Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism.

Nmp4/CIZ suppresses the response of bone to anabolic parathyroid hormone by regulating both osteoblasts and osteoclasts.

How parathyroid hormone (PTH) increases bone mass is unclear, but understanding this phenomenon is significant to the improvement of osteoporosis therapy. Nmp4/CIZ is a nucleocytoplasmic shuttling transcriptional repressor that suppresses PTH-induced osteoblast gene expression and hormone-stimulated gains in murine femoral trabecular bone. To further characterize Nmp4/CIZ suppression of hormone-mediated bone growth, we treated 10-week-old Nmp4-knockout (KO) and wild-type (WT) mice with intermittent human PTH(1-34) at 30 µg/kg daily or vehicle, 7 days/week, for 2, 3, or 7 weeks. Null mice treated with hormone (7 weeks) gained more vertebral and tibial cancellous bone than WT animals, paralleling the exaggerated response in the femur. Interestingly, Nmp4/CIZ suppression of this hormone-stimulated bone formation was not apparent during the first 2 weeks of treatment. Consistent with the null mice enhanced PTH-stimulated addition of trabecular bone, these animals exhibited an augmented hormone-induced increase in serum osteocalcin 3 weeks into treatment. Unexpectedly, the Nmp4-KO mice displayed an osteoclast phenotype. Serum C-terminal telopeptide, a marker for bone resorption, was elevated in the null mice, irrespective of treatment. Nmp4-KO bone marrow cultures produced more osteoclasts, which exhibited elevated resorbing activity, compared to WT cultures. The expression of several genes critical to the development of both osteoblasts and osteoclasts was elevated in Nmp4-KO mice at 2 weeks, but not 3 weeks, of hormone exposure. We propose that Nmp4/CIZ dampens PTH-induced improvement of trabecular bone throughout the skeleton by transiently suppressing hormone-stimulated increases in the expression of proteins key to the required enhanced activity and number of both osteoblasts and osteoclasts.

RAGE supports parathyroid hormone-induced gains in femoral trabecular bone.

Parathyroid hormone (PTH) restores bone mass to the osteopenic skeleton, but significant questions remain as to the underlying mechanisms. The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily; however, recent studies indicate a role in bone physiology. We investigated the significance of RAGE to hormone-induced increases in bone by treating 10-wk-old female Rage-knockout (KO) and wild-type (WT) mice with human PTH-(1-34) at 30 microg.kg(-1).day(-1) or vehicle control, 7 days/wk, for 7 wk. PTH produced equivalent relative gains in bone mineral density (BMD) and bone mineral content (BMC) throughout the skeleton in both genotypes. PTH-mediated relative increases in cortical area of the midshaft femur were not compromised in the null mice. However, the hormone-induced gain in femoral cancellous bone was significantly attenuated in Rage-KO mice. The loss of RAGE impaired PTH-mediated increases in femoral cancellous bone volume, connectivity density, and trabecular number but did not impact increases in trabecular thickness or decreases in trabecular spacing. Disabling RAGE reduced femoral expression of bone formation genes, but their relative PTH-responsiveness was not impaired. Neutralizing RAGE did not attenuate vertebral cancellous bone response to hormone. Rage-null mice exhibited an attenuated accrual rate of bone mass, with the exception of the spine, and an enhanced accrual rate of fat mass. We conclude that RAGE is necessary for key aspects of the skeleton's response to anabolic PTH. Specifically, RAGE is required for hormone-mediated improvement of femoral trabecular architecture but not intrinsically necessary for increasing cortical thickness.

Nonclinical Studies that Support Viral Vector-Delivered Gene Therapies: An EFPIA Gene Therapy Working Group Perspective.

Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered in vivo gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question.

Nmp4/CIZ suppresses parathyroid hormone-induced increases in trabecular bone.

The nucleocytoplasmic shuttling transcription factor Nmp4/CIZ (nuclear matrix protein 4/cas interacting zinc finger protein) is a ubiquitously expressed protein that regulates both cytoplasmic and nuclear activities. In the nucleus, Nmp4/CIZ represses transcription of genes crucial to osteoblast differentiation and genes activated by various anabolic stimuli, including parathyroid hormone (PTH). We investigated the role of Nmp4/CIZ in the PTH-induced increase in bone by engineering mice with loss-of-function mutations in the Nmp4/CIZ gene, and treating 10-week-old female mice with anabolic doses of human PTH (1-34) at 30 microg/kg/day, 7 day/week, for 7 weeks or vehicle control. The untreated, baseline phenotype of the Nmp4-null mice between 8 and 16 weeks of age included a modest but significant increase in bone mineral density (BMD) and bone mineral content (BMC) compared to wild-type (WT) mice. Type I collagen mRNA expression was moderately elevated in the femurs of the Nmp4-null mice. The Nmp4 mutant alleles decreased body weight by 4% when expressed on a mixed background but the same alleles on a pure B6 background yielded a significant, 15% increase in body weight among the KO mice, compared to their WT controls. Hormone treatment equally enhanced BMD and BMC over vehicle-treated mice in both the WT and Nmp4-null groups but Nmp4-KO mice exhibited a significantly greater PTH-induced acquisition of femoral trabecular bone as compared to WT mice. These data support our hypothesis that Nmp4/CIZ is a transcriptional attenuator that suppresses osteoid synthesis and PTH-mediated acquisition of cancellous bone. J. Cell. Physiol. 219: 734-743, 2009. (c) 2009 Wiley-Liss, Inc.

Potential mechanisms of target-independent uptake and toxicity of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a promising therapeutic modality for oncology indications. The concept of an ADC platform is to increase the therapeutic index (TI) of chemotherapeutics through more selective delivery of cytotoxic agents to tumor cells while limiting exposure to healthy normal cells. Despite the use of antibodies targeting antigens abundantly and/or exclusively expressed on cancer cells (i.e., target cells), dose limiting toxicities (DLTs) in normal cells/tissues are frequently reported even at suboptimal therapeutic doses. Although advancement of ADC technology has helped to optimize all three key components (i.e., mAb, linker, and payload), DLTs remain a key challenge for ADC development. Mechanisms of ADC toxicity in normal cells/tissues are not clearly understood, but the majority of DLTs are considered to be target-independent. In addition to linker-drug instability contributing to the premature release of cytotoxic drug (payload) in circulation, uptake/trafficking of intact ADCs by both receptor-dependent (FcγRs, FcRn and C-type lectin receptors), and-independent (non-specific endocytosis) mechanisms may contribute to off-target toxicity in normal cells. In this article, we review potential mechanisms of target-independent ADC uptake and toxicity in normal cells, as well as discuss components of ADCs which may influence these mechanisms. This information will provide a deeper understanding of the underlying mechanisms of ADC off-target toxicity and prove helpful toward improving the overall TI of the next generation of ADCs.

Preliminary evaluation of anti-pyretic and anti-ulcerogenic activities of Sida cordifolia methanolic extract.

The anti-pyretic and anti-ulcerogenic properties of methanolic extract of Sida cordifolia aerial parts (MESC) were investigated in rats. Oral dose of 500 mg/kg MESC significantly reduced pyrexia induced by TAB vaccine. MESC exhibited significant anti-ulcerogenic effect against aspirin and ethanol induced damage. Both these properties were comparable to the reference drugs.

Impact of repeated exposure on toxicity of perchloroethylene in Swiss Webster mice.

The aim was to study the subchronic toxicity of perchloroethylene (Perc) by measuring injury and repair in liver and kidney in relation to disposition of Perc and its major metabolites. Male SW mice (25-29g) were given three dose levels of Perc (150, 500, and 1000 mg/kg day) via aqueous gavage for 30 days. Tissue injury was measured during the dosing regimen (0, 1, 7, 14, and 30 days) and over a time course of 24-96h after the last dose (30 days). Perc produced significant liver injury (ALT) after single day exposure to all three doses. Liver injury was mild to moderate and regressed following repeated exposure for 30 days. Subchronic Perc exposure induced neither kidney injury nor dysfunction during the entire time course as evidenced by normal renal histology and BUN. TCA was the major metabolite detected in blood, liver, and kidney. Traces of DCA were also detected in blood at initial time points after single day exposure. With single day exposure, metabolism of Perc to TCA was saturated with all three doses. AUC/dose ratio for TCA was significantly decreased with a concomitant increase in AUC/dose of Perc levels in liver and kidney after 30 days as compared to 1 day exposures, indicating inhibition of metabolism upon repeated exposure to Perc. Hepatic CYP2E1 expression and activity were unchanged indicating that CYP2E1 is not the critical enzyme inhibited. Hepatic CYP4A expression, measured as a marker of peroxisome proliferation was increased transiently only on day 7 with the high dose, but was unchanged at later time points. Liver tissue repair peaked at 7 days, with all three doses and was sustained after medium and high dose exposure for 14 days. These data indicate that subchronic Perc exposure via aqueous gavage does not induce nephrotoxicity and sustained hepatotoxicity suggesting adaptive hepatic repair mechanisms. Enzymes other than CYP2E1, involved in the metabolism of Perc may play a critical role in the metabolism of Perc upon subchronic exposure in SW mice. Liver injury decreased during repeated exposure due to inhibition of metabolism and possibly due to adaptive tissue repair mechanisms.

Prior administration of a low dose of thioacetamide protects type 1 diabetic rats from subsequent administration of lethal dose of thioacetamide.

Previously, we reported that an ordinarily non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic rats due to inhibited liver tissue repair, whereas 30 mg TA/kg allows 100% survival due to stimulated although delayed tissue repair. Objective of this investigation was to test whether prior administration of a low dose of TA (30 mg/kg) would lead to sustainable stimulation of liver tissue repair in type 1 diabetic rats sufficient to protect from a subsequently administered lethal dose of TA. Therefore, in the present study, the hypothesis that preplacement of tissue repair by a low dose of TA (30 mg TA/kg, ip) can reverse the hepatotoxicant sensitivity (autoprotection) in type 1 diabetic rats was tested. Preliminary studies revealed that a single intraperitoneal (ip) administration of TA causes 90% mortality in diabetic rats with as low as 75 mg/kg. To establish an autoprotection model in diabetic condition, diabetic rats were treated with 30 mg TA/kg (priming dose). Administration of priming dose stimulated tissue repair that peaked at 72h, at which time these rats were treated with a single ip dose of 75 mg TA/kg. Our results show that tissue repair stimulated by the priming dose enabled diabetic rats to overexpress, calpastatin, endogenous inhibitor of calpain, to inhibit calpain-mediated progression of liver injury induced by the subsequent administration of lethal dose, resulting in 100% survival. Further investigation revealed that protection observed in these rats is not due to decreased bioactivation. These studies underscore the importance of stimulation of tissue repair in the final outcome of liver injury (survival/death) after hepatotoxicant challenge. Furthermore, these results also suggest that it is possible to stimulate tissue repair in diabetics to overcome the enhanced sensitivity of hepatotoxicants.

Priming dose of phenylhydrazine protects against hemolytic and lethal effects of 2-butoxyethanol.

Protection against a high dose of a toxicant by prior exposure to another toxicant is called heteroprotection. Our objective was to establish a heteroprotection model in RBCs. Female Sprague Dawley rats treated with an LD90 dose of 2-butoxyethanol (BE, 1500 mg/kg in water, 5 ml/kg po) 14 days after priming with 0.9% NaCl suffered 90% mortality by 15 days, whereas all rats receiving the LD90 dose of BE 14 days after priming with phenylhydrazine (PHZ, 125 mg/kg in 0.9% NaCl, 3 ml/kg po) survived. Hematocrit decreased from normal 45% to 24% by day 3 after PHZ priming and improved thereafter. Increasing the time interval between the priming and LD90 dose to 21 days abolished the heteroprotection. RBCs obtained on days 7 and 14 after PHZ priming unlike those on day 21 were resilient to the hemotoxic metabolite of BE, butoxyacetic acid (BAA). Unaltered hepatic alcohol and aldehyde dehydrogenase activities upon PHZ priming suggested that bioactivation of BE to BAA was unaffected. Lower renal (6 and 12 h) and hepatic (12 h) BAA levels and 3 fold higher excretion of BAA in PHZ-primed rat urine suggested a protective role of toxicokinetics. Higher erythropoietin, reticulocytes, and resiliency of PHZ-primed rat RBCs indicated that newly formed RBCs are resilient to hemolytic BAA. The antioxidant levels in the PHZ-primed rat RBCs did not indicate a protective role in heteroprotection. In conclusion, the resistance of PHZ-primed rats against BE-induced hemotoxicity and lethality is mediated by a combination of altered toxicokinetics, robust erythropoiesis, and resiliency of new RBCs.

Subchronic chloroform priming protects mice from a subsequently administered lethal dose of chloroform.

Protection offered by pre-exposure priming with a small dose of a toxicant against the toxic and lethal effects of a subsequently administered high dose of the same toxicant is autoprotection. Although autoprotection has been extensively studied with diverse toxicants in acute exposure regimen, not much is known about autoprotection after priming with repeated exposure. The objective of this study was to investigate this concept following repeated exposure to a common water contaminant, chloroform. Swiss Webster (SW) mice, exposed continuously to either vehicle (5% Emulphor, unprimed) or chloroform (150 mg/kg/day po, primed) for 30 days, were challenged with a normally lethal dose of chloroform (750 mg chloroform/kg po) 24 h after the last exposure. As expected, 90% of the unprimed mice died between 48 and 96 h after administration of the lethal dose in contrast to 100% survival of mice primed with chloroform. Time course studies indicated lower hepato- and nephrotoxicity in primed mice as compared to unprimed mice. Hepatic CYP2E1, glutathione levels (GSH), and covalent binding of (14)C-chloroform-derived radiolabel did not differ between livers of unprimed and primed mice after lethal dose exposure, indicating that protection in liver is neither due to decreased bioactivation nor increased detoxification. Kidney GSH and glutathione reductase activity were upregulated, with a concomitant reduction in oxidized glutathione in the primed mice following lethal dose challenge, leading to decreased renal covalent binding of (14)C-chloroform-derived radiolabel, in the absence of any change in CYP2E1 levels. Buthionine sulfoximine (BSO) intervention led to 70% mortality in primed mice challenged with lethal dose. These data suggest that higher detoxification may play a role in the lower initiation of kidney injury observed in primed mice. Exposure of primed mice to a lethal dose of chloroform led to 40% lower chloroform levels (AUC(15-360 min)) in the systemic circulation. Exhalation of (14)C-chloroform was unchanged in primed as compared to unprimed mice (AUC(1-6 h)). Urinary excretion of (14)C-chloroform was higher in primed mice after administration of the lethal dose. However, neither slightly higher urinary elimination nor unchanged expiration can account for the difference in systemic levels of chloroform. Liver and kidney regeneration was inhibited by the lethal dose in unprimed mice leading to progressive injury, organ failure, and 90% mortality. In contrast, sustained and highly stimulated compensatory hepato- and nephrogenic repair prevented the progression of injury resulting in 100% survival of primed mice challenged with the lethal dose. These findings affirm the critical role of tissue regeneration and favorable detoxification (only in kidney) of the lethal dose of chloroform in subchronic chloroform priming-induced autoprotection.

Adaptive tolerance in mice upon subchronic exposure to chloroform: Increased exhalation and target tissue regeneration.

The aims of the present study were to characterize the subchronic toxicity of chloroform by measuring tissue injury, repair, and distribution of chloroform and to assess the reasons for the development of tolerance to subchronic chloroform toxicity. Male Swiss Webster (SW) mice were given three dose levels of chloroform (150, 225, and 300 mg/kg/day) by gavage in aqueous vehicle for 30 days. Liver and kidney injury were measured by plasma ALT and BUN, respectively, and by histopathology. Tissue regeneration was assessed by (3)H-thymidine incorporation into hepato- and nephro-nuclear DNA and by proliferating cell nuclear antigen staining. In addition, GSH and CYP2E1 in liver and kidney were assessed at selected time points. The levels of chloroform were measured in blood, liver, and kidney during the dosing regimen (1, 7, 14, and 30 days). Kidney injury was evident after 1 day with all three doses and sustained until 7 days followed by complete recovery. Mild to moderate liver injury was observed from 1 to 14 days with all three dose levels followed by gradual decrease. Significantly higher regenerative response was evident in liver and kidney at 7 days, but the response was robust in kidney, preventing progression of injury beyond first week of exposure. While the kidney regeneration reached basal levels by 21 days, moderate liver regeneration with two higher doses sustained through the end of the dosing regimen and 3 days after that. Following repeated exposure for 7, 14, and 30 days, the blood and tissue levels of chloroform were substantially lower with all three dose levels compared to the levels observed with single exposure. Increased exhalation of (14)C-chloroform after repeated exposures explains the decreased chloroform levels in circulation and tissues. These results suggest that toxicokinetics and toxicodynamics (tissue regeneration) contribute to the tolerance observed in SW mice to subchronic chloroform toxicity. Neither bioactivation nor detoxification appears to play a decisive role in the development of this tolerance.