Drug-induced nephrotoxicity is a major concern, since many pharmacological compounds are filtered through the kidneys for excretion into urine. To discover biochemical biomarkers useful for early identification of nephrotoxicity, metabolomic experiments were performed on Sprague-Dawley Crl:CD (SD) rats treated with the nephrotoxins gentamicin, cisplatin, or tobramycin. Using a combination of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), a global, nontargeted metabolomics analysis was performed on urine and kidney samples collected after one, five, and twenty-eight dosing days. Increases in polyamines and amino acids were observed in urine from drug-treated rats after a single dose, and prior to observable histological kidney damage and conventional clinical chemistry indications of nephrotoxicity. Thus, these metabolites are potential biomarkers for the early detection of drug-induced nephrotoxicity. Upon prolonged dosing, nephrotoxin-induced changes included a progressive loss of amino acids in urine, concomitant with a decrease in amino acids and nucleosides in kidney tissue. A nephrotoxicity prediction model, based on the levels of branched-chain amino acids in urine, distinguished nephrotoxin-treated samples from vehicle-control samples, with 100%, 93%, and 70% accuracy at day 28, day 5, and day 1, respectively. Thus, this panel of biomarkers may provide a noninvasive method to detect kidney injury long before the onset of histopathological kidney damage.

The regulation of adipocyte lipolysis is increasingly recognized to play a part in the pathophysiology of obesity, insulin resistance and Type II (non-insulindependent) diabetes mellitus. Both non-esterified fatty acids (NEFA) and glycerol, the two end products of lipolysis, are potential mediators for metabolic (dys)regulation, which is associated with these common conditions [1,2]. Prolonged increases in NEFA concentrations could induce or aggravate skeletal muscle insulin resistance [3], impair pancreatic betacell function [4] and result in glucose overproduction [5,6]. Glycerol has recently been suggested to be used by skeletal muscle as a precursor for intra-muscular triacylglycerol synthesis [2], which in turn when excessive, could result in skeletal muscle insulin Diabetologia (2001) Abstract Aims/hypothesis. Adipose tissue lipolysis plays a central part in total body fuel metabolism. Our study was to assess the long-term regulation of glycerol and non-esterified fatty acid (NEFA) release by insulin or TNF-a. Methods. Fully differentiated 3T3-L1 adipocytes were exposed for up to 22 h to insulin or TNF-a. Results. Long-term insulin treatment resulted in increased basal glycerol release, reaching sixfold at 22 h with 1 nmol/l insulin. Partial inhibition was observed by pharmacologically inhibiting phosphatidylinositol 3-kinase or the mitogen-activated kinase kinase ± extracellular signal-regulated kinase cascades. This represented 50±60 % of the response induced by 1 nmol/l TNF-a and approximately 40 % of the glycerol release maximally stimulated by isoproterenol (1 mmol/l, 30 min). The cellular mechanism seemed to be distinct from that of TNF-a: First, glycerol release in response to long-term insulin was progressive with time and did not display a lag-time characteristic of the effect of TNF-a. Second, pretreatment and co-treatment of the cells with troglitazone greatly inhibited TNF-a-induced glycerol release (128.5 10.2 to 35.4 2.1 nmol/mg protein per h) but not the effect of insulin, which was exaggerated. Third, hormone-sensitive lipase protein content was decreased (45 %) by TNF-a but not following longterm insulin. Finally, TNF-a was associated with NEFA release to the medium, whereas long-term insulin treatment was not. Moreover, glycerol release during isoproterenol-stimulated lipolysis was additive to the effect of long-term insulin, whereas NEFA release was inhibited by nearly 90 %. Conclusions interpretation. Contradictory to its shortterm inhibitory effect, long-term insulin stimulates glycerol release with concomitant stimulation of NEFA re-esterification. [Diabetologia (2001)

Glatiramer acetate (GA), the active ingredient in Copaxone 1 , is a complex mixture of polypeptides used for the treatment of relapsing remitting multiple sclerosis. Glatiramoids are related mixtures that may differ in some characteristics of the prototype molecule. Our aim is to describe the long-term toxicity studies with protiramer (TV-5010), a new glatiramoid, in comparison with similar studies conducted with GA. The toxicity of twice-weekly subcutaneous injections of protiramer to Sprague-Dawley rats (twenty-six weeks) and cynomolgus monkeys (fifty-two weeks) was compared with similar studies done with daily subcutaneous injections of GA. Daily treatment with GA was safe and well tolerated, without systemic effects or death. Protiramer administration was not as well tolerated as GA and led to dose-and time-related mortalities, probably mediated through severe injection-site lesions both in rats and in monkeys. Bridging fibrosis in the liver and severe progressive nephropathy were seen in rats. A dose-related increase in eosinophils was observed in monkeys. The protiramer toxicity studies show that minor variations in the manufacturing of glatiramoids may lead to significant toxic effects. It is therefore essential that the safety of any new glatiramoid be studied in long-term preclinical studies before exposing humans.

Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα’s TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity.

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