bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic Nephropathy in Ad Libitum Overfed Sprague-Dawley Rats and Its Early Attenuation by Increasing Degrees of Dietary (Caloric) Restriction to Control Growth
Authors: Keenan Kevin P. , Coleman John B. , Mccoy Carol L. , Hoe Chao-Min , Soper Keith A. , Laroque Philippe
Published: February 2008

The early development and progression of chronic nephropathy and its amelioration by moderate and marked dietary restriction (DR) was determined in Sprague-Dawley (SD) rats at 20, 33, 60, and 113 weeks of age. Both sexes of SD rats were overfed ad libitum (AL) or DR-fed at 72-79%, 68-72%, or 47-48% of the adult AL intake. The AL-fed rats rapidly developed increased body and kidney size, increased glomerular area (GA) and urinary protein loss, followed by declining creatinine clearance. Early increased kidney growth and glomerular hypertrophy by 20 weeks preceded increases in glomerular sclerotic index (GSI), 7-day BrdU tubular labeling index (TLI), and the lesions associated with chronic nephropathy. The glomerular number (GN) or the number of nephrons did not differ between the groups over the course of the study. Moderate DR (68-79% of AL) prevented the increased kidney size and GA at 20 weeks and delayed increases in GSI and TLI until 60 weeks of age. Marked DR (47—48% of AL) prevented increases in kidney size, GA and TLI at 20 weeks, and GSI at 60 weeks of age. In AL-fed rats, the early increase in GA predicted the early onset of proteinuria and the later decrease in creatinine clearance, and increased GSI, TLI, and mortality from severe nephropathy. The temporal and dose-related effects of increasing degrees of DR demonstrated that while nephron numbers were unchanged with age, the early development of glomerular hypertrophy was the critical morphological biomarker predicting the progression and severity of chronic nephropathy. Caloric restriction by DR prevented or delayed the development of glomerulosclerosis, tubulointerstitial damage, functional changes, morbidity, and mortality associated with chronic nephropathy in AL-overfed SD rats by controlling initial body and kidney growth, glomerular size, and nephron hypertrophy. These results indicate that control of body and renal growth by DR may be essential to prevent the development and progression of glomerulosclerosis in spontaneous nephropathy of laboratory rats.
Aromatase Inhibitors Prevent Spontaneous Granular Cell Tumors in the Distal Female Reproductive Tract of Sprague-Dawley Rats
Authors: Markovits Judit E. , Sahota Pritam S.
Published: February 2008

We recently established diagnostic criteria for granular cell changes in the distal female reproductive tract of rats. In a review of control animals from 9 carcinogenicity studies, we found that approximately 23% of animals had granular cell alterations. Because estrogen may play a role in the pathogenesis of granular cell alterations, we reviewed tissue sections from carcinogenicity studies with 2 aromatase inhibitors and found compound-related decreases in the incidence of granular cell changes. Since these aromatase inhibitors selectively prevent the conversion of androgenic steroids to the corresponding estrogens, these data further suggest that estrogen may play a role in the pathogenesis of granular cell tumors of the reproductive tract of rats.
Age-Related Cell Proliferation and Apoptosis in the Kidney of Male Fischer 344 Rats With Observations on a Spontaneous Tubular Cell Adenoma
Authors: Cardani Rosanna , Zavanella Teresa
Published: February 2008

Cell proliferation rate and apoptosis were examined in archival kidneys from young, middle-aged, and old male F344 rats. Immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and apoptosis were quantified in the same cell populations of the proximal tubule epithelium. A total of 79 kidneys from 40 rats were examined. There was a progressive increase in cell proliferation rates in rats from 4 and 6-10 months of age. In 23-month-old rats, proliferative activity appeared to be reduced. No age-related variations in apoptotic indices were found. One of the 16 rats aged 23 months had a tubular cell adenoma. In the tumor-affected kidney, cell proliferation rate was dramatically higher than in the contralateral kidney as well as in all the other kidneys examined. This high proliferative activity was not balanced by variation in cell death.
Review Article: Skin Ulcers in Fish: Pfiesteria and Other Etiologies
Authors: Noga Edward J.
Published: February 2008

Skin ulcers on fish are one of the most well-recognized indicators of polluted or otherwise stressed aquatic environments. In recent years, skin ulcer epidemics have been either experimentally or epidemiologically linked to exposure to a number of xenobiotic chemicals as well as to biotoxins. Some of these agents, such as toxins produced by the dinoflagellate alga Pfiesteria, have led to serious concerns about the health of aquatic ecosystems, such as estuaries along the east coast of the United States. However, a number of other risk factors besides Pfiesteria have been shown to damage epithelium and may also play important roles in skin ulcer pathogenesis. In addition, increasing evidence indicates that not only may skin damage occur via direct contact with toxins, but it may also be induced indirectly from physiological changes that result from exposure not only to toxins but also to other environmental stressors, such as pH and temperature extremes. The multifactorial pathways that operate at both the ecological and the organismal levels as well as the nonspecific response of the skin to insults make it very challenging to link epidemic skin ulcers to any single cause in natural aquatic populations. Consequently, using pathology to unequivocally identify the specific cause of a lesion (eg, Pfiesteria exposure) is not a valid approach. Only with an increased understanding of the basic mechanisms leading to skin damage (including development of specific biomarkers for specific toxins), along with a better understanding of ecological processes operating in these environments, will we be able to discern the relative importance of various risk factors in skin ulcer development.
Combination of a Teratoma and Embryonal Carcinoma of the Testis in SD IGS Rats: A Report of Two Cases
Authors: Sawaki Masakuni , Shinoda Kazutoshi , Hoshuyama Satsuki , Kato Fumie , Yamasaki Kanji
Published: February 2008

Testicular tumors of germ cell origin are extremely rare in rats. We encountered 2 cases of teratoma and embryonal carcinoma in the testes of 8- and 10-week-old Sprague-Dawley IGS rats. A unilateral tumor mass with bilateral testicular atrophy was observed macroscopically in both cases. Microscopic examination revealed that the tumor mass had characteristic features of a teratoma and was composed of several types of differentiated cells and tissues at various stages of maturation. Embryonal carcinoma tissue, composed of undifferentiated cells with an embryonic and anaplastic appearance, was observed within the tumor mass. In addition, foci of intratubular teratomas and embryonal carcinomas were observed in the testis on the side without any obvious mass. No obvious germ cells were observed in the seminiferous tubules in the remnant nontumorous area. Furthermore, intratubular transition of cells was observed from the embryonal carcinoma tissue to the squamous epithelium. This finding indicates that an embryonal carcinoma differentiates toward a teratoma even at a very early stage of development of the germ cell tumor.
Using Cytochrome P-450 Gene Knock-Out Mice to Study Chemical Metabolism, Toxicity, and Carcinogenicity
Authors: Ghanayem Burhan I. , Hongbing Wang  , Sumner Susan
Published: February 2008

Cytochrome P-450 (CYP) enzymes are heme-containing proteins that carry out oxidative metabolism of a wide range of structurally diverse exogenous chemicals and therapeutic agents as well as endogenous compounds. For some of these xenobiotics, oxidative metabolism results in the formation of toxic, mutagenic, or carcinogenic metabolites. In the past, the role of CYP enzymes in metabolism and chemical-induced toxicity was studied indirectly through use of specific antibodies or inducers and inhibitors of these enzymes. Progress in molecular biology and the ability to bioengineer animal models that do not express CYP1A2, CYP1A1, CYP1B1, CYP2E1, or both CYP1A2 and CYP2E1 isozymes has allowed for direct investigations of the in vivo role of these enzymes in the metabolism, toxicity, and carcinogenicity of xenobiotics. This article reviews research conducted to date that utilizes these genetically bioengineered mice in metabolism, toxicity, or carcinogenicity studies of chemicals. Some studies showed a positive correlation between in vivo results and in vitro predictions for the role of a specific CYP in chemical-induced effects, whereas other studies did not support in vitro predictions. Work reviewed herein demonstrates the importance of using animal models for investigating the role of specific CYP enzymes in metabolism and chemical-induced toxicity or carcinogenicity rather than relying solely on in vitro techniques. Eventually, studies of this nature will facilitate a more accurate assessment of human risks with regard to chemicals by helping us to understand the relationships between chemical metabolism, carcinogenicity, and polymorphisms in CYP enzymes.
Pharmacology and Safety Assessment of Humanized Monoclonal Antibodies for Therapeutic Use
Authors: Klingbeil Corine , Hsu Di-Hwei
Published: February 2008

The humanization of monoclonal antibodies has generated a class of therapeutic products with improved safety, longer half-lives, and greatly diminished immunogenicity. These engineered proteins are highly species specific and in many cases only cross-react in humans. Where there is cross-reactivity in nonhuman primates or other species, it is not always clear that the pharmacologic effects reflect the potential actions in human volunteers or patients. As with other biologic products, the profile of humanized monoclonal antibodies dictates the preclinical strategy. The preclinical programs for the 2 humanized monoclonal antibodies described here, anti-HLA-DR (Hu1D10) and anti-CD3 (HuM291), demonstrate several unique aspects that affected their preclinical development strategy. Hu1D10 binds to a posttranslational form of HLA-DR and recognizes this antigen in some but not all human and nonhuman primates. The second antibody, HuM291, cross-reacts with CD3 only in the chimpanzee, which is not an optimal test species. In addition, a marketed anti-CD3 product exists (OKT3®), and in the preclinical development of our antibody during testing of efficacy and safety, we needed to focus on adverse effects that might be similar to those of OKT3®. In these studies, the safety, pharmacokinetics, immunogenicity, and pharmacology (B- and T-cell depletion and recovery) of the 2 antibodies were evaluated. The focus in this review is on the safety and pharmacology testing and the current status of each drug.
Preclinical Development Strategies for Novel Gene Therapeutic Products
Authors: Pilaro Anne M. , Serabian Mercedes A.
Published: February 2008

With over 220 investigational new drug applications currently active, gene therapy represents one of the fastest growing areas in biotherapeutic research. Initially conceived for replacing defective genes in diseases such as cystic fibrosis or inborn errors of metabolism with genes encoding the normal, or wild-type, gene product, gene therapy has expanded into other novel applications such as treatment of cancer or cardiovascular disease, where the risk: benefit profiles may be more acceptable in relation to the severity of the disease. Different types of vectors, including modified retroviruses, adenoviruses, adenovirus-associated viruses, and herpesviruses and plasmid DNA, are used to transfer foreign genetic material into patients' cells or tissues. Developing a toxicology program to determine the safety of these agents, therefore, requires a modified approach that encompasses the pharmacology and toxicity of both the gene product itself and the vector system used for delivery in the context of the application for the clinical trial. In general, the issues involved in designing and developing appropriate preclinical testing to determine the safety of these products are similar to those encountered for other recombinant molecules, including protein biotherapeutics. Limitations to some of the typical toxicology studies conducted for a traditional drug development program may exist for these agents, and nontraditional approaches may be required to demonstrate their safety. Many factors may affect the safety and clinical activity of these agents, including the route, frequency, and duration of exposure and the type of vector employed. Other safety considerations include quantitation of the duration and degree of expression of the vector in target and other tissues, the effects of gene expression on organ pathology and/or histology, evaluation of trafficking of gene-transduced cells or vector after injection, and interactions of the host immune system with the transduced cell population. Because of the unique concerns regarding each of these therapies, the Center for Biologics Evaluation and Research encourages sponsors to obtain toxicity data whenever possible while evaluating the pharmacologic activity of the vector in a species or animal model relevant to their clinical indication. Sponsors are encouraged to discuss preclinical study design and results with the Center during product development to facilitate early identification of safety concerns prior to entry of these novel agents into the clinical setting and to ensure an uninterrupted course of development while addressing issues required for licensure.
Synthetic Oligonucleotides: The Development of Antisense Therapeutics
Authors: Monteith David K. , Levin Arthur A.
Published: February 2008

Antisense therapeutics using synthetic oligodeoxynucleotides (ODNs) are currently being evaluated in clinical trials for cancer, inflammation, and viral diseases. These macromolecules afford a unique opportunity to treat disease at the molecular level. The specificity of these compounds is derived from the genetic code and Watson-Crick base pairing, utilizing an antisense paradigm for the inhibition of translation and the regulation of protein expression. Currently, most antisense ODNs in development contain a phosphorothioate (P=S) backbone. Additional modifications primarily involve the 2' position on the ribose or modification of the nucleotide linkages of the backbone. To date, no toxicities in animal models appear related to inhibition of the pharmacologic target, rather toxicities induced by P=S ODNs appear similar and are independent of pharmacologic target. In general, toxicities correlate well with pharmacokinetic or tissue distribution parameters. In primates, the primary acute effects are associated with complement activation and the systemic effects associated with accumulation of high concentrations of P=S ODNs in the kidneys. In rodents, the primary effect is an immune stimulation characterized by splenomegaly, lymphoid hyperplasia, and mononuclear cell infiltrates in multiple tissues. At extraordinarily high doses (15-50 times the targeted clinical doses), hepatocellular and renal tubular degeneration are evident in rodents. Second generation antisense compounds, new routes of administration, and new formulations appear to broaden and improve the application of antisense technology.
Regulatory Decision Strategy for Entry of a Novel Biologic Therapeutic with a Clinically Unmonitorable Toxicity into Clinical Trials: Pre-IND Meetings and a Case Example
Authors: Black Lauren E. , Bendele Alison M. , Bendele Ray A. , Zack Philip M. , Hamilton Marta
Published: February 2008

The following material was derived from a synthesis of case histories taken from investigational new drug (IND) applications and drug sponsors' experiences, utilizing fictionalized data to avoid any resemblance to any proprietary information; any such resemblance is accidental. These examples are used as an instructional scenario to illustrate appropriate handling of a difficult toxicology issue. In this scenario, a drug caused a toxicity in animals that was detected only by histopathologic analysis; if it were to develop in patients, no conventional clinical methods could be identified to monitor for it. It is not unusual for a firm to cancel clinical development plans for a lead drug candidate that causes such a toxicity, especially if such a drug is intended for use as a chronic therapeutic in a population of patients with a chronic disease. This case synthesis was inspired by a Food and Drug Administration (FDA) agreement to allow such a product to proceed into clinical trials after substantive pre-IND discussions and agreement on well-considered toxicology program designs. The scientists most closely involved in the strategy development included the sponsor's toxicologist, veterinary toxicologic pathologist, and pharmacokineticist, as well as the FDA's reviewing pharmacologist. The basis of this decision was thorough toxicity characterization (1-month studies in 2 species); correlating toxicities with a particular cumulative area under the curve (AUC) in both species; identification of the most sensitive species (the species that showed the lower AUC correlating with toxicity); allometric assessment of clearance of the drug in 3 nonhuman species; construction of a model of human kinetics (based on extrapolation from animal kinetics); and finally, estimation of clinical safety factors (ratios of the human estimated cumulative AUC at the proposed clinical doses, over the animal cumulative AUC that correlated with the no adverse effect levels). Industry and FDA scientists negotiated a joint assessment of risk and benefit in patients, resulting in the FDA permitting such a compound to enter into clinical trials for a serious autoimmune disease. Such constructive, early communication starts with the pre-IND meeting, and the conduct and planning for this meeting can be very important in establishing smooth scientific and regulatory groundwork for the future of a drug under IND investigation.
Flow Cytometry in the Preclinical Development of Biopharmaceuticals
Authors: Gossett Kent A. , Narayanan Padma K. , Williams Donna M. , Gore Elizabeth R. , Herzyk Danuta J. , Hart Timothy K. , Sellers Teresa S.
Published: February 2008

Novel biomarkers are often required in the preclinical development of biopharmaceuticals in order to characterize pharmacologic and toxicologic effects and to establish pharmacodynamic and pharmacokinetic relationships. Flow cytometry is uniquely suited for measurement of these biomarkers. Large numbers of single cells in a heterogeneous population can be rapidly identified and characterized with high accuracy and reproducibility. Cells are not damaged by the detection system and can be subsequently sorted for further morphologic or functional analysis. The availability of clinical instruments and a wide range of fluorescent probes have made this technology applicable for use in toxicologic clinical pathology. Flow cytometry has played an integral role in the development of a monoclonal antibody to human CD4 (keliximab, IDEC-CE9.1, SB 210396). Lymphocyte subset analysis and assays for expression, coating, and modulation of human CD4 were used for sequential assessment of the pharmacologic activity of keliximab in transgenic mice expressing human CD4.
Lesions and Identification of Crystalline Precipitates of Glycoprotein IIb-IIIa Antagonists in the Rat Kidney
Authors: Levin Stuart , Friedman Robert M. , Cortez Enriqueta , Hribar Jeremy , Nicholas Mark , Schlessinger Sally , Fouant Monique , Khan Nasir
Published: February 2008

Two glycoprotein IIb-IIIa antagonists (xemilofiban, SC-54684A, and orbofiban, SC-57099B), which are platelet aggregation inhibitors, caused crystalline precipitates in the kidney tubules of rats at high dosages. Dogs were not affected. Depending on the degree of the precipitation, which was dosage dependent, and the location, which differed somewhat between the two compounds, the lesions varied from acute obstruction with tubule cell necrosis, nephron dilation, and sudden death with no inflammation to severe chronic pyogranulomatous inflammation. In order to understand the relevance of the lesions, it was important to identify the precipitates. This was technically challenging because the crystals were water soluble (dissolving in routine fixing and staining techniques) and were present in insufficient quantity to physically isolate. Techniques were devised to evaluate the crystals in situ in unstained frozen sections prepared without directly embedding the tissues in supporting medium, which interfered with the analyses. The crystals were analyzed in situ by infrared and Raman spectroscopy and time-of-flight secondary ion mass spectroscopy (TOF-SIMS). Uroliths found in the renal pelvis of one animal were analyzed by liquid chromatography/mass spectrometry. The resulting spectra showed that the crystals were the de-esterified acids of the parent compounds. This knowledge allowed us to predict that the crystalline precipitates would not be a hazard to humans because of the large multiples of the human dosage at which they occurred and because of differences in renal physiology between rats, dogs, and humans.
Use of In Vivo Confocal Microscopy to Understand the Pathology of Accidental Ocular Irritation
Authors: Maurer J.K. , Jester J.V.
Published: February 2008

In vivo confocal microscopy (CM) provides a unique ability to section optically through living, intact tissues and organs to characterize qualitatively and quantitatively pathological changes in 4 dimensions (x, y, and z, and time). It involves the capture of real-time images without the need for excision, fixation and processing. In vivo CM principally has been used for evaluation of eyes in patients and laboratory animals but has potential application to studies of other tissues/organs. In vivo CM is being used in human ophthalmology clinics. It has been used as a research tool for quantitative, in situ measurement of corneal wound contraction, fibroblast migration, corneal endothelial cell migration, corneal epithelial cell size and desquamation following contact lens wear and surgery, and the assessment of corneal surface toxicity following application of commonly used ophthalmic preservatives. In vivo CM allows us to (a) characterize changes to a light microscopic (i.e., cellular) level; (b) quantify changes objectively; (c) conduct studies of injury and repair in the same animal and directly correlate microscopic changes to clinical observations over time as this technique is used in the living animal; and (d) conduct comparative studies in humans. Here we present a brief overview of in vivo CM and how we are using it to provide noninvasive, in situ qualitative and quantitative histopathologic characterization of accidental ocular irritation. Our intent is to provide an awareness of this relatively new methodology and one practical application of its use in research. The goal of our work is to provide objective, quantitiative data for use in developing and validating mechanistically based in vitro replacement tests.
Molecular Pathology in the Preclinical Development of Biopharmaceuticals
Authors: Gillett Nancy A. , Chan Curtis M.
Published: February 2008

Advances in cell and molecular biology have engendered a wide range of techniques that can be used to study the molecular events that underlie the cause of disease, thus producing a new field of study called "molecular pathology." These techniques can be either slide-based or non-slide-based (solution-based). The slide-based techniques include immunohistochemistry, in situ hybridization, and in situ polymerase chain reaction; pathologists play a unique role in the administration of these techniques because of their ability to interpret the end product (i.e., the slide). In this manuscript, we briefly discussed the use and impact of these slide-based techniques within all phases of drug development in the pharmaceutical industry.
Toxicologic Pathology of Cytokines, Cytokine Receptors, and Other Recombinant Human Proteins
Authors: Puri Raj K.
Published: February 2008

About 12,000 Americans are diagnosed with malignant astrocytoma each year. Despite surgery, radiotherapy, and chemotherapy, the prognosis of these patients remains poor. Targeted toxins based on the identification of novel antigens or receptors provide a promising new approach to treating cancer. We have identified one such cell surface protein in the form of interleukin (IL)-4 receptors (IL-4R) on human malignant astrocytoma. Normal brain tissues from frontal cortex and temporal lobe cortex do not express IL-4R. To target IL-4R, we generated a chimeric fusion protein composed of IL-4 and Pseudomonas exotoxin (IL4-PE). This toxin is highly cytotoxic to IL-4R-bearing human brain cancer cells. Preclinical toxicologic experiments were performed in mice, rats, and guinea pigs to determine an maximum tolerated dose. Intrathecal administration in cynomolgus monkeys produced high cerebrospinal fluid levels without any central nervous system or other abnormalities. When IL4-PE was injected into the right frontal cortex of rats, localized necrosis was observed at 1,000 but not ≤100 μg/ml doses. Intravenous administration of this biologic to monkeys produced reversible grade 3 or grade 4 elevations of hepatic enzymes in a dose-dependent manner. These results indicate that localized administration can produce nontoxic levels of IL4-PE that may have significant activity against astrocytoma. In vivo experiments with nude mice have demonstrated that IL4-PE has significant antitumor activity against human glioblastoma tumor model. Intratumor administration of IL4-PE has been initiated for the treatment of malignant astrocytoma in a phase I clinical trial.
The Toxicology of Interleukin-12: A Review
Authors: Car Bruce D. , Eng Vicki M. , Lipman Jack M. , Anderson Timothy D.
Published: February 2008

Recombinant murine interleukin (IL)-12 (rmIL-12) exhibits antitumor, antiviral, and antimicrobial activities and can modify allergic inflammatory reactions in animal models. Recombinant human IL-12 (rhIL-12) is currently in clinical trials for treatment of cancer, asthma, and viral hepatitis. Principally a phagocyte-derived cytokine, IL-12 targets natural killer cells and T lymphocytes, stimulating their activity and the secretion of interferon (IFN)-γ. An understanding of the toxicology of IL-12, due in part to effects mediated by IFN-γ, has emerged from preclinical safety and mechanistic studies and initial clinical trials. Target organs common to several animal species and humans include the lymphohematopoietic system, intestines, liver, and lung.
Preclinical and Early Clinical Development of Keratinocyte Growth Factor, an Epithelial- Specific Tissue Growth Factor
Authors: Danilenko Dimitry M.
Published: February 2008

Keratinocyte growth factor (KGF) is a 28-kDa heparin-binding member of the fibroblast growth factor (FGF) family (alternative designation = FGF-7) that specifically binds to the KGF receptor, a splice variant of FGF receptor 2, which is expressed only in epithelial tissues. KGF has been identified as an important paracrine mediator of proliferation and differentiation in a wide variety of epithelial cells, including hepatocytes and gastrointestinal epithelial cells, type II pneumocytes, transitional urothelial cells, and keratinocytes in all stratified squamous epithelia. Systemic administration of recombinant human KGF (rHuKGF) provides significant cytoprotection to epithelial tissues in a number of different animal models of epithelial/mucosal damage, including models of injury to the gastrointestinal tract, lung, urinary bladder, and hair follicles. The results obtained with these preclinical models prompted an investigation of the use of rHuKGF as a cytoprotective agent against radiation- and/or chemotherapy-induced oral and gastrointestinal mucositis. Several dose- and time-variable studies were conducted in normal rhesus macaques to determine the lowest dose and shortest duration of rHuKGF administration required to induce oral mucosal proliferation without other significant systemic effects. Numerous studies were also conducted in murine models of chemotherapy-induced mucositis to fine-tune the dosing schedule. These studies showed that 2-3 days of rHuKGF administration were sufficient to induce significant oral mucosal proliferation and to protect against gastrointestinal mucositis when administered prior to the initiation of chemotherapy. The results from these models were used to design a phase I study in normal human volunteers to evaluate the safety of rHuKGF and its ability to induce oral mucosal proliferation. rHuKGF was well tolerated and induced a significant increase in markers of oral mucosal proliferation following 3 days of administration at the highest doses. Phase I/II studies to evaluate the safety and efficacy of rHuKGF in the prevention of chemotherapy-induced mucositis are currently in progress.
Development of a Recombinant Growth Factor and Fusion Protein: Lessons from GM-CSF
Authors: Schuh Joann C.L. , Morrissey Philip J.
Published: February 2008

Several colony stimulating factors (CSFs) and cytokines have been successfully used to mobilize hematopoietic cells during myeloablative therapy, bone marrow failure, and transplantation and to provide supportive treatment during sepsis. The use of yeast-derived recombinant human granulocyte-macrophage CSF (rhuGM-CSF) and its interleukin-3 fusion protein, PIXY321, provides an example of issues associated with development programs for recombinant hematopoietic growth factors. Species specificity of rhuGM-CSF, different bioactivity of homologous molecules in mice, and production in laboratory animals of antibodies to human proteins limit preclinical evaluation of such molecules. In clinical trials, rhuGM-CSF was efficacious and well tolerated. The derivation of the recombinant molecule, optimal dosing, scheduling, and confounding effects of concurrent disease and treatments are factors that influence efficacy, adverse responses, and immunogenicity reported in patients treated with CSFs. In comparisons of yeast-derived with Escherichia coli-derived rhuGM-CSF, the reduced severity and frequency of all adverse events, preponderance of low-grade adverse events, and similarity of positive clinical response versus adverse events reported for granulocyte CSF support safety and efficacy of yeast-derived rhuGM-CSF Enhanced pharmacoeconomic evaluations are beginning to limit and redirect clinical applications in this class of biological agents.
BR96 sFv-PE40 Immunotoxin: Nonclinical Safety Assessment
Authors: Haggerty H.G. , Warner W.A. , Comereski C.R. , Peden W.M. , Mezza L.E. , Damle B.D. , Siegall C.B. , Davidson T.J.
Published: February 2008

BR96 sFv-PE40, a recombinant DNA-derived fusion protein composed of the heavy- and light-chain variable region domains of the monoclonal antibody BR96 and the translocation and catalytic domains of Pseudomonas exotoxin A, is being developed for the treatment of solid tumors expressing cell surface Lewisy-related antigens. Single- and repeat-dose intravenous toxicity studies in rats and dogs and a comparative ex vivo tissue-binding study with rat, dog, and human tissues were conducted to assess the toxicity of BR96 sFv-PE40 and to estimate a safe starting dose in humans. Additional studies were performed to investigate the prevention of pulmonary vascular-leak syndrome, the dose-limiting toxicity of BR96 sFv-PE40 in rats, and the immunogenicity of BR96 sFv-PE40. In single-dose studies in rats, the vascular leak appeared to be primarily confined to the lungs; however, with a repeat-dose regimen (every other day for 5 doses) other organs including the brain and heart were involved at lethal doses (12-15 mg/m2 cumulative). Single doses of 1.8 mg/m2 and a cumulative 3.8 mg/m2 dose (0.75 mg/m2, every other day for 5 doses) were generally well tolerated in rats. These doses are significantly greater than doses required to cure rodents bearing human tumor xenografts. In dogs, the major target organ following single or repeated doses (every 3 days for 5 doses) was the pancreas. Morphologic changes in the exocrine pancreas ranged from atrophy with single-cell necrosis to diffuse acinar necrosis. After a 1-mo dose-free observation period, no residual pancreatic toxicity was observed in dogs given single doses up to 6.0 mg/m2 or 5 doses of 2.4 mg/m2 (12 mg/m2 cumulative). No significant pancreatic toxicity was observed at doses <0.6 mg/m2 in high Lewisy-expressing dogs. Assessment of trypsinlike immunoreactivity was useful in monitoring changes in pancreatic function. The immunogenicity of BR96 sFv-PE40 could be inhibited by combined treatment with an immunosuppressant in dogs, thus maintaining exposure to BR96 sFv-PE40.
Safety Evaluation of Human Living Skin Equivalents
Authors: Nemecek Georgina M. , Dayan Anthony D.
Published: February 2008

Human living skin equivalents (LSEs) offer an alternative to the use of split-thickness autografts for the treatment of hard-to-heal wounds. LSEs consist of 4 active components: a well-differentiated stratum corneum derived from epidermal keratinocytes, dermal fibroblasts, and an extracellular collagen matrix. Neonatal foreskins are used as the source of keratinocytes and dermal fibroblasts for the manufacture of LSEs. Following isolation and expansion in vitro, the cells are cultured on a 3-dimensional scaffold to give an upper epidermal layer and supporting dermal layer. The resulting product has the appearance and handling characteristics of human skin. Safety evaluation of LSEs begins with insuring that foreskins are obtained only from healthy infants whose mothers are negative for a panel of adventitious agents. Keratinocyte and fibroblast cell banks are characterized using morphologic, biochemical, and histologic criteria; checked for the absence of contaminating cell types such as melanocytes, macrophages, lymphocytes, and Langerhans cells; subjected to rigorous microbiological testing (with any production materials of biological origin); and evaluated for in vivo tumorigenicity. The consistency of certain key morphologic and functional characteristics are regularly assessed. Because an LSE represents an allogeneic graft, preclinical safety studies include in vitro and in vivo determinations of its potential immunogenicity. Immunocompromised (SCID) mice reconstituted with human leukocytes or engrafted with human fetal hematolymphoid organs have been useful animal models for assessing possible immunologic responses to LSEs. Additional preclinical studies are being conducted to show that LSEs are noncytotoxic and lack allergenic, sensitizing, or irritation potential.
The Pathologist and Toxicologist in Pharmaceutical Product Discovery
Authors: Alden Carl L. , Sagartz John E. , Smith Peter F. , Wilson Alan G. , Bunch Roderick T. , Morris Dale L.
Published: February 2008

Significant change is occurring in the drug discovery paradigm; many companies are utilizing dedicated groups from the toxicology/ pathology disciplines to support early stage activities. The goal is to improve the efficiency of the discovery process for selecting a successful clinical candidate. Toxicity can be predicted by leveraging molecular techniques via rapid high-throughput, low-resource in vitro and in vivo test systems. Several important activities help create a platform to support rapid development of a new molecular entity. The proceedings of this symposium provide excellent examples of these applied concepts in pharmaceutical research and development. Leading biopharmaceutical companies recognize that a competitive advantage can be maintained via rapid characterization of animal models, the cellular identification of therapeutic targets, and improved sensitivity of efficacy assessment. The participation of the molecular pathologist in this quest is evolving rapidly, as evidenced by the growing number of pathologists that interact with drug discovery organizations.
Mechanisms of Disease and Injury: Utilization of Mutants, Monoclonals, and Molecular Methods
Authors: Schuh Joann C.L. , Harrington Kimberly A.
Published: February 2008

Rapid advances in our ability to localize and quantify macromolecular changes in health and disease are being brought about by the availability of genetically altered animals (mutants), purified reagents such as monoclonal antibodies, and new molecular methods. Targeted gene deletion (knockouts) and gene insertions (transgenics) in animals can allow identification of the importance and function of macromolecules. Monoclonal antibodies and fluorescent labels coupled with advances in microscopy provide exacting and multidimensional information about localization and cellular changes in proteins, carbohydrates, and lipids using immunohistochemistry, fluorescent activated cell sorting, and immunoprecipitation. Similarly, new applications of molecular methods can be used to identify and localize nucleic acids in tissues via in situ hybridization, polymerase chain reaction (PCR), reverse transcription (RT) PCR, differential display RT-PCR, RNase protection assays, and microchip arrays. The ligand for CD40 (CD40L), an important immunoregulatory molecule, is an example of the successful application of mutants, monoclonal antibodies, and molecular methods to cloning and biological characterization of new molecules. CD40L knockout mice, monoclonal antibodies, and several molecular methods were used to identify mutations in CD40L as the genetic basis for hyper-IgM syndrome in humans, to provide new insights into the pathobiology of Pneumocystis carinii infection, and to evaluate CD40L for immunotherapy of tumors and opportunistic infections.
Tumor Models: Assessing Toxicity in Efficacy Studies
Authors: Arp Lawrence H.
Published: February 2008

Efficacy studies in animal tumor models provide an early opportunity to collect preliminary information on toxicity. When screening and evaluating cytotoxic chemotherapeutic agents, efficacy studies usually include at least 1 dosage level that causes severe toxicity and death. Pathologic evaluation in early efficacy studies may reveal major target organs, dosage/schedule relationships, pharmacokinetic/toxicity relationships, effects of formulation and route of administration, maximum tolerated dose, cause of death, and reversibility of changes in normal tissues. Intraperitoneal formulations are frequently used to establish proof of concept for promising compounds (hits) from in vitro screens; however, these crude formulations may also induce intraperitoneal inflammation and confound the interpretation of both efficacy and toxicity. Efficacy studies conducted in the later stages of drug discovery may be used to refine the dose and schedule proposed for phase I clinical trials. Efficacy studies in animal tumor models provide useful toxicologic data for screening potential drug candidates, optimizing the therapeutic index, and designing both preclinical and clinical development programs.
The Role of IL-10 in Inflammatory Bowel Disease: "Of Mice and Men"
Authors: Leach Michael W. , Davidson Natalie J. , Fort Madeline M. , Powrie Fiona , Rennick Donna M.
Published: February 2008

Inflammatory bowel disease (IBD) is a generic term typically used to describe a group of idiopathic inflammatory intestinal conditions in humans that are generally divided into Crohn's disease and ulcerative colitis. Although the etiology of these diseases remains unknown, a number of rodent models of IBD have recently been identified, all sharing the concept that the development of chronic intestinal inflammation occurs as a consequence of alterations in the immune system that lead to a failure of normal immunoregulation in the intestine. On the basis of these models, it has been hypothesized that the development of IBD in humans may be related to a dysregulated immune response to normal flora in the gut. Immunodeficient scid mice injected with CD4+ CD45RBhigh T cells and mice deficient in interleukin (IL)-10 (IL-10-/-) are among the rodent models of IBD. In both models, there is inflammation and evidence of a Th1-like response in the large intestine, characterized by CD4+ T-cell and macrophage infiltrates, and elevated levels of interferon-γ. Because IL-10 is an immunomodulatory cytokine that is capable of controlling Thl-like responses, the role of IL-10 was investigated in these models. IL-10 was shown to be important in regulating the development of intestinal inflammation in both models. These results provided key data that supported initiation of clinical trials evaluating the efficacy of IL-10 in patients with IBD.
Animal Models of Arthritis: Relevance to Human Disease
Authors: Bendele Alison , Mccomb Jennifer , Gould TY , Mcabee Tracy , Sennello Gina , Chlipala Elizabeth , Guy Michael
Published: February 2008

Animal models of arthritis are used to evaluate potential antiarthritis drugs for clinical use. Therefore capacity of the model to predict efficacy in human disease is one of the most important criteria in model selection. Animal models of rheumatoid arthritis (RA) with a proven track record of predictability include rat adjuvant arthritis, rat type II collagen arthritis, mouse type II collagen arthritis, and antigen-induced arthritis in several species. Agents currently in clinical use (or trials) that are active in these models include corticosteroids, methotrexate, nonsteroidal anti-inflammatory drugs, cyclosporin A, leflunomide, interleukin-1 receptor antagonist, and soluble tumor necrosis factor receptors. For some of these agents, the models also predict that toxicities seen at higher doses for prolonged periods would preclude dosing in humans at levels that might provide disease-modifying effects. Animal models of osteoarthritis (OA) include mouse and guinea pig spontaneous OA, meniscectomy and ligament transection in guinea pigs, meniscectomy in rabbits, and meniscectomy and cruciate transection in dogs. None of these models have a proven track record of predictability in human disease because there are no agents that have been proven to provide anything other than symptomatic relief in human OA. Efficacy data and features of the various models of RA and OA are discussed with emphasis on their proven relevence to human disease.
Oncostatin M: Development of a Pleiotropic Cytokine
Authors: Loy James K. , Davidson Thomas J. , Berry Karen K. , Macmaster John F. , Danle Bharat , Durham Stephen K.
Published: February 2008

Oncostatin M (OM) is a member of the interleukin-6 (IL-6) cytokine subfamily. The binding of OM to its receptor initiates signal transduction through JAK-signal transducers and activators of transcription (STAT) pathways and activates transcription activators through mitogen-activated protein (MAP) kinases. Results of in vitro assays documented that OM modulates cytokine expression and alters the production of proteases that down-regulate inflammation. Administration of OM to lipopolysaccharide (LPS)-challenged mice lowered serum tumor necrosis factor-α (TNF-α) levels and decreased the lethal effects of LPS administration. OM also reduced inflammation in animal models of human disease, including inflammatory bowel disease, antibody-induced arthritis, and experimental autoimmune encephalomyelitis. Preclinical safety studies have been conducted in the mouse and monkey. Mice were administered OM (subcutaneously) at 72, 360, or 1,560 μg/kg/day in a 2-wk toxicity study. Decreased body weights occurred at 1,560 μg/kg. Drug-related changes at 360 and 1,560 μg/kg consisted of dermal irritation at the injection site, leukopenia, and thymic lymphoid depletion; all changes were reversible following a 2-wk recovery period. In a 2-wk subcutaneous study in monkeys, OM was administered at 1, 5, 15, 45, or 150 μg/kg/day. At all doses there was reversible, transient inappetence and dermal irritation at the injection site. Drug-related changes at 5, 15, 45, and 150 μg/kg consisted of reversible elevations in both serum amyloid A and IL-6, and reversible thymic lymphoid depletion. Transient increases in body temperature occurred at 15, 45, and 150 μg/kg. The observed spectrum of immunomodulatory effects suggests that OM may have therapeutic utility in treating chronic inflammatory diseases.
Coronary Arteriopathy in Monkeys Following Administration of CI-1020, an Endothelin A Receptor Antagonist
Authors: Albassam Mudher A. , Metz Alan L. , Gragtmans Norbert J. , King Lena M. , Macallum Grace E. , Hallak Hussein , McGuire Edward J.
Published: February 2008

A selective non-peptide endothelin A (ETA) receptor antagonist, CI-1020, was administered to cynomolgus monkeys intravenously (iv) for 2 or 4 wk and orally for 4 wk. Groups consisting of 3 animals of each sex received CI-1020 at 1, 5, and 10 mg/kg/hr (iv) or orally at 250, 500, and 750 mg/kg body weight for 4 wk. Control animals received the vehicle only. In a separate experiment, 1 male was infused with 10 mg/kg/hr for 2 wk, and Monastral blue dye was administered iv to facilitate localization of lesions to the vascular walls. One female was administered saline and the dye and served as a control. One female at 1 mg/kg/hr was found dead at week 2, and 1 female at 5 mg/kg/hr was euthanatized during week 4 as a result of severe thigh swelling at the catheter site. Macroscopically, extramural coronary arteries appeared thickened and nodular in the 4-wk iv study in the female found dead at 1 mg/kg/hr, in 1 male and 1 female at 5 mg/kg/hr, and in 2 females at 10 mg/kg/hr. Histologically, Monastral blue pigment trapped in the walls of coronary arteries with arteriopathy was observed in the male treated with CI-1020 at 10 mg/kg/hr for 2 wk. Extramural coronary arteriopathy occurred at all doses in the 4-wk iv study, with higher incidence occurring in females than in males (7 of 9 treated females compared with 3 of 9 treated males). In the oral study, 1 female at 500 mg/kg/day and 1 male and 2 females at 750 mg/kg/day had coronary arteriopathy. Histological changes after 2 wk of treatment were characterized by intimal thickening, fragmentation of the internal elastic lamina, necrosis and edema of the media, and mixed inflammatory-cell infiltrates in the intima, media, and adventitia. After 4 wk of iv administration, arteriopathy was characterized by segmental disruption of the elastic lamina and intimal and medial fibrosis with complete replacement of smooth muscle with fibrous tissue. The adventitia was thickened as a result of fibrosis and mixed or mononuclear inflammatory-cell infiltrates. CI-1020 concentrations were higher in males (1.57 to 29 μg/ml) than in females (0.974 to 24.4 μg/ml) in the iv study. Transient systemic exposure with high maximum plasma concentration (Cmax) (120-352 μg/ml) in the oral study was insufficient to provoke arterial changes of the same magnitude as those noted with continuous iv administration. The regeneration of the media by fibrous tissue and the disruption of the elastic lamina may weaken the arterial wall and increase the susceptibility of the artery to the development of aneurysm.