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

The purpose of this study is to compare the concentration of \[6R\] 5,10-methylene-THF, 5-formyl-THF, 5-methyl-THF and THF in the tumor, in adjacent mucosa and in plasma in patients with colon cancer receiving two different does of Modufolin® and Levoleucovorin (Isovorin®), respectively (60 and 200 mg/m2).

• 6R-MTHF (arfolitixorin) 200 mg/m2 (Drug), also known as Modufolin®, ISO-901, [6R] 5,10-methylenetetrahydrofolate, arfolitixorin: i.v. bolus injection
• Levoleucovorin 200 mg/m2 (Drug), also known as Isovorin®: i.v. bolus injection
• 6R-MTHF (arfolitixorin) 60 mg/m2 (Drug), also known as Modufolin®, ISO-901, [6R] 5,10-methylenetetrahydrofolate, arfolitixorin: i.v. bolus injection
• Levoleucovorin 60 mg/m2 (Drug), also known as Isovorin®: i.v. bolus injection

Primary outcomes

• Concentration of [6R]-5,10-methylene-THF in Tumor Tissue [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]: Comparison of concentration of the active substance in Modufolin: \[6R\]-5,10-methylene-THF in tumor tissue after the different treatments.
• Concentration of [6R]-5,10-methylene-THF in Adjacent Mucosa Tissue [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]: Comparison of concentration of the active substance in Modufolin: \[6R\]-5,10-methylene-THF in mucosa adjacent to the tumor after the different treatments.
• Concentration of [6S]-5-THF in Tumor Tissue [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]: Comparison of concentration of the metabolite \[6S\]-5-THF in tumor after the different treatments.
• Concentration of [6S]-5-THF in Adjacent Mucosa Tissue [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]: Comparison of concentration of the metabolite \[6S\]-5-THF in mucosa adjacent to the tumor after the different treatments.
• Concentration of [6S]-5-methyl-THF in Tumor Tissue [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]: Comparison of concentration of the metabolite \[6S\]-5-methyl-THF in tumor after the different treatments.
• Concentration of [6S]-5-methyl-THF in Adjacent Mucosa Tissue [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]: Comparison of concentration of the metabolite \[6S\]-5-methyl-THF in mucosa adjacent to the tumor after the different treatments.
• Concentration of [6S]-5-formyl-THF in Tumor Tissue [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]: Comparison of concentration of the metabolite \[6S\]-5-formyl-THF in tumor after the different treatments.
• Concentration of [6S]-5-formyl-THF in Adjacent Mucosa Tissue [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]: Comparison of concentration of the metabolite \[6S\]-5-formyl-THF in mucosa adjacent to the tumor after the different treatments.

Secondary outcomes

• AUC(0-2h) of [6R]-5,10-methylene-THF [Samples taken Day 1 (Day of surgery)]: 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
• AUC(0-2h) of [6S]-5-THF [Samples taken Day 1 (Day of surgery)]: Area under the plasma concentration versus time curve from time 0 to 2 hours, for the metabolite \[6S\]-5-THF
• AUC(0-2h) of [6S]-5-methyl-THF [Samples taken Day 1 (Day of surgery)]: Area under the plasma concentration versus time curve from time 0 to 2 hours, for the metabolite: \[6S\]-5-methyl-THF
• AUC(0-2h) of [6SR]-5-formyl-THF [Samples taken Day 1 (Day of surgery)]: Area under the plasma concentration versus time curve from time 0 to 2 hours, for the metabolite \[6S\]-5-formyl-THF
• AUC(Last) of [6R]-5,10-methylene-THF [Samples taken Day 1 (Day of surgery) and Day 2]: 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
• AUC(Last) of [6S]-5-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Area under the plasma concentration versus time curve, from time 0 to the last time point, for the metabolite \[6S\]-5-THF
• AUC(Last) of [6S]-5-methyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Area under the plasma concentration versus time curve, from time 0 to the last time point, for the metabolite \[6S\]-5-methyl-THF
• AUC(Last) of [6S]-5-formyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Area under the plasma concentration versus time curve, from time 0 to the last time point, for the metabolite \[6S\]-5-formyl-THF
• Cmax of [6R]-5,10-methylene-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Maximum concentration (Cmax) in plasma of the active substance in Modufolin: \[6R\]-5,10-methylene-THF
• Cmax of [6S]-5-THF in Plasma [Samples taken Day 1 (Day of surgery) and Day 2]: Maximum concentration (Cmax) in plasma of the metabolite \[6S\]-5-THF
• Cmax of [6S]-5-methyl-THF in Plasma [Samples taken Day 1 (Day of surgery) and Day 2]: Maximum concentration (Cmax) in plasma of the metabolite \[6S\]-5-methyl-THF
• Cmax of [6S]-5-formyl-THF in Plasma [Samples taken Day 1 (Day of surgery) and Day 2]: 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
• Tmax of [6R]-5,10-methylene-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Timepoint (tmax) when Cmax in plasma occurs of the active substance in Modufolin: \[6R\]-5,10-methylene-THF
• Tmax of [6S]-5-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Timepoint (tmax) when Cmax in plasma occurs for the metabolite \[6S\]-5-THF
• Tmax of [6S]-5-methyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Timepoint (tmax) when Cmax in plasma occurs for the metabolite \[6S\]-5-methyl-THF
• Tmax of [6S]-5-formyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Timepoint (tmax) when Cmax in plasma occurs for the metabolite \[6S\]-5-formyl-THF
• T(1/2) of [6R]-5,10-methylene-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Terminal plasma elimination half-life time for the active substance in Modufolin: \[6R\]-5,10-methylene-THF
• T(1/2) of [6S]-5-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Terminal plasma elimination half-life time for the metabolite \[6S\]-5-THF
• T(1/2) of [6S]-5-methyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Terminal plasma elimination half-life time for the metabolite \[6S\]-5-methyl-THF
• T(1/2) of [6S]-5-formyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: Terminal plasma elimination half-life time for the metabolite \[6S\]-5-formyl-THF
• T(Last) of [6R]-5,10-methylene-THF [Samples taken Day 1 (Day of surgery) and Day 2]: The time-point for the last measurable concentration of the active substance in Modufolin: \[6R\]-5,10-methylene-THF
• T(Last) of [6S]-5-THF [Samples taken Day 1 (Day of surgery) and Day 2]: The time-point for the last measurable concentration for the metabolite \[6S\]-5-TH in plasma
• T(Last) of [6S]-5-methyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: The time-point for the last measurable concentration for the metabolite \[6S\]-5-methyl-THF in plasma
• T(Last) of [6S]-5-formyl-THF [Samples taken Day 1 (Day of surgery) and Day 2]: The time-point for the last measurable concentration for the metabolite \[6S\]-5-formyl-THF in plasma
• Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6R]-5,10-methylene-THF [Samples taken Day 1 (Day of surgery)]: 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.
• Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-THF [Samples taken Day 1 (Day of surgery)]: 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.
• Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-methyl-THF [Samples taken Day 1 (Day of surgery)]: 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.
• Correlation Between Plasma and Tissue Concentration (Tumor and Mucosa) for [6S]-5-formyl-THF [Samples taken Day 1 (Day of surgery)]: 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.
• Gene Expression Ratios (Mucosa:Tumor) [Sample taken Day 1 (Day of Surgery)]: 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.
• Correlation of Gene Expression in Tumor and Adjacent Mucosa [Sample taken Day 1 (Day of Surgery)]: 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.
• Homocystein Concentration [Samples taken at Screening visit, Day 2, and End of Study (Day 5)]: Blood samples for folate biomarker (homocystein) analysis were collected at Screening, Day 2, and Day 5 (End of study visit).
• S-Folate Concentration [Samples taken at Screening visit, Day 2 and End of Study (Day 5)]: Blood samples for folate biomarker (S-folate) analysis were collected at Screening, Day 2 and Day 5 (End of study visit).
• Change in Homocystein Concentration From Screening [Samples taken at Screening visit, Day 2 and End of Study (Day 5)]: 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.
• Change in S-Folate Concentration From Screening [Samples taken at Screening visit, Day 2 and End of Study (Day 5)]: 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.
• Number of AEs Per Severity [Screening visit until end of study, Day 5]: Number of reported AEs per treatment with respect to severity

Sex: All, ages: 18 Years to any

Main Inclusion Criteria:

* Operable colon cancer amenable to curative surgery.
* Performance status of 0 to 1
* Informed consent form
* Patients must be at least 18 years of age.

Main Exclusion Criteria:

* Any concurrent other anti-tumor therapy
* Any prohibited concomitant medication within 30 days of surgery
* Pregnancy or breast-feeding.
* Second primary malignancy

Participant flow

Outcome results

• Concentration of [6R]-5,10-methylene-THF in Tumor Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 1871 +/- 1159; Levoleucovorin 60 mg/m2 959 +/- 417; Modufolin 200 mg/m2 4725 +/- 2210; Modufolin 60 mg/m2 2393 +/- 1920 (p 0.0323)
• Concentration of [6R]-5,10-methylene-THF in Adjacent Mucosa Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 1333 +/- 852; Levoleucovorin 60 mg/m2 626 +/- 439; Modufolin 200 mg/m2 5099 +/- 3927; Modufolin 60 mg/m2 3481 +/- 3926 (p 0.1480)
• Concentration of [6S]-5-THF in Tumor Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 1329 +/- 720; Levoleucovorin 60 mg/m2 933 +/- 598; Modufolin 200 mg/m2 4175 +/- 3127; Modufolin 60 mg/m2 2219 +/- 1995 (p 0.1182)
• Concentration of [6S]-5-THF in Adjacent Mucosa Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 1333 +/- 852; Levoleucovorin 60 mg/m2 626 +/- 439; Modufolin 200 mg/m2 5099 +/- 3927; Modufolin 60 mg/m2 3481 +/- 3926 (p 0.2716)
• Concentration of [6S]-5-methyl-THF in Tumor Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 3574 +/- 2415; Levoleucovorin 60 mg/m2 1904 +/- 918; Modufolin 200 mg/m2 4396 +/- 1858; Modufolin 60 mg/m2 1882 +/- 1204 (p 0.0092)
• Concentration of [6S]-5-methyl-THF in Adjacent Mucosa Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 3667 +/- 2043; Levoleucovorin 60 mg/m2 1216 +/- 455; Modufolin 200 mg/m2 2494 +/- 765; Modufolin 60 mg/m2 2066 +/- 1517 (p 0.0728)
• Concentration of [6S]-5-formyl-THF in Tumor Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 3611 +/- 3899; Levoleucovorin 60 mg/m2 512 +/- 259; Modufolin 200 mg/m2 100 +/- 47; Modufolin 60 mg/m2 57 +/- 48 (p 0.0728)
• Concentration of [6S]-5-formyl-THF in Adjacent Mucosa Tissue (Primary), Mean, pmol/g: Levoleucovorin 200 mg/m2 5456 +/- 3963; Levoleucovorin 60 mg/m2 1403 +/- 640; Modufolin 200 mg/m2 82 +/- 77; Modufolin 60 mg/m2 42 +/- 18 (p 0.0240)
• AUC(0-2h) of [6R]-5,10-methylene-THF (Secondary), Mean, mcg*h/L: L-LV(200) 326.40261 +/- 61.98416; L-LV(60) 239.90076 +/- 0; Mod(200) 7734.09113 +/- 1557.76552; Mod(60) 0 +/- 0 (p 0)
• AUC(0-2h) of [6S]-5-THF (Secondary), Mean, mcg*h/L: L-LV(200) 827.1083 +/- 178.23243; L-LV(60) 640.0489 +/- 0; Mod(200) 19408.1712 +/- 3000.64794; Mod(60) 5639.2700 +/- 885.82223 (p 0.0015)
• AUC(0-2h) of [6S]-5-methyl-THF (Secondary), Mean, mcg*h/L: L-LV(200) 2336.24432 +/- 829.18634; L-LV(60) 1258.60184 +/- 247.51353; Mod(200) 2277.89290 +/- 1132.95603; Mod(60) 997.74091 +/- 413.68052 (p 0.0728)
• AUC(0-2h) of [6SR]-5-formyl-THF (Secondary), Mean, mcg*h/L: L-LV(200) 29911.9184 +/- 6924.78031; L-LV(60) 7595.4599 +/- 764.27650; Mod(200) 0 +/- 0; Mod(60) 0 +/- 0 (p 0)
• AUC(Last) of [6R]-5,10-methylene-THF (Secondary), Mean, mcg*h/L: L-LV(200) 462.10746 +/- 221.85761; L-LV(60) 239.90076 +/- 0; Mod(200) 7910.61038 +/- 1623.24300; Mod(60) 2272.78010 +/- 448.01407 (p 0.0015)
• AUC(Last) of [6S]-5-THF (Secondary), Mean, mcg*h/L: L-LV(200) 2231.1443 +/- 1051.06650; L-LV(60) 640.0489 +/- 0; Mod(200) 28466.5389 +/- 5303.28705; Mod(60) 6766.9104 +/- 1484.91243 (p 0.0015)
• AUC(Last) of [6S]-5-methyl-THF (Secondary), Mean, mcg*h/L: L-LV(200) 32206.2771 +/- 9121.1818; L-LV(60) 7689.0921 +/- 5164.6098; Mod(200) 21737.2428 +/- 21757.1545; Mod(60) 5495.1924 +/- 6110.0065 (p 0.0240)
• AUC(Last) of [6S]-5-formyl-THF (Secondary), Mean, mcg*h/L: L-LV(200) 43429.1885 +/- 12411.2844; L-LV(60) 9637.0308 +/- 1637.2118; Mod(200) 75.6174 +/- 69.2176; Mod(60) 50.1858 +/- 0 (p 0)
• Cmax of [6R]-5,10-methylene-THF (Secondary), Mean, µg/L: L-LV(200) 195.0000 +/- 40.76763; L-LV(60) 123.0000 +/- 0; Mod(200) 22087.5000 +/- 5635.45853; Mod(60) 6452.8571 +/- 559.18477 (p 0.0014)
• Cmax of [6S]-5-THF in Plasma (Secondary), Mean, µg/L: L-LV(200) 577.6250 +/- 190.74436; L-LV(60) 335.0000 +/- 0; Mod(200) 13372.5000 +/- 2108.15932; Mod(60) 5015.7143 +/- 427.35064 (p 0.0015)
• Cmax of [6S]-5-methyl-THF in Plasma (Secondary), Mean, µg/L: L-LV(200) 2786.25000 +/- 822.6253; L-LV(60) 1078.00000 +/- 275.26206; Mod(200) 2020.50000 +/- 1142.25116; Mod(60) 800.42857 +/- 396.41849 (p 0.0240)
• Cmax of [6S]-5-formyl-THF in Plasma (Secondary), Mean, µg/L: L-LV(200) 32962.5000 +/- 8594.17369; L-LV(60) 10168.3333 +/- 2271.47896; Mod(200) 252.7500 +/- 141.51178; Mod(60) 261.0000 +/- 0 (p 0)
• 23 more outcome measures not shown.

Adverse events

Serious adverse events by term

Other adverse events by term

• Kristoffer Derwinger, MD PhD, Principal investigator, Sahlgrenska University Hospital