Pharmacokinetics of the hypoxic cell cytotoxic agent tirapazamine and its major bioreductive metabolites in mice and humans: retrospective analysis of a pharmacokinetically guided dose-escalation strategy in a phase I trial

 Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; SR 259075) is a selective hypoxic cell cytotoxic agent that is bioreductively activated in tumours to a reactive-drug free radical. Preclinically the agent has been shown to possess additive and synergistic anti-tumour activity in combination with radiotherapy and chemotherapy regimens. In the present study the pharmacokinetics and metabolism of tirapazamine were investigated in mice and patients as part of pre-clinical and phase I investigations. The objectives of this work were twofold; firstly, to evaluate retrospectively the utility of a pharmacokinetically guided dose-escalation (PGDE) strategy for tirapazamine, and secondly, to investigate if pharmacologically relevant plasma concentrations could be achieved at tolerable doses. Pharmacokinetic studies for PGDE were conducted in mice at four dose levels ranging from one-tenth of the LD₁₀ to the LD₅₀. The AUC at the LD₁₀ (2932 μg ml^-1min) was used to determine a target AUC value of 1173 μg ml^-1min (equivalent to 40% of the mouse LD₁₀ AUC) for clinical studies. A phase I study to investigate the tolerance of a single i.v. infusion of tirapazamine (once every 3 weeks) was initiated with close pharmacokinetic monitoring. The starting dose (36 mg/m²) was based on toxicity data obtained in the mouse, rat and dog. Doses were escalated by increases in the volume and duration of infusion. A retrospective analysis of the pharmacokinetic and toxicity data was then made to determine the utility of a PGDE approach. The drug exhibited a steep dose-lethality relationship in mice (LD₁₀ 294 mg/m², LD₅₀ 303 mg/m²). The major gross toxicities were body-weight loss (15–20%), pilo-erection and hypoactivity at all dose levels. Sporadic ptosis and conjunctivitis were observed at doses of >300 mg/m². The plasma elimination of tirapazamine fitted a monoexponential open model, with rapid elimination from the plasma (t _1/2=36±0.65 min) occuring at the LD₁₀ dose of 294 mg/m². A 10.3-fold increase in dose resulted in a 25.0-fold increase in AUC. Clinically, doses were escalated over the range of 36–450 mg/m². Ototoxicity (tinnitus and reversible hearing loss) was dose-limiting at 450 mg/m² and the MTD was 390 mg/m² for this schedule. Pharmacokinetic analyses in patients revealed that the elimination of tirapazamine in patients was generally bi-phasic, with low inter-patient variability being found in clearance. A 12.5-fold increase in dose resulted in a 19.0-fold increase in AUC. There was good quantitative agreement in metabolite formation between mice and humans with respect to the two- and four-electron bioreductive metabolites. AUC values recorded for tirapazamine at the MTD of 390 mg/m² (range 1035–1611 μg ml^-1min) were similar to the target AUC in mice. Importantly, these levels are consistent with the levels required for radiation-dose enhancement and effective combination with cisplatin in mice. Given (a) the similarities in plasma pharmacokinetics and metabolism observed at the target AUC/MTD in mice, rats, dogs and humans, (b) the similar degree of plasma protein binding seen between species and (c) the relatively low inter-patient variability noted in drug clearance, a successful PGDE approach should have been feasible. The results also indicate that potentially therapeutic levels of tirapazamine are achievable in patients at tolerable doses. Received: 27 May 1996 / Accepted: 30 September 1996     

Comparison of rates and risk factors for development of anaemia and erythropoiesis-stimulating agent utilization after radical or partial nephrectomy

Study Type – Therapy (case series) Level of Evidence 4 What's known on the subject? and What does the study add? The consequences and significance of iatrogenically‐induced CKD are poorly understood. Most data regarding risk of CKD and its complications are inferred from the medical literature. This is the first study to examine impact of surgical management of renal masses on development of anaemia. Patients who underwent radical nephrectomy had a significantly higher incidence of anaemia and ESA utilization than a contemporary well‐matched cohort that underwent partial nephrectomy. The results obtained add to the growing body of data supporting the use of partial nephrectomy in the management of clinically appropriate renal masses.

OBJECTIVE

• ? To examine the incidence of and risk factors for the development of anaemia and erythropoiesis‐stimulation agent (ESA) treatment in patients undergoing radical nephrectomy (RN) and partial nephrectomy (PN) because anaemia is a significant cause of morbidity in chronic kidney disease.

PATIENTS AND METHODS

• ? The study comprised a retrospective review of 905 patients (610 RN/295 PN; mean age, 57.5 years; mean follow‐up, 6.4 years) who underwent surgery for renal tumours at two institutions from July 1987 to June 2007.
• ? Demographics, disease characteristics and pre‐ and postoperative (i.e. renal function, metabolic parameters, anaemia and ESA treatment) were recorded.
• ? Data were analyzed within subgroups based on treatment (RN vs PN).
• ? Multivariate analysis was conducted to determine the risk factors for developing anaemia after surgery.

RESULTS

• ? Tumour size (cm) was significantly larger for RN (RN 7.0 vs PN 3.7; P < 0.001). No significant differences were noted with respect to demographics and preoperative anaemia (RN 16.4% vs PN 18.6%; P= 0.454) and ESA‐treatment (RN 0.7% vs PN 1.4%; P= 0.499).
• ? After surgery, significantly less de novo anaemia (PN 4.1% vs RN 17.5%; P < 0.001) and ESA utilization (PN 2.7% vs RN 13.4%; P < 0.001) occurred in the PN cohort.
• ? Multivariate analysis showed that age ≥60 years (odds ratio, OR, 1.62; P= 0.008), African American ethnicity (OR, 2.30; P < 0.001), smoking (OR, 1.60; P= 0.013), glomerular filtration rate (GFR) <60 mL/min/1.73 m² (OR, 4.09; P < 0.001), ≥1+ proteinuria (OR, 2.19; P < 0.03), metabolic acidosis (OR, 4.08; P= 0.007) and RN (OR, 2.58; P < 0.001) were significantly associated with de novo anaemia.

CONCLUSIONS

• ? Patients who underwent RN had a significantly higher prevalence of anaemia and ESA‐treatment compared to a well‐matched cohort that underwent PN.
• ? In addition to RN, age ≥60 years, African American ethnicity, history of smoking, GFR < 60 mL/min/1.73 m², proteinuria and metabolic acidosis were associated with developing anaemia.

     

Electrical coupling of astrocytes in rat hippocampal slices under physiological and simulated ischemic conditions

Mammalian protoplasmic astrocytes are extensively coupled through gap junction channels but the biophysical properties of these channels under physiological and ischemic conditions in situ are not well defined. Using confocal morphometric analysis of biocytin‐filled astrocytic syncytia in rat hippocampal CA1 stratum radiatum we found that each astrocyte directly couples, on average, to 11 other astrocytes with a mean interastrocytic distance of 45 μm. Voltage‐independent and bidirectional transjunctional currents were always measured between directly coupled astrocyte pairs in dual voltage‐clamp recordings, but never from astrocyte–NG2 glia or astrocyte–interneuron pairs. The electrical coupling ratio varied considerably among astrocytes in developing postnatal day 14 rats (P14, 0.5–12.4%, mean = 3.6%), but became more constant in young adult P21 rats (0.18–3.9%, mean = 1.6%), and the coupling ratio declined exponentially with increasing pair distance. Electrical coupling was not affected by short‐term oxygen–glucose deprivation (OGD) treatment, but showed delayed inhibition in an acidic extracellular pH of 6.4. Combination of acidic pH (6.4) and OGD, a condition that better represents cerebral ischemia in vivo, accelerated the inhibition of electrical coupling. Our results show that, under physiological conditions, 20.7–24.2% of K⁺ induced currents can travel from any astrocytic soma in CA1 stratum radiatum to the gap junctions of the nearest neighbor astrocytes, but this should be severely inhibited as a consequence of the OGD and acidosis seen in the ischemic brain. © 2009 Wiley‐Liss, Inc.     

Chronic dialysis‐associated anaemia in end‐stage renal disease: analysis of management in two French centres

Background: Treatment of anaemia in renal‐insufficient patients relies on the use of an erythropoiesis‐stimulating agent (ESA). This study aimed to compare the impact of two different strategies of ESA prescribing on variation in haemoglobin (Hb) concentration in end‐stage renal disease (ESRD) patients. Methods: Patients with ESRD, on haemodialysis, and who had received ESA for >3 months were recruited. Different parameters were analysed: demographics, Hb level the last day of the year before dialysis, the most recent weekly ESA dose, risk factors for resistance and cost. Each institution continued its local practice for achieving the desired Hb level: increasing the ESA dose to overcome resistance in one centre and defining an upper ESA‐dose limit in the other. Results: A total of 185 patients were recruited. No significant differences in the biological parameters were found between the two populations. In both centres, Hb levels were comparable and mean levels exceeded 11 g/dL, despite the higher ESA doses given in one centre to achieve this target. This finding also held true for the subgroups with greater than or equal to two resistance factors. These two strategies led to large between‐centre differences in treatment costs. Conclusion: The ESA‐use strategy difference probably indicates that erythropoietin‐resistance was not overcome with increased dosing. The Hb concentrations remained stable even when ESA doses were increased. On current evidence, the cheaper ESA‐dose limitation strategy is preferable but randomized controlled studies, including comparisons of alternative ESA formulations are necessary.     

Endocannabinoid involvement in endometriosis

Endometriosis is a disease common in women that is defined by abnormal extrauteral growths of uterine endometrial tissue and associated with severe pain. Partly because how the abnormal growths become associated with pain is poorly understood, the pain is difficult to alleviate without resorting to hormones or surgery, which often produce intolerable side effects or fail to help. Recent studies in a rat model and women showed that sensory and sympathetic nerve fibers sprout branches to innervate the abnormal growths. This situation, together with knowledge that the endocannabinoid system is involved in uterine function and dysfunction and that exogenous cannabinoids were once used to alleviate endometriosis-associated pain, suggests that the endocannabinoid system is involved in both endometriosis and its associated pain. Herein, using a rat model, we found that CB1 cannabinoid receptors are expressed on both the somata and fibers of both the sensory and sympathetic neurons that innervate endometriosis’s abnormal growths. We further found that CB1 receptor agonists decrease, whereas CB1 receptor antagonists increase, endometriosis-associated hyperalgesia. Together these findings suggest that the endocannabinoid system contributes to mechanisms underlying both the peripheral innervation of the abnormal growths and the pain associated with endometriosis, thereby providing a novel approach for the development of badly-needed new treatments.     

Unusual sonographic appearance of a huge angiomyolipoma of the liver

Hepatic angiomyolipoma is a rare, benign, hepatic mesenchymal neoplasm. We report a case of an extremely large hepatic angiomyolipoma in a 58‐year‐old woman with unusual sonographic features. The tumor was well demarcated from the surrounding liver and showed a heterogeneous cystic appearance with a thick echogenic rim. Color Doppler showed blood flow in the echogenic rim and around the lesion. Contrast sonography demonstrated strong enhancement in the solid rim from early arterial phase to the late phase and no enhancement in the central cystic part of the lesion. Spiral CT showed a well‐defined huge heterogeneous cystic mass with fatty density. At surgery the tumor was soft and encapsulated with a thin, smooth membrane. The cut surface of the tumor revealed a cystic appearance with necrotic tissue. The diagnosis of hepatic angiomyolipoma was confirmed by immunohistochemistry. © 2010 Wiley Periodicals, Inc. J Clin Ultrasound, 2010     

Limitations and caveats of magnetic cell labeling using transfection agent complexed iron oxide nanoparticles

Cell labeling with various types of nanomaterial, such as FDA‐approved iron oxide nanoparticles (IONPs) has become common practice in biomedical research. The low uptake of IONPs stimulates the use of transfection agents (TA), but the effect on stability of the IONPs and their cellular interactions has received minimal attention. In the present study, we evaluated the use of Lipofectamine as a commonly used TA and tested different ratios of TA and IONPs. While the TA–IONP complexes are stable in saline, at a high ratio of TA over IONP, substantial aggregation occurred in serum‐containing media. Even for the highest ratio, TA was unable to completely cover the IONPs, resulting in a net negative charge of all complexes. At high TA–IONP ratios, more complexes remained surface‐associated without internalization, resulting in cell death, while at lower TA–IONP ratios, complexes were more avidly taken up through fluid‐phase pinocytosis and clathrin‐mediated endocytosis. At later time points, the endocytosed complexes accumulated within the lysosomes and affected the appearance of lysosomal structures. The data indicate that TAs should be used with care as, depending on the ratio of TA and IONP, the complexes may aggregate, inducing cell death and preventing internalization. Copyright © 2012 John Wiley & Sons, Ltd.     

Percutaneous treatment of orofacial vascular malformations

The aim of this study was to evaluate the efficacy of fluoroscopy-guided percutaneous injection of bleomycin as the primary treatment for low-flow vascular malformations. A total of 34 patients (mean (range) age 24 (8–51) years) with orofacial vascular lesions were treated in the Department of Interventional Radiology and Maxillofacial Surgery. There were 20 low-flow venous malformations, 11 lymphatic malformations, and three of mixed type. All patients were treated by fluoroscopy-guided percutaneous injection of a mixture of bleomycin (mean (range) 15 (5–15) mg) and a radio-opaque agent (Ultravist^® (iopromide), Bayer)/session. The number of sessions ranged from one to six. The clinical response was complete in 21 patients, obvious in nine, and of clinical benefit in four. Patients were reviewed within the first week, third week, and at three-month periods until 24 months. There were no serious complications such as pulmonary fibrosis. Fluoroscopy-guided intralesional injection of bleomycin should be considered as the first-line treatment for lymphatic malformations because it is effective and reliable with few complications.     

How to assess cytotoxicity of (iron oxide‐based) nanoparticles. A technical note using cationic magnetoliposomes

The range of different types of nanoparticles and their biomedical applications is rapidly growing, creating a need to thoroughly examine the effects these particles have on biological entities. One of the most commonly used nanoparticle types is iron oxide nanoparticles, which can be used as MRI contrast agents. The main research topic is the in vitro labeling of cells with iron oxide nanoparticles to render the cells detectable for MRI upon in vivo transplantation. For the correct evaluation of cell function and behavior in vivo, any effects of the nanoparticles on the cells must be completely ruled out. The present work provides a technical note where a detailed overview is given of several assays that could be useful to determine nanoparticle toxicity. The assays described focus on (i) nanoparticle internalization, (ii) immediate cell toxicity, (iii) cell proliferation, (iv) cell morphology, (v) cell functionality and (vi) cell physiology. Potential pitfalls, appropriate controls and advantages/disadvantages of the different assays are given. The main focus of this work is to provide a detailed guide to help other researchers in the field interested in setting up nanoparticle‐toxicity studies. Copyright © 2010 John Wiley & Sons, Ltd.     

Surface functionalization of magnetic iron oxide nanoparticles for MRI applications – effect of anchoring group and ligand exchange protocol

Hydrophobic magnetite nanoparticles synthesized from thermal decomposition of iron salts must be rendered hydrophilic for their application as MRI contrast agents. This process requires refunctionalizing the surface of the nanoparticles with a hydrophilic organic coating such as polyethylene glycol. Two parameters were found to influence the magnetic behavior and relaxivity of the resulting hydrophilic iron oxide nanoparticles: the functionality of the anchoring group and the protocol followed for the functionalization. Nanoparticles coated with PEGs via a catecholate‐type anchoring moiety maintain the saturation magnetization and relaxivity of the hydrophobic magnetite precursor. Other anchoring functionalities, such as phosphonate, carboxylate and dopamine decrease the magnetization and relaxivity of the contrast agent. The protocol for functionalizing the nanoparticles also influences the magnetic behavior of the material. Nanoparticles refunctionalized according to a direct biphasic protocol exhibit higher relaxivity than those refunctionalized according to a two‐step procedure which first involves stripping the nanoparticles. This research presents the first systematic study of both the binding moiety and the functionalization protocol on the relaxivity and magnetization of water‐soluble coated iron oxide nanoparticles used as MRI contrast agents. Copyright © 2010 John Wiley & Sons, Ltd.