Absorption, Metabolism, and Excretion of Oral [14C] Radiolabeled Donafenib: An Open-Label, Phase I, Single-Dose Study in Humans

doi:10.21203/rs.3.rs-4485746/v1

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Absorption, Metabolism, and Excretion of Oral [14C] Radiolabeled Donafenib: An Open-Label, Phase I, Single-Dose Study in Humans

https://doi.org/10.21203/rs.3.rs-4485746/v1

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Donafenib, a deuterated derivative of Sorafenib, exhibited significant efficacy and improved safety for the treatment of hepatocellular carcinoma (HCC) in clinical trials. In this study, the absorption, metabolism, and excretion of donafenib were investigated in six healthy Chinese male volunteers after administration of a single oral dose of 300 mg of [¹⁴C]donafenib (120 µCi). Mean recovery of total radioactivity was 97.31% of the dose. A total of 6 metabolites were identified in plasma, urine and feces. The parent drug was the major radioactive component detected in the plasma and feces, accounting for 67.52% of total radioactivity and 83.17% of the dose, respectively. M2, the N-oxidation metabolite was a major metabolite in human plasma. Overall, donafenib was excreted mainly by feces through liver metabolism and less by kidney.

The metabolic pathways of donafenib identified were similar to those of sorafenib, but metabolite profiles in plasma, urine and feces were quantitatively different, and especially the amide hydrolysis metabolite M6 in sorafenib was not detectable in donafenib. The difference of metabolic characteristics between donafenib and sorafenib, due to the deuterium isotope effect, may partially explain the better safety and efficacy of donafenib as the treatment of advanced HCC.

[14C]donafenib

sorafenib

deuterium isotope effect

pharmacokinetics

excretion

metabolism

HCC

Hepatocellular carcinoma (HCC) is a primary malignancy of liver, accounting for ཞ90% of liver cancer that occurs predominantly in patients with chronic liver disease and cirrhosis¹. It is the fourth leading causes of cancer-related deaths worldwide, distributing mostly in Asia and sub-Saharan Africa, where the endemic high prevalence of hepatitis B and C strongly predisposes individuals to chronic liver disease and subsequent development of HCC². China has the highest incidence, reported more than 50% of the yearly new cases and deaths caused by liver cancer in the world³. In the United States and other countries, HCC is also expected to increase, due to the growing number of patients with hepatitis C virus infections⁴. Sorafenib, the first multi-kinase inhibitor approved by the Food and Drug Administration (FDA) for first-line use for advanced HCC in 2005, can block Raf-1 and receptor tyrosine kinases including VEGF receptors (VEGFR)-1/-2/-3, PDGF receptor-beta (PDGFR-β), c-Kit, Flt-3, and RET, resulting in inhibition of tumor cell proliferation and survival^5–8. It has showed considerable and positive efficacies combating this disease in the recent 15 years, for instance, prolonging the HCC patients’ overall survival (OS) and progress-free survival (PFS) ^9,10. However, in China and Asia-Pacific regions, sorafenib’s median OS is shorter than the rest of the world⁴. Besides, sorafenib is usually associated with quite a few uncomfortable or even intolerable side effects, such as diarrhea and severe hand-foot skin reaction¹¹. So, there is a growing need of new medications for HCC with potentially better efficacy and safety.

Donafenib developed by Zelgen Biopharmaceuticals is a deuterated derivative of sorafenib, substituting a trideuteriomethyl group for a methyl on the molecule¹². In general, deuteration in a drug molecule can slow down a metabolic reaction to a less active or inactive molecule(s) via an isotope effect, or to stabilize stereogenic centers to stereomutation in enantiomers and diastereomers, resulting in longer residence time of the drug in plasma so as to increase drug exposure and further achieve greater efficacy and/or to minimize adverse side effects generated by the metabolite(s)¹³. As is known, there are considerable deuterated compounds undergoing clinical research, and deutetrabenazine, the trideuteriomethoxy derivative of tetrabenazine, is the first deuterium drug granted marketing approval by FDA in 2017 for the treatment of chorea associated with Huntington’s disease and tardive dyskinesia¹⁴, while the others are still on their ways to new drug applications (NDA). Deutetrabenazine demonstrates a better tolerability and an improved dosing regimen compared with tetrabenazine for patients¹⁵.

Donafenib was reported to exhibit similar antiproliferative potency to sorafenib in multiple human cancer cell lines. The latest phase II/III trial demonstrated that donafenib 0.2g bid was associated with a significantly longer median OS compared with sorafenib at 0.4g bid (12.1 vs 10.3 months) and a nearly 17% decreased risk of death (HR = 0.831, 95% CI, 0.699–0.988, P = .0363), based primarily on a full analysis set (659 Chinese patients with unresectable or metastatic hepatocellular carcinoma). In addition, grade 3 or higher adverse reaction (ADRs) occurred significantly less frequently in the donafenib arm compared with the sorafenib arm (37.5% vs 49.7%, P = .0018), so is it for ADRs leading to treatment interruption (25.2% vs 36.1%; P = .0025) ¹⁶. Meanwhile, donafenib (0.2 g bid) resulted in higher systemic exposure at the steady state than sorafenib (0.4 g bid) for each parent drug (mean C_max, 6.55 ± 2.47 vs 4.98 ± 2.68 µg/mL; mean C_trough, 2.75 ± 1.11 vs 2.36 ± 1.21 µg/mL; mean AUC_ss, 45.38 ± 15.37 vs 38.13 ± 15.71 h·µg/mL) and the main active metabolite M2 (pyridine N-oxide) (mean C_max, 1.54 ± 0.91 vs 1.33 ± 0.79 µg/mL; mean AUC_ss, 11.44 ± 6.47 vs 10.53 ± 6.56 h·µg/mL)¹⁷. Recently, the National Medical Products Administration (NMPA) approved donafenib tosylate tablets for the market, and also Chinese Society of Clinical Oncology (CSCO) updated the guidelines for hepatocellular carcinoma (2020), adding donafenib as the level I recommendation for the first-line treatment for patients with advanced HCC.

In the light of its superior result of treatment trials, i.e., the better efficacy/safety than sorafenib, the absorption, distribution, metabolism, and excretion (ADME) profile in humans, including the routes of elimination and metabolic pathways in humans, need an elaborative study to support its development and regulatory registration. It is important to identify metabolic pathways and exposure of sorafenib from considerations of metabolites’ safety assessment and drug-drug interaction. Moreover, such information will illustrate how deuteration can affect safety and efficacy of donafenib by changing the ADME profile and pharmacokinetic characteristics.

In this study, the metabolism and disposition of [¹⁴C]donafenib were studied in healthy male volunteers after a single oral administration of its suspension solution, as well as the subjects’ safety were evaluated.

2.1 Standards and reagents

Carbon-14-labeled ([¹⁴C]) Donafenib Tosilate (Fig. 1) was synthesized by WuXi AppTec (Shanghai, China). The specific activity and radiochemical purity of the [¹⁴C]donafenib were 53.9 mCi/mmol and greater than 99%, respectively. Reference standards for donafenib and the metabolites, including M2, M3, M4, and M6 (Fig. 1), were supplied by Zelgen Biopharmaceuticals Co., Ltd. (Suzhou, China). β-Glucuronidase from Escherichia coli was obtained from sigma. All other reagents were of reagent grade or better and obtained from commercial suppliers.

2.2 Human ADME study design and dose administration

The radiolabeled human ADME study was designed to evaluate the absorption, metabolism and excretion of [¹⁴C]donafenib in healthy male volunteers (ClinicalTrials.gov identifier 2017-MB-DNFN-16), and conducted in accordance with Good Clinical Practices (GCP) and in compliance with the Declaration of Helsinki. The protocol and consent form were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (Suzhou, China). All participants provided written informed consent before any study-related procedures were performed.

Male healthy volunteers aged 18–45 with a body mass index (BMI) of 19–26 kg/m² were enrolled in this study. All these individuals were examined to be in good health. Subjects who had participated in a radiolabeled clinical study or were exposed to significant levels of radiation for any reasons within the 12 months prior to the study were also excluded. Subjects were also ineligible for inclusion if they had used of prescription or herbal products that may affect the drug metabolism within 30 days before the study.

Eligible volunteers entered the study center on day − 2. Volunteers fasted for at least 10 hour (h) prior to drug administration and then were given a single oral suspension. A standard lunch was provided 4 h after dosing. Each volunteers received a single 300 mg dose of [¹⁴C]donafenib (as the tosilate) containing a radioactivity dose of 120 µCi in 50 mL of water as an oral suspension followed by three additional water intakes (50 mL each). The residual radioactivity in the dosing vials was determined, the administered dose (114.5–117.3 µCi) was close to the target dose of 120 µCi. Calculations for percent of dose recovered in excreta were based on the actual administered doses. The whole body committed effective dose was estimated to be 0.05 mSv, which is acceptable according to the recommended limited for the public 1 mSv per year. The associated radiation exposure fell within International Commission on Radiological Protection Guidelines for Category I studies (< 0.1 mSv).

2.3 Sample collection

Blood samples were collected into heparinized tubes before dosing (0 h) and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216 and 240 h after administration. Aliquot of the blood samples were mixed gently and chilled immediately under − 20°C; the remainder was centrifuged at 1660 g for 10min at 4°C to separate the plasma samples. The blood and plasma samples were pre- stored at -20°C and then stored at -70°C until analyzed. Urine samples were obtained at the following collection periods, -12 to 0 h (pre-dose), 0–4, 4–8, 8–12, 12–24 h, and every 24 h there after. Fecal samples were collected pre-dose and in 24 h intervals after administration. Urine and feces samples were consecutively collected until the accumulated excreted radioactivity was over 80% of the dose and the excreted radioactivity was lower than 1% of the administered radioactivity over 2 consecutive days.

2.4 Measurement of radioactivity

All radioactivity measurements were performed by TriCarb model 3110 liquid scintillation counting (LSC, PerkinElmer, Wellesley, MA). An aliquot of plasma (0.3 mL) or duplicate aliquots of urine (5 mL) were directly mixed with scintillation cocktail and analyzed by LSC.

The feces were weighed and homogenized in the solution of 50% isopropanol at the ratio of 1:3 (weight: volume). Duplicate aliquots of feces (approximately 0.3 g) and an aliquot of blood (0.3 mL) were weighed and burned in OX-501 oxidizer (R.J. Harvey, Tappan, NY). Then, the resultant ¹⁴CO₂ was captured and assayed by the instrument. The efficiency of the oxidizer was assessed using ¹⁴C standards and was greater than 90%. Then the radioactivity values would then be corrected by the oxidizer efficiency.

For all matrices, the corresponding blank samples were also prepared and subjected to the instrument. Radioactivity measurements of all samples were corrected by background subtraction and converted to nanogram equivalents (ng-eq) per gram (plasma and blood ) or a percentage of dose recovery (urine and feces) using automatic quench correction.

2.5 Sample Preparation for Radiolabeled Metabolite Profiling

An equal volume of 0.75, 1.5, 2.5, 3.5, 4.5, 6, 8, 12, 24 and 48 h plasma sample were pooled across subjects to give pooled plasma samples (n = 10) for metabolite profiling. In addition, AUC pooled plasma samples (n = 1) were prepared by pooling 0, 0.75, 1.5, 2.5, 3.5, 4.5, 6, 8, 12, 24 and 48 h from individual subject using AUC¹⁸ pooling method. Each pooled sample was mixed with methanol (1/3, v/v). The mixture was vortexed for 10 min and was centrifuged at 4000 rpm for 10 min at 4°C. The residual was resuspended by an aliquot of water and extracted with methanol (1/2, v/v) twice. Three aliquots of the supernatant were mixed and evaporated with nitrogen. The residues were dissolved with the reconstituting solution consisting of methanol–water (1:1, v/v) for profiling.

Six pooled human urine samples (Subject 01 ~ 06) were pooled from 0 to 96 h based on equal volume ratios from the individual subject across time intervals. In addition, four pooled urine samples (0–12, 12–48, 48–96 and 0–96 h) (Subjects 01–06) were prepared by pooling human urine samples across subjects and time points. Each pooled urine sample was centrifuged at 10000 rpm for 10 min at 4°C, the supernatant was detected for profiling.

Six human fecal samples (0-168 h) were prepared by pooling feces from individual subjects (Subjects 01–06). In addition, four pooled human fecal samples (0–48, 48–96, 96–168 and 0-168 h) were prepared by pooling feces samples across subjects and time points. Each pooled sample was extracted using the same procedure of plasma.

2.6 Enzyme hydrolysis of urine samples

1 mL of 0–12 h pooled urine sample was added with 2.74 mg β-Glucuronidase, mixed well, then incubated in 37℃ water bath for ~ 43 h to get the hydrolyzed urine sample. The urine samples before and after hydrolysis were centrifuged, and the supernatant were detected for LC-HRMS.

2.7 Radiolabeled Metabolite Profiling

The total radioactivity in plasma, urine and feces samples was measured using liquid scintillation counting(LSC). Radioactivity profiles of pooled plasma, urine and feces samples were determined using HPLC coupled with off-line radioactivity detection and structures of radiolabeled metabolites were characterized using on-line high-resolution mass spectrometry (HRMS). The HPLC instrument consisted with Shimadzu UFLC-20A and an ACE 3 C18 (150 × 4.6 mm, 3µm) column with mobile phase A (0.4% formic acid in water, adjust pH = 3.2 with ammonium hydroxide) and B (acetonitrile) at the flow rate of 0.7 mL/min. The gradient method started as 0% B from 0 to 5 min, 0%-20% B from 5 min to 10 min, 20%-65% B from 10 min to 60 min, 65%-100% B from 60 min to 65 min, 100% B from 65 min to 70 min, 100%-0% B from 70 min to 72 min and 0% B from 72 min to 87 min. The column temperature was 35ºC. Radioactivity profiles were determined by collecting the HPLC effluent into 96-well LunaPlate™ at a rate of 0.25 min/fraction. The plates were dried by SpeedVac System, and then the radioactivity was determined using Packard TopCount® NXT™ Microplate Scintillation and Luminescence Counter technology. Radiochromatogram data was evaluated using the ARC data system. Meanwhile, Q Exactive (ThermoFisher) couple with HPLC (Shimadzu) System (LC-HRMS) was applied for the metabolite identification with electrospray ionization (ESI) source in positive ion mode, and the data were processed using Xcalibur software (version 2.0.7). Radiolabeled metabolite structures were determined based on their HRMS spectral data and comparing them with those of reference standards if available.

2.8 Metabolite structural characterization using LC-HRMS

Thermo Q Exactive MS/MS System was applied for the metabolite identification with electrospray ionization (ESI) source in positive ion mode, and the data were processed using Xcalibur software. LC-RAM/HRMS and LC-HRMS were used for metabolite detection and structure analysis. Structures of donafenib metabolites were identified or characterized based on the interpretation of accurate mass spectral data, including their molecular ions, elemental composition, and product ions as well as common biotransformation pathways. The structures of M2, M3 and M4 were further confirmed with reference standards.

2.9 Determination of donafenib in human plasma using LC-MS

The plasma concentration of donafenib was determined using a validated liquid chromatography-tandem mass spectrometry (LC–MS/MS) assay method. The LC–MS/MS system was conducted with a Sciex API 5000 tandem MS (Applied Biosystems, CA, USA) coupled with 30AD XR UFLC system (Shimadzu Co., Kyoto, Japan).

For each sample, 100 µL of plasma was mixed with 20 µL of IS (sorafenil, 100 ng/mL) and spiked with 300 µL of acetonitrile in a 96-well plate, followed by vortex mixing for 5 min and centrifuged at 4000 rpm for 15 min. Then, the 100 µL supernatant was transferred to another plate, and 100 µL water was added. After vortex mixing for 3 min and centrifuged at 3200 g for 5 min, 2 µL aliquot of the resulting solution was injected into the LC–MS/MS system to determine the concentration of donafenib.

The compounds were chromatographed on an ACE 5 C18-AR column (50 × 3.0 mm) with a gradient method that used mobile phase A (5 mM ammonium acetate containing 0.2% formic acid) and B (acetonitrile) at the flow rate of 0.6 mL/min. The gradient was delivered as follows: 60% B from 0 min to 1 min; 60% B- 95% B from 1 min to 2 min; 95% B from 2 min to 3 min; 95% B- 60% B from 3 min to 3.01 min; 60% B from 3.01 min to 4.0 min. The mass spectrometer was operated in positive ionization mode for donafenib and sorafenib (internal standard), using the selected transitions m/z 468.2 → m/z 273.2 and m/z 465.2 → m/z 270.2, respectively.

Linear calibration curves for donafenib were obtained in the concentration range from 10.0 to 10000 ng/mL (r² > 0.995), with the lower limit of quantitation of 10.0 ng/mL.

Pharmacokinetic parameters of parent donafenib including C_max, T_max, t_1/2, AUC_last, AUC_inf, CL, and Vz were derived from plasma concentration versus time data in each subject by non-compartmental analysis using WinNonlin (version 6.3).

3.1 Demographics, safety, and tolerability

Six male subjects were enrolled in the study, received a single dose of 300 mg donafenib blended with approximately 120 µCi [¹⁴C]donafenib as solution, and completed the study according to protocol. Subjects age ranged from 23 to 30 years [mean 26.5 years (SD 2.4)], body weight ranged from 54 to 69 kg [mean 60.7 kg (SD 5.0)], and body mass index ranged from 20.6 to 24.4 kg/m²[mean 22.2 kg/m² (SD 1.4)]. Only one of six subjects in the safety analysis set reported grade 1 blood bilirubin increased as a treatment-emergent adverse event. This event was considered unrelated to the study drug. No other clinically relevant changes were observed by physical examination, laboratory investigations, vital sign and twelve-lead electrocardiograms.

Pharmacokinetics of the total radioactivity and donafenib

The mean concentration-time profiles of the total radioactivity and the parent drug in plasma after a single oral dose of 300 mg (120 µCi) of [¹⁴C]donafenib in healthy volunteers are depicted in Fig. 2A. PK parameters are summarized in Table 1.

Table 1

Pharmacokinetic parameters for donafenib, and the total radioactivity in plasma and blood (n = 6)
Parameters	Donafenib	Total radioactivity
Parameters	Plasma	Plasma	Blood
T_max(h)	3.5(3.0, 4.5)	3.58(3.0, 4.5)	3.75(3.5, 4.0)
C_max(ng/mL)	2050 ± 1037	4221 ± 2358 ^a	3057 ± 1565 ^a
AUC_last(ng/mL·h)	65361 ± 29317	127195 ± 88632 ^b	104115 ± 66299 ^b
AUC_inf(ng/mL·h)	65860 ± 29417	149278 ± 93283 ^b	138756 ± 60490 ^b
MRT_0 − t (h)	34.4 ± 5.60	32.3 ± 4.35	26.6 ± 5.53
t_1/2 (h)	20.9 ± 1.24	26.0 ± 6.46	32.4 ± 14.90
Notes: The data for all parameters were presented as mean ± SD except T_max, which was presented as median (range).
a: ng eq./g
b: ng eq./g·h

3.2 Excretion of Radioactivity in Urine and Feces

After a single oral dose of 300 mg (120 µCi) [¹⁴C]donafenib, the cumulative recoveries of radioactivity in human urine and feces were determined and the results are shown in Fig. 2B. Average recovery of total radioactivity in the six volunteers was 97.31% of the dose over the period of 0 h to 240 h, ranging from 87.96–101.25%. (The mean radioactivity recovered in urine was 9.27% of the dose (range, 4.53%-13.84%), whereas the mean radioactivity recovered in feces was 88.04% of the dose (range, 74.12%-95.68%). The results indicated that hepatic excretion is the major route of drug elimination from human.

3.3 Radiolabeled metabolite detection and structural characterization

Radiolabeled metabolites and the parent drug in plasma, urine and feces were detected and quantitatively analyzed by LC-radioactivity analysis (Fig. 3 and Sup Fig. 1) and structurally characterized based on product ion spectral data acquired by LC-HRMS (Fig. 4 and Sup Fig. 2). As a result, six metabolites of donafenib, including M2, M3, M4, M5, M7 and M8 were found and identified in plasma and excreta. The identities of M2, M3 and M4 were further confirmed using their reference standards. Structures of these metabolites are displayed in Fig. 1 and their mass spectral data including pronated molecules and product ion spectra are summarized in Table 2. Spectral interpretation of the parent drug and its metabolites are also illustrated in Fig. 4 and Sup Fig. 2.

Table 2

Summary of donafenib metabolite quantification and identification in pooled plasma (0–48 h), pooled urine (0–96 h) and pooled feces (0-168 h) after a single oral dose of [¹⁴C]donafenib to volunteers
Drug-related compound	Metabolism Type	%AUC (0–48 h Plasma)	0–96 h Urine (8.43%dose)		0-168 h Feces (87.08%dose)		Urine + Feces	[M + H]⁺	Major fragments
Drug-related compound	Metabolism Type	%AUC (0–48 h Plasma)	% TRA	%dose	%TRA	%dose	%dose	[M + H]⁺	Major fragments
[¹⁴C]donafenib	Parent	67.52	+	+	94.47	83.17	83.17	468.1124	450, 406, 273, 255
M2	Pyridine N-oxidation	24.76	+	+	+	+	0.00	484.1073	406, 370, 289, 271
M3	Mono-hydroxylation	0.97	+	+	0.56	0.49	0.49	483.1010	406, 270
M4	N-Demethylation	+	+	+	+	+	0.00	451.0779	406, 256
M5	N-oxidation, N-Demethylation	+	-	-	-	-	0.00	467.0728	406, 272
M7	Glucuronic acid conjugation	4.82	77.00	7.13	3.89	3.42	10.55	644.1445	468, 450, 273, 255
M8	N-oxidation, glucuronic acid conjugation	0.64	14.09	1.30	+	+	1.30	660.1394	484, 289
Total		98.71	91.09	8.43	98.92	87.08	95.51
-: Not detected; +: Only detected by MS

M2 was an N-oxidation metabolite at the pyridine moiety of donafenib (Fig. 4B). Its protonated molecular ion at m/z 484.1074/486.1039 is consistent with its molecular formula C₂₁H₁₄D₃O₄N₄^35/37ClF₃ (mass errors: 0.0705 ppm and − 1.0045 ppm) and 16 Da greater than that of donafenib. M3 was a mono-hydroxylation product of donafenib at trideuteriomethyl (Fig. 4C), its protonated molecule at m/z 483.1012/485.0977, consistent with its molecular formula C₂₁H₁₅D₂O₄N₄^35/37ClF₃ (mass errors: 0.3042 ppm and − 0.8040 ppm). M4 was an N-demethylation product of donafenib (Fig. 4D), which has protonated molecule at m/z 451.0779/453.0749, consistent with its molecular formula C₂₀H₁₅O₃N₄^35/37ClF₃ (mass errors: -0.0600 ppm and − 0.2279 ppm). M5 was a N-demethylation and N-oxidation product of donafenib (Fig. 2A), which has protonated molecule at m/z 467.0729/469.0695, consistent with its molecular formula C₂₀H₁₅O₄N₄^35/37ClF₃ (mass errors: 0.1645 ppm and − 0.9211 ppm). As shown in its product ion spectrum, a loss of formamide and OH (61 Da) produced an intense fragment ion at m/z 406. Amide bond fracture produced product ion at m/z 272. M7, a product of glucuronic acid conjugation of donafenib (Fig. 2B), which has protonated molecule at m/z 644.1446/646.1417, consistent with its molecular formula C₂₇H₂₂D₃O₉N₄^35/37ClF₃ (mass errors: 0.1548 ppm and 0.1756 ppm). As shown in its product ion spectrum, a loss of glucuronic acid (176 Da) produced an intense fragment ion at m/z 468, further loss of H₂O produced an intense fragment ion at m/z 450. Amide bond fracture produced product ion at m/z 272, further loss of H₂O produced an intense fragment ion at m/z 255. M8, a product of N-oxidation and glucuronic acid conjugation of donafenib (Fig. 2D), which has protonated molecule at m/z 660.1394/662.1367, consistent with its molecular formula C₂₇H₂₂D₃O₁₀N₄^35/37ClF₃ (mass errors: 0.0394 ppm and 0.3194 ppm). As shown in its product ion spectrum, a loss of glucuronic acid (176 Da) produced an intense fragment ion at m/z 484, Amide bond fracture produced product ion at m/z 289. Furthermore, 0–12 h urine samples before and after hydrolyzed with glucuronase were analyzed for LC-HRMS (Fig. 5), Results indicated that M7 and M8 in urine was hydrolyzed to form parent drug and M2, respectively, so M8 was presumed to be the glucuronic acid conjugation of M2.

3.4 Radiolabeled metabolite profiles in human plasma

Radiolabeled metabolite profiles of pooled plasma samples at 0.75, 3.5 and 12 h post dose across six patients were presented in Fig. 3B, Sup Fig. 1A and Sup Fig. 1B. In addition, Fig. 3A showed the AUC (0–48 hours) plasma radioactivity of each metabolite. Relative concentrations of donafenib and its metabolites in the 0–48 h AUC-pooled plasma samples, which were determined by LC-radioactivity detection are summarized in Table 2. The parent drug was the single major component in human plasma, accounting for 67.52% of the total drug related components in the AUC pooled samples. M2 was a major metabolite in human plasma, accounted for 24.76% of the total drug related components (total plasma radioactivity) in the AUC pooled plasma. M7 was a minor metabolite in human plasma, accounted for 4.82% of the total radioactivity in the AUC pooled plasma. M3, M4, M5 and M8 were trace metabolites, accounting for less than 1% of the total radioactivity in the AUC pooled plasma.

3.5 Metabolite profiles in human urine and feces

Radiolabeled metabolite profiles of pooled human feces (0-168 h, 87.08% of dose) and pooled urine (0–96 h, 8.43% of dose) are presented in Fig. 3C and Fig. 3D, respectively. Relative concentrations (% the total radioactivity and % the dose) of individual metabolites in the pooled feces and urine samples are summarized in Table 2. The parent drug was a major drug-related component in feces, accounted for 83.17% of the dose. M7 was a minor metabolite, accounted 3.42% of the dose. Other metabolites in human feces were less than 1% of the dose. M7 and M8 were major drug-related components in pooled human urine, accounted for 77.00% and 14.09% of the total radioactivity (Table 2), but only accounted for 7.13% and 1.30% of the administered dose. Other metabolites in human urine were only detected in LC-HRMS.

The pharmacokinetics, metabolism and excretion of donafenib were investigated in this study. A single oral dose of 300 mg donafenib (120 µCi of [¹⁴C] donafenib) was administered to six healthy male volunteers. Over the 240 h of collection period, the mean total recovery of radioactivity in urine and feces was 97.31% of the administered dose, of which the major excretion route was via feces (the arithmetic means of 88.04% for feces, 83.17% as unchanged donafenib), and most of the recovery occurred within 168 h postdose, accounting for approximately 96.54%. As a comparison, based on the public information¹⁹, following the oral administration of [¹⁴C]sorafenib to healthy volunteers, recovery was 96% of total radioactivity within the respective time interval of 336 h in excreta, and 77% of the radioactivity was in feces (50.7% as unchanged drug). The difference in the recovery ratio of unchanged donafenib and sorafenib may be attributed to the deuterium isotope effect which makes the donafenib more metabolically stable.

Donafenib rapidly appeared in plasma with a median T_max of 3.5 h for both parent drug and total radioactivity. After reaching C_max, plasma concentrations of the parent compound declined with a mean half-life (t_1/2) of 20.9 ± 1.24 h, while the mean t_1/2 of total radioactivity in plasma was longer, around 26.0 ± 6.46 h, indicating the existence of some longer half-life metabolites in plasma. In addition, the mean values of C_max, AUC_last, AUC_inf of donafenib versus those of total radioactivity in plasma were, respectively, 2050 ± 1037 ng/mL vs 4221 ± 2358 ng eq./g, 65361 ± 29317 ng/mL·h vs 127195 ± 88632 µg eq./g·h, and 65860 ± 29417 µg/mL·h vs 149278 ± 93283 µg eq./g·h, exhibiting more general pharmacokinetic characteristics of donafenib and its metabolites with longer t_1/2. It is noteworthy that the secondary absorption peaks were also observed in human plasma after administration of donafenib at 8–12 and 24 hours postdose. This phenomenon was attributed to enterohepatic recirculation of donafenib after cleavage of the glucuronide conjugate or reduction of the N-oxidation metabolite M2 in the gastrointestinal tract by colonic bacteria. The blood-to-plasma AUC_inf ratio of the radioactivity was 0.93, which suggested donafenib and related metabolites were not distributed in blood cells. On the other hand, our study demonstrated that, donafenib had similar exposures in healthy volunteers and solid tumor patients at the single dose of 300 mg²⁰.

In this study, metabolite profiling was conducted in plasma, urine and feces samples. Six metabolites were identified, and unchanged donafenib was detected as the predominant drug related component in plasma (67.52% of the total radioactivity) and feces (83.17% of the dose). M2, the N-oxidation metabolite was a major metabolite in human plasma, accounting for 24.76% of the total radioactivity in the AUC pooled plasma, while other metabolites M3、M4、M5、M7、M8 accounting for less than 5% of the total radioactivity in the AUC pooled plasma. The major metabolite in feces was M7, a glucuronidation metabolite of donafenib, accounting 3.42% of the dose. And in urine, M7 and M8 were the two metabolites of donafenib, accounting for 7.13% and 1.30% of the administered dose. In our study, it is revealed that donafenib was excreted mainly by feces through liver metabolism and less by kidney (88.04% and 9.27%, respectively). The metabolic pathways of donafenib identified were similar to those of sorafenib, but each metabolite ratio was different and amide hydrolysis metabolite M6 was not found in donafenib, which the deuterium isotope effect may lead to metabolic pathways change.

Comparing to sorafenib, the proportion of parent drugs in AUC pooled plasma was lower in donafenib than that in sorafenib (67.52% vs 73.5%), but in fecal samples the parent proportion of donafenib was much higher than that of sorafenib (83.17% vs 50.7%)¹⁹. It was assumed that the large quantities of prototypes of donafenib in feces were mainly sourced from transformation of M2. What’s more, it is intriguing to find in our results that the proportion of metabolites related to deuterated groups gave result to significantly differences between donafenib and sorafenib. In specificity, the main active metabolite M2, which showed no biotransformation at the deuterated group site, was determined by higher proportion in donafenib plasma than sorafenib (24.76% vs 20.2%)¹⁹; the metabolite M7, i.e. the glucuronic acid conjugate of parent drug accounted for 4.82% in plasma and 3.4% in feces for donafenib, but only 0.5% in plasma and no detection in feces for sorafenib. The proportion of M7 in urine showed an inverse feature, that is to say, lower in donafenib than sorafenib (7.1% vs 14.8%)¹⁹. It is speculated that the above differences may be related to the more stable metabolic characteristics of deuterated methyl in donafenib structure. The metabolic pathways related to deuterated methyl in donafenib structure are inhibited, which leads to the transformation of donafenib metabolism to other pathways. The metabolite M6, in particular, was not detected in plasma and feces of donafenib, but 19.1% in feces of sorafenib¹⁹. Regarding the metabolite of M6 in body, which was reported to damage intestinal cells and cause diarrhea through further glucuronidation in vivo²¹, its significant decrease in donafenib might attribute to reduced adverse reactions and improved efficacy when compared to sorafenib.

Therefore, this ADME study elucidates the PK profile, the possible metabolic pathways and the excretion of donafenib in human. The difference of metabolic characteristics between donafenib and sorafenib, due to the deuterium isotope effect, may partially explain the better safety and efficacy of donafenib as the first line treatment of advanced HCC.

Acknowledgments

This work was sponsored and funded by Zelgen Biopharmaceuticals Co., Ltd. and supported by the National Key New Drug Creation Special Programs (No 2017ZX09304021), Jiangsu Provincial Medical Youth Talent (QNRC2016714), National Natural Science Foundation of China (No.81773820), the Jiangsu Provincial Medical Talent (ZDRCA2016048).

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Absorption, Metabolism, and Excretion of Oral [14C] Radiolabeled Donafenib: An Open-Label, Phase I, Single-Dose Study in Humans

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Standards and reagents

2.2 Human ADME study design and dose administration

2.3 Sample collection

2.4 Measurement of radioactivity

2.5 Sample Preparation for Radiolabeled Metabolite Profiling

2.6 Enzyme hydrolysis of urine samples

2.7 Radiolabeled Metabolite Profiling

2.8 Metabolite structural characterization using LC-HRMS

2.9 Determination of donafenib in human plasma using LC-MS

3. Results

3.1 Demographics, safety, and tolerability

3.2 Excretion of Radioactivity in Urine and Feces

3.3 Radiolabeled metabolite detection and structural characterization

3.4 Radiolabeled metabolite profiles in human plasma

3.5 Metabolite profiles in human urine and feces

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1