Good Laboratory Practice Standards Research Articles

Sensitivity assessment of Biomphalaria glabrata (SAY, 1818) using reference substance sodium dodecyl sulfate for ecotoxicological analyzes.

Sodium dodecyl sulfate (SDS) is a surfactant used and recommended by regulatory agencies as a reference substance in ecotoxicological analyzes. In this work, acute toxicity assays were performed with adults and embryos of the freshwater snail Biomphalaria glabrata, an endemic organism with environmental and public health importance, to evaluate the effects of the surfactant and establish a sensitivity control chart. The organisms were exposed to SDS for 24 h to a range of concentrations, as well as a control group. Six assays were performed to establish the control chart for adults (with a median LC50 of 36.87 mg L-1) and differential sensitivity was observed at each embryonic stage (EC50 = blastulae 33.03, gastrulae 35.03, trochophore 39.71 and veliger 72.55 mg L-1). The following behavioral responses were observed in exposed adult snails: release of hemolymph and mucus, body outside the shell, and penile overexposure. Embryos at the blastulae and gastrulae stages were more sensitive, and teratogenic effects were accentuated in the trochophore stage. The difference in results obtained between adults and embryos underscore the importance of carrying out analyzes at different developmental stages. The serial assays established with SDS for B. glabrata demonstrated efficiency and constancy conditions in the assays with good laboratory practice standards. The wide distribution of Biomphalaria species in several countries, their easy maintenance and cultivation in the laboratory, in addition to ecological importance, make them economical alternatives for ecotoxicological assays.

Dual-Purpose Aptamer-Based Sensors for Real-Time, Multiplexable Monitoring of Metabolites in Cell Culture Media.

The availability of high-frequency, real-time measurements of the concentrations of specific metabolites in cell culture systems will enable a deeper understanding of cellular metabolism and facilitate the application of good laboratory practice standards in cell culture protocols. However, currently available approaches to this end either are constrained to single-time-point and single-parameter measurements or are limited in the range of detectable analytes. Electrochemical aptamer-based (EAB) biosensors have demonstrated utility in real-time monitoring of analytes in vivo in blood and tissues. Here, we characterize a pH-sensing capability of EAB sensors that is independent of the specific target analyte of the aptamer sequence. We applied this dual-purpose EAB to the continuous measurement of pH and phenylalanine in several in vitro cell culture settings. The miniature EAB sensor that we developed exhibits rapid response times, good stability, high repeatability, and biologically relevant sensitivity. We also developed and characterized a leak-free reference electrode that mitigates the potential cytotoxic effects of silver ions released from conventional reference electrodes. Using the resulting dual-purpose sensor, we performed hourly measurements of pH and phenylalanine concentrations in the medium superfusing cultured epithelial tumor cell lines (A549, MDA-MB-23) and a human fibroblast cell line (MRC-5) for periods of up to 72 h. Our scalable technology may be multiplexed for high-throughput monitoring of pH and multiple analytes in support of the broad metabolic qualification of microphysiological systems.

Brain-targeted delivery of neuroprotective survival gene minimizing hematopoietic cell contamination: implications for Parkinson’s disease treatment

BackgroundNeurodegenerative diseases, including Parkinson's disease, Amyotropic Lateral Sclerosis (ALS) and Alzheimer's disease, present significant challenges for therapeutic development due to drug delivery restrictions and toxicity concerns. Prevailing strategies often employ adeno-associated viral (AAV) vectors to deliver neuroprotective survival genes directly into the central nervous system (CNS). However, these methods have been limited by triggering immunogenic responses and risk of tumorigenicity, resulting from overexpression of survival genes in peripheral blood mononuclear cells (PBMC), thereby increasing the risk of tumorigenicity in specific immune cells. Thus, by coding selectively suppressive microRNA (miRNA) target sequences in AAV genome, we designed CNS-targeted neuroprotective gene expression vector system without leakage to blood cells.MethodsTo minimize the potential for transgene contamination in the blood, we designed a CNS-specific AAV system. Our system utilized a self-complementary AAV (scAAV), encoding a quadruple repeated target sequence of the hematopoietic cell-specific miR142-3p at the 3' untranslated region (UTR). As a representative therapeutic survival gene for Parkinson’s disease treatment, we integrated DX2, an antagonistic splice variant of the apoptotic gene AIMP2, known to be implicated in Parkinson's disease, into the vector.ResultsThis configuration ensured that transgene expression was stringently localized to the CNS, even if the vector found its way into the blood cells. A single injection of scAAV-DX2 demonstrated marked improvement in behavior and motor activity in animal models of Parkinson’s disease induced by either Rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, comprehensive preclinical data adhering to Good Laboratory Practice (GLP) standards revealed no adverse effects in the treated animals.ConclusionsOur CNS-specific vector system, which encodes a survival transgene DX2, signifies a promising avenue for safe gene therapy, avoiding unintended expression of survival gene in blood cells, applicable to various neurodegenerative diseases.

Clear evidence of the carcinogenic potential of anthracene: A 2-year feeding study in rats and mice.

Anthracene is an environmental pollutant and its adverse effects on human health have long been a concern due to its persistence and bioaccumulation properties. However, there is insufficient evidence for its chronic toxicity, especially carcinogenicity, in either humans or experimental animals. In this study, its carcinogenicity and chronic toxicity were investigated in compliance with the OECD test guideline 451 (OECD TG 451) and Good Laboratory Practice Standards. Fifty male and 50 female F344 rats and 50 female BDF1 mice were administrated 0, 8000, 20000, or 50000 ppm anthracene in the diet for 104 weeks, and 50 male BDF1 mice were fed diets containing anthracene at 0, 3200, 8000, or 20000 ppm. Anthracene treatment had no adverse effect on either the survival rate or general condition of the rats or mice during the study period. Body weights were lower or tended to be lower in the anthracene-treated groups than in the control groups. Increased incidence of hepatocellular carcinoma and hepatocellular adenoma was observed in male rats and female mice. Renal cell carcinoma and renal cell adenoma, fibroadenoma in the mammary gland, and uterine endometrial stromal sarcoma were increased in female rats. Transitional cell carcinoma and transitional cell papilloma in the urinary bladder were also increased in male and female rats. In addition, several different pre-neoplastic lesions were increased in the anthracene-treated male and female rats and female mice. These results provide clear evidence that oral administration of anthracene for 104 weeks has a carcinogenic effect in male and female rats and female mice.

Estudio de propiedades y estructura de geles de aplicación tópica para uso en alvéolos dentarios postextracción

The process from an active ingredient to the final medicine requires pre-formulation and stability studies of the product developed under different conditions, as well as the standardization of the extract as raw material. The objective of this work was to characterize formulations for topical application (gels), for use in post-extraction tooth sockets. Materials and methods: The gels were made according to good laboratory practice standards. The pH of the gel formulations was measure with a pH meter (Eutech, Cyberscan) using 1% aqueous solutions of the gels at room temperature. Extensibility was determined with a wooden block and glass slide apparatus. The gel was added and the time it took too completely separate from the fixed was note. The spreading capacity was calculate using formulas (S = W × L / T) The measurement of the consistency of the gels was carried out by dropping a cone from a fixed distance of 10 cm in such a way that it lands in the center of a glass cup filled with the gel. Cone penetration was measured from the surface of the gel to the tip of the cone within the gel. The distance traveled by the cone after 10 seconds was recorded. To analyze the superficial topographic characteristics of the gels, photographs were taken with a confocal microscope. Results: All formulations are in a range of pH values close to neutrality (pH 6.4-6.9). The gel prepared with alendronate presented higher extensibility, while the others showed lower extensibility with similarities between them. The consistency analysis showed no differences between the formulations. The selected formulations presented a brown olive-green color with different intensities; they presented a homogeneous appearance to the naked eye, but heterogeneous when observed under a confocal microscope. Conclusions: In the characterization of the gels, show that they have a variable texture and consistency depends on the type and concentration of the active ingredient used. The most suitable gel formulation was alendronate.

The design and implementation of natural population cohort study Biobank: A multiple-center project cooperation with medical consortia in Southwest China.

The West China Hospital of Sichuan University collaborated with regional medical consortia in Sichuan Province to launch a natural population cohort study (NPCS) to investigate the health status of residents and collect public health data in southwest China. Up to 80,000 participants will be enrolled by the NPCS from 11 regional medical consortia over five years. Individuals are invited to visit one of 11 participating medical consortia to fill out questionnaires, receive a free health exam, and donate biospecimens upon enrolment. All participating medical facilities adhered to standard operating procedures for collecting and processing biospecimens to ensure uniformity (serum, lithium heparinized plasma, ethylene diamine tetraacetie acid plasma, and buffy coat). The Electronic Data Capture System, Picture Archiving and Communication System, Laboratory Information Management System, Biospecimen Quality Control System, Biobank Information Management System, and will be used to sort and classify clinical indices, imaging data, laboratory parameters, pre-analytical variables, and biospecimen information, respectively. All quality assurance and quality control procedures in the NPCS biobank adhered to the "DAIDS Guidelines for Good Clinical Laboratory Practice Standards". This project will integrate high-dimensional multi-omics data, laboratory data, clinical data, questionnaire data, and environmental risk factors. An estimated 2,240,000 aliquots of the sample will be stored by the end of the study. These samples are linked with comprehensively collected clinical indices, imaging data, and laboratory parameters. Big data analysis can be implemented to create predictive algorithms, explore pathogenesis mechanisms, uncover potential biomarkers, and provide information on public health. NPCS will provide an integrative approach to research risk factors and pathogenesis of major chronic or endemic diseases in Sichuan Province and provide key scientific evidence to support the formulation of health management policies in China.

Implementing OECD GLP principles for the evaluation of novel vector control tools: a case study with two novel LLINs, SafeNet® and SafeNet NF®

BackgroundTo sustain high universal Long-Lasting Insecticidal Nets (LLINs) coverage, affordable nets that provide equivalent or better protection than standard LLINs, are required. Test facilities evaluating new LLINs require compliance to Good Laboratory Practice (GLP) standards to ensure the quality and integrity of test data. Following GLP principles allows for the reconstruction of activities during the conduct of a study and minimizes duplication of efficacy testing. This case study evaluated the efficacy of two LLINs: SafeNet NF® and SafeNet® LLIN.MethodsThe study was conducted according to GLP principles and followed World Health Organization guidelines for evaluating LLINs. The LLINs were assessed in experimental huts against wild, pyrethroid-resistant Anopheles arabiensis mosquitoes. Nets were either unwashed or washed 20 times and artificially holed to simulate a used torn net. Blood-feeding inhibition and mortality were compared with a positive control (Interceptor® LLIN) and an untreated net.ResultsMosquito entry in the huts was reduced compared to negative control for the unwashed SafeNet NF, washed Safenet LLIN and the positive control arms. Similar exiting rates were found for all the treatment arms. Significant blood-feeding inhibition was only found for the positive control, both when washed and unwashed. All insecticide treatments induced significantly higher mortality compared to an untreated net. Compared to the positive control, the washed and unwashed SafeNet NF® resulted in similar mortality. For the SafeNet® LLINs the unwashed net had an equivalent performance, but the mortality for the washed net was significantly lower than the positive control.Internal audits of the study confirmed that all critical phases complied with Standard Operating Procedures (SOPs) and the study plan. The external audit confirmed that the study complied with GLP standards.ConclusionsSafeNet NF® and SafeNet® LLIN offered equivalent protection to the positive control (Interceptor® LLIN). However, further research is needed to investigate the durability, acceptability, and residual efficacy of these nets in the community. This study demonstrated that GLP-compliant evaluation of LLINs can be successfully conducted by African research institutions.

Accurate Quantification of AAV Vector Genomes by Quantitative PCR.

The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario.

Nonclinical Efficacy and Toxicity and Selection of a Safe Clinical Starting Dose for an NMT Inhibitor in Development for Hematological Malignancies

N-myristoylation is the addition of the 14-carbon fatty acid myristate to proteins. This plays a fundamental role in cell signaling: Over 500 proteins are myristoylated, including all 9 Src Family Kinases (SFK) Src, Lyn, Lck, Hck, and Fgr, as well as c-Abl, Gα subunits, caspase truncated (ct-) Bid and ct-PAK2, regulating cell growth and apoptosis. Human myristoylation is performed by two ubiquitously expressed N-myristoyltransferases NMT1 and NMT2. PCLX-001 is a new, orally bioavailable, small-molecule, dual NMT inhibitor under investigation as a novel and selective treatment for B-cell malignancies.In vitro, PCLX-001 inhibits the growth of hematological cancer cells at concentrations 10-fold lower than dasatinib and ibrutinib and prevents SFK recruitment to B cell receptor signaling complex, reducing survival signals and triggering apoptosis. PCLX-001 causes complete tumor regression in NOD/SCID mouse xenograft models of Acute Myeloid Leukemia (AML), Burkitt's Lymphoma (BL), and Diffuse Large B-cell Lymphoma (DLBCL), including drug-resistant human tumor in a PDX model. In mice, PCLX-001 produced complete remission of most xenografts at ≥35 mg/kg/day but also produced some deaths associated with bacterial infection and GI hemorrhage. In an AML xenograft mouse model, the drug exposure from oral dosing required to achieve 91% tumor inhibition was 3,423 ng*hr/mL (AUC (0-tlast)). When expressed on the basis of body surface area, the efficacious dose in mice was ≥105 mg/m2. To support clinical development, we evaluated PCLX-001 for safety in 4-week oral toxicity studies with 2-week recovery periods in rats at 50, 125, and 300 mg/kg/day and dogs at 2, 4, 8, and 12 mg/kg/day, performed to Good Laboratory Practice (GLP) standards. The highest non-severely toxic dose levels (HNSTDs) were 125 mg/kg/day for male rats, 300 mg/kg/day for female rats, and 4 mg/kg/day for dogs of both sexes. At higher dose levels, dose-limiting toxicity occurred within the first few days, was similar in both species, and was attributed to GI mucosal damage and decreased hematopoiesis, with secondary complications such as dehydration and inflammation. In dogs, systemic exposure to PCLX-001 increased more-than-proportionally with dose level after the first dose and did not change with repeated daily dosing. After the last dose at the HNSTD, Cmax averaged 651 ng/mL and 24-hour AUC averaged 3,760 h*ng/mL. In rats, systemic exposure to PCLX-001 after the first dose increased approximately proportionally with dose level and was much greater in males than females at all dose levels. With repeated daily dosing, exposure decreased dramatically in male rats but less so in female rats. As a result, exposure to PCLX-001 was similar in rats of both sexes after the last dose. After the last dose at the HNSTD in males and females, Cmax was 1650 and 8510 ng/mL, respectively, and 24-hour AUC averaged 6470 and 7590 h*ng/mL, respectively. When expressed on the basis of body surface area, the HNSTDs in male rats, female rats, and dogs were 750, 1800, and 80 mg/m2, respectively. As recommended in the ICH S9 Guidance, we chose a starting dose level of 20 mg/m2 for the first clinical trial because this was approximately 1/6th of the HNSTD in dogs. Given the dissimilarity of results between species, we compared the sequence of the NMTs in each since their inhibition was the target for PCLX-001. There was no significant difference between species in their sequence homology to humanNMT1(dog was 98%, rat 97% and mouse 99%), but dogNMT2had only 78% identity to humanNMT2while rat and mouseNMT2had 95% identity with humanNMT2. The significance of this dissimilarity is still under investigation. Disclosures Weickert: Pacylex Pharmaceuticals, Inc.:Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.Dillberger:Pacylex Pharmaceuticals, Inc.:Consultancy.Mackey:Pacylex Pharmaceuticals, Inc.:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.Berthiaume:Pacylex Pharmaceuticals, Inc.:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

A Circular Network of Coregulated Sphingolipids Dictates Lung Cancer Growth and Progression

Sphingolipid metabolism is one of the top dysregulated pathways in NSCLC. However, the molecular control of sphingolipid metabolic reprogramming in cancer progression remains unknown. Here, we first provide clinical evidence that expression of sphingolipid metabolic enzymes, B3GNT5 and GAL3ST1, but not the others, is most significantly associated with patient prognosis, whilst sphingolipidomics analysis of NSCLC patient sera identifies their metabolites, neolacto-series glycosphingolipid and sulfatide, as biomarkers that outperformed current clinical biomarkers for staging patients. Further network analysis of the sphingolipidomes reveals a circular network of coregulated sphingolipids, indicating that the neolacto-series glycosphingolipid/sulfatide balance functions as a checkpoint to determine sphingolipid metabolic reprograming during patient progression. Importantly, sphingolipidomics analysis of B3GNT5/GAL3ST1 genetically perturbed NSCLC cell lines confirms their key regulatory role in sphingolipid metabolism, while B3GNT5 and GAL3ST1 expression has an opposite role on tumorigenesis in vitro and in vivo. Overall, our results provide new insights whereby B3GNT5 and GAL3ST1 differentially regulate sphingolipid metabolism in lung cancer growth and progression. Funding Statement: This work was supported by the Natural Science Foundation of China (81920108028, 81872142); Guangzhou Science and Technology Program (201904020008); Young Teacher Foundation of Sun Yat-sen University (18ykzd07); Guangdong Science and Technology Department (2020B1212060018, 2019A1515011802, 2020A1515011280, 2020A0505100029); China Postdoctoral Science Foundation (2019M650226, 2019M650227). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: Serum samples were collected from healthy individuals and NSCLC patients (age-matched and body-mass index –matched) with informed consent, and the research program and all the related procedures were approved by the Ethics Committee of Sun Yat-sen Memorial Hospital (Guangzhou, China) and stored according to standard operating procedures for Good Clinical Laboratory Practice standards. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-Sen University.

Preclinical safety assessment of pathogen reduced red blood cells treated with amustaline and glutathione.

ABSTRACTBACKGROUNDThe nucleic acid targeted pathogen reduction (PR) system utilizing amustaline (S‐303) and glutathione (GSH) is designed to inactivate blood‐borne pathogens and leukocytes in red blood cell concentrates (PR‐RBCC). Inactivation is attained after amustaline intercalates and forms covalent nucleic acid adducts preventing replication, transcription, and translation. After pathogen inactivation, amustaline spontaneously hydrolyzes to S‐300, the primary negatively charged reaction product; amustaline is below quantifiable levels in PR‐RBCC. GSH quenches free unreacted amustaline.STUDY DESIGN AND METHODSThe genotoxic and carcinogenic potential of PR‐RBCC, the reaction by‐products, and S‐300 were assessed in accordance with the International Conference on Harmonization (ICH) guidelines and performed in compliance with the Food and Drug Administration (FDA) good laboratory practice standards, 21 CFR Part 58. in vitro bacterial reverse mutagenicity and chromosomal aberration assays were performed with and without exogenous S9 metabolic activation, and in in vivo clastogenicity and carcinogenic assays using validated murine models.RESULTSPR‐RBCCs were not genotoxic in vitro and in vivo and were non‐carcinogenic in p53+/− transgenic mice transfused over 26 weeks. Estimated safety margins for human exposure ranged from >90 to >36 fold for 2 to 5 PR‐RBCCs per day, respectively. PR‐RBCCs and S‐300 did not induce chromosome aberration in the in vivo murine bone marrow micronucleus assay at systemically toxic doses.CONCLUSIONSPR‐RBCCs did not demonstrate genotoxicity in vitro or in vivo and were not carcinogenic in vivo. These studies support the safety of PR‐RBCCs and suggest that there is no measurable genotoxic hazard associated with transfusion of PR‐RBCCs.

Conducting Assured Nonclinical Studies in the Pharmaceutical Industry: Good Laboratory Practice (GLP) Study, GLP Inspection, and Standards for Assurance

Many nonclinical and clinical studies are conducted to submit new drug applications for chemical entities. Nonclinical studies cover pharmacology, pharmacokinetic, and toxicology aspects and provide pharmacologic evidence as well as kinetic and toxicologic profiles of the compounds. The risks and benefits of compounds are evaluated based on these nonclinical studies, especially before initiation of the first human trials. Therefore, using adequate procedures, highly controlled, quality nonclinical data should be obtained. This section shares and discusses items required of good laboratory practice (GLP)-compliant organizations and management systems in GLP facilities in the pharmaceutical industry as well as those required for GLP inspections by the Japanese Pharmaceuticals and Medical Devices Agency. In addition, it explains standard operating procedures for conducting quality, non-GLP, pharmacology, and pharmacokinetic studies.

Good Laboratory Practice: Initial Development, Necessity, and Issues of Data Reliability in Basic Research

This review describes the initial development of good laboratory practice (GLP) and follows the discoveries of quality control problems in labs that conducted tests in U.S. pharmaceutical companies. In addition to introducing the essence of the GLP standards, how the GLP ensures the reconstructability and reproducibility of study results is explained in detail. Issues in nonclinical safety studies in drug development and approaches of the Japanese Pharmaceuticals and Medical Devices Agency to overcome them are also described. It is hoped that this review is helpful not only to those who work on drug development but also to faculties and students who work in academia and are involved in basic research when they attempt to resolve problems related to ensuring the reliability of basic research and research integrity.

Проблемы признания GLP-статусов отечественных испытательных центров и исследований за рубежом

For Russian pharmaceutical industry, the quality of non-clinical tests, which pursuant to internationally accepted requirements should be conducted in accordance with Good Laboratory Practice (GLP) standards, is of utmost importance. The best way to approximate problems in accepting the data generated by domestic test facilities is to directly refer to foreign competent authorities that both deal with foreign data and inspect foreign test facilities or audit foreign studies. In this paper, we highlight the general perception of the problem based on the communication with foreign government authorities responsible for enforcement of GLP rules.

A multi-visceral study comparing three proprietary microwave ablation devices

Objective: Microwave ablation (MWA) is the current standard electromagnetic energy source for tissue thermal ablation. This study was designed to evaluate the safety and effectiveness a next-generation MWA system (AngioDynamics Solero) compared to two standard MVA platforms (AngioDynamics Acculis and NeuWave Certus). Methods: Under good laboratory practice standards 15 female pigs received ablations in the liver, kidney, and/or lung utilizing the above MVA systems. All animals received a maximum of 3 ablations, with a maximum of 2 ablations per organ. Primary outcome was ablation zone size measured via MRI immediately (2 hours) following and 28-days post-ablation. Ablation zones were assessed at the largest 2 dimensional axes by a board certified radiologist. T-test and ANOVA were used where appropriate. All animals were necropsied after the 28 day imaging, with targeted tissue analyzed for histological treatment effect. Results: One animal required an emergent small bowel resection unrelated to the MVA. Mean ablation zones were similar across all three devices at day 0 for the liver, lung and kidney (table). Similarly, there were no differences among the three devices at day 28. Furthermore, the percent of decrease of the ablation zones were similar among all three devices. Histological analysis demonstrating zones of necrosis and fibrosis were consistent with the MRI findings above. Conclusion: All 3 microwave ablation devices were found to be effective and similar in regards to the zone of ablation as evaluated by MRI findings at 2 hours post-ablation and 28 days post-ablation.Tabled 1Mean ablation zone by MRINeuWaveSoleroAcculispDay 0 (cm2)Liver3.797.038.590.333Lung7.779.097.680.79Kidney6.663.36n/an/aDay 28 (cm2)Liver0.623.545.470.3Lung2.882.542.040.64Kidneyn/a1.75n/an/a% decrease (Day 0–28)Liver88.765.8680.52Lung75.176.187.90.57Kidney10068.3n/an/a Open table in a new tab

Non-clinical studies in the process of new drug development - Part II: Good laboratory practice, metabolism, pharmacokinetics, safety and dose translation to clinical studies.

The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using different protocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, such as determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME) and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacology and general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim at investigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for further decisions. There are, however, other studies that should be performed according to GLP standards and are mandatory for the safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must be conducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover all information needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverse effect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinical phase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.

Intubation-mediated intratracheal (IMIT) instillation: a noninvasive, lung-specific delivery system.

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a >98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a >98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Venovenous perfusion-induced systemic hyperthermia: Five-day sheep survival studies

Since hyperthermia selectively kills lung cancer cells, we developed a venovenous perfusion-induced systemic hyperthermia system for advanced lung cancer therapy. Our objective was to test the safety and accuracy of our venovenous perfusion-induced systemic hyperthermia system in 5-day sheep survival studies, following Good Laboratory Practice standards. Our venovenous perfusion-induced systemic hyperthermia system, which included a double-lumen cannula (Avalon Elite, Rancho Dominguez, Calif), a centrifugal pump (Bio-Pump 560; Medtronic Inc, Minneapolis, Minn), a heat exchanger (BIOtherm; Medtronic Perfusion Systems, Brooklyn Park, Minn), and a heater/cooler (modified Blanketrol IIIl Cincinnati Subzero, Cincinnati, Ohio), was tested in healthy adult sheep (n=5). The perfusion circuit was primed with prewarmed Plasma-Lyte A (Baxter Healthcare Corp, Deerfield, Ill) and de-aired. Calibrated temperature probes were placed in the right and left sides of the nasopharynx, bladder, and blood in/out tubing in the animal. The double-lumen cannula was inserted through the jugular vein into the superior vena cava, with the tip in the inferior vena cava. Therapeutic core temperature (42°C-42.5°C), calculated from the right and left sides of the nasopharynx and bladder temperatures, was achieved in all sheep. Heating time was 21±5 minutes. Therapeutic core temperature was maintained for 120 minutes followed by a cooling phase (35±6 minutes) to reach baseline temperature. All sheep recovered from anesthesia with spontaneous breathing within 4 hours. Arterial, pulmonary, and central venous pressures were stable. Transient increases in heart rate, cardiac output, and blood glucose occurred during hyperthermia but returned to normal range after venovenous perfusion-induced systemic hyperthermia termination. Electrolytes, complete blood counts, and metabolism enzymes were within normal to near normal range throughout the study. No significant venovenous perfusion-induced systemic hyperthermia-related hemolysis was observed. Neurologic assessment showed normal brain function all 5 days. Our venovenous perfusion-induced systemic hyperthermia system safely delivered the hyperthermia dose with no significant hyperthermia-related complications.

Development of methods for the bioanalysis of RRx-001 and metabolites.

Bioanalytical methods were required to study the novel anticancer drug, RRx-001 preclinically and for clinical pharmacokinetic analysis; however, RRx-001 quickly and completely disappeared on intravenous administration in preclinical species. Quantification of RRx-001 directly or by derivatization was unsuccessful. On exposure to whole blood, RRx-001 formed the glutathione (GSH) adduct very rapidly, suggesting this metabolite as the bioanalyte. However, rapid enzymatic degradation in the blood matrix of RRx-001-GSH posed significant technical problems. Herein, we describe a novel and broadly applicable solution to stabilize GSH conjugates in blood samples by inhibiting the degrading enzyme. Liquid chromatography-tandem mass spectrometry methods for analysis of RRx-001-GSH in rat, dog and human plasma were developed and successfully validated to good laboratory practice standards. Extensive breakdown of RRx-001-GSH was effectively stopped by addition of the enzyme inhibitor, acivicin. The developed liquid chromatography-tandem mass spectrometry assay for RRx-001-GSH was validated for use in preclinical toxicology studies and the Phase I first-in-human clinical trial.