Trial Outcomes & Findings for PK/PD Investigation of Modufolin (Arfolitixorin) in Plasma, Tumor and Adjacent Mucosa Adjacent Mucosa in Patients With Colon Cancer (NCT NCT01681472)

NCT ID: NCT01681472

Last Updated: 2020-09-25

Results Overview

Comparison of concentration of the active substance in Modufolin: \[6R\]-5,10-methylene-THF in tumor tissue after the different treatments.

• Recruitment status: COMPLETED
• Study phase: PHASE1/PHASE2
• Target enrollment: 32 participants
• Primary outcome timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration
• Results posted on: 2020-09-25

Participant Flow

Participant milestones

Measure	Levoleucovorin 200 mg/m2 Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 Levoleucovorin: i.v. bolus injection	6R-MTHF 200 mg/m2 \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	6R-MTHF 60 mg/m2 \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Overall Study STARTED	8	8	8	8
Overall Study COMPLETED	8	7	8	8
Overall Study NOT COMPLETED	0	1	0	0

Reasons for withdrawal

Measure	Levoleucovorin 200 mg/m2 Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 Levoleucovorin: i.v. bolus injection	6R-MTHF 200 mg/m2 \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	6R-MTHF 60 mg/m2 \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Overall Study Adverse Event	0	1	0	0

Baseline Characteristics

PK/PD Investigation of Modufolin (Arfolitixorin) in Plasma, Tumor and Adjacent Mucosa Adjacent Mucosa in Patients With Colon Cancer

Baseline characteristics by cohort

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Total n=32 Participants Total of all reporting groups
Age, Categorical <=18 years	0 Participants n=5 Participants	0 Participants n=7 Participants	0 Participants n=5 Participants	0 Participants n=4 Participants	0 Participants n=21 Participants
Age, Categorical Between 18 and 65 years	0 Participants n=5 Participants	3 Participants n=7 Participants	2 Participants n=5 Participants	2 Participants n=4 Participants	7 Participants n=21 Participants
Age, Categorical >=65 years	8 Participants n=5 Participants	5 Participants n=7 Participants	6 Participants n=5 Participants	6 Participants n=4 Participants	25 Participants n=21 Participants
Age, Continuous	80.5 years STANDARD_DEVIATION 9.15 • n=5 Participants	66.71 years STANDARD_DEVIATION 16.89 • n=7 Participants	72 years STANDARD_DEVIATION 15 • n=5 Participants	71.75 years STANDARD_DEVIATION 11.25 • n=4 Participants	72.74 years STANDARD_DEVIATION 13.07 • n=21 Participants
Sex: Female, Male Female	4 Participants n=5 Participants	5 Participants n=7 Participants	3 Participants n=5 Participants	4 Participants n=4 Participants	16 Participants n=21 Participants
Sex: Female, Male Male	4 Participants n=5 Participants	3 Participants n=7 Participants	5 Participants n=5 Participants	4 Participants n=4 Participants	16 Participants n=21 Participants
Region of Enrollment Sweden	8 Participants n=5 Participants	8 Participants n=7 Participants	8 Participants n=5 Participants	8 Participants n=4 Participants	32 Participants n=21 Participants

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the active substance in Modufolin: [6R]-5,10-methylene-THF in tumor tissue after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6R]-5,10-methylene-THF in Tumor Tissue	1871 pmol/g Standard Deviation 1159	959 pmol/g Standard Deviation 417	4725 pmol/g Standard Deviation 2210	2393 pmol/g Standard Deviation 1920

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the active substance in Modufolin: [6R]-5,10-methylene-THF in mucosa adjacent to the tumor after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6R]-5,10-methylene-THF in Adjacent Mucosa Tissue	1333 pmol/g Standard Deviation 852	626 pmol/g Standard Deviation 439	5099 pmol/g Standard Deviation 3927	3481 pmol/g Standard Deviation 3926

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the metabolite [6S]-5-THF in tumor after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6S]-5-THF in Tumor Tissue	1329 pmol/g Standard Deviation 720	933 pmol/g Standard Deviation 598	4175 pmol/g Standard Deviation 3127	2219 pmol/g Standard Deviation 1995

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the metabolite [6S]-5-THF in mucosa adjacent to the tumor after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6S]-5-THF in Adjacent Mucosa Tissue	1333 pmol/g Standard Deviation 852	626 pmol/g Standard Deviation 439	5099 pmol/g Standard Deviation 3927	3481 pmol/g Standard Deviation 3926

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the metabolite [6S]-5-methyl-THF in tumor after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6S]-5-methyl-THF in Tumor Tissue	3574 pmol/g Standard Deviation 2415	1904 pmol/g Standard Deviation 918	4396 pmol/g Standard Deviation 1858	1882 pmol/g Standard Deviation 1204

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the metabolite [6S]-5-methyl-THF in mucosa adjacent to the tumor after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6S]-5-methyl-THF in Adjacent Mucosa Tissue	3667 pmol/g Standard Deviation 2043	1216 pmol/g Standard Deviation 455	2494 pmol/g Standard Deviation 765	2066 pmol/g Standard Deviation 1517

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the metabolite [6S]-5-formyl-THF in tumor after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6S]-5-formyl-THF in Tumor Tissue	3611 pmol/g Standard Deviation 3899	512 pmol/g Standard Deviation 259	100 pmol/g Standard Deviation 47	57 pmol/g Standard Deviation 48

PRIMARY outcome

Timeframe: Sample taken Day 1 (Day of surgery) at 0 min (before anesthetics) and 5, 10, 20, 40, 60, 90, 120, 180, 240, 360 min after study drug administration, and 24 h after study drug administration

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Comparison of concentration of the metabolite [6S]-5-formyl-THF in mucosa adjacent to the tumor after the different treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Concentration of [6S]-5-formyl-THF in Adjacent Mucosa Tissue	5456 pmol/g Standard Deviation 3963	1403 pmol/g Standard Deviation 640	82 pmol/g Standard Deviation 77	42 pmol/g Standard Deviation 18

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve from time 0 to 2 hours, for the active substance in Modufolin: [6R]-5,10-methylene-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(0-2h) of [6R]-5,10-methylene-THF	326.40261 mcg*h/L Standard Deviation 61.98416	239.90076 mcg*h/L Standard Deviation 0	7734.09113 mcg*h/L Standard Deviation 1557.76552	0 mcg*h/L Standard Deviation 0

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve from time 0 to 2 hours, for the metabolite [6S]-5-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(0-2h) of [6S]-5-THF	827.1083 mcg*h/L Standard Deviation 178.23243	640.0489 mcg*h/L Standard Deviation 0	19408.1712 mcg*h/L Standard Deviation 3000.64794	5639.2700 mcg*h/L Standard Deviation 885.82223

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve from time 0 to 2 hours, for the metabolite: [6S]-5-methyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(0-2h) of [6S]-5-methyl-THF	2336.24432 mcg*h/L Standard Deviation 829.18634	1258.60184 mcg*h/L Standard Deviation 247.51353	2277.89290 mcg*h/L Standard Deviation 1132.95603	997.74091 mcg*h/L Standard Deviation 413.68052

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve from time 0 to 2 hours, for the metabolite [6S]-5-formyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(0-2h) of [6SR]-5-formyl-THF	29911.9184 mcg*h/L Standard Deviation 6924.78031	7595.4599 mcg*h/L Standard Deviation 764.27650	0 mcg*h/L Standard Deviation 0	0 mcg*h/L Standard Deviation 0

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve, from time 0 to the last time point, for the active substance in Modufolin: [6R]-5,10-methylene-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(Last) of [6R]-5,10-methylene-THF	462.10746 mcg*h/L Standard Deviation 221.85761	239.90076 mcg*h/L Standard Deviation 0	7910.61038 mcg*h/L Standard Deviation 1623.24300	2272.78010 mcg*h/L Standard Deviation 448.01407

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve, from time 0 to the last time point, for the metabolite [6S]-5-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(Last) of [6S]-5-THF	2231.1443 mcg*h/L Standard Deviation 1051.06650	640.0489 mcg*h/L Standard Deviation 0	28466.5389 mcg*h/L Standard Deviation 5303.28705	6766.9104 mcg*h/L Standard Deviation 1484.91243

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve, from time 0 to the last time point, for the metabolite [6S]-5-methyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(Last) of [6S]-5-methyl-THF	32206.2771 mcg*h/L Standard Deviation 9121.1818	7689.0921 mcg*h/L Standard Deviation 5164.6098	21737.2428 mcg*h/L Standard Deviation 21757.1545	5495.1924 mcg*h/L Standard Deviation 6110.0065

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Area under the plasma concentration versus time curve, from time 0 to the last time point, for the metabolite [6S]-5-formyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
AUC(Last) of [6S]-5-formyl-THF	43429.1885 mcg*h/L Standard Deviation 12411.2844	9637.0308 mcg*h/L Standard Deviation 1637.2118	75.6174 mcg*h/L Standard Deviation 69.2176	50.1858 mcg*h/L Standard Deviation 0

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Maximum concentration (Cmax) in plasma of the active substance in Modufolin: [6R]-5,10-methylene-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Cmax of [6R]-5,10-methylene-THF	195.0000 µg/L Standard Deviation 40.76763	123.0000 µg/L Standard Deviation 0	22087.5000 µg/L Standard Deviation 5635.45853	6452.8571 µg/L Standard Deviation 559.18477

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Maximum concentration (Cmax) in plasma of the metabolite [6S]-5-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Cmax of [6S]-5-THF in Plasma	577.6250 µg/L Standard Deviation 190.74436	335.0000 µg/L Standard Deviation 0	13372.5000 µg/L Standard Deviation 2108.15932	5015.7143 µg/L Standard Deviation 427.35064

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Maximum concentration (Cmax) in plasma of the metabolite [6S]-5-methyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Cmax of [6S]-5-methyl-THF in Plasma	2786.25000 µg/L Standard Deviation 822.6253	1078.00000 µg/L Standard Deviation 275.26206	2020.50000 µg/L Standard Deviation 1142.25116	800.42857 µg/L Standard Deviation 396.41849

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Maximum concentration (Cmax) in plasma of the active substance in Modufolin: [6R] 5,10- methylene-THF and the metabolites: [6S]-5-THF, [6S]-5-methyl-THF, and [6S]-5-formyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Cmax of [6S]-5-formyl-THF in Plasma	32962.5000 µg/L Standard Deviation 8594.17369	10168.3333 µg/L Standard Deviation 2271.47896	252.7500 µg/L Standard Deviation 141.51178	261.0000 µg/L Standard Deviation 0

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Timepoint (tmax) when Cmax in plasma occurs of the active substance in Modufolin: [6R]-5,10-methylene-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Tmax of [6R]-5,10-methylene-THF	1.14285857 h Standard Deviation 0.53079605	1.00000000 h Standard Deviation 0	0.08333000 h Standard Deviation 0.00000000	0.08333000 h Standard Deviation 0.00000000

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Timepoint (tmax) when Cmax in plasma occurs for the metabolite [6S]-5-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Tmax of [6S]-5-THF	2.02083375 h Standard Deviation 1.03677360	1.50000000 h Standard Deviation 0	0.38541750 h Standard Deviation 0.24775936	0.09523571 h Standard Deviation 0.03149956

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Timepoint (tmax) when Cmax in plasma occurs for the metabolite [6S]-5-methyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Tmax of [6S]-5-methyl-THF	4.75000000 h Standard Deviation 1.38873015	2.41666667 h Standard Deviation 0.66458007	3.75000000 h Standard Deviation 1.16496475	2.92857143 h Standard Deviation 0.93222724

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Timepoint (tmax) when Cmax in plasma occurs for the metabolite [6S]-5-formyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Tmax of [6S]-5-formyl-THF	0.12499750 h Standard Deviation 0.08908753	0.08333000 h Standard Deviation 0.00000000	0.10416500 h Standard Deviation 0.04167000	0.08333000 h Standard Deviation 0

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Terminal plasma elimination half-life time for the active substance in Modufolin: [6R]-5,10-methylene-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(1/2) of [6R]-5,10-methylene-THF	3.07069184 h Standard Deviation 2.48958159	9.12204918 h Standard Deviation 0	0.90893994 h Standard Deviation 0.62586420	0.34368840 h Standard Deviation 0.06725217

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Terminal plasma elimination half-life time for the metabolite [6S]-5-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(1/2) of [6S]-5-THF	5.59459251 h Standard Deviation 1.76982092	0 h Standard Deviation 0	1.24808943 h Standard Deviation 0.23995973	0.96378070 h Standard Deviation 0.19174152

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Terminal plasma elimination half-life time for the metabolite [6S]-5-methyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(1/2) of [6S]-5-methyl-THF	7.0955575 h Standard Deviation 1.18981597	10.7281060 h Standard Deviation 8.80812064	9.0743346 h Standard Deviation 3.84317226	8.5121573 h Standard Deviation 2.80644764

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

Terminal plasma elimination half-life time for the metabolite [6S]-5-formyl-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(1/2) of [6S]-5-formyl-THF	1.51328500 h Standard Deviation 0.34108131	0.98944069 h Standard Deviation 0.20217367	0.23180085 h Standard Deviation 0.13438901	0 h Standard Deviation 0

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

The time-point for the last measurable concentration of the active substance in Modufolin: [6R]-5,10-methylene-THF

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(Last) of [6R]-5,10-methylene-THF	3.00000000 h Standard Deviation 1.41421356	2.00000000 h Standard Deviation 0	3.25000000 h Standard Deviation 1.38873015	1.35714286 h Standard Deviation 0.24397502

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

The time-point for the last measurable concentration for the metabolite [6S]-5-TH in plasma

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(Last) of [6S]-5-THF	4.75000000 h Standard Deviation 1.48804762	2.00000000 h Standard Deviation 0	5.75000000 h Standard Deviation 0.70710678	3.42857143 h Standard Deviation 0.78679579

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

The time-point for the last measurable concentration for the metabolite [6S]-5-methyl-THF in plasma

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(Last) of [6S]-5-methyl-THF	24.0000000 h Standard Deviation 0.00000000	12.0000000 h Standard Deviation 9.29516003	15.0000000 h Standard Deviation 9.62140471	8.5714286 h Standard Deviation 6.80336051

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery) and Day 2

Population: Per protocol population, all patients who complete the trial without any major deviations from the protocol procedure.

The time-point for the last measurable concentration for the metabolite [6S]-5-formyl-THF in plasma

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
T(Last) of [6S]-5-formyl-THF	6.00000000 h Standard Deviation 0.00000000	8.33333333 h Standard Deviation 7.73735528	0.33333500 h Standard Deviation 0.23570226	0.16667000 h Standard Deviation 0

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Correlation between the exposure of [6R]-5,10-methylene-THF following 2 hours after dosing (AUC[0-2h]) and the concentration of [6R]-5,10-methylene-THF in the tumor or adjacent mucosa at surgery.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6R]-5,10-methylene-THF AUC(0-2h) vs. conc in tumor	0.54039 correlation coefficient	NA correlation coefficient Only one observation available for AUC(0-2h)	-0.21666 correlation coefficient	—
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6R]-5,10-methylene-THF AUC(0-2h) vs. conc in adjacent mucosa	0.68223 correlation coefficient	NA correlation coefficient Only one observation available for AUC(0-2h)	0.27618 correlation coefficient	—

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Correlation between the exposure of [6S]-5-THF following 2 hours after dosing (AUC[0-2h]) and the concentration of [6S]-5-THF in the tumor or adjacent mucosa at surgery.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-THF AUC(0-2h) vs. conc in tumor	-0.02188 correlation coefficient	NA correlation coefficient Only one observation available for AUC(0-2h)	0.27018 correlation coefficient	0.38298 correlation coefficient
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-THF AUC(0-2h) vs. conc in adjacent mucosa	0.07937 correlation coefficient	NA correlation coefficient Only one observation available for AUC(0-2h)	0.34905 correlation coefficient	0.45404 correlation coefficient

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Correlation between the exposure of [6S]-5-methyl-THF following 2 hours after dosing (AUC[0-2h]) and the concentration of [6S]-5-methyl-THF in the tumor or adjacent mucosa at surgery.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-methyl-THF AUC(0-2h) vs. conc in tumor	0.73743 correlation coefficient	0.44757 correlation coefficient	0.03292 correlation coefficient	0.09033 correlation coefficient
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-methyl-THF AUC(0-2h) vs. conc in adjacent mucosa	0.76502 correlation coefficient	0.34496 correlation coefficient	-0.66295 correlation coefficient	0.36854 correlation coefficient

SECONDARY outcome

Timeframe: Samples taken Day 1 (Day of surgery)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Correlation between the exposure of [6S]-5-formyl-THF following 2 hours after dosing (AUC[0-2h]) and the concentration of [6S]-5-formyl-THF in the tumor or adjacent mucosa at surgery.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-formyl-THF AUC(0-2h) vs. conc in adjacent mucosa	0.23512 correlation coefficient	0.02576 correlation coefficient	—	—
Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-formyl-THF AUC(0-2h) vs. conc in tumor	0.68778 correlation coefficient	0.11512 correlation coefficient	—	—

SECONDARY outcome

Timeframe: Sample taken Day 1 (Day of Surgery)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Concentration of different genes involved in folate transport and metabolism were analysed in both tumor and adjacent mucosa. The concentration in mucosa was divided by the concentration in tumor. A value above 1 indicate that the gene expression was higher in mucosa than in tumor and a value below 1 that the gene expression was higher in tumor than in mucosa.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=7 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=6 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Gene Expression Ratios (Mucosa:Tumor) FPGS	0.47 Ratio	0.70 Ratio	0.63 Ratio	0.83 Ratio
Gene Expression Ratios (Mucosa:Tumor) MTHFS	0.51 Ratio	0.87 Ratio	0.83 Ratio	0.72 Ratio
Gene Expression Ratios (Mucosa:Tumor) ABCC3	1.83 Ratio	3.37 Ratio	2.48 Ratio	4.15 Ratio
Gene Expression Ratios (Mucosa:Tumor) GGH	0.50 Ratio	0.47 Ratio	0.35 Ratio	0.31 Ratio
Gene Expression Ratios (Mucosa:Tumor) ABCC1	0.41 Ratio	0.55 Ratio	0.77 Ratio	0.43 Ratio
Gene Expression Ratios (Mucosa:Tumor) AMT	1.35 Ratio	5.60 Ratio	1.86 Ratio	3.33 Ratio
Gene Expression Ratios (Mucosa:Tumor) PCFT	2.03 Ratio	2.75 Ratio	2.92 Ratio	2.45 Ratio
Gene Expression Ratios (Mucosa:Tumor) MTHFD1L	0.15 Ratio	0.24 Ratio	0.29 Ratio	0.35 Ratio
Gene Expression Ratios (Mucosa:Tumor) SHMT1	0.89 Ratio	1.13 Ratio	0.72 Ratio	2.26 Ratio
Gene Expression Ratios (Mucosa:Tumor) SHMT2	0.31 Ratio	0.34 Ratio	0.38 Ratio	0.48 Ratio
Gene Expression Ratios (Mucosa:Tumor) RFC-1	0.48 Ratio	0.66 Ratio	0.53 Ratio	1.18 Ratio

SECONDARY outcome

Timeframe: Sample taken Day 1 (Day of Surgery)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Concentration of the gene expression was analysed in both tumor and adjacent mucosa. The presence of any correlation between the results (i.e., concentration of the gene expression in tumor versus adjacent mucosa) was evaluated for each treatment. No evaluation was done between treatments.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Correlation of Gene Expression in Tumor and Adjacent Mucosa AMT	-0.47193 Normalized concentration	-0.70730 Normalized concentration	0.75404 Normalized concentration	0.42972 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa PCFT	0.11497 Normalized concentration	0.89976 Normalized concentration	0.58502 Normalized concentration	0.88128 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa FPGS	0.17664 Normalized concentration	-0.64743 Normalized concentration	0.88682 Normalized concentration	0.97624 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa MTHFS	0.95840 Normalized concentration	0.58419 Normalized concentration	0.43194 Normalized concentration	0.94364 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa ABCC3	0.00322 Normalized concentration	0.77700 Normalized concentration	-0.25891 Normalized concentration	-0.38987 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa GGH	0.78928 Normalized concentration	-0.09737 Normalized concentration	0.46883 Normalized concentration	0.93711 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa ABCC1	0.74757 Normalized concentration	0.14436 Normalized concentration	-0.16724 Normalized concentration	0.82963 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa MTHFD1L	0.74459 Normalized concentration	0.20955 Normalized concentration	0.37160 Normalized concentration	0.32113 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa SHMT1	0.59183 Normalized concentration	0.05531 Normalized concentration	0.67853 Normalized concentration	0.50966 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa SHMT2	0.55508 Normalized concentration	-0.71666 Normalized concentration	0.86266 Normalized concentration	0.81040 Normalized concentration
Correlation of Gene Expression in Tumor and Adjacent Mucosa RFC-1	0.64565 Normalized concentration	-0.02450 Normalized concentration	0.23312 Normalized concentration	0.31800 Normalized concentration

SECONDARY outcome

Timeframe: Samples taken at Screening visit, Day 2, and End of Study (Day 5)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Blood samples for folate biomarker (homocystein) analysis were collected at Screening, Day 2, and Day 5 (End of study visit).

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Homocystein Concentration Concentration at Screening	12.59 μmol/L Standard Deviation 4.28	11.27 μmol/L Standard Deviation 3.98	14.53 μmol/L Standard Deviation 6.22	13.80 μmol/L Standard Deviation 3.83
Homocystein Concentration Concentration at Day 2	6.41 μmol/L Standard Deviation 2.32	6.40 μmol/L Standard Deviation 3.10	7.06 μmol/L Standard Deviation 1.97	8.14 μmol/L Standard Deviation 2.63
Homocystein Concentration Concentration at Day 5	9.04 μmol/L Standard Deviation 2.53	9.18 μmol/L Standard Deviation 4.65	10.20 μmol/L Standard Deviation 3.34	10.10 μmol/L Standard Deviation 2.64

SECONDARY outcome

Timeframe: Samples taken at Screening visit, Day 2 and End of Study (Day 5)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Blood samples for folate biomarker (S-folate) analysis were collected at Screening, Day 2 and Day 5 (End of study visit).

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=6 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=7 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
S-Folate Concentration Concentration at Screening	21.75 nmol/L Standard Deviation 8.88	21.00 nmol/L Standard Deviation 6.54	15.03 nmol/L Standard Deviation 5.20	18.71 nmol/L Standard Deviation 7.65
S-Folate Concentration Concentration at Day 2	45.00 nmol/L Standard Deviation 0.00	45.00 nmol/L Standard Deviation 0.00	45.00 nmol/L Standard Deviation 0.00	45.00 nmol/L Standard Deviation 0.00
S-Folate Concentration Concentration at Day 5	44.75 nmol/L Standard Deviation 0.71	40.80 nmol/L Standard Deviation 9.39	44.14 nmol/L Standard Deviation 2.27	40.29 nmol/L Standard Deviation 3.73

SECONDARY outcome

Timeframe: Samples taken at Screening visit, Day 2 and End of Study (Day 5)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Blood samples for folate biomarker (homocystein) analysis were collected at Screening, Day 2 and Day 5 (End of study visit). Changes from screening to later visits were counted.

The criteria for assessment categories "normal", "low" and "high" were based on the reference ranges for plasma-Homocystein as follows: "low" <4,7 mcmol/L; "normal" => 4,7 and <=16 mcmol/L; "high" >16 mcmol/L. Values were applicable for both male and female adults.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=7 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=6 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Change in Homocystein Concentration From Screening Normal at Screen and Normal at Day 2	5 Participants	4 Participants	4 Participants	4 Participants
Change in Homocystein Concentration From Screening Shift from Normal at Screen to Low at Day 2	0 Participants	1 Participants	2 Participants	2 Participants
Change in Homocystein Concentration From Screening Shift from High at Screen to Normal at Day 2	2 Participants	3 Participants	0 Participants	2 Participants
Change in Homocystein Concentration From Screening Normal at Screen and Normal at Day 5	5 Participants	5 Participants	4 Participants	6 Participants
Change in Homocystein Concentration From Screening Shift from Normal at Screen to Low at Day 5	0 Participants	0 Participants	1 Participants	0 Participants
Change in Homocystein Concentration From Screening Shift from High at Screen and Normal at Day 5	2 Participants	3 Participants	0 Participants	2 Participants
Change in Homocystein Concentration From Screening Missing data Screen to Day 5	0 Participants	0 Participants	1 Participants	0 Participants

SECONDARY outcome

Timeframe: Samples taken at Screening visit, Day 2 and End of Study (Day 5)

Population: The Per Protocol analysis set contained 29 of the 32 enrolled patients. One patient was withdrawn prior to study drug administration and two patients were excluded due to major protocol deviations.

Blood samples for folate biomarker (S-folate) analysis were collected at Screening, Day 2 and Day 5 (End of study visit). Changes from screening to later visits were counted.

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=7 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=6 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Change in S-Folate Concentration From Screening Shift from Normal at Screen to High at Day 2	7 Participants	8 Participants	5 Participants	8 Participants
Change in S-Folate Concentration From Screening Missing data Screen to Day 2	0 Participants	0 Participants	1 Participants	0 Participants
Change in S-Folate Concentration From Screening Normal at Screening and Normal at Day 5	3 Participants	1 Participants	1 Participants	0 Participants
Change in S-Folate Concentration From Screening Shift from Normal at Screen to High at Day 5	4 Participants	6 Participants	4 Participants	8 Participants
Change in S-Folate Concentration From Screening Missing data Screen to Day 5	0 Participants	1 Participants	1 Participants	0 Participants

SECONDARY outcome

Timeframe: Screening visit until end of study, Day 5

Population: The safety population contained 31 of the 32 enrolled patients. One patient randomized to L-LV 60 mg/m2 was withdrawn prior to study drug administration and thus excluded from the safety analysis set.

Number of reported AEs per treatment with respect to severity

Outcome measures

Measure	Levoleucovorin 200 mg/m2 n=8 Participants Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=7 Participants Levoleucovorin: i.v. bolus injection	Modufolin 200 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	Modufolin 60 mg/m2 n=8 Participants \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Number of AEs Per Severity AEs of mild severity	1 Events	0 Events	1 Events	5 Events
Number of AEs Per Severity AEs of moderate severity	3 Events	0 Events	7 Events	2 Events
Number of AEs Per Severity AEs of severe severity	0 Events	1 Events	2 Events	2 Events

Adverse Events

Levoleucovorin 200 mg/m2

Serious events: 0 serious events

Other events: 4 other events

Deaths: 0 deaths

Levoleucovorin 60 mg/m2

Serious events: 1 serious events

Other events: 2 other events

Deaths: 0 deaths

6R-MTHF 200 mg/m2

Serious events: 2 serious events

Other events: 4 other events

Deaths: 0 deaths

6R-MTHF 60 mg/m2

Serious events: 2 serious events

Other events: 5 other events

Deaths: 0 deaths

Serious adverse events

Measure	Levoleucovorin 200 mg/m2 n=8 participants at risk Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=7 participants at risk Levoleucovorin: i.v. bolus injection	6R-MTHF 200 mg/m2 n=8 participants at risk \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	6R-MTHF 60 mg/m2 n=8 participants at risk \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Respiratory, thoracic and mediastinal disorders Pulmonary embolism	0.00% 0/8	0.00% 0/7	0.00% 0/8	12.5% 1/8
Cardiac disorders Acute mycardial infarction	0.00% 0/8	0.00% 0/7	0.00% 0/8	12.5% 1/8
Injury, poisoning and procedural complications Postoperative wound complication	0.00% 0/8	0.00% 0/7	12.5% 1/8	0.00% 0/8
Injury, poisoning and procedural complications Anastomotic leak	0.00% 0/8	0.00% 0/7	12.5% 1/8	0.00% 0/8
Cardiac disorders Cardiac arrest	0.00% 0/8	14.3% 1/7	0.00% 0/8	0.00% 0/8

Other adverse events

Measure	Levoleucovorin 200 mg/m2 n=8 participants at risk Levoleucovorin: i.v. bolus injection	Levoleucovorin 60 mg/m2 n=7 participants at risk Levoleucovorin: i.v. bolus injection	6R-MTHF 200 mg/m2 n=8 participants at risk \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection	6R-MTHF 60 mg/m2 n=8 participants at risk \[6R\] 5,10-methylenetetrahydrofolate: i.v. bolus injection
Infections and infestations Pneumonia	0.00% 0/8	0.00% 0/7	12.5% 1/8 • Number of events 1	12.5% 1/8 • Number of events 1
Respiratory, thoracic and mediastinal disorders Cough	0.00% 0/8	0.00% 0/7	0.00% 0/8	12.5% 1/8 • Number of events 1
Gastrointestinal disorders Dyspepsia	0.00% 0/8	0.00% 0/7	0.00% 0/8	25.0% 2/8 • Number of events 2
Vascular disorders Flushing	0.00% 0/8	0.00% 0/7	0.00% 0/8	12.5% 1/8 • Number of events 1
Vascular disorders Hypotension	0.00% 0/8	0.00% 0/7	0.00% 0/8	12.5% 1/8 • Number of events 1
Gastrointestinal disorders Vomiting	0.00% 0/8	0.00% 0/7	12.5% 1/8 • Number of events 1	12.5% 1/8 • Number of events 1
Cardiac disorders Atrial fibrillation	25.0% 2/8 • Number of events 2	0.00% 0/7	25.0% 2/8 • Number of events 2	0.00% 0/8
Gastrointestinal disorders Nausea	0.00% 0/8	0.00% 0/7	12.5% 1/8 • Number of events 1	0.00% 0/8
Gastrointestinal disorders Abdominal pain	0.00% 0/8	0.00% 0/7	12.5% 1/8 • Number of events 1	0.00% 0/8
General disorders Pyrexia	0.00% 0/8	0.00% 0/7	12.5% 1/8 • Number of events 1	0.00% 0/8
Injury, poisoning and procedural complications Hepatic enzyme increased	0.00% 0/8	0.00% 0/7	12.5% 1/8 • Number of events 1	0.00% 0/8
Nervous system disorders Paralysis	0.00% 0/8	14.3% 1/7 • Number of events 1	0.00% 0/8	0.00% 0/8
Respiratory, thoracic and mediastinal disorders Dysphonia	0.00% 0/8	14.3% 1/7 • Number of events 1	0.00% 0/8	0.00% 0/8
Respiratory, thoracic and mediastinal disorders Dyspnoea	12.5% 1/8 • Number of events 1	0.00% 0/7	0.00% 0/8	0.00% 0/8
Gastrointestinal disorders Mouth haemorrhage	12.5% 1/8 • Number of events 1	0.00% 0/7	0.00% 0/8	0.00% 0/8

Additional Information

Anders Rabbe

Isofol Medical

Phone: +46 (0)707 64 65 00

Email: anders.rabbe@isofolmedical.com

Results disclosure agreements

• Principal investigator is a sponsor employee
• Publication restrictions are in place

Restriction type: OTHER