Vitamin D₃ analogs: Effect on leukemic clonal growth and differentiation, and on serum calcium levels

Abstract

In vitro, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) induces differentiation of HL-60 cells and inhibits their proliferation as well as the proliferation of leukemic cells from patients. In vivo, the survival of mice challenged with syngeneic leukemic cells is enhanced by treatment with 1,25(OH)₂D₃. Patients treated with 1,25(OH)₂D₃ develop hypercalcemia at a serum level of 2 × 10⁻¹⁰ mol/l which is a concentration too low to achieve an antileukemic effect in vitro. Several interesting vitamin D₃ analogs have recently been developed. We initially examined the effect of 1,25(OH)₂-16ene-23yne-19-nor-26,27-F₆-D₃ and 24a,26a,27a-tri-homo-22,24-diene-1-α,25-(OH) ₂-D₃ on clonal growth and differentiation of HL-60 cells. Each of the analogs had comparable effects on clonal growth with 50% inhibition (ED₅₀) at concentrations of 0.2–0.5 × 10⁻⁹ M; 1,25(OH)₂D₃ was about 20- to 50-fold less active in inhibiting growth. Differentiation was determined by induction of Superoxide production, as measured by nitroblue tetrazolium (NBT) reduction and by expression of a macrophage-specific enzyme (alpha napthyl acetate esterase (ANAE)). The 24a,26a,27a-tri-homo-22,24-diene-1-α,25-(OH)₂-D₃ and 1,25(OH)₂-16ene-23yne-19-nor-26,27-F₆-D₃ were about 5- to 14-fold more potent than 1,25(OH)₂D₃. The hypercalcemia inducing side-effects of these analogs and three other previously identified, extremely potent vitamin D₃ compounds, as well as 1,25(OH)₂D₃, were studied. The analogs were administered intraperitoneally every other day (qod) for 5 weeks; serum was collected weekly and Ca²⁺ measured by atomic absorption spectrophotometry. The highest tolerated dose of each analog leaving all mice alive was for 1,25(OH)₂D₃: 0.25 μg; 1,25(OH)₂-24a,26a,27a-tri-homo-22,24-diene-D₃: 0.25 μg; and 1,25(OH)₂-16ene-23yne-19-nor-26, 27-F₆-D₃: 0.0625 μg. Another hexafluoro compound with potent abilities to induce differentiation (1,25(OH)₂-16ene-23yne-26,27-F₆-D₃) was very toxic, all mice died in the second week while receiving 0.0625 μg qod. Prior studies showed that the most potent compound in inducing differentiation of HL-60 was 1,25(OH)₂-20-epi-D₃; but it is very toxic as only one mouse survived a dose of ⩾0.0125 μg qod for 5 weeks. 1,25(OH)₂-16ene-23yne-D₃ is an extremely active inducer of differentiation but, on the other hand, it has low potential to produce hypercalcemia; mice maintained normal serum calcium levels even while receiving 2 μg qod for 5 weeks. A prior study found 1,25(OH)₂-16ene-D₃ was also an active inducer of differentiation, but mice receiving more than 0.25 μg qod for 5 weeks developed prominent hypercalcemia. Therapeutic indices were calculated by comparing the concentrations of analogs required to induce differentiation and inhibit proliferation of HL-60 cells in relation to those that caused hypercalcemia; both 1,25(OH)₂-24a,26a,27a-tri-homo-22,24-diene-D₃ and 1,25(OH)₂-16ene-23yne-D₃ are the most promising vitamin D₃ analogs, and should be considered for clinical trials.