Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme ␣-galactosidase A (␣-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb 3; also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified ␣-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered ␣-gal A, (ii) to assess the pharmacokinetics of i.v.-administered ␣-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb3. ␣-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified ␣-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of ␣-gal A, nine of the 10 patients had significantly reduced Gb3 levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of ␣-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb 3 burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 microg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.

Forty-two chemicals were tested for their ability to induce cytogenetic change in Chinese hamster ovary cells using assays for chromosome aberrations (ABS) and sister chromatid exchanges (SCE). These chemicals were included in the National Toxicology Program's evaluation of the ability of four in vitro short-term genetic toxicity assays to distinguish between rodent carcinogens and noncarcinogens. The conclusions of this comparison are presented in Zeiger et al. [Zeiger E, Haseman JK, Shelby MD, Margolin BH, Tennant RW (1990): [Environ Molec Mutagen 16(Suppl 18): 1-14]. The in vitro cytogenetic testing was conducted at four laboratories, each using a standard protocol to evaluate coded chemicals with and without exogenous metabolic activation. Most chemicals were tested in a single laboratory; however, two chemicals, tribromomethane and p-chloroaniline, were tested at two laboratories as part of an interlaboratory comparison. Four chemicals (C.I. basic red 9 HCl, 2-mercaptobenzothiazole, oxytetracycline HCl, and rotenone) were tested for SCE in one laboratory and in a different laboratory for ABS. Tetrakis(hydroxymethyl)phosphonium sulfate was tested at one laboratory and the chloride form was tested at a different laboratory. Twenty-five of the 42 chemicals tested induced SCE. Sixteen of these also induced ABS; all chemicals that induced ABS also induced SCE. There was approximately 79% reproducibility of results in repeat tests, thus, we conclude that this protocol is effective and reproducible in detecting ABS and SCE.

This multicenter, open-label study evaluated pharmacokinetics, pharmacodynamics, and safety of agalsidase alpha in pediatric compared with adult patients with Fabry disease. The pharmacokinetic parameters of pediatric patients (19 boys, 5 girls, 6-18 years old; mean age, 11.8 years) were compared to those of adult male and female patients who participated in other clinical studies. All patients received agalsidase alpha at a dose of 0.2 mg/kg infused over 40 minutes every other week. Agalsidase alpha exhibited a biphasic serum elimination profile with a maximum serum concentration at the end of the 40-minute infusion; <1% of the maximum concentration was detected 8 hours after dosing. In children, serum clearance was 2.0 to 9.4 mL/min/kg and tended to decrease with increasing age. The average clearance in children, 3.7 +/- 1.5 mL/min/kg (mean +/- SD), was significantly greater than that measured in 33 adults (2.3 +/- 0.7 mL/min/kg, P < .0001). Mean terminal elimination half-life of agalsidase alpha was prolonged in week 25 compared with baseline (150 vs 66 minutes) in 8 of 19 male children. The magnitude of the reduction of plasma globotriaosylceremide was similar in all age groups and was independent of area under the curve and other pharmacokinetic parameters. Except for clearance in younger patients, agalsidase alpha appears to have comparable pharmacokinetic and pharmacodynamic profiles in pediatric and adult Fabry patients of both genders.