
2.2 to 5.6 h) with unlabeled Pacritinib.
Pacritinib was shown to be stable in human liver microsomes [3] indicating slow metabolic turnover. This was reflected in human patients, dosed with pacritinib at 100 and 600 mg, where the AUC of the major metabolites was < 10% of pacritinib. The 5 fold higher protein binding of pacritinib to human plasma relative to mouse plasma, the low metabolic clearance in HLM, the < 10 % relative abundance of metabolites in human plasma, and the observed biliary clearance in mice, suggest that biliary excretion may be a major route of elimination of Pacritinib in humans and the reason for its long terminal half-life. Mass balance studies are required to confirm the major route (s) of elimination for pacritinib in humans.
In summary, the physicochemical properties, the in vitro and in vivo nonclinical ADME, and vertical allometry of Pacritinib appear to reasonably explain its disposition- low oral clearance, long terminal t1/2, and high exposures in humans. Pacritinib is currently in two phase 3 trials for myelofibrosis [6, 7].

SUPPLEMENTARY MATERIAL
Supplementary material is available on the publisher’s web site along with the published article.

CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS
Declared none.

LIST OF ABBREVIATIONS

AAG = Alpha 1 acid glycoprotein
ADME = Absorption Distribution Metabolism and Excretion

AUC0-t = Area under the plasma concentration-
time curve from time zero to the last

[2] Brady, L.S.; Alison, R.M. Primary myelofibrosis and the myeloproliferative neoplasms: The role of individual variation.

measured non-zero concentration
AUC0-∞ = Area under the plasma concentration-
time curve from time zero to infinity
BLQ = Below limit of quantification
Cmax = Highest concentration of drug observed
in plasma following oral dosing

[3]
JAMA, 2010, 303(24), 2513-2518.
William, A.D.; Lee, A.C.; Blanchard, S.; Poulsen, A.; Teo, E.L.;
Nagaraj, H.; Tan, E.; Chen, D.; Williams, M.; Sun, E.T.; Goh,
K.C.; Ong, W.C.; Goh, S.K.; Hart, S.; Jayaraman, R.; Pasha, M.K.; Ethirajulu, K.; Wood, J.M.; Dymock, B.W. Discovery of the macrocycle 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza tetracyclo[19.3.1.1(2,6).1(8,12)] heptacosa-1(25), 2(26),3,5,8,10,12
(27),16,21,23-decaene (SB1518), a potent Janus kinase 2/fms-like tyrosine kinase-3 (JAK2/FLT3) inhibitor for the treatment of myelofibrosis and lymphoma. J. Med. Chem., 2011, 54(13), 4638-58.

Cl = Systemic clearance
Cl/F = Oral clearance CYP450 = Cytochrome P450
[4] Hart, S.; Goh, K.C.; Novotny-Diermayr, V.; Hu, C.Y.; Hentze, H.; Tan, Y.C.; Madan, B.; Amalini, C.; Loh, Y.K.; Ong, L.C.; William, A.D.; Lee, A.; Poulsen, A.; Jayaraman, R.; Ong, K.H.; Ethirajulu, K.; Dymock, B.W.; Wood, J.W. SB1518, a novel macrocyclic pyrimidine-based JAK2 inhibitor for the treatment of myeloid and

DLM = Dog Liver Microsomes
EMS = Enhanced Mass Spectrum
[5]
lymphoid malignancies. Leukemia, 2011, 25(11),1751-9. Derenzini, E.; Younes, A. Targeting the JAK-STAT pathway in lymphoma: a focus on pacritinib. Expert. Opin. Investig. Drugs, 2013, 22(6), 775-85.

EPI = Enhanced Product Ion
F = Oral bioavailability
Fa = Fraction absorbed
⦁ ClinicalTrials.gov, A service of the U.S. National Institutes of Health. http://clinicaltrials.gov/show/NCT01773187 (Accessed June 2nd, 2014)
⦁ ClinicalTrials.gov, A service of the U.S. National Institutes of Health. http://www.clinicaltrials.gov/ct2/show/NCT02055781

fu = Fraction of unbound drug in plasma HLM = Human Liver Microsomes
LBF = Liver Blood Flow
LC/MS/MS = Liquid chromatography with tandem mass spectrometry
LLOQ = Lower Limit of Quantitation
(Accessed June 2nd, 2014)
⦁ Collins, J.M.; Klecker, R.W. Jr. Evaluation of highly protein bound drugs: interspecies, intersubject, and related comparisons. J. Clin. Pharmacol., 2002, 42(9), 971-5.
⦁ Younes, A.; Romaguera, J.; Fanale, M.; McLaughlin, P.; Hagemeister, F.; Copeland, A.; Neelapu, S.; Kwak, L.; Shah, J.; de Castro Faria, S.; Hart, S.; Wood, J.; Jayaraman, R.; Ethirajulu, K.; Zhu, J. Phase I study of a novel oral Janus kinase 2 inhibitor, SB1518, in patients with relapsed lymphoma: evidence of clinical and biologic activity in multiple lymphoma subtypes. J. Clin.

MLM = Mouse Liver Microsomes PPB = Plasma Protein Binding

Oncol., 2012, 30(33), 4161-7.
Wilkinson, G.R. Cytochrome P4503A (CYP3A) metabolism: prediction of in vivo activity in humans. J. Pharmacokinet. Biopharm., 1996, 24(5), 475-90.

PK = Pharmacokinetic(s)
PK/PD = Pharmacokinetic/Pharmacodynamic RLM = Rat Liver Microsomes
Rt = retention time(s)
TIC = Total Ion Current
t1/2 = half-life
⦁ Bjornsson, T.D.; Callaghan, J.T.; Einolf, H.J.; Fischer, V.; Gan, L.; Grimm, S.; Kao, J.; King, S.P.; Miwa, G.; Ni, L.; Kumar, G.; McLeod, J.; Obach, R.S.; Roberts, S.; Roe, A.; Shah, A.; Snikeris, F.; Sullivan, J.T.; Tweedie, D.; Vega, J.M.; Walsh, J.; Wrighton,
S.A. The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug Metab. Dispos., 2003, 31(7), 815-32.
⦁ Guidance for Industry: Safety testing of drug metabolites. U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER).

Vss = Volume of distribution at steady state
V/F = Apparent volume of distribution
XIC = Extracted ion chromatogram


Pharmacology and Toxicology, 2008.
Davies, B.; Morris, T. Physiological parameters in laboratory animals and humans. Pharm. Res., 1993, 10(7), 1093-1095.
Yang, J.; Jamei, M.; Yeo, K.R.; Rostami-Hodjegan, A.; Tucker,
G.T. Misuse of the well-stirred model of hepatic drug clearance.
Drug Metab. Dispos., 2007, 35(3), 501-2.

UIM = Unidentified molecule
⦁ Smith, D.A.; Jones, B.C.; Walker, D.K. Design of drugs involving the concepts and theories of drug metabolism and

REFERENCES
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Received: July 24, 2014 Revised: December 09, 2014 Accepted: January 04, 2015

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