Leucovorin is a mixture of the diastereoisomers of the 5-formyl derivative of tetrahydrofolic acid (THF). The biologically active compound of the mixture is the (-)-l-isomer, known as Citrovorum factor or (-)-folinic acid. Leucovorin does not require reduction by the enzyme dihydrofolate reductase in order to participate in reactions utilizing folates as a source of “one-carbon” moieties. l-Leucovorin (l-5-formyltetrahydrofolate) is rapidly metabolized (via 5, 10-methenyltetrahydrofolate then 5, 10-methylenetetrahydrofolate) to l,5-methyltetrahydrofolate. l,5-Methyltetrahydrofolate can in turn be metabolized via other pathways back to 5, 10-methylenetetrahydrofolate, which is converted to 5-methyltetrahydrofolate by an irreversible, enzyme catalyzed reduction using the cofactors FADH2 and NADPH. 

Administration of leucovorin can counteract the therapeutic and toxic effects of folic acid antagonists such as methotrexate, which act by inhibiting dihydrofolate reductase. 

In contrast, leucovorin can enhance the therapeutic and toxic effects of fluoropyrimidines used in cancer therapy, such as 5-fluorouracil. Concurrent administration of leucovorin does not appear to alter the plasma pharmacokinetics of 5-fluorouracil. 5-Fluorouracil is metabolized to fluorodeoxyuridylic acid, which binds to and inhibits the enzyme thymidylate synthase (an enzyme important in DNA repair and replication). 

Leucovorin is readily converted to another reduced folate, 5,10-methylenetetrahydrofolate, which acts to stabilize the binding of fluorodeoxyuridylic acid to thymidylate synthase and thereby enhances the inhibition of this enzyme. 

The pharmacokinetics after intravenous, intramuscular and oral administration of a 25 mg dose of leucovorin were studied in male volunteers. After intravenous administration, serum total reduced folates (as measured by Lactobacillus casei assay) reached a mean peak of 1259 ng/mL (range 897 to 1625). The mean time to peak was 10 minutes. This initial rise in total reduced folates was primarily due to the parent compound 5-formyl-THF (measured by Streptococcus faecalis assay) which rose to 1206 ng/mL at 10 minutes. A sharp drop in parent compound followed and coincided with the appearance of the active metabolite 5-methyl-THF which became the predominant circulating form of the drug. 

The mean peak of 5-methyl-THF was 258 ng/mL and occurred at 1.3 hours. The terminal half-life for total reduced folates was 6.2 hours. The area under the concentration versus time curves (AUCs) for l-leucovorin, d-leucovorin and 5-methyltetrahydrofolate were 28.4 ± 3.5, 956 ± 97 and 129 ± 12 (mg/min/L ± S.E.). When a higher dose of d,l-leucovorin (200 mg/m2) was used, similar results were obtained. The d-isomer persisted in plasma at concentrations greatly exceeding those of the l-isomer. 

After intramuscular injection, the mean peak of serum total reduced folates was 436 ng/mL (range 240 to 725) and occurred at 52 minutes. Similar to IV administration, the initial sharp rise was due to the parent compound. The mean peak of 5-formyl-THF was 360 ng/mL and occurred at 28 minutes. The level of the metabolite 5-methyl-THF increased subsequently over time until at 1.5 hours it represented 50% of the circulating total folates. The mean peak of 5-methyl-THF was 226 ng/mL at 2.8 hours. The terminal half-life of total reduced folates was 6.2 hours. There was no difference of statistical significance between IM and IV administration in the AUC for total reduced folates, 5-formyl-THF, or 5-methyl-THF. 

After oral administration of leucovorin reconstituted with aromatic elixir, the mean peak concentration of serum total reduced folates was 393 ng/mL (range 160 to 550). The mean time to peak was 2.3 hours and the terminal half-life was 5.7 hours. The major component was the metabolite 5-methyltetrahydrofolate to which leucovorin is primarily converted in the intestinal mucosa. The mean peak of 5-methyl-THF was 367 ng/mL at 2.4 hours. The peak level of the parent compound was 51 ng/mL at 1.2 hours. The AUC of total reduced folates after oral administration of the 25 mg dose was 92% of the AUC after intravenous administration. 

Following oral administration, leucovorin is rapidly absorbed and expands the serum pool of reduced folates. At a dose of 25 mg, almost 100% of the l-isomer but only 20% of the d-isomer is absorbed. Oral absorption of leucovorin is saturable at doses above 25 mg. The apparent bioavailability of leucovorin was 97% for 25 mg, 75% for 50 mg, and 37% for 100 mg. 

In a randomized clinical study conducted by the Mayo Clinic and the North Central Cancer Treatment Group (Mayo/NCCTG) in patients with advanced metastatic colorectal cancer three treatment regimens were compared: Leucovorin (LV) 200 mg/m2 and 5-fluorouracil (5-FU) 370 mg/m2 versus LV 20 mg/m2 and 5-FU 425 mg/m2 versus 5-FU 500 mg/m2. All drugs were administered by slow intravenous infusion daily for 5 days repeated every 28 to 35 days. Response rates were 26% (p=0.04 versus 5-FU alone), 43% (p=0.001 versus 5-FU alone) and 10% for the high dose leucovorin, low dose leucovorin and 5-FU alone groups respectively. Respective median survival times were 12.2 months (p=0.037), 12 months (p=0.050), and 7.7 months. The low dose LV regimen gave a statistically significant improvement in weight gain of more than 5%, relief of symptoms, and improvement in performance status. The high dose LV regimen gave a statistically significant improvement in performance status and trended toward improvement in weight gain and in relief of symptoms but these were not statistically significant. 

In a second Mayo/NCCTG randomized clinical study the 5-FU alone arm was replaced by a regimen of sequentially administered methotrexate (MTX), 5-FU, and LV. Response rates with LV 200 mg/m2 and 5-FU 370 mg/m2 versus LV 20 mg/m2 and 5-FU 425 mg/m2 versus sequential MTX and 5-FU and LV were respectively 31% (p=<0.01), 42% (p=<0.01), and 14%. Respective median survival times were 12.7 months (p=<0.04), 12.7 months (p=<0.01), and 8.4 months. No statistically significant difference in weight gain of more than 5% or in improvement in performance status was seen between the treatment arms. 

The pharmacokinetics of 200 mg doses of leucovorin administered intravenously and orally (reconstituted powder, not tablets) have been evaluated in healthy male subjects. The serum clearance corrected for bioavailability, terminal half-life, and apparent volume of distribution of total folate were not significantly different between routes of administration. The oral bioavailability of the 200 mg dose was 31%. Eighty-three percent of the biologically active IV dose was recovered in the urine within 24 hours, 31% as 5-methyltetrahydrofolate. Twenty percent of the same oral dose was excreted in 24 hours, 16% as 5-methyltetrahydrofolate.