Human urinary excretion of doxifluridine and metabolites during a 5-day chemotherapeutic schedule using fluorine-19 nuclear magnetic resonance spectrometry

The urinary excretion of doxifluridine (5 dFUrd) and its metabolites was determined during five days of chemotherapy using fluorine-19 nuclear magnetic resonance spectrometry. The daily urinary excretion of 5 dFUrd and its metabolites was high (-100% of the 5 dFUrd administered) and nearly constant through out the treatment. By far the major excreted compounds were unchanged 5 dFUrd and -fluoro--alanine which made up respectively -40% and -50% of the total. Neither accumulation of 5 dFUrd nor significant modifications in its metabolism were observed during the treatment.     

Urinary stability of carboxycyclophosphamide and carboxyifosfamide, two major metabolites of the anticancer drugs cyclophosphamide and ifosfamide

Phosphorus-31 nuclear magnetic resonance spectroscopy was used to evaluate the stability of carboxycyclophosphamide (CXCP) and carboxyifosfamide (CXIF) in human urine at pH 7.0 and 5.5 at 25°, 8°, −20°, and −80 °C. At 25 °C and pH 7.0, CXCP and CXIF are relatively stable (≈10% degradation in 24 h). In contrast, they are much less stable at pH 5.5 (≈80% degradation of CXIF and ≈50% degradation of CXCP in 24 h). The rate of degradation of CXCP and CXIF was a function of the storage temperature of the urine samples but, even at −80 °C, was not negligible: ≈30% degradation for CXCP irrespective of pH and ≈40% and 50% degradation for CXIF at pH 7.0 and 5.5, respectively, after storage for 6 months. CXCP was more stable than CXIF at either pH (7.0 or 5.5) and at all storage temperatures (8°, −20°, or −80 °C) of the urine samples. CXCP and CXIF were more stable at pH 7.0 than at pH 5.5, although this difference fell with decreasing temperatures to be almost negligible at −80 °C. To ensure a true estimate of CXCP and CXIF levels, urine samples must be frozen and stored at −80 °C within a few hours of micturition. CXCP and CXIF assays should also be carried out within 2 months and 1 month of storage, respectively. Received: 13 July 1996 / Accepted: 20 January 1997     

Serum-free liquid marrow culture in patients with acute lymphoblastic leukaemia: a potential application to purge marrow for autologous transplantation

We have previously established a serum-free (SF) culture medium, which supports normal haemopoietic progenitor cell growth for at least 4 weeks as does conventional serum dependent (SD) medium. In the present study, we investigated the efficacy of such a defined SF liquid medium which sustained in vitro residual normal haemopoietic proliferation of marrow derived from ALL patients and which was detrimental for the leukaemic population. Evidence for a potential selective effect of SF culture was obtained by a leukaemic progenitor cell assay (ALL-CFU) and the detection of the bcr/abl translocation by polymerase chain reaction (PCR). In 13 experiments including 12 patients, morphological blast cells and ALL-CFU were dramatically reduced within 3 weeks of incubation in both SF and SD cultures. Likewise, in 5/5 experiments in SD and 2/5 experiments in SF conditions, leukaemic cells expressing the bcr/abl fusion gene disappeared within 3-4 weeks. In contrast, the absolute numbers of supernatant cells harvested weekly from SF and SD cultures were similar. No difference in CFU-GM production was detected for the two culture systems. Erythropoiesis in SF medium exhibited a slower decline than that found in SD. These results indicate that liquid marrow culture may selectively deplete leukaemic lymphoblastic cells and enable repopulation by residual normal haemopoietic cells. This technique may be useful to purge leukaemic cells for clinical autologous bone marrow transplantation in patients with ALL.     

Prevalence of sporadic medullary thyroid carcinoma: the importance of routine measurement of serum calcitonin in the diagnostic evaluation of thyroid nodules

OBJECTIVE The prevalence of sporadic forms of medullary thyroid carcinoma (MTC) has been studied In patients living in an area of moderate iodine deficiency. Such forms of MTC are usually diagnosed after surgery and have little chance of definitive cure. Using the measurement of basal serum calcitonin (CT) levels in a large series of patients with both thyroid disease and normal 24-hour urinary Iodine excretion, we assessed the prevalence of MTC and, in patients affected with the disease, we also evaluated the stage of the disease according to surgical findings and post surgical plasma CT levels. PATIENTS A prospective study of 657 patients with thyroid disease (469 with nodular and 188 with non-nodular thyroid disease). As controls, 40 normal subjects were also studied. MEASUREMENTS In all patients: (1) measurement of basal serum CT, free T4, total T3, TSH levels and serum TSH-receptor, peroxidase and thyroglobulin (Tg) antibody concentrations, (2) thyroid ultrasonography, (3) fine needle aspiration cytology (FNAC). In patients with Increased basal CT levels: (1) measurement of serum CT levels during pentagastrin test prior to surgery, (2) histological examination and immunostaining with both anti-CT and anti-Tg antibodies of all the nodular thyroid tissue surgically removed, (3) measurement of basal and pentagastrin stimulated serum CT values after surgery. RESULTS All the patients with non-nodular thyroid disease had normal basal CT levels. Four patients (0.84%) with nodular thyroid disease (2 with uninodular and 2 with multinodular goitre) had both elevated basal and pentagastrin stimulated CT levels. In the two patients with uninodular goitre, FNAC was suggestive of MTC in 1 (nodular diameter 8.0 cm) and of follicular carcinoma In 1 (nodular diameter 2.5 cm). Histological examination of the nodules confirmed these histotypes. Immunostaining with anti-CT antibodies was positive In the former patient but also In the latter. FNAC was suggestive of benign adenomatous tissues in the two patients with multinodular goitre. Histological examination of all the thyroid nodules confirmed the cytological findings. However, serial sections through the gland in each of these two patients showed an occult follicular carcinoma which had, however, positive staining with anti-CT antibodies. Furthermore, immunostaining with anti-Tg antibodies was negative In the patient with MTC but positive in the 3 patients with follicular carcinoma. Finally, both basal and pentagastrin stimulated CT levels remained elevated after total thyroidectomy only in the patient with FNAC suggesting MTC. CONCLUSIONS This study demonstrates a surprisingly high prevalence of sporadic forms of medullary thyroid carcinoma in patients with nodular thyroid disease. Such forms of medullary thyroid carcinoma seem to be unrelated to iodine intake and may be pure or mixed with a follicular carcinoma. In these mixed thyroid carcinomas, only the neoplastic follicular pattern was seen on both cytological and histological examination. Routine measurements of serum calcitonin levels should therefore be considered an integral part of the diagnostic evaluation of thyroid nodules. Indeed, increasing the accuracy of diagnosis of medullary thyroid carcinoma encourages the surgeon to perform more radical treatment, thus achieving more frequent normalization of post-operative serum calcitonin levels.     

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

Background

Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors.

Design and Methods

We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation.

Results

We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice.

Conclusions

These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.Key words: human induced pluripotent stem cells, terminal maturation, erythropoietic differentiation     

Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons

Projection neurons destined for the cortical plate are generated sequentially from the proliferative ventricular and subventricular zones (VZ/SVZ) of the pallium. However, the respective contribution of both proliferative zones to the generation of cortical plate neurons is better established in humans and non-human primates than in rodents. We identified Cux2 as a new marker for murine cortical subpopulations and used it to provide new insights to the development of the mouse cortex. Cux2 is an orthologue of the Drosophila cut gene, which encodes a homeodomain protein involved in neuronal specification. During cortical development Cux2 identifies two subpopulations with different spatial origins, migratory behaviours and phenotypic characteristics: (i) a population of interneurons, which invades the pallium from the subpallium; and (ii) a neuronal population produced in the pallium around embryonic day 11.5, which divides in the SVZ and accumulates in the intermediate zone (IZ). Subsequently, Cux2 is a marker of upper cortical layers. Using different molecular markers and Pax6-deficient mice, we provide data that suggest a relationship between the early-determined Cux2-positive neuronal precursors in the SVZ/IZ and upper layer neurons. This suggests that laminar determination of upper cortical layer neurons occurs during the earliest stages of corticogenesis.     

Fluorouracil prodrugs for the treatment of proliferative vitreoretinopathy: formulation in silicone oil and in vitro release of fluorouracil

Three new N(1)-alkylcarbonyl-5-fluorouracil derivatives that are prodrugs of 5-fluorouracil (FU), one of them being a co-drug FU-retinoic acid (RA), were studied as potentially effective drugs against postsurgical proliferative vitreoretinopathy (PVR). The stability of N(1)-octenoylFU (3), N(1)-lauroylFU (2), and N(1)-retinoylFU (4) in aqueous medium, their solubility in silicone oil (SiO), the kinetics of FU release in an in vitro system were determined. Compound 3 is very rapidly soluble in SiO. Its saturation concentration, reached after 6h, is 233 +/- 13 microg g(-1) SiO. Compound 2 is not very soluble in SiO but its kinetic of solubilization is fast. Its saturation concentration, reached after 2 days, is 27 +/- 2 microg g(-1) SiO. Compound 4 is poorly soluble in SiO. A concentration plateau, with a mean value of 4 microg g(-1) SiO, is reached after 4 days. The addition in SiO of 5% of a perfluorinated perhydrogenated alkene greatly improves the solubilization of compound 4. Two different types of FU release are observed. For compound 3, the release is fast and is achieved after 1 day. For compounds 2 and 4, the release is slower and is ended at 10 and 27 days, respectively. The solubility of the prodrugs in SiO is not correlated with their lipophilicity, whereas the release rate of FU decreased with increased lipophilicity of the prodrug. The most promising prodrug is compound 4 that slowly releases two active drugs (FU and RA) with a t (1/2 release) of 5.8 days. It might be interesting for the treatment of PVR. However, an in vivo study on an animal model of PVR is necessary to prove the efficacy of this formulation and to study its toxicity.     

Design, synthesis, and pharmacological evaluation of new farnesyl protein transferase inhibitors.

New CA(1)A(2)X peptidomimetics are described as Ras farnesyl transferase inhibitors (FTIs). They include cysteine and methionine as mimetics of the C-terminus sequence of farnesylated proteins. Furthermore, cysteine was replaced by heterocycles, taking into account the role of zinc and the metabolic instability of amino acids. The molecular docking of 8 in the active site of the enzyme and the pharmacological evaluation of the compounds are illustrative of a new class of FTIs.