Three‐Period Bioequivalence Study of Sodium Levofolinate Injection With Calcium Levofolinate for Injection and Sodium Folinate for Injection in Healthy Chinese Subjects

The aim of this study was to compare the bioequivalence and safety of test preparation sodium levofolinate injection with reference preparations of calcium levofolinate for injection and sodium folinate for injection in China. A single‐center, randomized, open‐label, 3‐period, crossover test was conducted on 24 healthy subjects. Plasma concentration of levofolinate, dextrofolinate, and their metabolites l‐5‐methyltetrahydrofolate and d‐5‐methyltetrahydrofolate were quantified by a validated chiral–liquid chromatography–tandem mass spectrometry method. All adverse events (AEs) were documented to evaluate safety as they occurred and evaluated descriptively. Pharmacokinetic parameters (maximum plasma concentration, time to maximum concentration, area under the plasma concentration–time curve over the dosing interval, area under the plasma concentration–time curve from time 0 to infinity, terminal elimination half‐life, and terminal rate constant) of 3 preparations were calculated. A total of 8 subjects (10 cases) of AEs occurred in this trial. No serious AEs or unexpected serious adverse reactions were observed. Sodium levofolinate was bioequivalent to calcium levofolinate and sodium folinate in Chinese subjects, and the 3 preparations were all well tolerated.

Folinic acid (Figure 1), the secondary metabolite of folic and 1 of the active forms of folic acid ( Figure 2) in vivo, has a certain control effect on the toxic reaction caused by methotrexate. [1][2][3][4][5] Folinic is the first chemical protective agent used as a rescue agent for high-dose methotrexate. 6 It can also improve the efficacy of 5-fluorouracil (5-FU) and act as a biochemical modulator. 7 Folinic acid is composed of 2 corresponding isomers in the same proportion, of which levofolinate was a eutomer. 8 L-formyltetrahydrofolate is the active ingredient of levofolinate, which can metabolize to L-5-methyltetrahydrofolate. Dextrofolinate is the same. 9 Plasma concentrations of L-formyltetrahydrofolate and D-formyltetrahydrofolate and their metabolites, L-5-methyltetrahydrofolate and D-5-methyltetrahydrofolate, were quantified. Most of the folinate drugs self-developed in China are racemic. Levofolinate synthesized by splitting technology removed ineffective components of the original racemate, which could achieve the same or even better efficacy with only a half-dose. 10,11   Levofolinate reduces the medication risk of distomer as well as improves therapeutic efficacy, which is in line with the current development trend of drug research (high curative effect, small side effects, and low dosage).
Sodium levofolinate (Na-Lev) has similar pharmacological properties to calcium levofolinate (Ca-Lev) 7,12 and had advantages over Ca-Lev in terms of the preparation process and administration. 10,13 Ca-Lev was the first-line therapy regimen for colon cancers. 7 However, simultaneous infusion of 5-FU and calcium folinate (Ca-FA) mixed in the same infusion pump was much more likely hindered by crystallization of calcium salts, which eventually led to catheter obstruction and damage, 7,9,12,14 increasing the risk of bacterial infection during surgery. 7 However, Na-Lev could be safely dosed with 5-FU simultaneously without the above disadvantages. 7 Time of treatment could be shortened to 1 day with Na-Lev compared to 2 days of FOLFOX-4 (a chemotherapy regimen for gastric cancer) with Ca-Lev, which showed higher efficacy, lower side effects, shorter dosing time, and better patient compliance. 7,12,15 A combination of Na-Lev and 5-FU showed synergistic actions, enhanced antitumor activity, and reduced toxicity, while Ca-Lev had just an additive effect on 5-FU activity. 7,12 Due to better safety and effectiveness and fewer disadvantages, Na-Lev showed a much better clinical application prospect. 16 Ca-Lev and sodium folinate (Na-F) was already marketed in China, so the aim of this study was to find, using pharmacokinetic (PK) parameters as indicators, whether Na-Lev was equivalent to Ca-Lev or Na-F in Chinese volunteers.
This study was the first comparison of PK behaviors, structure transformations, and safety studies of Na-Lev, Ca-Lev, and Na-F in the healthy Chinese population, which could provide a reference for the development and marketing of Na-Lev injection in China.

Study Design Subjects
A single-center, randomized, open-label, 3-preparation, 3-period and crossover trial was conducted on 24 healthy subjects according to "Technique Guideline for Human Bioavailability and Bioequivalence Studies on Chemical Drug Products" to avoid the differences of subjects interfering in the experiment. The main information of participants is shown in Table S2. This study was implemented after the approval of the Ethics Committee on drugs, Instruments and New Technologies, Yijishan Hospital of Wannan Medical College. Volunteers voluntarily participated in the trial screening procedure after signing an informed consent form and informing of the trial process and possible adverse reactions to the drugs.
The trial was conducted on 24 healthy subjects to compare in vivo PK behavior as well as the safety of 3 preparations (Na-Lev, Ca-Lev, and Na-F). Subjects were admitted to the phase I ward on day -1 of each period. Volunteers were randomly divided into groups A, B, C, D, E, and F (4 people per group) and were assigned numbers based on randomization. Subjects fasted for >10 hours before the trial.
Vital signs, including respiration, blood pressure, and heart rate, were collected before and 4, 8, 12, 24, 36, 48, 60, 72, 84, and 96 hours after administration. We set a 7-day washout period to avoid the effects of the drugs between periods.
Safety was evaluated on the basis of adverse events (AEs), physical examinations, vital signs, and 12-lead electrocardiogram. AEs were summarized by severity and relationship to the study drug, according to Common Terminology Criteria for Adverse Events version 5.0.

Blood Sampling
Four milliliters of blood was collected from contralateral forearm vein. Blood sampling collection time points were -0.5, 0, 0.25, 0.5, 1, 1.5, 2, 2.25, 2.5, 2.75, 3, 3.5, 4 4.5, 5, 6, 7, 8, 12, 24, 36, and 48 hours. Collected blood samples were placed into a 5-mL disposable sodium citrate anticoagulation negative pressure blood collection tube. Then all tubes were repeatedly turned over 6 times gently and stored in an ice bath before centrifugation. Blood samples were centrifuged at 3099 × g for 10 minutes at 4°C within 1 hour. All sample-handling procedures were carried out under yellow light. A methanolic solution containing 50 μg/mL of ascorbic acid and 50 μg/mL of mercaptoethanol was added to separated plasma samples, then placed in 1.5 mL tubes, and stored in the refrigerator at -70°C.
All samples (200 μL) were added to 1.5-mL centrifuge tubes. A double blank sample added 20 μL of 50% methanol-water solution including antioxygen; other samples added 20 μL of internal standard solution (1 μg/mL). Centrifuge tubes were eddied at 1937 × g with 500 μL methanol for 3 minutes. After that, centrifuge tubes were centrifuged for 10 minutes at 930 × g and 4°C. A total of 610 μL of supernatant was drawn into a clean 96-well plate. Ammonium acetate (600 μL, 8 mM, pH 3.5, and containing antioxygen) was added a 96-well plate to redissolve after the 96-well plate was blow-dried with a nitrogen blower and then eddied at 1937 × g for 3 minutes. The solid phase extraction column was activated with 1 mL of methanol, 1 mL of pure water and 2 mL of ammonium acetate, 8 mM, pH 3.5, and containing antioxygen) at low flow rate of 5 psig. Redissolved solution in the 96-well plate was transferred to the solid-phase extraction column for loading at a low flow rate of 3 psig. One milliliter of pure water was sucked for leaching at a low flow rate of 3 psig. A total of 500 μL of methanol (containing antioxygen) was sucked for leaching at a low flow rate of 2 psig to a 96well plate, and then a 16 μL sample was injected for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
Methotrexate was used as an internal standard to establish a simultaneous determination of Lformyltetrahydrofolate, D-formyltetrahydrofolate, L-5-methyltetrahydrofolate, and D-5-methyltetrahydrofolate in human plasma by a validated chiral-LC-MS/MS method. The liquid-phase condition is shown in Table S3. Separation was performed on AstecCHI-ROBIOTIC (25 cm*4.6 mm, 5 μm) using mobile phase A with 10 mM of ammonium trifluoroacetate-water (containing 0.5% acetic acid) and mobile phase B with 10 mM of ammonium trifluoroacetate-methanol (containing 0.5% acetic acid) in a gradient elution flow rate of 0.8 mL/min. Detection was performed by MS/MS using an API 4000 electrospray ionization mass spectrophotometer (Applied Biosystems/Sciex, Redwood City, California). The monitored reaction ion pairs m/z were 474.1→327.3 for L/D-formyltetrahydrofolate, 460.2→313.2 for L/D-5-methyltetrahydrofolate, and 455.2→308.2 for methotrexate (internal standard), respectively.

Statistical Processing
The PK profile after natural logarithmic transformation was analyzed using a linear mixed-effects model with WinNonlin software version 7.0 (Certara, Princeton, New Jersey). Statistical analysis was performed with SAS 9.4 (SAS Institute, Cary, North Carolina). Using PK parameters as indicators, the bioequivalence (BE) of Na-Lev with Ca-Lev or Na-F was calculated. If the 90%CIs for the geometric mean ratio of the PK parameters (maximum plasma concentration [C max ], area under the plasma concentration-time curve

Pharmacokinetics
The concentrations of L-formyltetrahydrofolate, Dformyltetrahydrofolate, L-5-methyltetrahydrofolate, and D-5-methyltetrahydrofolate in plasma were determined by a chiral-LC-MS/MS method. D-5methyltetrahydrofolate was not quantified in plasma samples, with concentrations all below the lower limit of quantification. Therefore, only the PK parameters of L-formyltetrahydrofolate, D-formyltetrahydrofolate, and L-5-methyltetrahydrofolate were analyzed. Results are shown in Table 1 and the PK parameters of 3 preparations are shown in Table 2.
The main parameters of the regression curve are shown in Table S6. The mean plasma concentrationtime profiles of L-formyltetrahydrofolate, L-5methyltetrahydrofolate, and D-formyltetrahydrofolate are shown in  No conversion between levorotatory and dextrorotatory was seen for both pro-drugs and metabolites in vivo. Dextrofolinate was seen only after administration of injectable sodium folinate acid and D-5-methyltetrahydrofolate was lower than the lower limit of quantification after administration of all 3 preparations. Comparisons of BE of 3 preparations were evaluated by L-formyltetrahydrofolate and L-5-methyltetrahydrofolate.
The main parameters (C max , AUC 0-t , and AUC 0-∞ ) of Na-Lev were a little bit higher than Ca-Lev or Na-F. Table 3 shows the 90%CI results of C max , AUC 0-t , and AUC 0-∞ of L-formyltetrahydrofolate and L-5-methyltetrahydrofolate after Na-Lev and reference preparation Ca-Lev or Na-F were administrated. The listed 90%CIs were all within the range of 80.0%-125.0%, indicating that the Na-Lev was equivalent to the Ca-Lev or the Na-F. Plasma sampling procedures were carried out under yellow light because folinate is sensitive to light and air, so this trial was conducted away from sunlight. A total of 50 μg/mL of ascorbic acid was added to eliminate sample matrix interference and background interference and 50 μg/mL of mercaptoethanol was added to avoid oxidation of samples.

Safety and Tolerability
All documented AEs included dizziness, stomachache, elevated triglyceride, scratch on the second toe of the right foot, rash on chest area, red and swollen tip of the tongue, finger entrapment, and other adverse reactions  during the trial. There were no serious AEs or AEs leading to early withdrawal, death, or serious diseases. All AEs were level I and relieved without sequelae. The relationship with drugs were "may be related" and "unrelated." Safety data showed that 3 kinds of injections were well tolerated among healthy Chinese volunteers.

Discussion
We studied the BE of Na-Lev, Ca-Lev, and Na-F by conducting a single-center, randomized, open-label, 3preparation 3-period, crossover test in 24 healthy subjects. A Chiral-LC-MS/MS method we established. This study concluded that Na-Lev was bioequivalent to Ca-Lev and Na-F.
Levofolinate (L-formyltetrahydrofolate) is rapidly metabolized to L-5-methyltetrahydrofolate. And the left-handed form of folinate seemed unable to be converted into the right-handed form. Nearly none of the D-5-methyltetrahydrofolate was quantified in any of the 3 preparations in this study. No dextrophiles appeared in the metabolites of both Na-Lev and Ca-Lev in this study, which was the same as experiments of Taflin et al. 17 A large number of prior studies reported that Na-Lev shows great advantages in terms of safety and efficacy. 5-FU combined with Na-Lev had better safety profiles than 5-FU combined with Ca-Lev in mice. 7 Other clinical trials had also demonstrated 5-FU together with Na-Lev showed better efficacy and lower toxicity in various chemotherapy dosing regimens. [18][19][20] A phase II randomized study of 57 subjects reported by Bleiberg et al 21 showed that the objective response rate of the patients was 55% using Na-Lev and 61% using Ca-Lev. Median overall survival durations were 22.9 months and 11.9 months, respectively. Progression-free survival was 11.5 and 8.0 months. In addition, grade 3 AEs were 64% and 46%, indicating that treatments of Na-Lev were better than Ca-Lev.
This study was the first comparison of PK behaviors, structure transformations, and safety studies of Na-Lev, Ca-Lev, and Na-F in Chinese. Na-Lev seemed to be the most convenient, safe, and time-saving drug and a suitable selection for application as examined in all reported experiments. However, Na-Lev with obvious advantages had not been produced or imported in China. Thus, promotion of Na-Lev offered a vital treatment option for patients with colorectal cancer 9 and also fill the gap in the Chinese pharmaceutical market. This trial only compared the BE of Na-Lev, Ca-Lev, and Na-F, as well as its evaluated safety and tolerability. Further studies are necessary to clearly assess drug-drug-interaction, nationalities, or races in the PK characteristics of Na-Lev and provide a theoretical basis for individualized clinical medication use.

Conclusion
Na-Lev was bioequivalent to the Ca-Lev and Na-F in healthy Chinese volunteers. Moreover, the 3 preparations were well tolerated. Replacing Ca-Lev with Na-Lev is feasible. It may represent a possible new standard of therapy when administering 5-FU-based therapy. Administration method of group A: On the first day of period 1, 62.5 mg/m 2 (calculated by levofolinate) of Na-Lev was used, diluted to 0.4 mg/mL with normal saline and intravenously infused for 2 hours. In period 2, the same operation procedure as period 1 was conducted except that Na-Lev was replaced by Ca-Lev. In period 3, Na-F was used for injection with 125 mg/m 2 body surface area (calculated as folinate) and diluted with normal saline to 0.8 mg/mL, then intravenous infusion for 2 hours.
The administration methods were as follows: for group B: Ca-Lev, Na-F, and Na-Lev; for group C: Na-F, Na-Lev, and Ca-Lev; for group D: Na-Lev, Na-F, and Ca-Lev; for group E: Ca-Lev, Na-Lev, and Na-F; and for group F: Na-F, Ca-Lev, and Na-Lev.
Main inclusion criteria included the following: age ≤18 years; body mass index 19-26 kg/m 2 , male weight ≥ 50 kg and female weight ≥ 45 kg; no parental scheme, no sperm donation plan, and taking reliable contraception (both volunteers and their spouses) from the first day of medication to 1 month after drug withdrawal; comprehension of the procedure and method of this study; and willingness to obey the rules of this clinical trial and sign the informed consent.
Exclusion criteria included the following: allergic to the study drug or any component of study drug, history of allergy to other drugs or history of drug dependence; abnormal results of physical examinations, laboratory examinations, 12-lead electrocardiogram, type-B ultrasonography and radiograph; hepatitis B surface antigen positive, hepatitis C antibody positive, HIV antibody positive and syphilis antibody positive; abnormal blood pressure or other severe diseases; history of drug clinical trials within 3 months before screening; history of medical treatment including serious infections, trauma, or major surgery within 3 months before screening; history of blood donations of >400 mL or hemorrhage with great blood loss no less than 400 mL, and no receptions of transfusion within 3 months; medication history of any drugs including Chinese medicinal herbs, dietary supplements, and vitamins within 2 weeks; history of smoking (≥5 cigarettes per day) and drinking (≥5 units per week or ≥1 unit per day [1 unit = 45 mL high-alcohol liquor/150 mL common-alcohol liquor/360 mL beer); positive results of urine drug test; and consumption of foods that may contain caffeine (such as coffee, cola, and tea), alcohol, or beverage and fruits like grape, grapefruit, pitaya, and juice made of these fruits.