Rat Model Of Human Disease Research Articles

Salt-Sensitive Hypertension and Renal Damage are Amplified by Adoptive Transfer of CD4+ and Not CD8+ T Cells: A Role of NADPH Oxidase 2 (NOX2)

Infiltration of T cells into the kidney of the Dahl Salt Sensitive (SS) rat model of human disease accompanies the development of salt-sensitive hypertension and renal damage. Deletion of T cells on the Dahl SS genetic background (SSCD247−/−) attenuates hypertension and renal damage, and subsequent replacement of T cells by splenocyte transfer restores the disease phenotype. The present studies were performed to assess if the T cell subtype (CD4+ vs CD8+) is important in Dahl SS hypertension and interrogate the mechanism of action. In initial experiments, adoptive transfer of purified CD4+ or CD8+ T cells (~5 million) from splenocytes isolated from the Dahl SS (CD4+ SS→CD247 or CD8+ SS→CD247) or PBS vehicle (PBS→CD247) into the SSCD247−/− rat was performed on postnatal day 5. Animals were recovered and instrumented with radiotelemeters at 7 weeks of age. After a week of recovery, baseline measurements of blood pressure and albumin excretion were obtained while rats were fed a low salt (LS, 0.4% NaCl) diet. Rats were then switched to a high salt (HS, 4.0% NaCl) diet for 21 days, with continuous blood pressure measurements and urine collections every 7 days. On day 21, the rats were euthanized, and their kidneys were perfused and collected for immune cell analysis by flow cytometry. During the LS baseline period there was no significant difference observed in mean arterial pressure (MAP) between the CD4+ SS→CD247, CD8+ SS→CD247, or PBS→CD247 groups (113±1, 115±1, 114±1 mmHg; n=21, 10, 24, respectively). After three weeks of HS, rats receiving CD4+ T cells from the SS (CD4+ SS→CD247) demonstrated an amplified salt-sensitive hypertension (154±3 mmHg) and albuminuria (184±22 mg/day) compared to blood pressure (140±3 and 145±3 mmHg) and albuminuria (82±9 and 85±8 mg/day) in the CD8+ SS→CD247 or PBS→CD247 groups, respectively. Since the deletion of the p67phox subunit of NADPH oxidase 2 (NOX2) in adoptively transferred splenocytes attenuated Dahl SS hypertension and kidney damage, parallel studies examined the influence of deletion of p67phox in CD4+ (n=14) and CD8+ T cells (n=8). The adoptive transfer of either CD4+ or CD8+ T cells lacking functional NOX2 did not alter the LS or HS blood pressure or albuminuria when compared to the PBS control group. Finally, a flow cytometric analysis at the end of the experiment confirmed the absence of CD3+ T cells in the PBS→CD247 and documented the reconstitution of equal numbers of CD4+ cells in the blood and kidney of the rats receiving CD4+ cells with intact or deficient NOX2. Interestingly, no difference was observed in CD8+ T cells in the kidney and blood of rats receiving CD8+ cells with intact or deficient NOX2, but a significant number of CD4+ T cells were also observed in the SSCD247−/− rats receiving the purified CD8+ T cells. In summary, these studies demonstrate that CD4+ T cells amplify salt-sensitive hypertension and renal damage in the Dahl SS via a NOX2-dependent mechanism while CD8+ T cells do not alter disease phenotypes when compared to PBS control. GRANTS: HL161231, HL166458, Georgia Research Alliance. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P320: Transgenic Cre Drivers For Harvesting Podocytes And Endothelial Cells From Dahl Salt-sensitive Rats

Complex phenotypes such as blood pressure regulation involve multiple organ systems, tissues, and specific cell types. Transgenic rodent models with the expression of fluorescent proteins under the control of tissue- and cell-type-specific promoters are powerful tools for identifying and isolating specific cells but have been primarily limited to mice. Our long-term goal is to establish a Cre-driver rat resource for conditional and physiologically predictive rat models of human diseases. We developed two Cre-driver transgenic strains of rats (SS/PodocinCreERT2 and SS/VECadCre) capable of activating a second Cre-sensitive tdTomato reporter transgene integrated into the rat Rosa26 locus in the Dahl salt-sensitive hypertension model background. Cells with reporter activation were identified under fluorescence microscopy or harvested using fluorescence-activated cell sorting (FACS). A tamoxifen-inducible Cre transgene under the control of the Nphs2 (podocin) gene promoter was used to harvest fluorescently labeled podocytes after tamoxifen injection. A constitutive Cre under the control of the Cdh5 (VE-Cadherin) gene promoter was used for the isolation of endothelial cells. Correspondingly, tens to hundreds of thousands of podocytes or endothelial cells were isolated from a single kidney or mesenteric artery. Isolated cells were enriched for specific marker genes and could be used in downstream assays including single-cell transcriptional profiling. These new Cre/reporter dual transgenic rat models provide a much-needed resource for the biomedical research community to study the role of these cell types in a broad range of health and disease modeling studies.

Rat Models of Human Diseases and Related Phenotypes: A Novel Inventory of Causative Genes

The laboratory rat (Rattus norvegicus) has been used for a long time as the model of choice in several biomedical disciplines. In 2020, I made an inventory of rat genes that had been identified as underlying diseases or playing a key role in critical biological processes that are altered in diseases. Over 350 genes could be found, a significant number of which have similar effects in rat and humans (Szpirer in J Biomed Sci 27:84-155, 2020). However, a few rat disease genes were unintentionally overlooked; in addition, since this review was published, numerous rat genes were inactivated by targeted mutations, revealing their potential role in diseases. It thus seems appropriate to update these data, which is the aim of this paper.

Establishment of a Cre-rat resource for creating conditional and physiological relevant models of human diseases

The goal of this study is to establish a Cre/loxP rat resource for conditional and physiologically predictive rat models of human diseases. The laboratory rat (R. norvegicus) is a central experimental animal in several fields of biomedical research, such as cardiovascular diseases, aging, infectious diseases, autoimmunity, cancer models, transplantation biology, inflammation, cancer risk assessment, industrial toxicology, pharmacology, behavioral and addiction studies, and neurobiology. Up till recently, the ability of creating genetically modified rats has been limited compared to that in the mouse mainly due to lack of genetic manipulation tools and technologies in the rat. Recent advances in nucleases, such as CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9), as well as TARGATT™ integrase system enables fast, efficient and site-specific introduction of exogenous genetic elements into the rat genome. Here, we report the generation of a collection of tissue-specific, inducible transgenic Cre rats as tool models using TARGATT™, CRISPR/Cas9 and random transgenic approach. More specifically, we generated Cre driver rat models that allow controlled gene expression or knockout (conditional models) both temporally and spatially through the Cre-ERT2/loxP system. A total of 10 Cre rat lines and one Cre reporter/test line were generated, including eight (8) Cre lines for neural specific and two (2) lines for cardiovascular specific Cre expression. All of these lines have been deposited with the Rat Resource and Research Center and provide a much-needed resource for the bio-medical community who employ rat models for their studies of human diseases.

Genome Editing in Rats Using TALE Nucleases.

The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

Recent Developments in Small Animal Cardiovascular MRI

This review is intended to give a comprehensive overview over new cardiovascular magnetic resonance (CMR) method developments and refinements dedicated to the fully non-invasive in vivo exploration of the rodent heart. Unlike other cardiovascular imaging techniques, CMR techniques exist in many modalities giving access to parameters characterizing morphology, global and regional function, blood flow, myocardial structure, cell damage, metabolism and other molecular processes in mouse and rat models of human disease. But even in healthy animals, small animal CMR techniques can help exploring general physiological and biochemical mechanisms in vivo. New magnetic resonance imaging methods and imaging protocols are actively being developed by the entire CMR community with the goal of widening the spectrum of observable and measurable myocardial properties. This report also includes a selection of application studies using recent CMR methodology in this field. Beyond giving new insights into pathophysiologic processes, these studies underline the growing usefulness of CMR in a small animal research context.

Whole-Genome Sequences of DA and F344 Rats with Different Susceptibilities to Arthritis, Autoimmunity, Inflammation and Cancer

DA (D-blood group of Palm and Agouti, also known as Dark Agouti) and F344 (Fischer) are two inbred rat strains with differences in several phenotypes, including susceptibility to autoimmune disease models and inflammatory responses. While these strains have been extensively studied, little information is available about the DA and F344 genomes, as only the Brown Norway (BN) and spontaneously hypertensive rat strains have been sequenced to date. Here we report the sequencing of the DA and F344 genomes using next-generation Illumina paired-end read technology and the first de novo assembly of a rat genome. DA and F344 were sequenced with an average depth of 32-fold, covered 98.9% of the BN reference genome, and included 97.97% of known rat ESTs. New sequences could be assigned to 59 million positions with previously unknown data in the BN reference genome. Differences between DA, F344, and BN included 19 million positions in novel scaffolds, 4.09 million single nucleotide polymorphisms (SNPs) (including 1.37 million new SNPs), 458,224 short insertions and deletions, and 58,174 structural variants. Genetic differences between DA, F344, and BN, including high-impact SNPs and short insertions and deletions affecting >2500 genes, are likely to account for most of the phenotypic variation between these strains. The new DA and F344 genome sequencing data should facilitate gene discovery efforts in rat models of human disease.

Abstract 353: Rapid Gene Editing in Rat Models of Human Disease Using Targeted Nucleases

Previous investigations into mechanisms of genes in animal models have primarily used the mouse because of the availability of technologies for targeted manipulation of its genome in embryonic stem (ES) cells. To accelerate functional mechanistic studies in other species where ES technology is not yet widespread, we recently developed an alternative and rapid method for generating targeted mutations in rat genes by the application of Zinc Finger Nucleases (ZFNs). When introduced into an embryo, ZFNs target and induce a chromosome double strand DNA break at the investigator-specified locus, stimulating cellular responses which can result in knock out or knock in modifications at the locus. Using commercially available reagents, we targeted and disrupted more than 100 protein-coding genes across 13 different inbred, consomic, and outbred genetic backgrounds with a 97% (102/105) success rate in approximately 30 months. ZFNs can therefore access nearly every gene in the rat genome and can induce multiple different alleles at the target locus. The majority of mutations, 81% (355/437) were simple deletions ranging from 1-228 base pairs (median 13-bp) and upon breeding, 94% (178/189) of mutations were transmitted to the next generation with little evidence of germline mosaicism. Many of these strains are now available for functional mechanistic studies of candidate human disease genes for hypertension and chronic kidney disease. Co-introduction of a homologous gene template with the ZFNs could stimulate knockin at three different loci in the rat genome. Gene knock into the rat Rosa26 locus allows for uniform expression of a gene in all cells and tissues of the adult animal as well as germline-competent ES cells derived from the Fawn Hooded Hypertensive strain. This knockin technology in embryos and ES cells offers many new opportunities to make very precise modifications to the rat genome and is a key step toward creating conditional gene alleles in the rat. Transcriptional activator-like effector nucleases (TALENs) are also active in the rat embryo and we have disrupted 70% (7/10) genes with this new technology. In sum, targeted nuclease technology is enabling many new genetic approaches to the rat and other model systems to create even better human disease models.

Development of monoclonal antibodies and ELISA specific for the mouse vascular endocan

Human vascular endocan is a proteoglycan exhibiting tumorigenic activity through both its glycan and protein cores. Endocan mRNA is identified as being one of the most significant molecular signatures defining a poor prognosis in lung, breast, kidney, prostate, and thyroid malignancies. The survival inversely correlates with endocan expression in tumor tissue from hepatocarcinoma, and in serum from lung cancer. In mouse, endocan mRNA is also increased in tumor vessels. However, mouse endocan has not yet been fully characterized. Here, we produced a panel of rat monoclonal antibodies directed against mouse endocan, leading to the development of a specific mouse/rat endocan ELISA. Mouse endocan serum level was measured at a median of 0.96ng/mL and 1.08ng/mL in 129Sv mice and C57bl6, respectively. These results also provide new tools to characterize and explore the role of endocan in mouse and rat models of human diseases. These results present mouse vascular endocan as a circulating molecule similar to human endocan.

Cardiovascular Magnetic Resonance of Myocardial Structure, Function, and Perfusion in Mouse and Rat Models

This review summarizes small-animal cardiovascular magnetic resonance (CMR) techniques that are being actively developed at present. Taking into account with few exceptions only literature of the past 2 years it shows that small-animal CMR has become an important and versatile analysis tool in many biomedical studies. The relatively complex signal formation and detection in magnetic resonance offers numerous ways of creating and modulating image contrast as a function of the specific needs. Although most new small-animal CMR developments are done within the scientific MR community, the MR manufacturers have readily contributed in making these techniques robust and available for routine application studies. Unlike other cardiovascular imaging techniques, CMR is used in many facets to assess morphology, global and regional function, blood flow, myocardial structure, cell damage, metabolism, and other molecular processes for studying mouse and rat models of human disease as well as general biochemical mechanisms in vivo.

Cardioprotective and hepatoprotective effects of ellagitannins from European oak bark (Quercus petraea L.) extract in rats

Red wine contains many potentially bioactive polyphenols including resveratrol, catechins, anthocyanins and flavonoids as well as tannins derived from oak during maturation. This study examined the effects of a mixture of ellagitannins from oak bark (Quercus petraea L.) on cardiovascular, metabolic and liver changes in high-carbohydrate, high-fat diet-fed rats and in Spontaneously Hypertensive Rats (SHR). First, 8-week-old male Wistar rats were divided into four groups and given either cornstarch diet, cornstarch diet + oak bark extract (0.5 mL/kg food), high-carbohydrate, high-fat diet or high-carbohydrate, high-fat diet + oak bark extract (0.5 mL/kg food) for 16 weeks. Oak bark extract was added to the diets for last 8 weeks of the feeding period. Secondly, SHR aged 42 weeks fed on standard chow diet were divided into two groups with and without oak bark extract treatment for 12 weeks (0.5 mL/kg food). The high-carbohydrate, high-fat diet induced signs of metabolic syndrome along with cardiovascular remodelling and non-alcoholic steatohepatitis. Oak bark extract attenuated the signs of metabolic syndrome in high-carbohydrate, high-fat diet-fed rats and improved the structure and function of the heart and the liver. SHR after oak bark extract treatment for 12 weeks showed lower systolic blood pressure, lower cardiac fibrosis and cardiac stiffness and improved vascular reactivity. Oak bark extract containing ellagitannins improved cardiovascular, metabolic and liver parameters in these rat models of human disease, suggesting that part of the benefits attributed to red wine may be produced by these ellagitannins.

Sequence Analysis and Characterization of a Transferable Hybrid Plasmid Encoding Multidrug Resistance and Enabling Zoonotic Potential for Extraintestinal Escherichia coli

ColV plasmids of extraintestinal pathogenic Escherichia coli (ExPEC) encode a variety of fitness and virulence factors and have long been associated with septicemia and avian colibacillosis. These plasmids are found significantly more often in ExPEC, including ExPEC associated with human neonatal meningitis and avian colibacillosis, than in commensal E. coli. Here we describe pAPEC-O103-ColBM, a hybrid RepFIIA/FIB plasmid harboring components of the ColV pathogenicity island and a multidrug resistance (MDR)-encoding island. This plasmid is mobilizable and confers the ability to cause septicemia in chickens, the ability to cause bacteremia resulting in meningitis in the rat model of human disease, and the ability to resist the killing effects of multiple antimicrobial agents and human serum. The results of a sequence analysis of this and other ColV plasmids supported previous findings which indicated that these plasmid types arose from a RepFIIA/FIB plasmid backbone on multiple occasions. Comparisons of pAPEC-O103-ColBM with other sequenced ColV and ColBM plasmids indicated that there is a core repertoire of virulence genes that might contribute to the ability of some ExPEC strains to cause high-level bacteremia and meningitis in a rat model. Examination of a neonatal meningitis E. coli (NMEC) population revealed that approximately 58% of the isolates examined harbored ColV-type plasmids and that 26% of these plasmids had genetic contents similar to that of pAPEC-O103-ColBM. The linkage of the ability to confer MDR and the ability contribute to multiple forms of human and animal disease on a single plasmid presents further challenges for preventing and treating ExPEC infections.

Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

BackgroundAlthough the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc.Methodology/Principal FindingsWe report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype.Conclusions and SignificanceThe efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

MRI‐based large deformation high dimensional mapping of the hippocampus in rats: Development and validation of the technique

To report the detection of structural and functional biological changes in living animals using small animal in vivo MRI that complements traditional ex vivo histological techniques. We report the development and validation of the application of large deformation high dimensional mapping (HDM-LD) segmentation for the hippocampus in the rat. High resolution volumetric T2 weighted MRI images were acquired at 4.7 Tesla from six male in-breed nonepileptic Wistar rats. Two HDM-LD segmentations of the hippocampus (automated 1 and automated 2) were compared with the manual segmentations of two investigators who independently segmented the hippocampi (manual 1 and manual 2). The mean overlap for the hippocampi between automated 1 and automated 2 for the right hippocampi was 94.4% (SD 1.0) and for the left hippocampi was 94.3% (SD 2.5), while the mean overlap between automated 1 and manual 1 for the right hippocampi was 91.4% (SD 1.3) and for the left hippocampi was 91.9% (SD 1.4). Mean values for absolute differences for comparisons of all the segmentations were the following: automated 1 versus automated 2, 3.2% (SD 1.0); manual 1 versus manual 2 6.82% (SD 5.22); automated 1 versus manual 1 13.0% (SD 1.8). HDM-LD can be applied to obtain accurate and reproducible three-dimensional segmentations of the hippocampus from rat MR images. HDM-LD will be a useful tool for investigations of hippocampal structural changes in vivo in rat models of human disease.

Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 microg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 microg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.

Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease

We aimed to identify genetic variants associated with heart failure by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses between spontaneously hypertensive heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2 segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload-induced heart failure and cardiac arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species role for Ephx2 in this complex disease.

Soluble epoxide hydrolase (Ephx2) is a susceptibility gene for heart failure in a rat model of human disease 3044

We aimed to idenify heart failure genes by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses using spontaneously hypertensive heart failure rats (SHHF) and reference strains. We combined linkage analyses with genome wide expression profiling and identified Ephx2 as heart failure susceptibility gene in SHHF. We demonstrate that cis‐variation at Ephx2 co‐segregated with heart failure, increased transcript expression, protein expression, and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we next tested the role of Ephx2 in heart failure by using knockout mice. We show that Ephx2 gene ablation protects from pressure overload‐induced heart failure and cardiac arrhythmias. We further demonstrate differential regulation of Ephx2 in human heart failure suggesting a cross‐species role for Ephx2 in this complex disease.

Adrenalectomy Blocks the Compensatory Increases in UT-A1 and AQP2 in Diabetic Rat Kidney

In normal rats we showed that glucocorticoids participate in the downregulation of UT-A1 protein abundance in the inner medullary tip and in lowering of basal and vasopressin-stimulated facilitated urea permeability in terminal IMCDs. To examine the relevance of this response to a rat model of human disease, we studied rats with uncontrolled diabetes mellitus (DM) induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We found that at 3 days of DM, UT-A1 protein abundance is downregulated in the inner medullary tip compared to pair-fed control rats, while DM for more than 7 days caused an increase in UT-A1. To test whether adrenal steroids could be a mechanism contributing to the latter increase, we studied adrenalectomized rats (ADX), ADX rats given STZ to induce diabetes (ADX + STZ), and ADX + STZ rats receiving exogenous aldosterone or dexamethasone. In contrast to control rats, UT-A1 protein abundance was not increased by prolonged DM in the ADX rats. Aquaporin 2 (AQP2) was not increased in the inner medullas of 10-day DM rats either. However, UT-A1 protein abundance was significantly reduced in the inner medullary tips from both diabetic aldosterone-treated (40 +/- 2%) and dexamethasone-treated (43 +/- 2%) ADX rats compared to diabetic ADX rats without steroid replacement. AQP2 was unaffected by steroid hormone treatments. Thus, both mineralocorticoids and glucocorticoids downregulate UT-A1 protein abundance in rats with uncontrolled diabetes mellitus for 10 days. These results suggest that: 1) the increase in UT-A1 observed in DM is dependent upon having adrenal steroids present; and 2) adrenal steroids are not sufficient to enable the compensatory rise in UT-A1 to a steroid-deficient diabetic animal.

Expression, purification, and characterization of rat interferon-β, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters

To identify potential new clinical uses and routes of administration for human interferon-β-1a (IFN-β-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-β (IFN-β) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-β signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to >99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1 × 10 8 U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-β and human IFN-β-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-β in which a 20 kDa methoxy polyethylene glycol (PEG)-propionaldehyde was attached to the N-terminal α-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-β will be useful for testing in rat models of human disease.

N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine -1(2H)-carbox-amide (BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic properties: I. in vitro characterization and pharmacokinetic properties.

Vanilloids such as capsaicin have algesic properties and seem to mediate their effects via activation of the vanilloid receptor 1 (VR1), a ligand-gated ion channel highly expressed on primary nociceptors. Although blockade of capsaicin-induced VR1 activation has been demonstrated in vitro and in vivo with the antagonist capsazepine, efficacy in rat models of chronic pain has not been observed with this compound. Here, we describe the in vitro pharmacology of a highly potent VR1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC). Similar to capsazepine, this compound inhibits capsaicin-induced activation of rat VR1 with an IC50 value of 35 nM. Interestingly however, BCTC also potently inhibits acid-induced activation of rat VR1 (IC50 value of 6.0 nM), whereas capsazepine is inactive. Similarly, in the rat skin-nerve preparation both BCTC and capsazepine block capsaicin-induced activation, whereas the response to acidification is inhibited by BCTC, but not by capsazepine. Specificity for VR1 was demonstrated against 63 other receptor, enzyme, transporter, and ion channel targets. BCTC was orally bioavailable in the rat, demonstrating a plasma half-life of approximately 1 h and significant penetration into the central nervous system. Thus, BCTC is a high potency, selective VR1 antagonist that, unlike capsazepine, has potent blocking effects on low pH-induced activation of rat VR1. These properties make it a more suitable candidate than capsazepine for testing the role played by VR1 in rat models of human disease.