Search

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.

Rapid and durable recovery of hematopoietic function after hematopoietic stem cell transplantation (HSCT) requires the infusion of a sufficient number of hematopoietic stem cells (HSC). Granulocyte colony-stimulating factor (G-CSF), either alone or with chemotherapy, has been the traditional backbone of regimens used to mobilize HSC. Plerixafor (previously known as AMD3100), a selective antagonist of CXCR4, has recently been approved for autologous HSC mobilization in combination with G-CSF. The current study assessed the safety and efficacy of plerixafor as a single agent when given subcutaneously and followed by apheresis 6 hours later for the mobilization of HSC for transplantation in 9 patients with multiple myeloma (MM). All patients mobilized enough cells for at least 1 transplant, and demonstrated prompt recovery of hematopoietic function. Median time to engraftment was 10.5 days for neutrophils and 21 days for platelets. Significant adverse events were not observed. Recovery of peripheral blood cell counts was durable in all surviving patients. Despite these successes, mobilization with plerixafor alone was modest. However, in clinical circumstances where G-CSF or chemotherapy based-mobilization should not be used, mobilization with plerixafor alone may be required and effective. Further research into single agent use should focus on alternate route of administration as well as adjustment of the timing of the apheresis to improve cell HSC yield., (Copyright 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

This report describes the first investigational use of plerixafor in Europe and the determination of tumor cell mobilization by polymerase chain-reaction after plerixafor treatment in a subset of patients with multiple myeloma (MM). Thirty-five patients (31 MM and 4 NHL) received granulocyte colony-stimulating factor (G-CSF) (10 microg/kg) each morning for 4 days. Starting the evening of Day 4, patients recieved plerixafor 0.24 mg/kg. Apheresis was initiated 10-11 h later, in the morning of Day 5. This regimen of G-CSF treatment each morning before apheresis and plerixafor treatment in the evening was repeated for up to 5 consecutive days. Mobilization with plerixafor and G-CSF resulted in a median 2.6-fold increase in peripheral blood (PB) CD34+ cell count compared with before plerixafor treatment. All patients collected > or =2 x 10(6) CD34+ cells/kg and 32 of 35 patients collected > or =5 x 10(6) CD34+ cells/kg. After plerixafor treatment, 3 of 7 patients had a small increase and 4 of 7 patients had a small decrease in PB tumor cells. No G-CSF was given post transplant. The median number of days to polymorphonuclear leukocyte and platelet engraftment was 14.0 and 11.0, respectively. There were no reports of graft failure. Plerixafor was generally well tolerated. Mobilization of PB CD34+ cells was consistent with previous clinical trials. The addition of plerixafor did not significantly increase the relative number of PB MM tumor cells.

Plerixafor is a selective antagonist of CXCR4 used for mobilization of hematopoietic stem cells (HSCs) for autologous stem cell transplantation (SCT) in patients with multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). This Phase 1 open-label study in healthy subjects was conducted to evaluate the pharmacokinetic characteristics of plerixafor in subjects with renal impairment. All subjects received a single 0.24 mg/kg subcutaneous dose of plerixafor. Subjects were stratified into 4 cohorts based on creatinine clearance determined from a 24-hour urine collection: control (>90 mL/min), mild renal impairment (51-80 mL/min), moderate renal impairment (31-50 mL/min), and severe renal impairment (<31 mL/min, not requiring dialysis). Eleven female subjects (48%) and 12 male subjects (52%), ranging in age from 35 to 73 years, were enrolled. Plerixafor clearance was reduced in subjects with renal impairment and was positively correlated with creatinine clearance. The mean area under the concentration- versus-time curve from time 0 to 24 hours postdose of plerixafor in subjects with mild, moderate, and severe renal impairment was 7%, 32%, and 39% higher, respectively, than that in subjects with normal renal function. Renal impairment had no effect on maximal plasma concentrations. The safety profile was similar among subjects with renal impairment and controls. No renal impairment-related trends in the incidence of adverse events were apparent. A plerixaflor dose reduction to 160 microg/kg in patients with a creatinine clearance value

Plerixafor, a novel CXCR4 inhibitor, is effective in mobilizing PBSCs particularly when used in conjunction with G-CSF. In four cohorts, this pilot study explored the safety of plerixafor mobilization when incorporated into a conventional stem cell mobilization regimen of chemotherapy and G-CSF. Forty (26 multiple myeloma and 14 non-Hodgkin's lymphoma) patients were treated with plerixafor. Plerixafor was well tolerated and its addition to a chemo-mobilization regimen resulted in an increase in the peripheral blood CD34+ cells. The mean rate of increase in the peripheral blood CD34+ cells was 2.8 cells/microl/h pre- and 13.3 cells/microl/h post-plerixafor administration. Engraftment parameters were acceptable after myeloblative chemotherapy, with the median day for neutrophil and plt engraftment being day 11 (range 8-20 days) and day 13 (range 7-77 days), respectively. The data obtained from the analysis of the cohorts suggest that plerixafor can safely be added to chemotherapy-based mobilization regimens and may accelerate the rate of increase in CD34+ cells on the second day of apheresis. Further studies are warranted to evaluate the effect of plerixafor in combination with chemomobilization on stem cell mobilization and collection on the first and subsequent days of apheresis, and its impact on resource utilization.

Background: The X4 Antagonist Concept Trial investigates the safety and antiviral activity of AMD11070, a potent inhibitor of X4-tropic human immunodeficiency virus (HIV) in vitro in HIV-infected patients harboring X4-tropic virus., Methods: Patients enrolled in the study had an X4 virus population 2000 relative luminescence units (rlu; by the Monogram Trofile Assay) and an HIV-1 RNA level 5000 copies/mL. Patients received AMD11070 monotherapy for 10 days. Coreceptor tropism, plasma HIV-1 RNA level, and CD4 cell count were measured at study entry, on day 5, and on day 10. Daily predose and serial samples on the last day of treatment were obtained for determination of plasma AMD11070 concentration., Results: Ten patients were given AMD11070 monotherapy (200 mg to 8 patients and 100 mg to 2 patients) twice daily for 10 days. The median baseline CD4 cell count was 160 cells/mm(3), and the median HIV-1 RNA level was 91,447 copies/mL. Four of 9 evaluable patients achieved a reduction in X4 virus population of >or= rlu. The median change in X4 virus population at the end of treatment was -0.22 log(10) rlu (range, -1.90 to 0.23 log(10) rlu). Three of 4 patients who responded to therapy showed a tropism shift from dual- or mixed-tropic viruses to exclusively R5 virus by day 10. There were no drug-related serious adverse events, adverse events of greater than grade 2, or laboratory abnormalities., Conclusion: These results demonstrate the activity of AMD11070, the first oral CXCR4 antagonist, against X4-tropic HIV-1. The drug was well tolerated, with no serious safety concerns. AMD11070 is on clinical hold because of histologic changes to the liver observed in long-term animal studies; additional preclinical safety assessments are pending.

We investigated the efficacy and toxicity of combining granulocyte-colony stimulating factor (G-CSF) at standard doses with plerixafor, a CXCR4 inhibitor, to mobilize stem cells in patients with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). Patients with NHL and MM underwent mobilization with G-CSF (10 microg/kg/day) for up to 9 days and plerixafor (240 microg/kg/day), which started on the evening of day 4. Apheresis began on day 5 and continued daily until either >or= 5 x 10(6) CD34/kg were collected or to a maximum of 5 aphereses. Toxicities, increase in circulating CD34 cells/microL before and after the first dose of plerixafor, percentage of patients collecting >or= 5 x 10(6) CD34/kg, total CD34 cells/kg collected, engraftment, and exploratory efficacy analyses in heavily pretreated patients were examined. Six sites enrolled 49 patients (NHL, 23; MM, 26). All completed mobilization and 47 of 49 (96%) underwent transplant. Circulating CD34 cells/microL increased by 2.5-fold (1.3-6.0-fold) after the first plerixafor dose. The median CD34 cells/kg collected was 5.9 x 10(6) (1.5-22.5) in 2 (1-5) days of aphereses. Median days to neutrophil and platelet engraftment were 11 (8-16) and 14.5 (7-39) days, respectively. Adverse events primarily were mild nausea and diarrhea (n=24). Twenty-eight (57%) were identified as heavily pretreated patients. Their median fold increase in circulating CD34 cells/microL was 2.5 (1.4-5.0) after plerixafor, similar to minimally pretreated patients. Plerixafor and G-CSF increased circulating CD34 cells/microL and led to the adequate collection of stem cells for autotransplant in 96% of the patients. This combination may have particular value in heavily pretreated patients.

Collection of an adequate number of hematopoietic stem cells can be difficult in patients with relapsed or refractory Hodgkin lymphoma (HL) who are candidates for autologous stem cell transplantation (ASCT). Plerixafor (AMD3100), an inhibitor of the interaction between stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4, is an effective hematopoietic stem cell mobilization agent in patients with multiple myeloma and non-Hodgkin lymphoma (NHL). This study was undertaken to investigate the efficacy and safety of hematopoietic stem cell mobilization with plerixafor in patients with HL. Twenty-two patients with HL who were candidates for ASCT underwent hematopoietic stem cell mobilization with a combination of granulocyle-colony stimulating factor (G-CSF), 10 microg/kg daily, and plerixafor, 240 microg/kg subcutaneous, 10-11 hours prior to apheresis. The primary endpoint was the proportion of patients who collected>or=5x10(6) CD34+ cells/kg. Outcomes were compared to a historical control population of HL patients mobilized with G-CSF alone. Pharmacokinetic (PK) analysis of plerixafor was performed in a subset of patients. Fifteen patients (68%) collected>or=5x10(6) CD34+ cells/kg, and 21 patients (95%) achieved the minimum collection of >or=2x10(6) CD34+ cells/kg, in a median 2 apheresis procedures. Both the proportion of patients collecting>or=5x10(6) CD34+ cells/kg and the median CD34+ cells collected in days 1-2 of apheresis were significantly improved over historical controls. The PK of plerixafor in this patient population was similar to that previously seen in healthy volunteers. The regimen was generally safe and well tolerated.

AMD3100 given with G-CSF has been shown to mobilize CD34+ cells in non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), and Hodgkin's disease (HD) patients who could not collect sufficient cells for autologous transplant following other mobilization regimens. These poor mobilizers are usually excluded from company-sponsored trials, but have been included in an AMD3100 Single Patient Use protocol, referred to as a Compassionate Use Protocol (CUP). A cohort of 115 data-audited poor mobilizers in CUP was assessed, with the objective being to collect > or =2 x 10(6) CD34+ cells per kg following AMD3100 plus G-CSF mobilization. The rates of successful CD34+ cell collection were similar for patients who previously failed chemotherapy mobilization or cytokine-only mobilization: NHL -- 60.3%, MM -- 71.4% and HD -- 76.5%. Following transplant, median times to neutrophil and PLT engraftment were 11 days and 18 days, respectively. Engraftment was durable. There were no drug-related serious adverse events. Of the adverse events considered related to AMD3100, two (1.6%) were severe (one patient -- headache, one patient -- nightmares). Other AMD3100-related adverse events were mild (84.8%) or moderate (13.6%). The most common AMD3100-related adverse events were gastrointestinal reactions, injection site reactions and paresthesias. AMD3100 plus G-CSF offers a new treatment to collect CD34+ cells for autologous transplant from poor mobilizers, with a high success rate.

AMD3100 is a drug capable of mobilizing peripheral blood stem cells (PBSCs) in donors and in cancer patients as a single agent or in combination with granulocyte-colony-stimulating factor (G-CSF). We initiated a phase II study of 11 refractory or relapsed non-Hodgkin's lymphoma (NHL) patients, receiving 16 microg/kg daily of G-CSF for 4 days followed by 240 microg/kg of AMD3100 given subcutaneously on a new schedule of 9-10 h before apheresis collection on day 5. Our aims were to assess the effect of AMD3100 on the mobilization of CD34+ cells, dendritic cells (DCs) and lymphoma cells. Administration of G-CSF and AMD3100 were continued daily until >or=2 x 10(6) CD34+ cells/kg were collected. Adequate collection of the target of CD34+ cells was achieved in all but 1 patient within 2 days, and 10/11 patients were transplanted within 2 months. All transplanted patients engrafted with a mean of 10 and 12 days for neutrophils and platelets, respectively. Addition of AMD3100 to G-CSF resulted with >2.5-fold increase in CD34+ cells/microl (p = 0.0001) and in a >2-fold increase in pDC1 and pDC2 cells/microl (p = 0.003). Adverse events related to AMD3100 were minimal. AMD3100 was generally safe and improved PBSC and DC cell mobilization with no apparent contamination of lymphoma cells.

Hematopoietic progenitor cells (HPCs) traffic to and are retained in the marrow through the trophic effects of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) binding to its receptor, CXC chemokine receptor 4 (CXCR4). AMD3100 reversibly inhibits SDF-1alpha/CXCR4 binding, and AMD3100 administration mobilizes CD34(+) cells into the circulation. We therefore tested the hypotheses that the combination of AMD3100 plus granulocyte colony-stimulating factor (G-CSF) (hereafter A + G) would be superior to G-CSF alone (hereafter G) in mobilizing hematopoietic progenitor cells (HPCs) and that A + G-mobilized cells would engraft as well as G-mobilized cells. The primary objective was to determine whether patients mobilized more progenitor cells per unit of blood volume of apheresis after A + G administration versus G alone. Secondary objectives were to determine whether patients mobilized with A + G compared with G alone required fewer apheresis procedures to reach the target level at least 5 x 10(6) CD34(+) cells/kg for transplantation and to determine whether patients mobilized with A + G had at least a 90% success rate of autologous transplantation as assessed by neutrophil engraftment by day 21. Each patient served as his or her own control in a sequential mobilization design. All study objectives were met without significant toxicity. The results demonstrate that the combination of A + G is generally safe, effective, and superior to G alone for autologous HPC mobilization.

Background: AMD3100 is a small-molecule CXCR4 antagonist that has been shown to induce the mobilization of CD34 + hematopoietic progenitor cells from bone marrow to peripheral blood. AMD3100 has also been shown to augment the mobilization of CD34 + cells in cancer patients when administered in combination with granulocyte colony-stimulating factor (G-CSF) (filgrastim). The purpose of this study was to characterize the exposure-response relationship of AMD3100 in mobilizing CD34 + cells when administered as a single agent in healthy volunteers., Methods: AMD3100 concentrations and CD34 + cell counts obtained from 29 healthy subjects in a single-dose, intensively sampled pharmacokinetic/pharmacodynamic (PK-PD) study were analyzed by use of nonlinear mixed effects regression with the software NONMEM. FOCE (first order conditional estimation) with interaction was the estimation method, and simultaneous PK-PD fitting was adopted., Results: The pharmacokinetics of AMD3100 was described by a 2-compartment model with first-order absorption. The population estimates (+/-SE) for clearance and central volume of distribution were 5.17 +/- 0.49 L/h and 16.9 +/- 3.79 L, respectively. CD34 + cell mobilization was best described by an indirect effect model that stimulates the entry process of CD34 + from bone marrow to peripheral blood in the form of a sigmoid maximum effect model. The population estimates (+/-SE) of maximum effect, concentration causing 50% of maximum response, and equilibration time were 12.6 +/- 4.89, 53.6 +/- 11.9 mug/L, and 5.37 +/- 1.31 hours, respectively., Conclusions: This study characterizes the exposure-response relationship of AMD3100 in mobilizing CD34 + cells after subcutaneous administration. This PK-PD model will be useful in assessing relevant covariates and for optimizing the use of AMD3100 in various patient populations.

Background: AMD3100, a selective antagonist of CXCR4, rapidly mobilizes CD34+ hematopoietic progenitor cells (HPCs) from marrow to peripheral blood with minimal side effects., Study Design and Methods: To further investigate potential clinical utility of AMD3100 for CD34+ cell mobilization and collection, a Phase I study in normal volunteers was performed examining single-dose administration of AMD3100 alone and in combination with a standard 5-day granulocyte-colony-stimulating factor (G-CSF) regimen., Results: AMD3100 (160 microg/kg x 1 on Day 5) significantly increased both G-CSF-stimulated (10 microg/kg/day) mobilization of CD34+ cells (3.8-fold) and leukapheresis yield of CD34+ cells. Moreover, collection of CD34+ cells was comparable between individuals mobilized by a single-dose regimen of AMD3100 (240 microg/kg) and individuals mobilized with a 5-day regimen of G-CSF. AMD3100-mobilized leukapheresis products contained significantly greater numbers of T and B cells compared to G-CSF-stimulated leukapheresis products., Conclusion: These findings indicate that AMD3100 can be used alone or as an adjunct to G-CSF to mobilize cells for HPC transplantation.

AMD3100 is a CXCR4 receptor inhibitor with anti-HIV-1 activity in vitro. We tested the safety, pharmacokinetics, and antiviral effect of AMD3100 administered for 10 days by continuous intravenous infusion in an open-label dose escalation study from 2.5 to 160 microg/kg/h. Forty HIV-infected patients with an HIV RNA level >5000 copies/mL on stable antiretroviral (ARV) regimens or off therapy were enrolled. Syncytium-inducing (SI) phenotype in an MT-2 cell assay was required in higher dose cohorts. Most subjects were black (55%), male (98%), and off ARV therapy. HIV phenotype was SI (30%), non-SI (45%), or not tested (25%). One patient (5 microg/kg/h) had serious and possibly drug-related thrombocytopenia. Two patients (40 and 160 microg/kg/h) had unexpected, although not serious, premature ventricular contractions. Most patients in the 80- and 160-microg/kg/h cohorts had paresthesias. Steady-state blood concentration and area under the concentration-time curve were dose proportional across all dose levels; the median terminal elimination half-life was 8.6 hours (range: 8.1-11.1 hours). Leukocytosis was observed in all patients, with an estimated maximum effect of 3.4 times baseline (95% confidence interval: 2.9-3.9). Only 1 patient, the patient whose virus was confirmed to use purely CXCR4 and who also received the highest dose (160 microg/kg/h), had a significant 0.9-log10 copies/mL HIV RNA drop at day 11. Overall, however, the average change in viral load across all patients was +0.03 log10 HIV RNA. Given these results, AMD3100 is not being further developed for ARV therapy, but development continues for stem cell mobilization.

Purpose: Interactions between the chemokine receptor CXCR4 and its ligand stromal derived factor-1 regulate hematopoietic stem-cell trafficking. AMD3100 is a CXCR4 antagonist that induces rapid mobilization of CD34+ cells in healthy volunteers. We performed a phase I study assessing the safety and clinical effects of AMD3100 in patients with multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL)., Patients and Methods: Thirteen patients (MM, n=7; NHL, n=6) received AMD3100 at a dose of either 160 microg/kg (n=6) or 240 microg/kg (n=7). WBC and peripheral blood (PB) CD34+ cell counts were analyzed at 4 and 6 hours following injection., Results: AMD3100 caused a rapid and statistically significant increase in the total WBC and PB CD34+ counts at both 4 and 6 hours following a single injection. The absolute CD34+ cell count increased from a baseline of 2.6 +/- 0.7/microL (mean +/- SE) to 15.6 +/- 3.9/microL and 16.2 +/- 4.3/microL at 4 hours (P=.002) and 6 hours after injection (P =.003), respectively. The absolute CD34+ cell counts observed at 4 and 6 hours following AMD3100 were higher in the 240 microg/kg group (19.3 +/- 6.9/microL and 20.4 +/- 7.6/microL, respectively) compared with the 160 microg/kg group (11.3 +/- 2.7/microL and 11.3 +/- 2.5/microL, respectively). The drug was well tolerated and only grade 1 toxicities were encountered., Conclusion: AMD3100 appears to be a safe and effective agent for the rapid mobilization of CD34+ cells in patients who have received prior chemotherapy. Further studies in combination with granulocyte colony-stimulating factor in patients with lymphoid malignancies are warranted.