Phase I/II multisite trial of optimally dosed clofarabine and low-dose TBI for hematopoietic cell transplantation in acute myeloid leukemia

Clofarabine is an immunosuppressive purine nucleoside analog that may have better anti-leukemic activity than fludarabine. We performed a prospective phase I/II multisite trial of clofarabine with 2 Gy total body irradiation as non-myeloablative conditioning for allogeneic hematopoietic cell transplantation in adults with acute myeloid leukemia who were unfit for more intense regimens. Our main objective was to improve the 6-month relapse rate following non-myeloablative conditioning, while maintaining historic rates of non-relapse mortality (NRM) and engraftment. Forty-four patients, 53 to 74 (median: 69) years, were treated with clofarabine at 150 to 250 mg/m², of whom 36 were treated at the maximum protocol-specified dose. One patient developed multifactorial acute kidney injury and another developed multiorgan failure, but no other grade 3 to 5 non-hematologic toxicities were observed. All patients fully engrafted. The 6-month relapse rate was 16% (95% CI, 5%-27%) among all patients and 14% (95% CI, 3%-26%) among high-risk patients treated at the maximum dose, meeting the pre-specified primary efficacy endpoint. Overall survival was 55% (95% CI, 40%-70%) and leukemia-free survival was 52% (95% CI, 37%-67%) at 2 years. Compared to a historical high-risk cohort treated with the combination of fludarabine at 90 mg/m² and 2 Gy TBI, protocol patients treated with the clofarabine-TBI regimen had lower rates of overall mortality (HR of 0.50, 95% CI, 0.28-0.91), disease progression or death (HR 0.48, 95% CI, 0.27-0.85), and morphologic relapse (HR 0.30, 95% CI, 0.13-0.69), and comparable NRM (HR 0.85, 95% CI 0.36-2.00). The combination of clofarabine with TBI warrants further investigation in patients with high-risk AML.     

Vascular changes play a role in the pathogenesis of necrotizing enterocolitis in asphyxiated newborn pigs

Necrotizing enterocolitis (NEC) is the most common acquired gastrointestinal emergency in neonates. We have developed an animal model of NEC in asphyxiated newborn pigs and investigated the effects of asphyxia on blood flow in superior mesenteric artery and abdominal aorta, cardiovascular data, arterial acid-base and blood gas parameters, and endothelial cytoskeletal structure in mesenteric microvasculature. Anesthetized, mechanically ventilated newborn pigs were included in two groups: piglets underwent severe asphyxia, and sham-operated control animals. A cardiovascular and metabolic failure developed in asphyxiated piglets approximately 1 h after the induction: severe hypotension and bradyarrhythmia were seen and significant reductions of the blood flow were measured in the superior mesenteric artery and abdominal aorta during the critical phase. Rearrangement of cytoskeletal actin structure corresponding to enhanced vascular permeability was seen with bodipy phallacidin in mesenterial endothelium of asphyxiated piglets after a 24-h recovery period. In conclusion, severe vasomotor changes during asphyxia may result in mesenteric endothelial dysfunction implicated in increased vascular permeability, edema formation, and development of NEC in asphyxiated piglets.     

Effect of myelopoietic and pleiotropic cytokines on colony formation by blast cells of children with acute lymphoblastic leukemia

The aim of this study was to see whether pleiotropic or myeloid hematopoietic growth factors, which do not stimulate normal lymphoid cells, can induce proliferation of blast cells of the acute lymphoid leukemia (ALL) of childhood. Bone marrow cells of 13 children with untreated ALL (nine common ALL, two myeloid antigen positive ALL and two early T-cell ALL) formed colonies of leukemic blast cells in primary methylcellulose cultures. Spontaneous growth was observed in three of 13 cases, whereas phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM), a conventional source of various natural human cytokines, induced colony formation in ten of 13 cases. A similar rate of responsiveness was seen with recombinant human granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF); a combination of these three cytokines induced colony formation in all cases studied. The effect of these growth factors on colony formation seemed to be dose-dependent in some cases. Of the stimuli studied, GM-CSF induced the smallest number of colonies, whereas the effects of G-CSF, SCF and PHA-LCM were similar in this respect. Combination of cytokines proved to be even more efficient in inducing clonal proliferation of leukemic lymphoblasts. In double combinations, G-CSF and GM-CSF as well as G-CSF and SCF were able to potentiate each other's effects. Triple combination of these cytokines mediated the most potent growth stimulus. Our results demonstrate that myeloid and pleiotropic cytokines are able to stimulate clonal proliferation of pediatric leukemic lymphoblasts. This may present a potential hazard to children with ALL while on adjuvant therapy with hematopoietic growth factors. In vitro colony assays performed prior to or in parallel with the administration of hematopoietic growth factors to ALL patients may help to forecast their possible effects on leukemic cells in vivo.     

Effects of PACAP on Mitochondrial Apoptotic Pathways and Cytokine Expression in Rats Subjected to Renal Ischemia/Reperfusion

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly efficient cytoprotective actions. Its neuroprotective effects are well-known, but PACAP is able to exert similar actions in non-neuronal cells. Recently, we have shown that PACAP prolongs renal ischemic time, decreases mortality, and attenuates tubular degeneration in a rat model of renal ischemia/reperfusion, but the mechanism of renoprotection is not yet known. Therefore, the aim of the present study was to obtain further insight into the renoprotective effects of PACAP by examining its direct effects of PACAP on mitochondrial permeability transition in vitro and on the expression of the anti-apoptotic Bcl-2 and cytokines/chemokines in kidney tissues following 45 and 60 min renal ischemia and reperfusion in vivo. We found that PACAP did not have any direct effect on mitochondrial permeability transition. Cytokine array revealed that the expression of a few cytokines/chemokines was strongly increased after ischemia/reperfusion, which was ameliorated by PACAP treatment. Furthermore, in rats subjected to renal ischemia, PACAP treatment counteracted the ischemia/reperfusion-induced decrease of the anti-apoptotic Bcl-2, both after 45 and 60 min ischemia, as analyzed by Western blot. In summary, we showed that PACAP could attenuate tissue injury involving both anti-inflammatory and anti-apoptotic effects, but not directly acting on mitochondrial permeability transition.     

Pulmonary targeting of sustained release formulation of budesonide in neonatal rats

Systemic corticosteroids are widely used for the treatment/prevention of chronic lung disease (CLD) in premature infants. The use of the inhalation route for delivering corticosteroids has been widely recognized, however so far pre-term babies continue to be treated with oral glucocorticoids, such as dexamethasone. We hypothesize that the pulmonary administration of sustained release formulations of inhaled corticosteroids to pre-term infants will result in a higher benefit/risk ratio as compared to traditional inhalation therapy. To achieve a slow release formulation budesonide particles were coated with a very thin film of polylactic acid using a pulse laser ablation technique. Coated material was characterized with respect to the dissolution behavior and particle size. Ex vivo receptor binding studies were performed to monitor the cumulative lung, liver and brain receptor occupancies after adminstration in neonatal (10-11 days old) rats after intratracheal instillation of either uncoated budesonide or poly (l-lactic acid) (PLA) coated budesonide. The mean dissolution timed for the uncoated and the polymer coated formulations were 1.2 +/- 0.5 and 4.7 +/- 0.1 h, respectively (p < 0.05). No significant differences in the respirable fraction were found between coated and uncoated formulation (p>0.05). The average receptor occupancies in the lung, liver and brain after administration of uncoated budesonide were 58.4 +/- 12.9, 56.4 +/- 6.8 and 38.3 +/- 6.7%, respectively. However, after administration of PLA coated budesonide, the average AUC estimates in the lung, liver and brain were 75.8 +/- 3.7%, 46.6 +/- 14.5 and 29 +/- 7, respectively. The results from our study suggest sustained receptor occupancy in the lungs of neonatal rats after administration of PLA coated budesonide results in lower systemic exposure (as indicated by low liver receptor occupancy). The data strongly underscore the urgent need to develop sustained release pulmonary-targeted delivery systems of corticosteroids for the treatment of CLD in pre-term infants. The administration of inhaled corticosteroids using targeted drug-delivery systems will potentially result in higher local effects and a reduction in systemic exposure.