Safety Findings of Dosing Gene Therapy Vectors in NHP With Pre-existing or Treatment-Emergent Anti-capsid Antibodies.

Replication-incompetent adeno-associated virus (AAV)-based vectors are nonpathogenic viral particles used to deliver therapeutic genes to treat multiple monogenic disorders. AAVs can elicit immune responses; thus, one challenge in AAV-based gene therapy is the presence of neutralizing antibodies against vector capsids that may prevent transduction of target cells or elicit adverse findings. We present safety findings from two 12-week studies in nonhuman primates (NHPs) with pre-existing or treatment-emergent antibodies. In the first study, NHPs with varying levels of naturally acquired anti-AAV5 antibodies were dosed with an AAV5-based vector encoding human factor VIII (hFVIII). In the second study, NHPs with no pre-existing anti-AAV antibodies were dosed with an AAV5-based vector carrying the beta subunit of choriogonadotropic hormone (bCG); this led to the induction of high-titer antibodies against the AAV5 capsid. Four weeks later, the same NHPs received an equivalent dose of an AAV5-based vector carrying human factor IX (hFIX). In both of these studies, the administration of vectors carrying hFVIII, bCG, and hFIX was well-tolerated in NHPs with no adverse clinical pathology or microscopic findings. These two studies demonstrate the safety of AAV-based vector administration in NHPs with either low-titer pre-existing anti-AAV5 antibodies or re-administration, even in the presence of high-titer antibodies.

Safety, pharmacokinetics and CNS distribution of tralesinidase alfa administered via intracerebroventricular infusion to juvenile cynomolgus monkeys.

Mucopolysaccharidosis Type IIIB (MPS IIIB) is an ultrarare, fatal pediatric disease with no approved therapy. It is caused by mutations in the gene encoding for lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Tralesinidase alfa (TA) is a fusion protein comprised of recombinant NAGLU and a modified human insulin-like growth factor 2 that is being developed as an enzyme replacement therapy for MPS IIIB. Since MPS IIIB is a pediatric disease the safety/toxicity, pharmacokinetics and biodistribution of TA were evaluated in juvenile non-human primates that were administered up to 5 weekly intracerebroventricular (ICV) or single intravenous (IV) infusions of TA. TA administered by ICV slow-, ICV isovolumetric bolus- or IV-infusion was well-tolerated, and no effects were observed on clinical observations, electrocardiographic or ophthalmologic parameters, or respiratory rates. The drug-related changes observed were limited to increased cell infiltrates in the CSF and along the ICV catheter track after ICV administration. These findings were not associated with functional changes and are associated with the use of ICV catheters. The CSF PK profiles were consistent across all conditions tested and TA distributed widely in the CNS after ICV administration. Anti-drug antibodies were observed but did not appear to significantly affect the exposure to TA. Correlations between TA concentrations in plasma and brain regions in direct contact with the cisterna magna suggest glymphatic drainage may be responsible for clearance of TA from the CNS. The data support the administration of TA by isovolumetric bolus ICV infusion to pediatric patients with MPS IIIB.

Lack of germline transmission in male mice following a single intravenous administration of AAV5-hFVIII-SQ gene therapy.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype five gene therapy under investigation for the treatment of hemophilia A. Herein, we assessed the potential for germline transmission of AAV5-hFVIII-SQ in mice. Male B6.129S6-Rag2tm1Fwa N12 mice received a single intravenous dose of vehicle or 6 × 1013 vg/kg AAV5-hFVIII-SQ. Vehicle and AAV5-hFVIII-SQ-treated mice were mated with naïve females 4 days after dosing, when the concentration of vector genomes was expected to be at its peak in semen, and 37 days after dosing, when a full spermatogenesis cycle was estimated to be complete. Quantitative PCR was used to evaluate the presence of transgene DNA in liver and testes from F0 males dosed with AAV5-hFVIII-SQ and liver tissue of F1 offspring. Transgene DNA was detected in liver and testes of all F0 males dosed with AAV5-hFVIII-SQ, confirming successful transduction. Importantly, no transgene DNA was detected in any tested F1 offspring derived from F0 males dosed with AAV5-hFVIII-SQ. Using a novel 2-stage statistical model that takes into account the number of males dosed with AAV5-hFVIII-SQ and the number of offspring sired by these males, we estimate that the risk of germline transmission is <5% with a 99.2% confidence level.

Brain Alterations in Aged OVT73 Sheep Model of Huntington's Disease: An MRI Based Approach.

BACKGROUND: Huntington's disease (HD) is a fatal neurodegenerative autosomal dominant disorder with prevalence of 1 : 20000 that has no effective treatment to date. Translatability of candidate therapeutics could be enhanced by additional testing in large animal models because of similarities in brain anatomy, size, and immunophysiology. These features enable realistic pre-clinical studies of biodistribution, efficacy, and toxicity. OBJECTIVE AND METHODS: Here we non-invasively characterized alterations in brain white matter microstructure, neurochemistry, neurological status, and mutant Huntingtin protein (mHTT) levels in cerebrospinal fluid (CSF) of aged OVT73 HD sheep. RESULTS: Similar to HD patients, CSF mHTT differentiates HD from normal sheep. Our results are indicative of a decline in neurological status, and alterations in brain white matter diffusion and spectroscopy metric that are more severe in aged female HD sheep. Longitudinal analysis of aged female HD sheep suggests that the decline is detectable over the course of a year. In line with reports of HD human studies, white matter alterations in corpus callosum correlates with a decline in gait of HD sheep. Moreover, alterations in the occipital cortex white matter correlates with a decline in clinical rating score. In addition, the marker of energy metabolism in striatum of aged HD sheep, shows a correlation with decline of clinical rating score and eye coordination. CONCLUSION: This data suggests that OVT73 HD sheep can serve as a pre-manifest large animal model of HD providing a platform for pre-clinical testing of HD therapeutics and non-invasive tracking of the efficacy of the therapy.

Young mice administered adult doses of AAV5-hFVIII-SQ achieve therapeutic factor VIII expression into adulthood.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels.

Tralesinidase Alfa Enzyme Replacement Therapy Prevents Disease Manifestations in a Canine Model of Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects. SIGNIFICANCE STATEMENT: This work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.

Natural history of Tay-Sachs disease in sheep.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disease caused by a deficiency of the enzyme ß-N-acetylhexosaminidase A (HexA). TSD naturally occurs in Jacob sheep is the only experimental model of TSD. TSD in sheep recapitulates neurologic features similar to juvenile onset and late onset TSD patients. Due to the paucity of human literature on pathology of TSD, a better natural history in the sheep TSD brain, which is on the same order of magnitude as a child's, is necessary for evaluating therapy and characterizing the pathological events that occur. To provide clinicians and researchers with a clearer understanding of longitudinal pathology in patients, we compare spectrum of clinical signs and brain pathology in mildly symptomatic (3-months), moderately symptomatic (6-months), or severely affected TSD sheep (humane endpoint at ~9-months of age). Increased GM2 ganglioside in the CSF of TSD sheep and a TSD specific biomarker on MRS (taurine) correlate with disease severity. Microglial activation and reactive astrocytes were observed globally on histopathology in TSD sheep with a widespread reduction in oligodendrocyte density. Myelination is reduced primarily in the forebrain illustrated by loss of white matter on MRI. GM2 and GM3 ganglioside were increased and distributed differently in various tissues. The study of TSD in the sheep model provides a natural history to shed light on the pathophysiology of TSD, which is of utmost importance due to novel therapeutics being assessed in human patients.

Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor.

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.

Intravitreal enzyme replacement preserves retinal structure and function in canine CLN2 neuronal ceroid lipofuscinosis.

CLN2 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disorder characterized by progressive vision loss, neurological decline, and seizures. CLN2 disease results from mutations in TPP1 that encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Children with CLN2 neuronal ceroid lipofuscinosis experience ocular disease, characterized by progressive retinal degeneration associated with impaired retinal function and gradual vision loss culminating in total blindness. A similar progressive loss of retinal function is also observed in a dog CLN2 model with a TPP1 null mutation. A study was conducted to evaluate the efficacy of periodic intravitreal injections of recombinant human (rh) TPP1 in inhibiting retinal degeneration and preserving retinal function in the canine model. TPP1 null dogs received periodic intravitreal injections of rhTPP1 in one eye and vehicle in the other eye beginning at approximately 12 weeks of age. Ophthalmic exams, in vivo ocular imaging, and electroretinography (ERG) were repeated regularly to monitor retinal structure and function. Retinal histology was evaluated in eyes collected from these dogs when they were euthanized at end-stage neurological disease (43-46 weeks of age). Intravitreal rhTPP1 dosing prevented disease-related declines in ERG amplitudes in the TPP1-treated eyes. At end-stage neurologic disease, TPP1-treated eyes retained normal morphology while the contralateral vehicle-treated eyes exhibited loss of inner retinal neurons and photoreceptor disorganization typical of CLN2 disease. The treatment also prevented the development of disease-related focal retinal detachments observed in the control eyes. Uveitis occurred secondary to the administration of the rhTPP1 but did not hinder the therapeutic benefits. These findings demonstrate that periodic intravitreal injection of rhTPP1 preserves retinal structure and function in canine CLN2 disease.

Effects of a selective androgen receptor modulator (SARM), GSK2849466A, on stress urinary incontinence and bladder activity in rats with ovariectomy-induced oestrogen deficiency.

OBJECTIVES: To report the effect of a selective androgen receptor modulators (SARMs) on the urethral continence mechanisms in a rat model of stress urinary incontinence (SUI) induced by bilateral ovariectomy (OVX). MATERIALS AND METHODS: Female Sprague-Dawley rats with bilateral OVX were used. Rats were divided into five groups; sham operated, vehicle-treated OVX, low-dose SARM-treated OVX (GSK2849466A: 0.005 mg/kg/day, per os [p.o.]), high-dose SARM-treated OVX (GSK2849466A: 0.03 mg/kg/day, p.o.) and dihydrotestosterone (DHT)-treated OVX (1 mg/kg/day, subcutaneous) groups. After 4 weeks of SARM treatments or 3 weeks of DHT treatment (6 weeks after OVX), rats were subjected to evaluation of the sneeze-induced continence reflex using microtransducer-tipped catheter methods, sneeze-induced leak-point pressure, and continuous cystometry measurements, followed by histological analyses of urethral tissues. RESULTS: (i) OVX significantly impaired urethral continence function after 6 weeks to induce SUI during sneezing. (ii) Low-dose SARM treatment restored urethral baseline pressure (UBP) without affecting the amplitude of urethral response during sneezing (A-URS), partially reversing OVX-induced SUI during sneezing. (iii) High-dose SARM treatment reversed decreases in both UBP and A-URS, more effectively preventing SUI during sneezing. (iv) DHT treatment only restored A-URS without affecting UBP, partially preventing OVX-induced SUI during sneezing. (v) The high-dose SARM treatment induced hypertrophy of the striated and smooth muscle around the urethra. (vi) SARM treatment did not affect bladder function in sham or OVX rats. CONCLUSION: Treatment with SARMs could be a more effective modality for the treatment of SUI than DHT, without affecting bladder function, by enhancing smooth- and striated muscle-mediated urethral function under stress conditions such as sneezing.

A Safe and Reliable Technique for CNS Delivery of AAV Vectors in the Cisterna Magna.

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

Ex Vivo Gene Therapy: Graft-versus-host Disease (GVHD) in NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) Mice Transplanted with CD34+ Human Hematopoietic Stem Cells.

A therapeutic option for monogenic disorders is gene therapy with ex vivo-transduced autologous hematopoietic stem cells (HSCs). Safety or efficacy studies of ex vivo-modified HSCs are conducted in humanized mouse models after ablation of the murine bone marrow and transfer of human CD34+ HSCs. Engrafted human CD34+ cells migrate to bone marrow and differentiate into various human hematopoietic lineages. A 12-week study was conducted in NSG™ mice to evaluate engraftment, differentiation, and safety of human CD34+ cells that were transduced (ex vivo) with a proprietary lentiviral vector encoding a human gene (BMRN-1) or a mock (green fluorescent protein) vector. Several mice intravenously injected with naive CD34+ cells or transduced CD34+ cells had variable lymphohistiocytic inflammatory cell infiltrates and microgranulomas in the liver and lungs consistent with graft-versus-host disease (GVHD). Spleen, bone marrow, stomach, reproductive tract, but not the skin had similar inflammatory changes. Ex vivo viral transduction of CD34+ cells did not impact engraftment or predispose to xenogeneic GVHD.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Scientific and Regulatory Policy Committee Points to Consider: Histopathologic Evaluation in Safety Assessment Studies for PEGylated Pharmaceutical Products.

Colorless, intracytoplasmic vacuoles occur in multiple tissues in animals following repeated administration of polyethylene glycol (PEG)-conjugated molecules. The extent of vacuolation depends on physical characteristics and molecular backbone of the PEG and the dose, product, drug target/pharmacology, and duration of exposure. The collective experience gathered from multiple nonclinical toxicology studies of PEGylated biopharmaceuticals indicates that in general, PEG-related vacuolation is not associated with demonstrable cell and tissue damage or dysfunction and is reversible with sufficient duration of drug-free periods. Existing data are insufficient to predict whether nonclinical animal species differ in their sensitivity to develop PEG-associated vacuoles; however, recent data suggest that there may be species differences. Recent comprehensive reviews have addressed the basic challenges in developing PEGylated pharmaceutical products, including general reference to and description of PEG-associated tissue findings. These manuscripts have identified gaps in our current understanding of PEG-associated vacuolation, including the lack of a widely accepted standardized histological terminology and criteria to record and grade the severity of vacuolation as well as insufficient knowledge regarding the nature of the contents of these vacuoles. The goal of this article is to help address some of the gaps identified above by providing points to consider, including a pictorial review of PEG-associated microscopic findings, when evaluating and reporting the extent, severity, and significance (adversity or lack of adversity) of PEG-associated cytoplasmic vacuolation in safety assessment studies. [Box: see text].

Dermal toxicity studies: factors impacting study interpretation and outcome.

The field of dermal toxicity continues to evolve in order to accurately predict dermal (and systemic) responses in humans to topically applied chemicals. Although the testing methods have undergone extensive refinements, idiosyncrasies and unexpected issues during the conduct of these studies are not unusual due to the plethora of new vehicles available for formulating test substances, changing regulatory requirements, and introducting new strain and/or species of laboratory animals as no single species or method seems to suffice for evaluating skin toxicity. The objective of this article is to illustrate some pragmatic issues that should be considered during the conduct as well as interpretation of dermal toxicity studies. Routine procedure-related issues such as hair clipping, tape stripping, and wrapping the animal's torso to prevent oral ingestion can influence the interpretation. Excipients used in dermal toxicity studies may be nontoxic when used alone but complex dermal formulations can result in unexpected irritation and toxicity. In conclusion, interpretation and risk assessment of dermal toxicity studies should be done in a comprehensive manner, taking into account procedure-related impact on study results, unique species susceptibility, limitation of gross visual (naked eye) observation for evidence of toxicity, and normal anatomical variation.

Effect of estrous cycle phase on clinical pathology values in beagle dogs.

BACKGROUND: In dogs, the diestrus phase is considerably longer than in most domestic animals, and is characterized by high circulating progesterone concentrations that may influence clinical pathology values. OBJECTIVE: The objective of this retrospective study was to investigate differences in clinical pathology data in dogs in diestrus compared with data from dogs in all other phases of the estrous cycle. METHODS: Phase of the estrous cycle was determined by histologic evaluation of reproductive tissues from 86 control female Beagles that had participated in 23 toxicity studies. Serum biochemical, hematologic, and urinalysis values from dogs in diestrus were compared with data from dogs in all other estrous cycle phases using a 2-tailed t-test. RESULTS: In Beagles in diestrus (n = 38), serum cholesterol concentrations and eosinophil counts were 35% (P < .0001) and 45.8% (P = .0035) higher, respectively, than for Beagles in all other phases of the estrous cycle (n = 48). Furthermore, Beagles in diestrus had 14% lower AST activity (P = .0011), 1% lower chloride concentration (P = .0224), 7.8% lower hemoglobin concentration (P < .0001), 7.8% lower RBC count (P < .0001), and 7.6% lower hematocrit (P < .0001) compared with female dogs in all other phases of the estrous cycle. Urine values did not differ significantly between groups. CONCLUSIONS: Differences in clinical pathology values between dogs in different phases of the estrous cycle could potentially confound interpretation of data in toxicity studies, which often have small group sizes. Interpretation of clinical pathology data in female dogs should be performed with due consideration given to the phase of the estrous cycle.

Cyclic morphological changes in the beagle mammary gland.

Interpretation of chemical-induced effects on the female beagle mammary gland can be difficult owing to the wide variation of normal glandular morphology. In this retrospective study, morphological features of the gland in four (proestrus, estrus, diestrus, and anestrus) phases of the cycle are described. The gland was quiescent (inactive) in proestrus and estrus. In diestrus, with the rise of progesterone, four (I-IV) distinct morphological changes were evident. In phase I, there was exuberant stromal and ductal proliferation. In phase II, there was early lobular development with branching ducts and alveolar proliferation. In phase III, there was an abundance of glandular tissue with large lobules containing secretory material, whereas phase IV had features of early regression, increased interlobular connective tissue, and eosinophilic secretions in distended ducts and acini. In early anestrus, ducts were distended, with eosinophilic secretions with alveolar regression, whereas regression was complete in late anestrus. Glandular morphology was slightly variable in the mammary chain within the same dog. Progesterone receptor expression was prominent in estrus and early diestrus, and peak estrogen receptor expression was noted in diestrus II. Expression of proliferation marker ki-67 was highest in diestrus I, followed by diestrus II. There was excellent concordance between the estrous stage and the glandular morphology.

Chemical carcinogenesis of the gastrointestinal tract in rodents: an overview with emphasis on NTP carcinogenesis bioassays.

Cancers of the stomach and large intestine (LI) are the second and fourth leading causes of human cancer mortality. A review of the National Toxicology Program (NTP) database and the Carcinogenic Potency Database (CPDB) reveals that chemically induced neoplasms of the gastrointestinal tract (GIT) are relatively common. Within the GIT, epithelial tumors of the forestomach in mice and rats and LI of the rat are most common. Generally, there is a high species concordance for forestomach with at least 26 chemicals inducing tumors in both species. Glandular stomach tumors are rare, and the few reported are usually neuroendocrine tumors (carcinoids) originating from the enterochromaffin-like (ECL) cells. Of 290 carcinogenic agents identified by the NTP, 19 (7%) caused intestinal neoplasia, 14 in the rat and 5 in the mouse. Neoplasms occurred in both males and females, exclusively in the small intestine (SI) of the mouse and in the LI or both SI and LI in the rat. Enteric carcinogens (NTP) frequently induced neoplasms at other alimentary sites (oral cavity, esophagus, and stomach). In conclusion, the most common induced GIT tumors are squamous neoplasms of the forestomach, glandular neoplasms of the stomach are rare, and rats appear more prone to developing LI (colorectal) cancer compared to mice.

Frequency of different estrous stages in purpose-bred beagles: a retrospective study.

The beagle is a monoestric, nonseasonal breeder with a long estrous cycle. Owing to lengthy stages in individual phases of the estrous cycle, limited group size, and typical group assignment focused on homogenized body weight, dogs in the same stage of the cycle can be inadvertently assigned to one treatment group in toxicity studies potentially leading to erroneous interpretation. This study was conducted to better understand the frequency of the different stages of the cycle and review the associated histological features. Histologic sections of reproductive tissues were reviewed from 102 control dogs from thirty-two GLP studies. The average age of dogs at necropsy was 14.38 months, and the mean terminal body weight was 6.87 kg. Based on histological classification, fifty-five dogs were in anestrus, twenty-eight in diestrus, nine in estrus, five in proestrus, and five were classified as immature. Mean ovarian weights were higher in the estrus stage. This review indicates that more than 80% of the dogs in this study were in the anestrus-diestrus stage, and a small percentage of dogs were immature. Interpretation of drug-induced effects on the morphologic changes in the reproductive tract should be performed with due consideration given to the stage of the cycle and the potential for nonuniform assignment to drug treatment groups.

Induction of transitional cell hyperplasia in the urinary bladder and aberrant crypt foci in the colon of rats treated with individual and a mixture of drinking water disinfection by-products.

Cancer of the urinary bladder and colon are significant human health concerns. Epidemiological studies have suggested a correlation between these cancers and the chronic consumption of chlorinated surface water containing disinfection by-products (DBPs). The present study was designed to determine if exposure to DBPs would cause preneoplastic or neoplastic lesions in the urinary bladder and colon of rats, and what effect a mixture of DBPs would have on these lesions. Male and female Eker rats were treated via drinking water with low and high concentrations of potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, or bromodichloromethane individually or in a mixture for 10 months. The urinary bladders and colons were examined for the presence of preneoplastic lesions. Cell proliferation in the urothelium was examined using immunohistochemical staining for bromodeoxyuridine. Aberrant crypt foci (ACF), as well as the number of individual crypts in each ACF, were identified and counted microscopically after staining with 0.2% methylene blue. Colon crypt cell proliferation and mitotic index were determined using immunohistochemical staining for proliferating cell nuclear antigen. Labeling indexes for the urinary bladder and colon were calculated based on the percentage of positively labeled cells. Treatment with the high dose of MX caused transitional epithelial hyperplasia and cell proliferation in the rat urinary bladder, and this effect was diminished in the high dose mixture animals. Treatment with 4 individual DBPs, as well as a mixture of them, caused the development of ACF, the putative preneoplastic lesion of colon cancer.