Katherine P Hoyes

Subcellular distribution of desferrioxamine and hydroxypyridin-4-one chelators in K562 cells affects chelation of intracellular iron pools

Summary The interactions of iron chelators with intracellular iron pools have been examined by measuring the subcellular distribution of radiolabelled desferrioxamine (DFO) and the orally active hydroxypyridinone (HPO) chelator 1, 2‐diethyl‐3‐hydroxypyridin‐4‐one (CP94), as well as the ability of these chelators to modify the subcellular distribution of 59Fe delivered by the receptor mediated endocytosis of transferrin. K562 cells were pulsed with 59Fe transferrin and challenged with DFO or CP94 (100 μm IBE) for 20 or 240 min and then subjected to subcellular fractionation. At 20 min there was a significant decrease (P <0.45) in both lysosomal/particulate 59Fe (75% of control) and cytosolic 59Fe ferritin (50% of control) in cells incubated with CP94, unlike cells treated with DFO where no decrease was observed. By 240 min, in addition to the above, 59Fe accumulation was significantly decreased in the nuclear, mitochondrial, and low molecular weight cytosolic fractions with CP94 (P < 0.05). With DFO a significant decrease in 59Fe in only the lysosomal/particulate and cytosolic ferritin compartments was observed at 240 min (P <045). At this time, however, there was a significant accumulation of both cytosolic low molecular weight 59Fe and cytosolic DFO. The relatively rapid decrease of 59Fe within intracellular compartments seen with CP94 compared to DFO was paralleled by a significantly higher accumulation of CP94 than DFO in nuclear, lysosomal/particulate and low molecular weight cytosolic compartments at 20 min (P <0 05). These results suggest that transferrin derived endosomal iron may be chelated by HPOs, unlike DFO, due to their faster uptake into these organelles. The more rapid access of HPOs than DFO to certain intracellular iron pools may explain the greater possibility of HPOs to inhibit proliferation of cells in vivo.

Contrasting interspecies efficacy and toxicology of 1, 2 ‐diethy 1–3 ‐hydroxypyridin‐4‐one, CP9 4, relates to differing metabolism of the iron chelating site

Summary. In order to define a predictive animal model for the effects of hydroxypyridinone (HPO) iron chelators in humans, we have compared the 28 d oral efficacy and toxicology of the HPO, 1,2‐diethyI‐3–hydroxypyridin‐4–one (CP94) in rats and guinea‐pigs and related the results to the contrasting metabolism of this compound in the two species. CP94 was highly effective at mobilizing liver iron in rats but showed toxicity at higher doses, whereas in the guinea‐pig the compound lacked toxicity but was ineffective at mobilizing liver iron. These differences can be explained by the contrasting metabolism of the drug between the two species. In rats, at the top dose of 300 mg/kg intragastrically, all animals died before the end of the study, with no deaths or weight loss at lower doses. At 100 mg/kg, rat liver non‐haem iron concentrations were reduced by 53% and 44% in females and males respectively (P<0.001). At this dose, adrenal medullary cell vacuolation, increased mammary secretory activity, vacuolation of corpora luteal cells and single cell hepatocyte necrosis were seen. There were no reductions in the white cell count. At 50 mg/kg rat liver non‐haem iron concentrations were decreased by 50% and 34% in females and males respectively (P<0.02). In female rats this was associated with increased mammary secretory activity. In iron‐overloaded rats given 100 mg/kg by gavage for 28 d, liver non‐haem iron concentration was reduced by 39% (P<0.01) and serum ferritin by 71% (P<0.001). Ovarian and mammary changes were not influenced by iron loading. In guinea‐pigs, CP94 was evaluated at 50 mg/kg, 100 mg/kg or 200 mg/kg by oral insufflation for 28 d. No reduction in liver iron was seen and no systematic dose related histological, biochemical or haematological effects were observed. Whereas in guinea‐pigs 99% of urinary recovery following an oral dose of CP94 (100 mg/kg) was as the inactive glucuronide metabolite, in the rat only 23% of the dose was excreted in the urine as the glucuronide with remainder as the free drug or an iron binding metabolite. The lack of both efficacy and toxicity in the guinea‐pig may therefore be explained by the rapid inactivation of CP94 by glucuronidation. This metabolism of CP94 in the guinea‐pig is closer to humans than the rat, suggesting that both the efficacy and toxicity of this compound in humans may also be limited by glucuronidation.

Lessons from Preclinical and Clinical Studies with 1,2-Diethyl-3-Hydroxypyridin-4-One, CP94 and Related Compounds

In clinical use, the therapeutic safety margin with any iron chelator is likely to be narrow compared with commonly used pharmaceuticals. This is because there is inevitably a fine balance between excess iron and iron depletion within cells. This safety margin may be increased in the presence of iron overload but as not all cells will be equally iron overloaded, some will be more susceptible to iron deprivation than others. It is likely that minor modifications to chelator structure will make important differences to the therapeutic safety margin in clinical practice. The approach of our group has therefore been to identify the principles determining the efficacy and/or toxicity of iron chelators so that compounds which can be effective at lower doses or have less toxicity, than for example L1, can be identified. The hydroxypyridin-4-ones (HPOs) (Hider et al, 1982) are an appropriate group of compounds for such studies as their relatively simple structure allows a systematic investigation of structure-function relationships. The problems in identifying models which will predict the behaviour of chelators in iron overloaded humans has not helped in the development of iron chelators; indeed the preclinical and clinical experience with the HPOs has highlighted these shortcomings.

Secretion of neutrophil secondary granules occurs during granulocyte‐macrophage colony stimulating factor induced margination

The effects of recombinant human granulocytemacrophage colony‐stimulating factor (rhGM‐CSF) on neutrophil lactoferrin (LF) and transcobalamin (TC) 1 and 3 secretion were determined in vitro and during in vivo administration in humans. In whole blood, in vitro incubation with GM‐CSF reproducibly produced a rise in plasma LF concentration (P<0.05) whereas in purified neutrophils the results were variable. Exposure of whole blood to GM‐CSF also resulted in a significant rise in plasma TC 1 and 3 (190.60%, P<0.05). The response was dose dependent with maximal effect at GM‐CSF concentrations of 10 ng/ml and above. rhGM‐CSF was administered on seven occasions to six patients with malignant disease prior to chemotherapy. Plasma LF and unsaturated TC 1 and 3 levels rose significantly in each patient studied and the rise coincided with the initial neutropenia due to margination that occurs during infusions of rhGM‐CSF. Patients receiving rhGM‐CSF may therefore have hypofunctional neutrophils due to secondary granule depletion.

Effect of bcl-2 deficiency on the radiation response of clonogenic cells in small and large intestine, bone marrow and testis

PURPOSE Overexpression of bcl-2 protects against radiation induced apoptosis in lymphohaematopoietic cell types in vivo, whilst bcl-2 deficiency radiosensitizes murine T-lymphocytes in vitro. However, there are few data regarding the influence of bcl-2 deficiency on the radiosensitivity of non-lymphoid cell types. The purpose of this study was to investigate the role of bcl-2 in the clonogenic radiation response of intestinal crypts, bone marrow progenitor cells and testicular stem cells. METHOD Survival curves were obtained for each cell type from bcl-2 null (-/-), heterozygote (+/-) and wild type (+/+) mice. Crypt survival in the small and large intestine was assessed using the crypt microcolony assay. Committed haemopoietic progenitors were assayed using in vitro colony-forming cell (CFC) assays and survival of clonogenic spermatogonia was assessed by scoring regenerative tubules at 35 days post-irradiation. RESULTS There was no difference in small intestine crypt survival between the three genotypes. In the colon, there was a tendency towards lower clonogen survival in the +/- and -/- animals. Haemopoietic in vitro CFC from -/- animals showed lower survival in comparison to +/+ mice, but spermatogonial stem cells were comparatively more radioresistant. CONCLUSIONS Deficiencies in bcl-2 affect the radiation response of different cell populations in small but different ways. This may be due to variations between cells in their innate capacity for apoptosis, their dependence on different members of the bcl-2 family gene and their cell-cycle status and p53 expression.Purpose : Overexpression of bcl -2 protects against radiation induced apoptosis in lymphohaematopoietic cell types in vivo, whilst bcl -2 deficiency radiosensitizes murine T-lymphocytes in vitro. However, there are few data regarding the influence of bcl-2 deficiency on the radiosensitivity of non-lymphoid cell types. The purpose of this study was to investigate the role of bcl-2 in the clonogenic radiation response of intestinal crypts, bone marrow progenitor cells and testicular stem cells. Method : Survival curves were obtained for each cell type from bcl -2 null (-/-), heterozygote (+/-) and wild type (+/+) mice. Crypt survival in the small and large intestine was assessed using the crypt microcolony assay. Committed haemopoietic progenitors were assayed using in vitro colony-forming cell (CFC) assays and survival of clonogenic spermatogonia was assessed by scoring regenerative tubules at 35 days post-irradiation. Results : There was no difference in small intestine crypt survival between the three genotypes. In the colon, there was a tendency towards lower clonogen survival in the +/- and -/- animals. Haemopoietic in vitro CFC from -/- animals showed lower survival in comparison to +/+ mice, but spermatogonial stem cells were comparatively more radioresistant. Conclusions : Deficiencies in bcl -2 a ffect the radiation response of different cell populations in small but different ways. This may be due to variations between cells in their innate capacity for apoptosis, their dependence on different members of the bcl -2 family gene and their cell-cycle status and p53 expression.

Transferrin-mediated uptake of plutonium by spermatogenic tubules

Using isolated rat seminiferous tubules as an in vitro model, we have found that 238Pu can cross the blood-tubule barrier and accumulate within tubules in a time dependent manner. Furthermore, similar to 59Fe, tubule 238Pu uptake was inhibited by the addition of excess transferrin, suggesting that plutonium may utilize the physiological iron-transferrin pathway to cross the blood-tubule barrier. However unlike 59Fe, 238Pu was only transiently associated with the tubules, suggesting differences in the intracellular processing of these radionuclides. The assumptions made in the estimation of doses to the human testis from incorporated plutonium are considered.

Modification of murine adult haemopoiesis and response to methyl nitrosourea following exposure to radiation at different developmental stages.

PURPOSE To investigate the hypothesis that the developmental phase at which an individual encounters radiation damage affects its long-term sensitivity to a subsequent tumourigenic insult. MATERIALS AND METHODS Either the pregnant C57B16 mouse was exposed to 137Cs gamma-rays at 4 or 15 days post-conception (embryonic and foetal stages respectively) or BDF1 offspring were irradiated at 4 or 21 days of age (neonatal and juvenile stages). Offspring were either assayed for changes in bone marrow stem cells and committed progenitors at 6, 12 and 18 weeks of age, or injected with the chemical carcinogen methyl nitrosourea (MNU) at 10 weeks of age and monitored for onset of neoplasia. RESULTS Gamma-irradiation induced a persistent long-term deficit in stem cells in all irradiated animals, with the foetal stage appearing most radiosensitive. However, femoral cellularity, committed progenitor cell numbers and peripheral blood counts were unaffected. When offspring were exposed to MNU, the incidence of malignancy was significantly enhanced in animals irradiated at the foetal, neonatal and juvenile stages. CONCLUSIONS This study has shown that exposure to ionizing radiation at the foetal, neonate or juvenile stages of development induces residual haemopoietic damage and increases oncogenic susceptibility to a subsequent exposure to MNU.Purpose: To investigate the hypothesis that the developmental phase at which an individual encounters radiation damage affects its long-term sensitivity to a subsequent tumourigenic insult. Materials and methods: Either the pregnant C57Bl6 mouse was exposed to 137Cs gamma-rays at 4 or 15 days post-conception (embryonic and foetal stages respectively) or BDF1 offspring were irradiated at 4 or 21 days of age (neonatal and juvenile stages). Offspring were either assayed for changes in bone marrow stem cells and committed progenitors at 6, 12 and 18 weeks of age, or injected with the chemical carcinogen methyl nitrosourea (MNU) at 10 weeks of age and monitored for onset of neoplasia. Results: gamma-Irradiation induced a persistent long-term deficit in stem cells in all irradiated animals, with the foetal stage appearing most radiosensitive. However, femoral cellularity, committed progenitor cell numbers and peripheral blood counts were unaffected. When offspring were exposed to MNU, the incidence of malignancy was significantly enhanced in animals irradiated at the foetal, neonatal and juvenile stages. Conclusions: This study has shown that exposure to ionizing radiation at the foetal, neonate or juvenile stages of development induces residual haemopoietic damage and increases oncogenic susceptibility to a subsequent exposure to MNU.

Autologous lymphocytes as vectors to target therapeutic radiation, using indium‐114m, in patients with lymphoid cell malignancy

Summary. Autologous lymphocytes provide a potential vector for the delivery of a cytotoxic agent in patients with lymphoid cell malignancy. This report describes a phase I–II study using autologous lymphocytes to target the radionuclide indium‐114m (114mIn) in patients with refractory chronic lymphocytic leukaemia or small lymphocytic non‐Hodgkins lymphoma. Nineteen patients, the majority of whom had been heavily pretreated with conventional chemotherapy and radiotherapy, received between 69 and 211 MBq 114mIn‐labelled autologous lymphocytes. Approximately 80% of the administered activity was localized in the liver and spleen, with around 5% accumulating in the bone marrow. Ten patients (53%) responded (one complete response and nine partial responses). The median duration of response was 7 months. The median survival for the responders was 14 months and for the non‐responders was 3 months. The first notable response in every patient was a fall in peripheral lymphocyte count. The indium treatment was not associated with any subjective toxicity, although all patients suffered from myelosuppression, with thrombocytopenia being the dose‐limiting factor. This study has demonstrated a significant anti‐tumour effect in a group of patients with late‐stage highly resistant disease.