Animal Model For Preclinical Evaluation Research Articles

A rhesus macaque intragastric challenge model for evaluating the safety, immunogenicity, and efficacy of live-attenuated Shigella dysenteriae 1 vaccine candidates.

Shigellosis remains a significant global health challenge, particularly in Asia and Africa, where it is a major cause of morbidity and mortality among children. Despite the urgent need, the development of a licensed Shigella vaccine has been hindered, partly due to the lack of suitable animal models for preclinical evaluation. In this study, we used an intragastric adult rhesus macaque challenge model to evaluate the safety, immunogenicity, and efficacy of five live-attenuated Shigella dysenteriae 1 vaccine candidates, all derived from the 1617 parent strain. The vaccine strains included WRSd1, a previously tested candidate with deletions in virG(icsA), stxAB, and fnr, and four other strains-WRSd2, WRSd3, WRSd4, and WRSd5-each containing deletions in virG and stxAB, but retaining fnr. Additionally, WRSd3 and WRSd5 had further deletions in the Shigella enterotoxin gene senA and its paralog senB, with WRSd5 having an extra deletion in msbB2. Rhesus monkeys were immunized three times at two-day intervals with a target dose of 2 × 1010 CFU of the vaccine strains. Thirty days after the final immunization, all monkeys were challenged with a target dose of 2 × 109 CFU of the S. dysenteriae 1 1617 wild-type strain. Safety, immunogenicity, and efficacy were assessed through physical monitoring and the evaluation of immunologic and inflammatory markers following immunization and challenge. Initial doses of WRSd1, WRSd3, and WRSd5 led to mild adverse effects, such as vomiting and loose stools, but all five vaccine strains were well tolerated in subsequent doses. All strains elicited significant IgA and IgG antibody responses, as well as the production of antibody-secreting cells. Notably, none of the vaccinated animals exhibited shigellosis symptoms such as vomiting or loose/watery stool post-challenge, in stark contrast to the control group, where 39% and 61% of monkeys exhibited these symptoms, respectively. The aggregate clinical score used to evaluate Shigella attack rates post-challenge revealed a 72% attack rate in control animals, compared to only 13% in vaccinated animals, indicating a relative risk reduction of 81%. This study highlights the potential of this NHP model in evaluating the safety, immunogenicity, and efficacy of live-attenuated Shigella vaccine candidates, offering a valuable tool for preclinical assessment before advancing to Phase 1 or more advanced clinical trials.

Experimental Animal Models for Preclinical Evaluation of Symmetrical Diiodinated Squaraine Dye

Experimental Animal Models for Preclinical Evaluation of Symmetrical Diiodinated Squaraine Dye

Animal model considerations to evaluate prosthetic tricuspid valve implants

AimsDetermining tricuspid valve comparative anatomy and appropriate animal models for preclinical evaluation of prosthetic tricuspid valve implants. Methods and resultsWe described and measured 81 heart specimens: 12 humans, 22 dogs, 21 sheep and 26 pigs. Tricuspid annulus circumference varied in humans from 109 to 149 mm, in pigs from 85 to 140 mm, and were ≤125 mm in dogs and sheep. Tricuspid leaflet demarcation in dogs is similar to humans, while in pigs and sheep we observed three distinct leaflets. In humans, sheep and pigs, papillary muscle positions are similar. In dogs they are all based on the septum. A moderator band was observed in all species, but was of consistent thickness only in sheep. ConclusionsSheep and pigs are relevant animal models for evaluating prosthetic tricuspid valve implants. Seventy to 90 kg pigs have a tricuspid annulus size comparable to that in a dilated human heart, but due to possible fast growth leading to sizing incompatibilities, this represents a model for short-term study. Sheep are more stable in size for long term study, however, their tricuspid annulus size is the most similar to that in a healthy, non-dilated human heart. Dogs are not a suitable model due to their significantly different sub-valvular anatomy and smaller size.

A Novel Rat Model to Test Intra-Abdominal Anti-adhesive Therapy.

Background: Adhesion formation after abdominal surgery is considered almost inevitable and a major cause of morbidity. Novel treatments have been proposed, however there is a lack of suitable small animal models for pre-clinical evaluation, mainly due to inconsistency in adhesion formation in positive control animals. Here, we propose a new rat model of abdominal adhesions using Kaolin as the adhesion-inducing agent at an optimized dosage for testing newer agents in respect to their anti-adhesive property.Materials and Methods: Twenty-five adult (8–10 week old) male Wistar albino rats underwent midline laparotomy and caecal abrasion and were randomized to receive topical applications of normal saline or different concentrations and volumes of a Kaolin-based formulation. At day 14 rats were humanely killed, and adhesions graded macroscopically by an investigator blinded to the treatment groups, using pre-determined adhesion scores and microscopically using histopathology.Results: Kaolin at 0.005 g/mL caused consistent adhesions without compromising rat viability. At higher doses significant morbidity and mortality was observed in the animals treated.Conclusions: Kaolin induced adhesion in a rat abdominal surgery model is reliable and can be safely used to test the efficacy of novel anti-adhesive formulations to prevent intra-abdominal adhesions.

2202. Validation of a Rabbit Model of Pseudomonas aeruginosa Acute Pneumonia

BackgroundSeverity and antimicrobial resistance of P. aeruginosa (PA) hospital-acquired pneumonia led the FDA to encourage the development of animal models for preclinical evaluation of new therapeutic strategies. We present here the validation of a rabbit model of PA acute pneumonia.MethodsRabbits were infected by endotracheal instillation of 1.8 mL of a standardized inoculum containing 9 × 107 CFU of PA clinical strain 6206 (predetermined 100% lethal dose). The natural history of the disease was described by the following parameters evaluated at 3, 4, 5, 6, 10 hours post-infection (hpi) and at the time of spontaneous death (6 rabbits/group): lung-to-body weight ratio (LW/BW), pulmonary, splenic and renal bacterial counts, pulmonary histology and blood markers (blood cell counts, blood gas and IL-8). Three groups of 12 rabbits were then treated with saline (controls), tobramycin or meropenem at doses determined by PK/PD analysis to confirm the efficacy of a human-equivalent dosing regimen.ResultsPA strain 6,206 caused fatal pneumonia in 13–23 hours by acute respiratory distress syndrome (pulmonary edema and necrosis with LW/BW > 10, pO2 <40 mmHg) and/or sepsis (hyperlactatemia, hypoglycemia, cytopenias). LW/BW and pulmonary bacterial counts increased significantly over time. The splenic and renal bacterial spread was constant after 6 hpi. Hypoxemia <60 mmHg appeared at 5 hpi for 4/6 rabbits, associated with elevated plasma IL-8 concentration, massive neutrophilic influx into the airspace, lung necrosis, hemorrhage, and pulmonary edema formation. Consequently, 5 hpi appeared as the most appropriate time to trigger a therapeutic intervention. Meropenem (80 mg/kg/q2h) or tobramycin (1 injection of 2.5 mg/kg, then saline/q2h) showed superiority over saline, with a mortality rate of 33% and 17% vs. 100%, and an LW/BW ratio of 8.53 and 8.54 vs. 13.9, respectively. Tobramycin was less effective than meropenem in clearing bacteria, with, respectively, 1 and 9 out of 12 rabbits having sterile samples.ConclusionThis rabbit model of PA acute pneumonia is a reliable evaluation tool for new therapeutic strategies. Our study also provides guidance for the development of animal models by describing the natural history of disease and therapeutic validation. Disclosures All authors: No reported disclosures.

Promise, progress, and problems in whole disc tissue engineering.

Intervertebral disc degeneration is frequently implicated as a cause of back and neck pain, which are pervasive musculoskeletal complaints in modern society. For the treatment of end stage disc degeneration, replacement of the disc with a viable, tissue‐engineered construct that mimics native disc structure and function is a promising alternative to fusion or mechanical arthroplasty techniques. Substantial progress has been made in the field of whole disc tissue engineering over the past decade, with a variety of innovative designs characterized both in vitro and in vivo in animal models. However, significant barriers to clinical translation remain, including construct size, cell source, culture technique, and the identification of appropriate animal models for preclinical evaluation. Here we review the clinical need for disc tissue engineering, the current state of the field, and the outstanding challenges that will need to be addressed by future work in this area.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Sensitivity of osteosarcoma cells to HDAC inhibitor AR-42 mediated apoptosis

BackgroundOsteosarcoma (OS) is the most common primary bone tumor in both humans and dogs and is the second leading cause of cancer related deaths in children and young adults. Limb sparing surgery along with chemotherapy has been the mainstay of treatment for OS. Many patients are not cured with current therapies, presenting a real need for developing new treatments. Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer agents. In this study, we investigated the activity of the novel HDAC inhibitor AR-42 in a panel of human and canine OS cell lines.MethodsThe effect of AR-42 and suberoylanilide hydroxamic acid (SAHA) alone or in combination with doxorubicin on OS cell viability was assessed. Induction of histone acetylation after HDAC inhibitor treatment was confirmed by Western blotting. Drug-induced apoptosis was analyzed by FACS. Apoptosis was assessed further by measuring caspase 3/7 enzymatic activity, nucleosome fragmentation, and caspase cleavage. Effects on Akt signaling were demonstrated by assessing phosphorylation of Akt and downstream signaling molecules.ResultsAR-42 was a potent inhibitor of cell viability and induced a greater apoptotic response compared to SAHA when used at the same concentrations. Normal osteoblasts were much less sensitive. The combination of AR-42 with doxorubicin resulted in a potent inhibition of cell viability and apparent synergistic effect. Furthermore, we showed that AR-42 and SAHA induced cell death via the activation of the intrinsic mitochondrial pathway through activation of caspase 3/7. This potent apoptotic activity was associated with the greater ability of AR-42 to downregulate survival signaling through Akt.ConclusionsThese results confirm that AR-42 is a potent inhibitor of HDAC activity and demonstrates its ability to significantly inhibit cell survival through its pleiotropic effects in both canine and human OS cells and suggests that spontaneous OS in pet dogs may be a useful large animal model for preclinical evaluation of HDAC inhibitors. HDAC inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in both canine and human OS.

Development of bioluminescence imaging of respiratory syncytial virus (RSV) in virus-infected live mice and its use for evaluation of therapeutics and vaccines

Respiratory Syncytial virus (RSV) is one of the leading causes of pneumonia among infants with no human vaccine or efficient curative treatments. Efforts are underway to develop new RSV vaccines and therapeutics. There is a dire need for animal models for preclinical evaluation and selection of products against RSV. Herein, we developed a whole body bioluminescence imaging to follow replication of RSV A2 virus strain expressing firefly luciferase (RSVA2-line19-FFL) in live BALB/c mice that can be used as an extremely sensitive readout for studying effects of antiviral and vaccines in living mice. Strong bioluminescence signal was detected in the nasal cavity and in the lungs following intranasal infection of mice with RSVA2-line19-FFL. The kinetics of viral replication in lungs quantified by daily live imaging strongly correlated with viral titers measured by ex-vivo plaque assay and by assessing viral RNA by qRT-PCR. Vaccination of mice with a pre-fusion F protein elicited high neutralizing antibody titers conferring strong protective immunity against virus replication in the nasal cavity and lungs. In contrast, post-challenge treatment of mice with the monoclonal antibody Palivizumab two days after infection reduced viral replication in the nasal cavity at day 4, but only modestly reduced virus loads in the lungs by day 5. In contrast to RSV bioluminescence, plaque assay did not detect viral titers in lungs on day 5 in Palivizumab-treated animals. This difference between viral loads measured by the two assays was found to be due to coating of virions with the Palivizumab that blocked infection of target cells in vitro and shows importance of live imaging in evaluation of RSV therapeutics. This recombinant RSV based live imaging animal model is convenient and valuable tool that can be used to study host dissemination of RSV and evaluation of antiviral compounds and vaccines against RSV.

Oseltamivir Population Pharmacokinetics in the Ferret: Model Application for Pharmacokinetic/Pharmacodynamic Study Design.

The ferret is a suitable small animal model for preclinical evaluation of efficacy of antiviral drugs against various influenza strains, including highly pathogenic H5N1 viruses. Rigorous pharmacokinetics/pharmacodynamics (PK/PD) assessment of ferret data has not been conducted, perhaps due to insufficient information on oseltamivir PK. Here, based on PK data from several studies on both uninfected and influenza-infected groups (i.e., with influenza A viruses of H5N1 and H3N2 subtypes and an influenza B virus) and several types of anesthesia we developed a population PK model for the active compound oseltamivir carboxylate (OC) in the ferret. The ferret OC population PK model incorporated delayed first-order input, two-compartment distribution, and first-order elimination to successfully describe OC PK. Influenza infection did not affect model parameters, but anesthesia did. The conclusion that OC PK was not influenced by influenza infection must be viewed with caution because the influenza infections in the studies included here resulted in mild clinical symptoms in terms of temperature, body weight, and activity scores. Monte Carlo simulations were used to determine that administration of a 5.08 mg/kg dose of oseltamivir phosphate to ferret every 12 h for 5 days results in the same median OC area under the plasma concentration-time curve 0–12 h (i.e., 3220 mg h/mL) as that observed in humans during steady state at the approved dose of 75 mg twice daily for 5 days. Modeling indicated that PK variability for OC in the ferret model is high, and can be affected by anesthesia. Therefore, for proper interpretation of PK/PD data, sparse PK sampling to allow the OC PK determination in individual animals is important. Another consideration in appropriate design of PK/PD studies is achieving an influenza infection with pronounced clinical symptoms and efficient virus replication, which will allow adequate evaluation of drug effects.

Methods in Molecular Biology, Volume 716: Drug Design and Discovery: Methods and Protocols

Over the past few years, small molecule screening has undergone a dramatic shift from simple ‘cheap and fast’ high-throughput assay systems to more complex screening setups where one aims to use more physiologically relevant systems. The new edition of ‘Methods in Molecular Biology: Drug Design and Discovery’ aims to cover aspects of this new approach with a comprehensive volume about various strategies in drug discovery. This volume is an easily accessible collection of protocols covering a broad range of different screening methodologies. The book is divided into 16 chapters that cover topics such as the design of large therapeutic molecules (DNA or peptides), revised protocols for the use of conventional assays in primary screening, computational methods and animal models for pre-clinical evaluation of drug candidates. One particular emphasis is the use of computational methods in drug discovery, which is covered in the first four chapters of the book. Chapter 4 (‘Methods for evaluation of structural and biological properties of anti-invasive natural products’) deserves particular mention in this regard, and the authors provide protocols for isolation and purification of compounds, the use of 3D model systems in cell culture and 3D QSAR modelling applications. Another major emphasis of this book is the use of animal models in primary screening (such as chapter 12, ‘Chemical screening with zebrafish embryos’ and chapter 10, ‘Imaging NFkB signaling in mice for screening anticancer drugs’) or in pre-clinical testing (chapter 15, ‘Evaluation of antibacterial activity of proteins and peptides using a specific animal model for wound healing’). Other noteworthy areas covered are the use of mass spectroscopy, in vivo imaging in drug discovery, and the use of in vitro phage display for selection of peptide ligands in human placental transcytosis systems (chapter 8; clearly a gem in this book). The strong academic background of the authors makes this book highly recommendable to everyone involved in drug screening and has a particular appeal to an academic audience. Written in the highly successful format of the Methods in Molecular Biology series, each chapter includes a background on the topic, a list of materials required, a detailed step-by-step description of the methods and a notes section that draws attention to potential pitfalls and highlights some of the key steps in the procedure. All protocols provide very detailed instructions, a characteristic of the Methods in Molecular Biology series. The only minor drawback of this volume is the slightly misleading title. This collection of protocols touches very briefly on drug design. The book rather focuses on novel approaches to drug screening and physiological validation. It is highly recommended for all those who are performing drug screening or who aim to be doing so in the near future.

Enhancing translation: Guidelines for standard pre-clinical experiments in mdx mice

Duchenne Muscular Dystrophy is an X-linked disorder that affects boys and leads to muscle wasting and death due to cardiac involvement and respiratory complications. The cause is the absence of dystrophin, a large structural protein indispensable for muscle cell function and viability. The mdx mouse has become the standard animal model for pre-clinical evaluation of potential therapeutic treatments. Recent years have seen a rapid increase in the number of experimental compounds being evaluated in the mdx mouse. There is, however, much variability in the design of these pre-clinical experimental studies. This has made it difficult to interpret and compare published data from different laboratories and to evaluate the potential of a treatment for application to patients. The authors therefore propose the introduction of a standard study design for the mdx mouse model. Several aspects, including animal care, sampling times and choice of tissues, as well as recommended endpoints and methodologies are addressed and, for each aspect, a standard procedure is proposed. Testing of all new molecules/drugs using a widely accepted and agreed upon standard experimental protocol would greatly improve the power of pre-clinical experimentations and help identifying promising therapies for the translation into clinical trials for boys with Duchenne Muscular Dystrophy.

Pseudoanaphylactoid reaction analysis of Chinese herbal injections in Beagle dogs

To investigate the preclinical evaluation method of pseudoanaphylactoid reactions for Chinese herbal injections. Beagle dogs were divided into control group (C), 0.5% tween 80 group (T), Yuxincao injection containing 0.5% tween 80 (YT), distilled solution from Yuxincao (Y). Various groups of Beagle dogs were given 3 mL x kg(-1) of the test articles intravenously. The anaphylactoid reactions were observed immediately, while blood pressure, respiratory frequencies and heart rates were tested at 10 min and 30 min after administration. A variety of symptoms that range from cutaneous and mucosa signs to bronchospasma and cardiovascular collapse, including angioedema at lip, conjunctiva, ear and circumoral skin, somnolence, lethargy, breathless or dyspnea, severe hypotension etc were observed in T and TY groups from immediately post-injection to at least 30 min after administration. These reactions occurred at both first injection or repeated injections at 24 weeks intervals, manifesting that it was pseudoanaphylactoid reaction mediated by non-immune mechanisms. Beagle dogs could be used as an animal model for preclinical evaluation of pseudoanaphylactoid reactions of Chinese herbal injection with sensitivity, reproducibility, and high clinic consistency.

Preclinical Testing of Human Recombinant von Willebrand Factor: ADAMTS13 Cleavage Capacity in Animals as Criterion for Species Suitability

Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 ( A Disintegrin and Metalloproteinase with Thrombo Spondin repeats, number 13) that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. In vitro and ex vivo studies have shown that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine ADAMTS13. In contrast, rabbit and cynomolgus ADAMTS13 is able to cleave human rVWF. These findings were consistent with in vivo results showing distinct pharmacological behavior of human rVWF depending on the cleaving capacity of ADAMTS13 present in the species tested. Studies were performed using three mouse strains (ADAMTS13 deficient, C57BL/6J [wild type], VWF deficient), rats, rabbits, and cynomolgus monkeys. All animals were infused once with different doses of human rVWF and, in addition, 14 daily doses were given to rats and cynomolgus monkeys. Exaggerated pharmacological effects were observed in mice, with the ADAMTS13 knockout mouse being the most sensitive strain. Similar findings with decreased incidence and severity were seen in normal C57BL/6J mice and also in VWF-deficient mice, where they were least pronounced. In rats, exaggerated pharmacological effects were observed only after 14 doses. Rabbits and cynomolgus monkeys showed no exaggerated pharmacological effects. These differences between species and between mouse strains suggest that the efficiency of ADAMTS13 to cleave rVWF determines the severity of clinical, laboratory and pathohistological findings. These observations highlight the importance of evaluating species' suitability for the generation of meaningful preclinical data for determining the therapeutic safety margins for human patients. Only animals with a sufficient rVWF cleavage capacity by endogenous ADAMTS13 (rabbits and cynomolgus monkeys) are considered appropriate animal models for preclinical evaluation of the rVWF product.

In vivo preclinical evaluation of the accuracy of toroidal-shaped HIFU treatments using a tumor-mimic model

The pig is an ideal animal model for preclinical evaluation of HIFU treatments, especially in the liver. However, there is no liver tumor model available for pigs. In this work, we propose to study an in vivo tumor-mimic model as a tool for evaluating if a sonographycally guided HIFU treatment, delivered by a toroidal-shaped device dedicated for the treatment of liver metastases, is correctly located in the liver. One centimeter tumor-mimics were created in liver tissues. These tumor-mimics were detectable on ultrasound imaging and on gross pathology. Two studies were carried out. First, an in vivo study of tolerance at mid-term (30 days, 10 pigs) revealed that tumor-mimics are suitable for studying HIFU treatments at a preclinical stage, since local and biological tolerances were excellent. The dimensions of the tumor-mimics were reproducible (diameter at day 0: 9.7 ± 2.0 mm) and were the same as a function of time (p = 0.64). A second in vivo study was carried out in ten pigs. Tumor mimics were used as targets in liver tissues in order to determine if the HIFU treatment is correctly located in the liver. A procedure of extensive HIFU ablation using multiple HIFU lesions juxtaposed manually was then tested on eight tumor-mimics. In 88% of the cases (seven out of eight), tumor-mimics were treated with negative margins (⩾1 mm) in all directions. On average, negative margins measured 10.0 ± 6.7 mm. These tumor-mimics constitute an excellent reference for studying in vivo the accuracy of HIFU therapy in the liver.

Flow cytometric assays for quantifying activated ovine platelets.

Ovines are a common animal model for preclinical evaluation of cardiovascular devices including heart valves, endovascular grafts, and ventricular assist devices. Biocompatibility is essential to the success of these devices; however, tools to assess biocompatibility in ovines are limited. To address this need, antibodies that bind to activated human and bovine platelets and annexin V protein were evaluated for potential cross-reactivity to activated ovine platelets. These candidate markers were incubated with stimulated and quiescent ovine whole blood, and binding to platelets was quantified by flow cytometry. Several antihuman CD62P antibodies including one polyclonal antibody, three monoclonal antibodies, and annexin V selectively bound to activated ovine platelets. An assay to quantify platelet microaggregates was also developed. The availability of assays to quantify ovine platelet activation can increase the quality of biocompatibility data obtainable during preclinical development of artificial organs in the ovine model, potentially aiding in the evaluation of design refinements to enhance device biocompatibility.

Pharmacokinetic–pharmacodynamic modeling of diltiazem in spontaneously hypertensive rats: A microdialysis study

Introduction The aim of the present work was to study the applicability of a modified E max pharmacodynamic model for the pharmacokinetic-pharmacodynamic (PK–PD) modeling of diltiazem in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Methods A “shunt” microdialysis probe was inserted in a carotid artery of anaesthetized SHR and WKY rats for simultaneous determination of unbound plasma concentrations of diltiazem and their effects on mean arterial pressure (MAP) and heart rate (HR) after the intravenous application of 1 and 3 mg kg − 1 of the drug. Correlation between diltiazem plasma levels and their cardiovascular effects was established by fitting the data to a conventional and modified E max model. Results Volume of distribution and clearance of diltiazem was greater in SHR than in WKY animals. A proportional increase of area under curve with dose increment was observed in WKY animals but not in SHR. A good correlation between plasma unbound concentrations of diltiazem and their hypotensive and chronotropic effects was found in both experimental groups using both PK–PD models. The application of the modified E max model for PK–PD modeling of diltiazem allowed a more accurate and precise estimation of PK–PD parameters than the E max equation do. Chronotropic effect of 3 mg kg − 1 diltiazem was lower in SHR compared to WKY animals. Initial sensitivity ( S 0) to diltiazem chronotropic effect was greater in SHR with regards to WKY animals after administration of 1 mg kg − 1 . S 0 to diltiazem hypotensive effect was greater in SHR with regards to WKY animals after administration of both doses of diltiazem. Discussion Microdialysis sampling is a useful technique for the pharmacokinetic study and pharmacokinetic–pharmacodynamic (PK–PD) modeling of diltiazem. The modified E max model allows an accurate estimation of drug sensitivity in conditions when maximal pharmacological response can not be attained. Genetic hypertension induced changes in the pharmacokinetic and PK–PD behavior of diltiazem suggesting that SHR is an interesting animal model for pre-clinical evaluation of calcium channel blockers.

Lentiviral Vectors Encoding HIV-1 Polyepitopes Induce Broad CTL Responses In Vivo

Lentiviral Vectors Encoding HIV-1 Polyepitopes Induce Broad CTL Responses In Vivo

Improvement of Canine XSCID by In Vivo Gene Therapy Using RD114/TR-Pseudotyped SIV Lentiviral Vectors.

X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency (dysfunctional B cells; absence of T and NK cells) and early mortality, caused by mutations in the IL2RG gene encoding the common gamma chain (γc) of receptors for interleukins (IL)-2,-4,-7,-9,-15 and -21. The standard therapy for XSCID in infants is a T cell depleted bone marrow transplant with either none or very modest conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution that may be limited to only the T cell lineage. Ex vivo autologous stem cell gene therapy has emerged as an alternate treatment capable of achieving substantial to complete immune reconstitution in infants without a sibling donor. However, some children have developed lymphocytic leukemia, which appears in part to be related to vector insertional mutagenesis. The gammaretroviruses currently in use have potent enhancers in the LTR and have a predilection to insert at the 5′ end of genes. Self-inactivating (SIN) lentivirus vectors may be advantageous because they do not show this property and can be constructed with internal promotors that have less enhancer activity together with insertion of insulators. We have constructed SIN simian immunodeficiency viral vectors (SIVmac) encoding human γc (hγc), with or without a double copy chicken insulator core element in the 3′ LTR. In addition we have pseudotyped the vector with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicity of the VSV-G envelope traditionally used with lentivectors. We have previously shown that it is possible to correct a dog model of XSCID using a RD114-pseudotyped gammaretroviral vector encoding the dog γc for in vivo gene therapy. Unlike the mouse model of XSCID which lacks B cells, the dog model more closely resembles the phenotype in humans. We decided to use our in vivo dog model for preclinical testing of safety and efficacy of our SIV hγc vectors. Viral particles were produced by transient transfection of 293T cells with a 4-plasmid system and concentrated by high-speed centrifugation. 30 mls (average 2.4–3×107 viral particles in total) was injected IV into 2–5 day-old pups. Transgene marking in blood lymphocytes was detected as early as 2 wks after treatment, increased within the first 6–8 wks and became relatively stable thereafter. The absolute lymphocyte count was normalized in one dog (injected on the second day after birth) by wk 6 (4000, 76% hγc+), and improved in two other dogs (422, 29% hγc+ at 18 wks; 498, 27% hγc+, at 16 wks) which had received the same amount of virus over 2 days. Furthermore, up to 5% of the myeloid lineage showed gene marking at 10–18 wks after viral delivery, indicating that early committed progenitors or HSCs had been transduced. It is noteworthy that our finding that the hγc can improve the disease phenotype in XSCID dogs makes it an excellent large animal model for preclinical evaluation of vectors. In summary, we have demonstrated that in vivo delivery of SIV lentiviral vectors expressing hγc efficiently reconstitute the T-lymphoid compartment in the XSCID canine model. We plan to monitor these dogs closely for potential adverse events.

Tissue distribution of octreotide binding receptors in normal mice and strains prone to autoimmunity.

Somatostatin has diverse functions, including immunomodulatory functions. In humans, sites of active inflammation can be visualized by the administration of 111In-DTPA(0)-octreotide, a radiolabelled somatostatin analogue. We wished to establish an animal model for preclinical evaluation of the effects of somatostatin analogues on the immune system. However, most animal models for immunological diseases are murine. This report is a preliminary study of the distribution of somatostatin receptors in mouse tissues, with emphasis on the immune system. Tissue distribution of octreotide binding receptors in normal (BALB/c) mice was determined in vivo by receptor binding of 111In-DTPA(0)-octreotide and in vitro and ex vivo by receptor autoradiography. Additionally, we investigated the tissue distribution of octreotide binding receptors in inflammatory lesions in a murine model of immune mediated disease, i.e. pre-diabetic pancreatic infiltration in the non-obese diabetic mouse strain. High specific uptake of radioactivity was seen in the thymus (range 1-1.7% ID/g) and the pituitary (1-1.6% ID/g) in all mouse strains. Specific uptake was also found in the stomach (0.1-0.7% ID/g), in the adrenal glands (0.1-0.3% ID/g) and in the pancreas (0.1-0.3% ID/g). However, we did not detect increased uptake of radiolabelled octreotide in the pancreas of pre-diabetic NOD mice. Autoradiography on tissue sections confirmed the presence of octreotide binding sites in the tissues that showed specific uptake. Moreover, by using autoradiography we could localize the cortex of the thymus and the anterior part of the pituitary as the localization of specific and high affinity, octreotide binding sites. A high, but not a receptor mediated, uptake of radioactivity was seen in the kidneys and was significantly higher in females than in males (12-19% vs 4% ID/g, respectively). Our results point to profound species differences in the tissue distribution of octreotide binding receptors. Of particular interest is the high uptake of 111In-DTPA(0)-octreotide in the cortex of the mouse thymus. This offers perspectives for the use of this animal in studies concerning the effect of somatostatin analogues on the immune system. To our knowledge, this is the first report on the tissue distribution of octreotide binding receptors in mice.