Effect Of Busulphan Research Articles

Development of an assay for cellular efflux of pharmaceutically active agents and its relevance to understanding drug interactions.

Drug interactions may dictate the failure or success of a treatment. Patients undergoing hematopoietic stem cell transplantation (HSCT) are exposed to various types of drugs, and understanding how these drugs interact is of the utmost importance. The pharmacokinetics of busulfan, melphalan, and cyclophosphamide, drugs commonly used for HSCT, are known to be affected by a variety of other drugs with differing molecular structures. We hypothesized that these structurally unrelated drugs affect the transport of DNA-alkylating agents. To test this hypothesis, we developed a flow cytometry assay that used 5-carboxyfluorescein diacetate acetoxymethyl ester, which is cleaved by nonspecific intracellular esterases to 5-carboxyfluorescein (5-CF), a fluorescent ligand for the drug transporter MRP1. A decreased 5-CF efflux in the presence of a test compound suggests competitive inhibition. We demonstrated that chlorambucil, 4-hydroperoxycyclophosphamide, ketoconazole, ethacrynic acid, everolimus, and sirolimus strongly inhibited 5-CF efflux in lymphoma and leukemia cell lines. The efflux of these drugs partially depends on the glutathione (GSH) level, and their cytotoxicity is synergistic with inhibited GSH synthesis. This is consistent with the hypothesis that their GSH-conjugated products are ligands of a common cellular drug transporter. Our results may explain clinical observations on the effects of various drugs on the pharmacokinetics and pharmacodynamics of alkylating agents, and the assay may be used to deduce interaction mechanisms of drugs transported by a common system.

Very Small Embryonic-Like Stem Cells Survive and Restore Spermatogenesis after Busulphan Treatment in Mouse Testis

Study Objectives: Adult mammalian testes harbor a novel population of quiescent, pluripotent, very small embryonic-like stem cells (VSELs) along with spermatogonial stem cells (SSCs). Present study was undertaken to (i) characterize testicular VSELs (ii) investigate differential effect of chemotherapy on VSELs and SSCs and (iii) to restore the differentiation ability of surviving VSELs by providing healthy somatic microenvironment. Methods: Effect of busulphan (25 mg/Kg) was studied on mouse testes. Syngenic Sertoli cells(105 cells per testis) and bone marrow derived mesenchymal cells (104 cells per testis) were transplanted separately through intertubular route. Effect of niche reconstruction was studied two months later by histology and immuno-localization of germ cell markers MVH and PCNA. Caudal sperm were evaluated for their ability to fertilize oocytes in vitro. Results: VSELs were 2-6 μm in size, SCA-1+/CD45-/LIN-, had high nucleo-cytoplasmic ratio and comprised 0.03% of testicular cells whereas SSC specific marker GFRa localized on a distinct, larger cell population. Busulphan selectively destroyed SSCs and other germ cells however, nuclear OCT-4A, Nanog, Sox-2 and SCA-1 positive VSELs (0.06%) survived. Persisting VSELs were unable to differentiate possibly because chemotherapy also affected the ‘niche’ comprising Sertoli cells. Complete restoration of spermatogenesis was observed two months post transplantation of Sertoli and mesenchymal cells. Transplanted cells formed neo-tubules in the vicinity of surviving tubules and were possibly a source of growth factors essential for VSELs proliferation and differentiation. Both MVH and PCNA showed increased staining in the transplanted group. qRT-PCR studies revealed existence of a meiotic block in busulphan treated testis which was overcome after transplantation. Resulting sperm progressed to epididymis, showed normal motility and ability to fertilize in vitro. Conclusion: Results show that VSELs survive chemotherapy and can restore spermatogenesis in germ cells depleted mice. Results have direct relevance to address fertility issues of cancer survivors.

Long-term effects of busulphan on lymphocyte subpopulations in female B6C3F1 mice

Female B6C3F1 mice (12–14 weeks old) were dosed with busulphan (BU) by the intraperitoneal route at a range of dose levels between 10 and 40 mg/kg on four occasions at 14 day intervals. Six weeks after the final dose, mice were given normal drinking water or drinking water containing chloramphenicol succinate (CAP.S) at 4 mg/ml. Animals were killed at eight timepoints after the final BU dose, the final samples being taken at day 485 or 497. A range of haematological parameters were determined and lymphocyte subsets analysed. The inclusion of CAP.S in the drinking water did not affect the changes to haematological parameters or lymphocyte subsets induced by BU. Lymphocyte count was reduced to 51%–66% of control values in animals receiving 40 mg BU/kg on all occasions except day 85. However, the lymphocyte count was increased to 107%–143% of control values in animals receiving 10 mg BU/kg on all occasions except day 85. The large unstained cell (LUC) count was reduced to between 25%–47% of control values in animals receiving 40 mg BU/kg on all occasions, however, the LUC count was not increased at low BU dosage. At day 485/497, B lymphocyte counts were reduced to 48% (p<0.01), 64% (NS) and 55% (p<0.05) of control values in mice treated with 40, 33 and 25 mg BU/kg, respectively. T lymphocyte counts were not affected by BU treatment. We have, therefore, demonstrated in the present study that administration of BU at high levels to the B6C3F1 mouse induces a long-lasting and specific depletion of peripheral B lymphocytes; also, BU at low dose levels causes a mild but persistent lymphocytosis.

Clastogenic and aneuploidizing effects of antiblastic busulphan revealed by kinetochore immunofluorescence in CHO cells

We utilized, in CHO cells, the cytoplasm preservation technique to evaluate the micronucleus frequency at different busulphan concentrations, and the indirect immunofluorescence technique, using sera obtained from patients with scleroderma (CREST variant), to analyze if busulphan-induced micronuclei have kinetochores. Results show that this alkylating agent is capable of causing a significant increase of micronuclei in vitro, a great part (40%) of them having CREST-positive kinetochores. These findings confirm the clastogenic effect of busulphan and reveal a considerable capability of this agent to induce aneuploidy. These results are examined taking into account the high incidence of secondary neoplasias induced by chemotherapy with alkylating agents utilized against primary neoplasias.

Effects of high dose busulphan on leukaemic progenitor cells in chronic myeloid leukaemia.

Six patients with Philadelphia positive chronic myeloid leukaemia (CML) were treated with single high doses of busulphan. The action of busulphan on the in vivo kinetics of circulating progenitor cells (colony forming cells) was measured using an agar culture system which involved scoring of total colonies and clusters at 7 days and of granulocyte, monocyte and eosinophil colonies at 14 days. High dose busulphan was found to be effective in suppressing circulating granulocyte, monocyte and eosinophil progenitor cells. The effect of busulphan on progenitor cells was rapid and their levels fell by at least 85% within five days. By contrast, the white blood cells fell by only 9% and the platelets fell by 10% over this time. Subsequently, the white cell count and platelet count fell to near normal levels. The progenitor cell levels began to rise again at a mean of 35 days following busulphan treatment and the white blood cells at a mean of 39 days in four patients. One patient remained in haematological remission for six months following 100 mg of busulphan with less than 1 progenitor per 5 x 10(5) peripheral blood nucleated cells. One patient in myeloblastic transformation had a previously not described culture pattern consisting of a high cluster to colony ratio at 7 days and of an increased number of predominantly eosinophilic colonies at 14 days. There was no significant fall in progenitor cell levels following busulphan and this patient died.

The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose-response was a clearly defined linear relationship. Therefore a standard SCE dose-response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.

The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose-response was a clearly defined linear relationship. Therefore a standard SCE dose-response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.

The effects of busulphan, hydroxyurea and cytosine arabinoside on the colony forming cells in chronic granulocytic leukaemic and non leukaemic marrow

This study investigated the effects of drugs on the colony forming cells in normal marrow and in the marrow and peripheral blood of patients with CGL. Normal and CGL cells showed similar sensitivities to single doses of busulphan, but CGL cells were more sensitive than normal to hydroxyurea and did not show the characteristic plateau on the dose-response curve. The studies using ara-C indicate that, in CGL, the circulating colony forming cells are proliferating more slowly than those in the marrow.

COMPARATIVE EFFECTS OF NITROGEN MUSTARD (HN2), CHLORAMBUCIL (CB 1348), AND BUSULPHAN (MYLERAN) ON THE PERIPHERAL BLOOD AND THE FERROKINETIC PATTERNS IN DOGS

Three alkylating agents, HN2 (methyl-bis(betachlorethyl)amine), chlorambucil (p-(di-2-chlorethylamino)-phenylbutyric acid), and busulphan (1,4-dimethanesulphonoxybutane) were studied with regard to their effects on leukocyte and red cell production in dogs. This was assessed by estimation of the peripheral blood leukocyte and reticulocyte changes as well as alterations in the plasma iron, Fe59 disappearance rate, plasma iron turnover, and red cell incorporation of radioactive iron. The two chlorethylamines had a similar effect on both leukocyte and red cell production. The effect on peripheral leukocyte counts was evident in 24 hours and the reticulocyte response indicated maximum interference with erythropoiesis on the third to seventh day following the drug. The effect of busulphan on both granulocytes and lymphocytes was slower to appear and slowly progressive. A difference in ferrokinetic pattern between the chlorethylamines and busulphan was also noted. The dissimilarity in ferrokinetic patterns may indicate a difference not only in mechanism of these agents on proliferating normoblasts but also in the inhibitory effects on the acceptance of iron by the red cell precursors and other body iron pools.

COMPARATIVE EFFECTS OF NITROGEN MUSTARD (HN2), CHLORAMBUCIL (CB 1348), AND BUSULPHAN (MYLERAN) ON THE PERIPHERAL BLOOD AND THE FERROKINETIC PATTERNS IN DOGS

Three alkylating agents, HN2 (methyl-bis(betachlorethyl)amine), chlorambucil (p-(di-2-chlorethylamino)-phenylbutyric acid), and busulphan (1,4-dimethanesulphonoxybutane) were studied with regard to their effects on leukocyte and red cell production in dogs. This was assessed by estimation of the peripheral blood leukocyte and reticulocyte changes as well as alterations in the plasma iron, Fe59 disappearance rate, plasma iron turnover, and red cell incorporation of radioactive iron. The two chlorethylamines had a similar effect on both leukocyte and red cell production. The effect on peripheral leukocyte counts was evident in 24 hours and the reticulocyte response indicated maximum interference with erythropoiesis on the third to seventh day following the drug. The effect of busulphan on both granulocytes and lymphocytes was slower to appear and slowly progressive. A difference in ferrokinetic pattern between the chlorethylamines and busulphan was also noted. The dissimilarity in ferrokinetic patterns may indicate a difference not only in mechanism of these agents on proliferating normoblasts but also in the inhibitory effects on the acceptance of iron by the red cell precursors and other body iron pools.