Cyclin D1 targets hexokinase 2 to control aerobic glycolysis in myeloma cells.

Cancer cells are characterized by the Warburg effect, a shift from mitochondrial respiration to oxidative glycolysis. We report here the crucial role of cyclin D1 in promoting this effect in a cyclin-dependent kinase (CDK)4/6-independent manner in multiple myeloma (MM) cells. We show that the cyclin D1 oncoprotein targets hexokinase 2 (HK2), a major glycolysis regulator, through two original molecular mechanisms in the cytoplasmic and nuclear compartments. In the cytoplasm, cyclin D1 binds HK2 at the outer mitochondrial membrane, and in the nucleus, it binds hypoxia-inducible factor-1α (HIF1α), which regulates HK2 gene transcription. We also show that high levels of HK2 expression are correlated with shorter event-free survival (EFS) and overall survival (OS) in MM patients. HK2 may therefore be considered as a possible target for antimyeloma therapy.

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis.

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability.

HIF-2alpha specifically activates the VE-cadherin promoter independently of hypoxia and in synergy with Ets-1 through two essential ETS-binding sites.

The mechanisms that are responsible for the restricted pattern of expression of the VE-cadherin gene in endothelial cells are not clearly understood. Regulation of expression is under the control of an approximately 140 bp proximal promoter that provides basal, non-endothelial specific expression. A larger region contained within the 2.5 kb genomic DNA sequence located ahead of the transcription start is involved in the specific expression of the gene in endothelial cells. We show here that the VE-cadherin promoter contains several putative hypoxia response elements (HRE) which are able to bind endothelial nuclear factors under normoxia. The VE-cadherin gene is not responsive to hypoxia but hypoxia-inducible factor (HIF)-2alpha specifically activates the promoter while HIF-1alpha does not. The HRE, that are involved in this activity have been identified. Further, we show that HIF-2alpha cooperates with the Ets-1 transcription factor for activation of the VE-cadherin promoter and that this synergy is dependent on the binding of Ets-1 to DNA. This cooperative action of HIF-2alpha with Ets-1 most probably participates to the transcriptional regulation of expression of the gene in endothelial cells. This mechanism may also be involved in the expression of the VE-cadherin gene by tumor cells in the process of vascular mimicry.

The Ets family contains transcriptional activators and repressors involved in angiogenesis.

The Ets family contains a growing number of transcriptional activators and inhibitors, which activity is regulated by phosphorylation and protein-protein interactions. Among these factors, Ets1, Erg1 and Fli1 are expressed in endothelial cells during angiogenesis in normal and pathological development. The expression of these transcription factors is regulated by angiogenic factors in cultured endothelial cells, as well as by various stresses occurring during angiogenesis. Transfection experiments and transgenic mice analysis revealed that Ets family members are involved in the transcriptional regulation of endothelial specific genes such as those encoding Tie1 and -2, VEGFR1 and -2 and VE-Cadherin. In vitro studies plead for a role of Ets family members in endothelial cell adhesion, spreading and motility. Gene inactivation experiments show that Ets1 is dispensable for embryonic development. The phenotype of knocked-out embryos indicates that Tel is required for maintenance of the developing vascular network in the yolk sac. Altogether, we suggest that Ets family members act both positively and negatively during the different steps of the angiogenic process. The regulation of the initiation of gene transcription arises from the combined activity of different transcriptional regulators. Therefore very few transcription factors are specific for a physiological process, or a given cell type. The transcriptional network that regulates blood vessel formation involves transcription factors which are expressed in a variety of situations. The Lung Kruppel Like Factor (LKLF) which is required for blood vessel stabilisation during murine development is also expressed in the primitive vertebrae and in the lung of the adult (C.T. Kuo, M.L. Veselits, K.P. Barton, M.M. Lu, C. Clendenin, J.M. Leiden, The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilisation during murine embryogenesis, Genes Dev. 11 (22) (1997) 2996-3006). Scl/Tal1 which is essential for angiogenic remodelling of the yolk sac capillary network (J.E. Visvader, Y. Fujiwara, S.H. Orkin, Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development, Genes Dev. 12 (4) (1998) 473-479), is involved in blood cell development and is also expressed in the developing brain. The EPAS transcription factor which was thought to be endothelial cell specific in the mouse embryo (H. Tian, S.L. McKnight, D.W. Russell, Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells, Genes Dev. 11 (1) (1997) 72-82) is also expressed in the liver, kidney and cells of the sympathetic nervous system of the chick embryo (J. Favier, H. Kempf, P. Corvol, J.M. Gasc, Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase, FEBS Lett. 462 (1-2) (1999) 19-24). Ets1, which expression was originally detected in lymphoid cells of adult tissues, has been the first transcription factor to be identified in endothelial cells during angiogenesis in the embryo (B. Vandenbunder, L. Pardanaud, T. Jaffredo, M.A. Mirabel, D. Stehelin, Complementary patterns of expression of c-etsl, c-myb and c-myc in the blood-forming system of the chick embryo, Development 107 (1989) 265-274 [5]) and in tumours (N. Wernert, M.B. Raes, P. Lassalle, M.P. Dehouck, B. Gosselin, B. Vandenbunder, D. Stehelin, The c-ets 1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularisation and other forms of angiogenesis in man, Am. J. Path. 140 (1992) 119-127 [6]). Since then, the Ets family has extended and this review will emphasise the relationships between these factors and angiogenesis.

Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.

The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine.

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.

[Role of the ETS transcription factors in the control of endothelial-specific gene expression and in angiogenesis]. / Rôle des facteurs de transcription ETS dans le contrôle de la spécificité d'expression endothéliale et l'angiogenèse.

The transcription factors of the ETS family are involved in the control of the endothelial-specific expression of genes that are important for the formation of new blood vessels. The analysis of the expression pattern of ets1, the gene inactivation of tel and fli1, the in vitro analysis of potential target genes of ETS factors in endothelial cells, the in vivo studies of the promoter regions of endothelial-specific genes all demonstrate a role for ETS factors in this specificity. However, the precise role of individual ETS factors in the endothelial identity and in angiogenesis in general remains difficult to understand in vivo.

CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of various cell types in the peripheral and central nervous systems. Its receptor complex consists of a non-signaling alpha chain, CNTFR, and two signaling beta chains, gp130 and the leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second, developmentally important ligand. We have identified this factor as a stable secreted complex of cardiotrophin-like cytokine (CLC) and the soluble receptor cytokine-like factor-1 (CLF). CLF expression was required for CLC secretion, and the complex acted only on cells expressing functional CNTF receptors. The CLF/CLC complex activated gp130, LIFR and signal transducer and activator of transcription 3 (STAT3) and supported motor neuron survival. Our results indicate that the CLF/CLC complex is a second ligand for CNTFR with potentially important implications in nervous system development.

ETS1 lowers capillary endothelial cell density at confluence and induces the expression of VE-cadherin.

Ets1 is a transcription factor expressed in endothelial cells during angiogenesis but its target genes and function in blood vessel formation are still unknown. We have over-expressed Ets1 as a tagged protein in brain capillary endothelial cells and in 3T3 fibroblasts using a retroviral vector. Over-expression of Ets1 reduced by nearly half cell density at confluence of endothelials but not of fibroblasts. As density at confluence is controlled in part by cadherins, this growth arrest could be due to the up-regulation of these cell contact molecules. Indeed, Ets1 increased the expression of the endothelial-specific VE-cadherin without affecting N-cadherin expression levels. In parallel, both a dominant negative mutant of Ets members and an Ets1 anti-sense oligonucleotide inhibited VE-cadherin expression in endothelial cells. Ets1 bound to two Ets-binding sites located in the proximal region of the VE-cadherin promoter. Mutation of these sites abolished Ets1-induced transactivation of the promoter. The present work is the first demonstration of a function of Ets1 in the regulation of a specific endothelial marker based on its endogenous gene and protein expression.

Identification of a gp130 cytokine receptor critical site involved in oncostatin M response.

Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.

Structural and functional properties of membrane and secreted IgD.

More than 35 years ago, study of an unknown immunoglobulin (Ig) in the serum from a myeloma patient led to the discovery of IgD. Subsequently, the finding that it also exists as a membrane-bound Ig stimulated a large number of studies during the 70s. Then, the interest on IgD shrank, largely because of the lack of known function of secretory IgD (secIgD) and of a stagnating knowledge of the functions of surface IgD. In the recent years, very significant advances followed the tremendous accumulation of data on the physiology of the B cell receptor, of which IgD is the major component, on the role of secIgD in normal and diseased individuals. This review, which is focused on human IgD but integrates data in the mouse and other species when needed, summarizes present data on the structure, synthesis and functions of both membrane and secIgD, IgD receptors and the involvement of IgD in various diseases, especially the hyperIgD syndrome.

T(h)2 cytokine dependence of IgD production by normal human B cells.

IgD is a minor component of serum Ig and the control of IgD secretion is virtually unknown. We measured concentrations of IgD (and IgE and IgM as controls) in culture supernatants of peripheral blood mononuclear cells (PBMC) from 60 normal donors as well as mononuclear cells from 10 tonsils following culture in the absence or presence of CD40 mAb and cytokines. Low levels of IgD were measured in cultures of PBMC, either unstimulated or stimulated by anti-CD40 antibodies. IL-4 and IL-10 significantly increased IgD production by CD40 mAb-stimulated cells in the majority of normal subjects studied, whereas in a limited number of individuals, spontaneous IgD production was either low or high, but with no increase upon stimulation. Spontaneous IgD production by tonsil-derived mononuclear cells was higher than by PBMC and increased after CD40 stimulation and even more in the presence of IL-10, but not IL-4. IL-2 and IFN-gamma exerted a dose-dependent inhibition on spontaneous as well as CD40- and cytokine-induced IgD production by PBMC, but not by tonsil mononuclear cells. Activation by IL-4 of CD40-stimulated purified B cells from tonsil and PBMC, and by IL-10 of tonsil B cells increased IgD production, whereas IL-2 and IFN-gamma had no detectable inhibitory effect. This suggests that accessory cells indirectly regulate IgD synthesis. IgD production induced in PBMC by IL-4 or IL-10 appeared to result from an active synthesis, and correlated with an increase in the number of IgD-containing plasma cells as demonstrated by immunofluorescence and increased expression of secreted IgD transcripts. These findings suggest that IgD production by normal peripheral blood human B cells is regulated positively by T(h)2 cytokines and negatively by T(h)1 cytokines.

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6.

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.

Preincubation of human resting T cell clones with interleukin 10 strongly enhances their ability to produce cytokines after stimulation.

Interleukin 10 (IL-10) has been described as a cytokine inhibitory factor downregulating IL-2 secretion and inducing T cell anergy. The data reported in this study show that preincubation of resting T cells from the human CD4+ clone SP-B21 (and clone TA-23.6) with IL-10 strongly enhances their capacity to further produce IL-2, interferon gamma (IFN-gamma), IL-4 and tumour necrosis factor alpha (TNF-alpha) after subsequent activation. In contrast, when IL-10 was added during the activation step, the previously reported specific inhibition of IL-2 synthesis was observed. Flow cytometric analysis of intracellular IL-2- and IL-4-producing cells revealed that preincubation with IL-10 increased the number of cytokine-producing cells, but did not affect their individual ability to produce these cytokines. We further show that IL-10 plays a dose-dependent role of viability maintenance factor. This effect relates to a direct anti-apoptotic effect of IL-10, which is likely independent of the expression of bcl-2, bcl-x and fas. Such paradoxal properties of IL-10 on T cells should be considered when aiming at using IL-10 as an immunosuppressive molecule in the treatment of diseases.

Radioimmunoassay for the measurement of S9788 in serum and microdialysis samples.

S9788, 6-[4-(2,2-di-(fluorophenyl)-ethylaminol-1-piperdinyl]-N,N'-d i-2-propenyl-1, 3,5-triazine- 2, 4-diamine, is a novel compound designed to reverse tumour multidrug resistance associated with cancer chemotherapy. A specific and sensitive radioimmunoassay has been developed for the analysis of S9788 in serum samples and adapted for samples obtained by microdialysis. The limit of quantitation is 0.2 ng ml-1 in perfusion medium and there is no cross reactivity of the antibody with known metabolites of the parent compound or with certain cytotoxic compounds likely to be coadministered with S9788. Maximum probe recovery during microdialysis was 66% at a flow of 1 microliter min-1, using Ringer/BSA (70 mg ml-1) as the perfusion medium. The assay has sufficient sensitivity, precision, accuracy and specificity for the analysis of rat and human serum and microdialysis perfusate samples. The assay has been successfully applied to the determination of S9788 in rat plasma (total concentration) and the microdialysate of the same samples.

IL-6 and soluble IL-6 receptors (sIL-6R and sgp130) in human pleural effusions: massive IL-6 production independently of underlying diseases.

IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130) levels were measured in sera and pleural effusions from 42 patients with metastatic carcinoma, non-Hodgkin's lymphoma, tuberculosis, cardiac failure and miscellaneous diseases. Pleural IL-6 levels measured by ELISA were very high in all patient groups (mean 34.8 +/- 15.3 ng/ml) without significant difference according to diseases. IL-6 was shown to be biologically active in a proliferative assay. Serum IL-6 levels were low (0.049 +/- 0.014 ng/ml) and did not correlate with pleural fluid levels. Pleural IL-6 levels correlated with the number of polymorphonuclear cells in pleural fluid (P < 0.03). Pleural sIL-6R levels (76 +/- 8 ng/ml) were always lower than serum levels (196 +/- 12 ng/ml; P < 0.0001) but correlated with them (P < 0.01). Pleural sIL-6R and albumin levels correlated (P < 0.01), suggesting a transudation of sIL-6R from the serum. Pleural sgp130 levels (10.9 +/- 1.0 ng/ml) were lower than serum levels (24.6 +/- 2.8 ng/ml; P < 0.002). After gel filtration of pleural fluid, the bulk of IL-6 (> 90%) was recovered in a 15,000-30,000 fraction, corresponding to the expected mol. wt of free IL-6. These results suggest a production and a sequestration of IL-6 in the pleural cavity in all studied conditions.

Enzyme immunoassays for a new angiotensin-converting enzyme inhibitor, zabicipril, and its active metabolite in human plasma: application to pharmacokinetic studies.

Zabicipril (S 9650) is a new angiotensin-converting enzyme inhibitor whose hydrolysis in vivo produces the pharmacologically active metabolite zabiciprilat (S 10211). Two competitive enzyme immunoassays specific for either zabicipril or zabiciprilat have been developed using acetylcholinesterase (E.C. 3.1.1.7) as label. Antibodies were raised in rabbits after immunization with lysil derivatives of zabicipril or zabiciprilat coupled with bovine serum albumin. Assays were performed in 96-well microtiter plates coated with a monoclonal antibody raised against rabbit immunoglobulin G, thus ensuring rapid separation of free and bound fractions of the tracer. The analysis does not require any extraction step. In the case of the assay of zabiciprilat, interference generated by endogenous angiotensin-converting enzyme (ACE) was eliminated by the addition of perindoprilat, another ACE inhibitor. Perindoprilat was not recognized by the antibodies (cross-reactivity < 0.01%) and did not affect assay efficiency. The specificity of the assays was checked by high-performance liquid chromatography of human plasma samples obtained after oral administration of 2 mg of zabicipril. No metabolites or endogenous substances were detected. The mean reproducibility was 15% for the assay of zabicipril and 19% for the assay of zabiciprilat. The quantification limits were 1.2 ng/ml for the zabicipril assay and 0.8 ng/ml for the zabiciprilat assay. These assays are therefore suitable for pharmacokinetic studies and drug monitoring in clinical studies.

Human pharmacokinetics of a new Vinca alkaloid S 12363 with use of a monoclonal antibody-based radio- or enzyme immunoassay.

S 12363 is a new Vinca alkaloid derivative, characterized by the grafting of an alpha-aminophosphonate, onto the Vinca nucleus, facilitating drug penetration and increasing intracellular drug retention. As a high cytotoxic activity had been demonstrated in in vitro and in vivo models recommended by the National Cancer Institute, a phase I trial was initiated in cancer patients. In order to quantify S 12363 systemic levels in humans, two monoclonal antibody-based immunoassays, RIA (radio-) and EIA (enzyme immunoassay) were developed. The gamma-emitting probe used in the RIA, 125I-(deacetyl-O4-vinblastine)-tyramine, bound very tightly to the monoclonal antibody (dissociation constant, Kd = 2.5 x 10(-11) M), demonstrating a high affinity mainly directed toward the catharantine nucleus (vindesine, vincristine, vinblastine, 100% cross-reactivity; vinorelbine, 0.3% cross-reactivity). In the EIA, a deacetyl O4-vinblastine/ovalbumine conjugate was used as the competing antigen. Its binding to the monoclonal antibody was revealed by an anti-mouse immunoglobulin G conjugated to biotin which interacts with streptavidin labeled with alkaline phosphatase. This method permitted obtaining nearly the same sensitivity and reproducibility with EIA as with RIA, their respective minimum quantitation limits being 0.100 and 0.040 ng/ml (106 and 42 pM) of S 12363 in plasma. These assays allowed the study of S 12363 systemic pharmacokinetics in cancer patients during a phase I trial up to 72 h after dosing. As determined by RIA, the S 12363 plasma profile was triphasic with a terminal half-life; t1/2 gamma = 49 +/- 16 h, a plasma clearance, CL = 0.14 +/- 0.04 liter/h/kg, and a volume of distribution at steady state, Vdss = 5.0 +/- 2.8 liter/kg. The pharmacokinetics of S 12363 is linearly related to dose when increased from 0.08 up to 0.84 mg/m2 in humans. Its plasma profile and pharmacokinetic parameters are close to those of other Vinca alkaloids with clearance and terminal half-life being intermediate between those of vinblastine and vincristine. Therapeutic doses are 4 to 10 times lower and should be a direct consequence of the higher uptake and retention by the cells of this new aminophosphonate Vinca alkaloid derivative.

Value of early pharmacodynamic and pharmacokinetic investigations with anticancer drugs: data from phase I tolerance studies on a new vinca alkaloid derivative.

1. A novel anticancer vinca alkaloid derivative (I) has been given as an i.v. bolus to cancer patients, using four different dosage regimens with dose levels ranging from 0.04 to 0.84 mg/m2 (equivalent to between 0.12 and 1.35 mg per dose), and the pharmacokinetics determined up to 72 h after dosing. In addition, secondary effects of leukopenia and neutropenia, were related to drug exposure using a sigmoid Emax model. 2. Plasma levels of I declined in a triphasic manner with a terminal half-life of approximately 50 h; most drug elimination (55%) being associated with the terminal phase. 3. Clearance of I was relatively low (245 +/- 160 ml/min) and remained constant with increasing doses. Initial distribution volume was low (approximately 71) but once distribution was complete, it was comparatively high (327 +/- 2121). 4. Both leukopenia and neutropenia were fitted successfully to a sigmoid Emax model showing that these effects were related to the total exposure to the drug. The Hill constant was less than 1, indicating a relatively shallow exposure/response curve and a predictable, graded increase in response with increasing I exposure, rather than a sudden quantal response. 5. Pharmacokinetically, I shows some similarities to other vinca alkaloids in its plasma level decline profile, although there are some notable differences which can be exploited clinically. In addition, the ability to model both leukopenia and neutropenia to the exposure to I, provides a valuable tool in the design of the most appropriate dosage regimen for the drug, as well as for dose adjustment taking into account inter-individual variations.