Diane E. Hasz

Interleukin‐2 liposome inhalation therapy is safe and effective for dogs with spontaneous pulmonary metastases

Systemic in vivo toxicity of interleukin‐2 (IL‐2) has been problematic. Antineoplastic activity of IL‐2 has been modest. The authors have previously demonstrated the biologic activity and safety of aerosols of IL‐2 liposomes in normal dogs. They now report objective regression of naturally occurring pulmonary metastases in dogs after 1 month of nebulized IL‐2 liposome therapy.

Cytokines in liposomes: Preliminary studies with IL-1, IL-2, IL-6, GM-CSF and interferon-γ

A simple, generally applicable method to incorporate cytokine proteins into multilamellar liposomes is presented. A variety of human cytokines including granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins 1 alpha, 2 and 6 (IL-1 alpha, IL-2, IL-6) and interferon-gamma (IFN-gamma) were incorporated into liposomes containing a single saturated synthetic lipid, dimyristoyl phosphatidyl choline (DMPC). Sterile cytokine liposomes were produced by gamma irradiation of DMPC lipid powder prior to use in cytokine liposome synthesis. A highly sensitive and reliable fluorescamine assay to detect microgram quantities of cytokine protein associated with liposomes is also described. When a high lipid:aqueous ratio [e.g. 300 mg DMPC lipid:1.0 ml aqueous cytokine solution] was utilized, aqueous cytokines (1 mg/ml) could be incorporated with efficiencies ranging from 19% (IL-1) to > 80% (IL-2). Combinations of cytokines (e.g. IL-2 + GM-CSF) were also co-incorporated into liposomes. Experiments with IL-2, IL-6, and GM-CSF demonstrated that these cytokines remain stably associated with the DMPC lipid and do not significantly leak from liposomes when stored at 4 degrees C for at least 3 months. Washing IL-6 liposome or GM-CSF liposome preparations reliably increased the proportion of cytokine protein associated with the liposome pellet compared to free cytokine in the supernatant of centrifuged specimens. For example the proportion of GM-CSF associated with the lipid carrier increased from 34.8% (SD 2.6%) in the original preparation to 98.0% (SD 0.6%) after three washes. Differences in the pharmacokinetics of subcutaneous (sc) free GM-CSF and GM-CSF liposomes (14 mcg/mouse) were studied in BALB/c mice. Both free GM-CSF and free GM-CSF mixed with saline loaded liposomes exhibited biphasic pharmacokinetics with very high peak levels 1 and 2 h after sc injection of 14 mcg the rapid decline to very low levels after 24 h. In contrast, sc GM-CSF liposomes provided sustained and stable levels of cytokine in the serum (approximately 100 pg/ml) for 24 h. Intraperitoneal injection of GM-CSF liposomes had > 10-fold more cytokine in the peritoneal wash than free GM-CSF mixed with saline loaded liposomes. In summary, the liposome synthesis procedure described is simple and utilizes a single synthetic lipid to reliably produce sterile cytokine preparations with in vivo depot effects after either sc or ip administration. Furthermore, the method is feasible for quantities of sterile cytokine liposomes sufficient for in vivo experiments.

Depot characteristics and biodistribution of interleukin-2 liposomes: Importance of route of administration

Due to rapid clearance of interleukin-2 (IL-2), it has had limited effective use as an in vivo immunostimulant. Current experimental and clinical protocols generally must utilize large doses, multiple injections, or continuous infusions of IL-2 in order to achieve significant immunostimulation, often at the expense of systemic toxicity. Therefore, the pharmacodynamics of IL-2 liposomes were investigated. IL-2 liposome incorporation efficiency was 80.4% (SD 5.5); vesicle diameter was 1.65 microns (SD 0.09) as determined by fluorescence-activated cell sorting (FACS). Both formulation (free cytokine vs. IL-2 liposomes) and route of administration were important variables in determination of the biodistribution and pharmacokinetic characteristics of IL-2. When free [125I]IL-2 was given i.v. to mice, only 6.5% was in the blood and 3% in liver and spleen 2 h after injection; on the other hand, at 2 h greater than 70% of i.v. [125I]IL-2 liposomes were detected in the blood, liver, spleen, and lungs. Mean i.v. elimination t1/2 from the blood of rats given 20 x 10(6) U/kg free cytokine or IL-2 liposomes was 41 versus 102 min, respectively, as measured by bioassay and 59 and 119 min as measured by enzyme immunoassay (EIA). After i.v. administration, the estimated Vd of IL-2 liposomes was 13-fold smaller than the free cytokine. Intrathoracic (i.tx.), i.p., and s.c. administration of [125I]IL-2 to mice also demonstrated significant depot effects when IL-2 was incorporated into liposomes. These data suggest IL-2 liposomes may provide in vivo immunostimulation superior to the free cytokine due to biodistribution and depot characteristics.

Antitumor mechanisms of attenuated Salmonella typhimurium containing the gene for human interleukin-2: A novel antitumor agent?☆☆☆

Currently, there is no long-term effective treatment for unresectable hepatic malignancies. Salmonella species are known to naturally track to the liver during active infection. To develop a biological vector for delivery of interleukin-2 (IL-2) to the liver for antitumor purposes, the thi 4550 attenuated strain of Salmonella typhimurium was used as a vector for IL-2. The gene for human IL-2 was cloned into plasmid pYA292 and inserted into the attenuated S typhimurium and renamed (thi 4550(pIL-2)]. MCA-38 murine adenocarcinoma cells were injected intrasplenically into C57BL/6 mice to produce hepatic metastases that were subsequently enumerated after 12 days. We previously have demonstrated that the thi 4550(pIL-2) produces biologically active IL-2 and that a single gavage feeding of 10(7) thi 4550(pIL-2) significantly reduced the number of hepatic metastases when compared with animals fed salmonella lacking the IL-2 gene or nontreated controls. The aims of the current studies were to determine which host effector cell populations were responsible for the antitumor effect seen with thi 4550(pIL-2) by depletion of natural killer (NK), cytotoxic T lymphocytes (CD8+), T helper (CD4+) cells, and Kupffer cells. Multiple experiments were conducted for each host effector cell population depleted. We found a consistent reduction in the mean number of hepatic metastases in animals fed thi 4550(pIL-2) (55.6 metastases; n = 54) when compared with controls (162.3 metastases; n = 53) (P < .0001). Depletion of NK cells and CD8+ T cells significantly inhibited the antitumor effect of thi 4550(pIL-2) (analysis of variance [ANOVA], P < .01). Elimination of CD4+ T cells and Kupffer cells had no significant impact on the antitumor effect of thi 4550(pIL-2) (ANOVA, P value was not significant). Salmonella IL-2 may represent a novel form of in vivo biotherapy for unresectable hepatic malignancies that employs the oral route of administration. Furthermore, both NK cells or CD8+ cells are required for the antitumor effect seen while CD4+ T cells and Kupffer cells do not appear to be as essential.

Nebulized Interleukin 2 Liposomes: Aerosol Characteristics and Biodistribution

Although interleukin 2 (IL‐2) has been associated with modest anti‐tumour responses in man, treatment‐related toxicity has limited its widespread use. The local delivery of liposomal formulations of interleukin 2 to the lung as aerosols has been demonstrated to be non‐toxic, biologically active, and associated with regression of spontaneous pulmonary metastases in dogs. This study was undertaken to evaluate the physical and biological characteristics of nebulized interleukin 2 liposomes.

Nf1 gene inactivation in acute myeloid leukemia cells confers cytarabine resistance through MAPK and mTOR pathways

Nf1 gene inactivation in acute myeloid leukemia cells confers cytarabine resistance through MAPK and mTOR pathways

Stat5 is critical for the development and maintenance of myeloproliferative neoplasm initiated by Nf1 deficiency

Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients.

Nf1 mutant mice with p19ARF gene loss develop accelerated hematopoietic disease resembling acute leukemia with a variable phenotype

Juvenile Myelomonocytic Leukemia (JMML) is a relentlessly progressive myeloproliferative/myelodysplastic (MPD/MDS) hematopoietic disorder more common in patients with any one of at least three distinct genetic lesions, specifically NF1 gene loss and PTPN11 and NRAS mutations. NF1 and PTPN11 are molecular lesions associated with Neurofibromatosis Syndrome Type I (NF1 Syndrome) and Noonans Syndrome, respectively. The occurrence of JMML is rare; even among those predisposed with these syndromes to development of disease, and secondary genetic events likely contribute to the development and progression of disease. In NF1 syndrome, loss of p53 function is a common event in solid tumors, but uncommon in JMML, suggesting that the p53 pathway may be modified by other events in this hematopoietic disorder. The work presented here investigates the possible role of the p19Arf (p19) tumor suppressor in development of MPD associated with Nf1 gene loss in mice. We find that Nf1 mutant hematopoietic cells with loss of p19 develop accelerated hematopoietic disease similar to acute leukemia with a variable phenotype. This suggests that p19 may play a role in development of JMML and evaluation of the human p19 homolog (p14ARF) in JMML may be informative. Am. J. Hematol. 86:579–585, 2011.

Erratum: A microarray study of altered gene expression in Ara-C resistance in acute myeloid leukemia (Leukemia (2007) 21 (1093-1097))

A microarray study of altered gene expression in Ara-C resistance in acute myeloid leukemia

A microarray study of altered gene expression in Ara-C resistance in acute myeloid leukemia

A microarray study of altered gene expression in Ara-C resistance in acute myeloid leukemia