The in vitro and in vivo effects of stem cell factor on human hematopoiesis.

The c-kit ligand or stem cell factor (SCF) and the c-kit ligand receptor (KR) are thought to play pivotal roles in the regulation of human hematopoiesis. When added to interleukin 3 (IL-3) and/or granulocyte-macrophage colony stimulating factor (GM-CSF), SCF has an especially profound effect on the in vitro proliferation of several classes of primitive hematopoietic progenitor cells including the burst forming unit megakaryocyte (BFU-MK), the high proliferative potential colony forming cell (HPP-CFC) and the long-term bone marrow culture-initiating cell (LTBMC-IC). These primitive hematopoietic progenitor cells are present in a CD34+HLA-DR- fraction of marrow which has in vivo marrow populating ability and thereby resembles the pluripotent hematopoietic stem cell. Furthermore, the CD34+HLA-DR- marrow subpopulation which expresses KR contains virtually all of the marrow BFU-MK, HPP-CFC and LTBMC-IC, indicating that the human stem cell is KR positive. The addition of SCF, IL-3 and GM-CSF to suspension cultures initiated with CD34+HLA-DR- cells results in an exponential expansion of the numbers of hematopoietic progenitor cells. Large numbers of such progenitor cells generated ex vivo may be useful as transfusion products for the treatment of chemotherapy induced cytopenias. The therapeutic potential of the in vivo administration of SCF has also been evaluated in a phase I trial of recombinant methionyl SCF. SCF administration led to an increase in both differentiated and primitive hematopoietic progenitor cells within the marrow. Such studies suggest that in vivo SCF administration may be useful for improving the quality of bone marrow grafts to be used either for autologous or allogeneic bone marrow transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioequivalence of two leucovorin calcium tablet formulations.

In a randomized, single-dose, two-way crossover study, 36 male volunteers received 25 mg each of two oral formulations of leucovorin calcium. Reduced serum folate concentrations were determined over the 24 hours after dosing. There were no statistically significant differences in areas under the serum concentration-time curves for total L-tetrahydrofolates, L-leucovorin (L-5-formyltetrahydrofolate), and L-5-methyltetrahydrofolate (the active metabolite of leucovorin and the predominant circulating form of reduced folate after oral administration). The peak serum concentrations and times to peak serum concentrations were also not significantly different. We conclude that the two leucovorin calcium 5 mg tablet formulations are bioequivalent.

Pharmacokinetics of leucovorin calcium after intravenous, intramuscular, and oral administration.

The pharmacokinetics of leucovorin was evaluated after intravenous, intramuscular, and oral administration in a randomized crossover study of 37 healthy men. A single 25-mg dose of leucovorin calcium was administered intravenously, intramuscularly, or orally to the subjects. Blood samples were obtained immediately before and at 13 time points up to 24 hours after the leucovorin dose. The three treatment phases were separated by one-week intervals. Bioavailability was assessed by measuring over 24 hours the blood concentrations of total folates, the parent compound 5-formyltetrahydrofolate, and the metabolite 5-methyltetrahydrofolate, using differential microbiologic assays with Lactobacillus casei and Streptococcus faecalis. Both intravenous and intramuscular administration produced rapid increases in serum concentrations of biologically active folates; these rises were sustained over time and were still detectable at 24 hours after drug administration. The bioavailability of intravenous and intramuscular doses was comparable based on area under the serum concentration-time curve, although for intramuscular administration, the peak concentration was lower and the time to peak concentration was longer. The initial rise in serum folate with intravenous and intramuscular dosing represented 5-formyltetrahydrofolate; this fell concomitantly with the appearance of 5-methyltetrahydrofolate. Oral leucovorin was 92% bioavailable compared with intravenous administration and produced a predictably different pattern of circulating folates, 5-methyltetrahydrofolate being the predominant form. Terminal elimination half-life, apparent volume of distribution, and clearance of total folate were not significantly different among the three treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption kinetics of orally administered leucovorin calcium.

Oral dose proportionality and pharmacokinetics of leucovorin [(d,l)-5-formyltetrahydrofolate (5-formyl-THF)] were studied in 30 healthy male subjects. In a randomized cross-over design, 24 fasted subjects were given 4 of a series of 5 single test doses between 20 and 100 mg, at 1-week intervals, of 5-formyl-THF as an oral solution of leucovorin calcium. Six separate subjects received 200 mg iv and po in a 2-way crossover. Blood and urine samples were collected over 24 hours for differential microbiological folate assays using Lactobacillus casei and Streptococcus faecalis. Using L casei activity to measure total serum folates, the area under the concentration-time curve from 0 to infinite time (AUC[0-infinity]) was calculated. Relative bioavailabilities were 78%, 62%, 49%, and 42% for the 40-, 60-, 80-, and 100-mg doses, respectively. Both the AUC and peak concentration (CPEAK) of total folates (consisting predominantly of the major metabolite, 5-methyltetrahydrofolate (5-methyl-THF], displayed significant deviation from linearity consistent with a saturation of folate absorption. Absolute bioavailability of the 200-mg oral dose of leucovorin based on AUC was 31% compared with that of the iv dose (6,848 vs. 22,298 ng.hr/ml, respectively). Total clearance, terminal half-life, and apparent volume of distribution of total folate at the 200-mg dose were not significantly different between the two routes of administration. Eighty-three percent of the biologically active iv dose was recovered in the urine within 24 hours, 31% as 5-methyl-THF. Twenty percent of the same oral dose was excreted in 24 hours, 16% as 5-methyl-THF. In contrast to the nondose-proportionality observed in total serum folates, AUC of the small component of S faecalis activity, which appeared earlier than 5-methyl-THF, displayed linear kinetics, suggestive of a distinct mechanism of uptake. As dose increased, S faecalis activity increased in relative proportion to L casei, indicating that saturation of the enzymatic bioconversion to 5-methyl-THF may also be occurring. In light of the demonstrated nondose-proportionality of total folates with oral leucovorin in this dose range, consideration should be given to parenteral administration in regimens employing higher doses. Oral administration should be at a level consistent with the capacity for efficient folate uptake.

Autoradiographic localization of serum retinol-binding protein in rat testis.

An interstitial target site in the rat testis for vitamin A has been detected using radioactively labeled rretinol-binding protein (RBP) in autoradiography at the light microscope level. RBP purified from rat serum was iodinated by the lactoperoxidase procedure and was also complexes with [3H]-retinol. The radioactively labeled RBP was administered to rat testis tissue by a direct intratesticular injection, an iv injection, and through an in vitro incubation of decapsulated tissue in medium containing the labeled protein. Localization on the autoradiograms of both [125I]RBP and [3H]retinol-RBP was interstitial; there was no interaction of RBP with cells in or on the seminiferous tubules. The patterns of localization were identical in normal rats and vitamin A-deficient rats. [3H]etinol uncomplexed with RBP (administered with rat serum albumin) was distributed evenly throughout testicular tissue, with no specific localization. Although the labeling of the cells on the autoradiograms was not visible diminished by the presence of excess unlabeled RBP, an in vitro binding assay of a crude interstitial cell preparation using [125I]RBP displayed saturable binding, indicative of receptors for RBP. An in vitro binding assay of Sertoli cells in culture with [125I]RBP demonstrated no specific binding, nor did [125I]RBP in autoradiography of Sertoli cell monolayers display interaction with these cells. Results from this investigation show that the plasma transport protein for vitamin A, RBP, interacts initially with cells in the interstitium of the testis. (Endocrinology 108: 658, 1981)

Three-step purification of retinol-binding protein from rat serum.

Rat serum retinol-binding protein has been purified to apparent homogeneity in high yield by a new procedure utilizing three simple steps: DEAE-cellulose chromatography at pH 6.0, Sephadex G-75 gel filtration in the presence of 3.0 M urea, and finally DEAE-cellulose chromatography at pH 8.3. The second step accomplished the dissociation and separation of retinol-binding protein from its complex with prealbumin; this represents a substantial improvement over published procedures, in which sample recycling and preparative polyacrylamide gel electrophoresis were necessary. The purified retinol-binding protein was characterized by molecular weight measurement, fluorescence spectra and immunological properties.