ALBERT

Description: Introduction: Long-term red blood cell transfusions effectively sustains patients who have β-thalassemia, sickle cell anemia, and myelodysplastic syndromes but they also lead to excess iron accumulation in the body. Iron overload is a major cause of morbidity and mortality in transfusion dependent patients. Chelation therapy reverses iron accumulation but marketed chelators have drawbacks such as: long infusions of deferoxamine (DFO, Novartis), large oral tablets with adverse effects (Exjade, Novartis), or twice daily oral dosing (Ferriprox, ApoPharma). These attributes contribute to poor compliance and poor outcomes in iron overload patients. To overcome long infusions and high doses of current therapies we have devised a stable nanoliposome encapsulated DFO (LDFO) for the treatment of iron overload. Methods: LDFO composed of saturated soy phosphatidylcholine and cholesterol (3/2 molar ratio) is manufactured using a proprietary remote loading method that provides high encapsulation of DFO in 90 nm diameter liposomes. For pharmacokinetics and bioavailability studies, DFO and lipid concentrations in CF-1 mice plasma and tissues were analyzed by HPLC utilizing an in-house method. For iron removal efficacy studies, CF-1 mice were overloaded with iron dextran and after 10 days washout were treated with 100 mg/kg LDFO or unencapsulated DFO. Animals were sacrificed 5 days post treatment and tissue iron was measured by a ferrozine based spectroscopic assay. Results: The manufacturing method to prepare LDFO results in a 300 g DFO/mole lipid encapsulation ratio. The formulation has greater than 6 months stability at 4 ºC. LDFO is long circulating and the DFO is bioavailable. At 24 hr post I.V. injection, there is 30% ID DFO in plasma and 10% ID DFO/g in liver whereas unencapsulated DFO is not detectable. Preclinical single dose safety studies in CF-1 mice indicate that LDFO is well tolerated at 300 mg/kg I.V. and 1250 mg/kg I.P. In the iron dextran overload model, LDFO greatly reduces iron levels in the liver and spleen. The absolute efficiency of LDFO is greater than 50% on a mole LDFO injected /mole iron removed from the liver (P

Description: Introduction: We describe a low heat nitric acid digestion and colorimetric ferrozine-based iron assay that provides a fast, inexpensive and accurate alternative to high temperature animal tissue processing and inductively coupled plasma mass spectrometry (ICP). This technique is useful for the quantification of iron in iron overloaded animal models and diseases such as β-thalassemia, sickle cell anemia, and myelodysplastic syndromes. We applied it to evaluate iron removal by iron chelation therapies such as liposome-encapsulated deferoxamine (LDFO). Methods: CF-1 mouse liver, spleen, heart, plasma, urine and feces were digested in nitric acid (70%) at 65 °C for 1-2 hours. For large tissues such as the liver, tissues were sectioned at 50 mg fractions (n = 4) to also assess iron homogeneousness. Digested samples were bleached with hydrogen peroxide (30%) and diluted with water before iron quantification by ICP or the ferrozine-based assay. For the ferrozine-based assay, nitric acid was neutralized with ammonium acetate and iron was reduced with ascorbic acid before reaction with ferrozine for the colorimetric assessment of the ferrozine-ferrous iron complex at 550 nm. For iron overloaded models, CF-1 mice (n = 4) were loaded I.V. with iron dextran, iron sucrose, liposome encapsulated iron, or horse ferritin for over a week before sacrifice. Iron removal studies of tissues and excreta from iron overloaded CF-1 (n = 4) started 2 weeks after iron dextran overloading. Animals were dosed with either deferoxamine (DFO) and LDFO by I.V. and Exjade by oral gavage. Urine and feces were collected daily at the start of treatment. Animals were sacrificed 7 days post treatment. Tissues and excrements were digested and measured for iron by the ferrozine-based assay. Results: The ferrozine-based tissue iron quantification assay yields high iron recovery from mouse tissue. CF-1 liver spiked with iron dextran or horse ferritin had an iron recovery of 99±2% and 97±2%, respectively. Liver iron measurements of iron dextran overloaded mouse models resulted in identical iron measurements for the ferrozine-based assay and ICP. For CF-1 mice treated with iron dextran I.V. (0, 100, 300, 600 mg/kg, n = 4), liver iron is highly correlative between the two iron quantification techniques with a slope of 1.03 and R2 of 0.999. In addition, there is a linear iron overloading effect in both the liver (R2 = 0.99) and spleen (R2 = 0.98). CF-1 mice were also iron overloaded with iron sucrose, liposome encapsulated iron, and horse ferritin with dose dependent tissue overload. For all iron overloaded models tested, liver iron and spleen iron were homogenous per tissue when multiple sections were analyzed with an average low 6% difference in iron content. Despite high liver and spleen overloading, none of these iron carriers resulted in statistically significant heart iron overload. Iron dextran overloaded CF-1 mice (100 mg/kg) were treated with LDFO, DFO, and Exjade. LDFO at 100 mg/kg I.V. greatly reduces iron levels in the liver (p = 0.019) and spleen (p = 0.014) compared to non-effective no treatment, free DFO (p = 0.3), and empty liposomes (p = 0.1). Exjade at 30 mg/kg by oral gavage did not result in statistically significant iron removal in the liver or spleen (p 〈 0.2). Over the first four days, urine and feces were collected daily and also analyzed for iron. Results revealed that iron clearance by LDFO is primarily in the urine (p = 0.022 urine; p = 0.8 feces) while Exjade removed iron appeared in the feces (p = 0.06 feces; p = 0.013 urine). During this short period, drug efficiency in iron excretion (5%) from one dose of the novel LDFO at 100 mg/kg was equivalent to four daily doses of the Exjade at 50 mg/kg/dose. Conclusion: The low heat nitric acid digestion and ferrozine-based tissue iron quantification assay is a simple, precise, highly reproducible tool for the assessment of tissue and excretion iron. The assay enabled the rapid, low cost evaluation of novel iron chelation therapies. We gratefully acknowledge support by NIH SBIR Phase 1 Grant 1R43HD075429-01 and NIH SBIR Phase 2 Grant 2R44HD075429-02. Disclosures Tran: Zoneone Pharma, Inc.: Employment. Petersen:Zoneone Pharma, Inc.: Employment. Noble:Zoneone Pharma, Inc.: Employment, Equity Ownership. Hayes:Zoneone Pharma, Inc.: Employment, Equity Ownership. Working:Zoneone Pharma, Inc.: Consultancy, Equity Ownership. Szoka:Zoneone Pharma, Inc.: Consultancy, Equity Ownership.

Description: Introduction: Patients who have β-thalassemia, sickle cell anemia, and myelodysplastic syndromes are sustained by long-term blood transfusion therapy. Transfusion therapy results in iron accumulation initially in the liver, spleen and bone marrow. These are the organs responsible for eliminating apoptotic red blood cells. To prevent iron overload, iron must be removed using iron chelators, administered either orally or infused on a daily basis. One strategy to more effectively deliver chelators to the sites of iron accumulation is to encapsulate them in liposomes. Liposomes also accumulate in the liver, spleen and bone marrow; therefore LDFO targets the chelator directly to the iron stores. We tested the hypothesis that the LDFO approach provides highly efficient (moles chelator administered/moles iron removed) iron chelation. If this hypothesis is supported by the data, lower amounts of chelator could be administered to patients, on a less frequent dosing schedule. This would enable LDFO to be used alone or as part of a combination chelation regime to minimize the need for high daily chelator doses. Methods: We prepared two LDFO formulations composed of (hydrogenated soy phosphatidylcholine, HSPC)/cholesterol (60/40) or (palmitoyloleoylphosphatidylcholine, POPC)/cholesterol (55/45). Animal protocols were approved by the institutional review board. We determined the pharmacokinetics of the encapsulated deferoxamine (DFO) in CF-1 female mice by analyzing serum plasma DFO concentrations by HPLC, after dosing LDFO. CF-1 female mice were overloaded with iron by administering IV, hydrogenated iron dextran 100 mg/kg four times, at three day intervals. Twenty-one days after the last iron dose, mice (n=10) were administered the first dose of the LDFO. The LDFO formulations were administered IV three times at 200 mg/kg at 14-day intervals. A control group (n=10) of non-encapsulated DFO was administered by SC infusion from an implantable minipump at 20 mg/kg/day for a total dose of 280 mg/kg DFO over 14 days. Saline controls were dosed on the same schedule as LDFO. Every 24 h, five mice from each group were alternated from the regular cages into metabolic cages and urine and feces collected. The iron concentration in urine and feces was measured by a modified ferrozine-based spectroscopic assay. At the study end, blood was drawn form the mice and standard blood substances were analyzed as an indicator of organ function. Results: The HSPC/cholesterol and POPC/cholesterol LDFO liposomes had 88 nm and 119 nm diameters and encapsulated 354 and 266 g DFO/mole phospholipid respectively. At 6 and 24 h post IV injection, there is 67.0% and 27.2% ID DFO in plasma for HSPC liposomes and 54.2% and 18.1% ID DFO in plasma for POPC liposomes. In treating iron overloaded mice, IV administered LDFO removed 2.3-2.8 times more iron than deferoxamine mesylate (DFO) given by SC continuous infusion. After LDFO treatment, iron was continuously eliminated for 14 days post dosing. At day 14 LDFO-HSPC and LDFO-POPC cumulatively had a 3.1 and 3.5-fold higher iron elimination in urine compared to SC infused DFO and 1.8 and 1.3-fold higher in feces respectively, corrected for background iron using the saline control group. The second and third dose of LDFO at 14-day intervals showed similar iron elimination patterns to the first dose. The iron removal efficiencies were 68% for LDFO-HSPC, 55% for LDFO-POPC and 24% for Free DFO. The liposome composition shifted the relative iron elimination profiles within the liposome groups. Of the two formulations, LDFO-HSPC produced more fecal iron removal while the LDFO-POPC group gave higher urinary iron removal. The iron elimination curves for both liposome formulations were statistically different than the infused DFO curve (p

Description: ▪ Abstract  Acetylation is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways. Although histones are the most thoroughly studied of acetylated protein substrates, histone acetyltransferases (HATs) and deacetylases (HDACs) are also responsible for modifying the activity of diverse types of nonhistone proteins, including transcription factors and signal transduction mediators. HDACs have emerged as uncredentialed molecular targets for the development of enzymatic inhibitors to treat human cancer, and six structurally distinct drug classes have been identified with in vivo bioavailability and intracellular capability to inhibit many of the known mammalian members representing the two general types of NAD+-independent yeast HDACs, Rpd3 (HDACs 1, 2, 3, 8) and Hda1 (HDACs 4, 5, 6, 7, 9a, 9b, 10). Initial clinical trials indicate that HDAC inhibitors from several different structural classes are very well tolerated and exhibit clinical activity against a variety of human malignancies; however, the molecular basis for their anticancer selectivity remains largely unknown. HDAC inhibitors have also shown preclinical promise when combined with other therapeutic agents, and innovative drug delivery strategies, including liposome encapsulation, may further enhance their clinical development and anticancer potential. An improved understanding of the mechanistic role of specific HDACs in human tumorigenesis, as well as the identification of more specific HDAC inhibitors, will likely accelerate the clinical development and broaden the future scope and utility of HDAC inhibitors for cancer treatment.