Preliminary Safety Evaluations Research Articles

Synthesis, Anticonvulsant, Antinociceptive Activity and Preliminary Safety Evaluations of Novel Hybrid Imidazolidine-2,4-Dione Derivatives with Morpholine Moiety.

This study aimed to design new hybrid compounds with imidazolidin-2,4-dione and morpholine rings as broad spectrum anticonvulsants. To achieve this goal, all compounds were evaluated in animal seizure models, namely the maximal electroshock (MES), the subcutaneous pentylenetetrazole (scPTZ), and selected in the 6 Hz (32 mA) tests. The most promising compound, 5-isopropyl-3-(morpholinomethyl)-5-phenylimidazolidine-2,4-dione (19), demonstrated broader anticonvulsant activity than phenytoin or levetiracetam, with ED50 of 26.3 mg/kg (MES), 11.1 mg/kg and 40.9 mg/kg (6 Hz, 32 and 44 mA, respectively). Compared to phenytoin, compound 19 was active in both MES and 6 Hz (32 and 44 mA) tests. It showed nearly 1.5-fold higher efficacy than levetiracetam in the 6 Hz (32 mA test), but unlike levetiracetam, it was also active in 6 Hz (44 mA) test. Moreover, compound 23 (3-(morpholinomethyl)-5,5-diphenylimidazolidine-2,4-dione), displayed similar anticonvulsant efficacy to phenytoin and slightly higher activity than levetiracetam in 6 Hz (32 mA) test. In vitro binding studies showed that compound 23 inhibited weakly sodium and calcium channels, whereas compound 19 did not exhibit this effect. Importantly, both 19 and 23 showed no cytotoxicity in HepG2 cells (MTT test). Unfortunately, these compounds didn't show antinociceptive activity in the oxaliplatin-induced neuropathic pain model.

Discovery, Expression, and In Silico Safety Evaluation of Honey Truffle Sweetener, a Sweet Protein Derived from Mattirolomyces terfezioides and Produced by Heterologous Expression in Komagataella phaffii.

Honey truffle sweetener (HTS), a 121 amino acid protein is identified as a high-intensity sweetener found naturally occurring in the Hungarian Sweet Truffle Mattirolomyces terfezioides, an edible mushroom used in regional diets. The protein is intensely sweet, but the truffle is difficult to cultivate; therefore, the protein was systematically characterized, and the gene coding for the protein was expressed in a commonly used host yeast Komagataella phaffii. The heterologously expressed protein maintained the structural characteristics and sweet taste of the truffle. Preliminary safety evaluations for use as a food ingredient were performed on the protein including digestibility and in silico approaches for predicting the allergenicity and toxicity of the protein. HTS is predicted to be nonallergenic, nontoxic, and readily digestible. This protein is readily produced by precision fermentation of the host yeast, making it a potential replacement for both added sugars and small molecule high-intensity sweeteners in food.

Nanostructured Dense Collagen-Polyester Composite Hydrogels as Amphiphilic Platforms for Drug Delivery.

Associating collagen with biodegradable hydrophobic polyesters constitutes a promising method for the design of medicated biomaterials. Current collagen‐polyester composite hydrogels consisting of pre‐formed polymeric particles encapsulated within a low concentrated collagen hydrogel suffer from poor physical properties and low drug loading. Herein, an amphiphilic composite platform associating dense collagen hydrogels and up to 50 wt% polyesters with different hydrophobicity and chain length is developed. An original method of fabrication is disclosed based on in situ nanoprecipitation of polyesters impregnated in a pre‐formed 3D dense collagen network. Composites made of poly(lactic‐co‐glycolic acid) (PLGA) and poly(lactic acid) (PLA) but not polycaprolactone (PCL) exhibit improved mechanical properties compared to those of pure collagen dense hydrogels while keeping a high degree of hydration. Release kinetics of spironolactone, a lipophilic steroid used as a drug model, can be tuned over one month. No cytotoxicity of the composites is observed on fibroblasts and keratinocytes. Unlike the incorporation of pre‐formed particles, the new process allows for both improved physical properties of collagen hydrogels and controlled drug delivery. The ease of fabrication, wide range of accessible compositions, and positive preliminary safety evaluations of these collagen‐polyesters will favor their translation into clinics in wide areas such as drug delivery and tissue engineering.

Nanosized functional miRNA liposomes and application in the treatment of TNBC by silencing Slug gene.

Background: Neo-adjuvant chemotherapy is an effective strategy for improving treatment of breast cancers. However, the efficacy of this treatment strategy is limited for treatment of triple negative breast cancer (TNBC). Gene therapy may be a more effective strategy for improving the prognosis of TNBC.Methods: A novel 25 nucleotide sense strand of miRNA was designed to treat TNBC by silencing the Slug gene, and encapsulated into DSPE-PEG2000-tLyp-1 peptide-modified functional liposomes. The efficacy of miRNA liposomes was evaluated on invasive TNBC cells and TNBC cancer-bearing nude mice. Furthermore, functional vinorelbine liposomes were constructed to investigate the anticancer effects of combined treatment.Results: The functional miRNA liposomes had a round shape and were nanosized (120 nm). Functional miRNA liposomes were effectively captured by TNBC cells in vitro and were target to mitochondria. Treatment with functional liposomes silenced the expression of Slug and Slug protein, inhibited the TGF-β1/Smad pathway, and inhibited invasiveness and growth of TNBC cells. In TNBC cancer-bearing mice, functional miRNA liposomes exerted a stronger anticancer effect than functional vinorelbine liposomes, and combination therapy with these two formulations resulted in nearly complete inhibition of tumor growth. Preliminary safety evaluations indicated that the functional miRNA liposomes did not affect body weight or cause damage to any major organs. Furthermore, the functional liposomes significantly increased the half-life of the drug in the blood of cancer-bearing nude mice, and increased drug accumulation in breast cancer tissues.Conclusion: In this study, we constructed novel functional miRNA liposomes. These liposomes silenced Slug expression and inhibited the TGF-β1/Smad pathway in TNBC cells, and enhanced anticancer efficacy in mice using combined chemotherapy. Hence, the present study demonstrated a promising strategy for gene therapy of invasive breast cancer.

CNS Delivery and Anti-Inflammatory Effects of Intranasally Administered Cyclosporine-A in Cationic Nanoformulations.

The main objective of this study was to develop and evaluate the CNS delivery efficiency, distribution, therapeutic efficacy, and safety of cyclosporine A (CSA) using a cationic oil-in-water nanoemulsion system upon intranasal administration. An omega-3 fatty acid-rich, flaxseed oil-based nanoemulsion was used for intranasal delivery to the brain, and further magnetic resonance imaging (MRI) was used to evaluate and confirm the transport of the positively charged CSA nanoemulsion (CSA-NE) in CNS. Furthermore, the anti-inflammatory potential of CSA peptide was evaluated using the lipopolysaccharide (LPS) model of neuroinflammation in rats. CSA-NE showed a good safety profile when tested in vitro in RPMI 2650 cells. Upon intranasal administration in rats, the nanoemulsion delivery system showed higher uptake in major regions of the brain based on changes in MRI T1 (longitudinal relaxation time) values. Additionally, CSA nanoemulsion showed improved therapeutic efficacy by inhibiting proinflammatory cytokines in the LPS-stimulated rat model of neuroinflammation compared with solution formulation. Preliminary safety evaluations show that the nanoemulsion system was well tolerated and did not cause any acute negative effects in rats. Based on these results, intranasal delivery of CSA and other "neuroprotective peptides" may provide a clinically translatable strategy for treating neurologic diseases.

Safety of transtympanic application of probiotics in a chinchilla animal model

BackgroundChronic suppurative otitis media can be recalcitrant and difficult to treat, particularly with the increasing occurrence of antibiotic resistance. Lactobacillus plantarum is a probiotic that has been shown to decrease S. aureus and P. aeruginosa growth in wounds, making it a good candidate for the treatment of chronic suppurative otitis media. However, before it can be applied in the ear, its ototoxicity potential must be evaluated.MethodsA prospective controlled trial was conducted in a chinchilla animal model at the Animal care research facilities of the Montreal Children’s Hospital Research Institute to determine whether Lactobacillus plantarum is ototoxic when applied transtympanically. Ten chinchillas each had one ear randomly assigned to receive 109 CFU/mL of Lactobacillus plantarum solution, while the contralateral ear received saline. Auditory brainstem responses were measured bilaterally at 8, 20, 25 kHz before, at 7–10 days after application, and at 28 days after application of probiotic or saline. Facial nerve and vestibular function were assessed clinically.ResultsThere were no statistically significant differences in hearing thresholds between control and experimental ears at 28 days after application. A difference of 11 dB was noted in the 25 kHz range at day 7–10, but resolved by day 28. No animals receiving probiotics developed vestibular nerve dysfunction. There was no histologic evidence of auditory hair cell damaged evidenced by scanning electron microscopy.ConclusionOur study suggests that a single application of Lactobacillus plantarum at 109 CFU/mL does not cause ototoxicity in a chinchilla animal model. These preliminary safety evaluations and the pathogen inhibitory effects of L. plantarum demonstrated by previous studies present this probiotic as a candidate of interest for further investigation.

Long-circulating lipoprotein-mimic nanoparticles for smart intravenous delivery of a practically-insoluble antineoplastic drug: Development, preliminary safety evaluations and preclinical pharmacokinetic studies

Chlorambucil (CHL) is a water-insoluble antineoplastic drug having a short elimination half-life. It suffers from remarkable differences in pharmacokinetics following oral administration. The current work aimed to assess safety and pharmacokinetics of CHL-loaded, lipoprotein-mimic, nanoparticles (NPs) following intravenous administration. The design of NPs was based on complexation between egg yolk lecithin (EYL) and bovine serum albumin (BSA). The NPs were preliminary evaluated via FT-IR, DSC and P-XRD. The NPs were characterized for particle size, zeta potential, morphology and drug entrapment efficiency (EE%). The best achieved NP dispersion (LP6) and CHL solution were challenged for in vitro hemolytic potential, in vivo vascular irritation studies in rabbits and in vivo pharmacokinetics following intravenous administration in rats. The results confirmed that NPs were stabilized by hydrophobic-attractions and hydrogen-bondings between CHL, BSA and EYL. The amorphous dispersion of CHL within NPs was revealed. LP6 dispersion displayed monodispersed nano-spherical particles (144.33±2.17nm). It possessed the highest negative zeta potential (−30.55±0.24mV) and the largest EE% (86.35±2.33%). The significantly (P<0.05) prolonged MRT(0−∞), longer elimination t50% and reduced plasma clearance highlighted the long-circulating characteristics of LP6. The preliminary safety evaluations and the seven-fold increase in bioavailability elucidated potentiality for smart intravenous delivery of CHL.

77. Clinical Scale Zinc Finger Nuclease (ZFN)-Driven Gene-Editing of PD-1 in Tumor Infiltrating Lymphocytes (TIL) for the Potential Treatment of Metastatic Melanoma

Multiple inhibitory pathways are exploited by a number of cancers to block the body's immune response which may limit the effectiveness of adoptive cell transfer (ACT). Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and is expressed on activated T cells. Importantly, its ligands, PDL-1 and PDL-2 are expressed on a variety of tumor cells, including melanoma. The binding of PD-1 to PDL-1 inhibits T cell effector function, and represents an important mechanism for PDL-1 expressing tumors to evade the host immune response to cancer. PD-1 thus represents an attractive target for gene-editing of tumor targeted T cells prior to ACT. Here we describe the elimination of PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PDCD1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 specific ZFNs, the clinical scale TIL production process yielded efficient modification of the PDCD1 gene locus (mean = 74.8% of alleles, n=3, range = 69.9 – 84.1%), which resulted in a 76% reduction in PD-1 surface-expression. Importantly, the PD-1 modified TIL products displayed an effector memory phenotype and expanded approximately 500 – 2000 fold during a rapid cell expansion. In addition, PD-1 modified and unmodified TILs showed equivalent levels of engraftment in a NSG-mouse model. PD-1 modified TILs demonstrated improved in vitro T cell effector functions relative to controls (statistically significant greater TNFα, GM-CSF and IFNγ cytokine secretion when co-cultured with MART-1+ tumor cells). Preliminary safety evaluations showed that PD-1 modified TILs maintained IL-2 dependent growth in vitro, and no tumors were observed in an NSG-mouse tumorigenicity study. These data support the further study of the efficacy and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma.

Analgesic efficacy and safety of DALDA peptide analog delivery to the brain using oil-in-water nanoemulsion formulation.

The main objective of this study was to develop and evaluate therapeutic efficacy and safety following systemic delivery of a peptide analgesic into the CNS using an oil-in-water nanoemulsion system. We have formulated a safe and effective, omega-3 rich polyunsaturated fatty acid containing oil-in-water nanoemulsion formulation, for encapsulating and delivering chemically-modified DALDA, a potent mu-opioid peptide analogue, to the CNS. One of the challenges with CNS delivery is the lack of a non-invasive bioanalytical technique to confirm CNS uptake and therapeutic efficacy. Using blood oxygen-level dependent (BOLD) functional magenetic resonance imaging (fMRI), we provide quantitative evidence of nanoemulsion-based delivery and analgesic activity of DALDA analogue in capsaicin-induced awake rat model of pain. Nanoemulsion formulation effectively encapsulated the modified analgesic peptide and demonstrated efficacy in the capsaicin- pain induced functional magnetic resonance imaging model in rodents. Preliminary safety evaluations show that the nanoemulsion system was well tolerated and did not cause any acute negative effects. Overall, these results show tremendous opportunity for the development of modified peptide analgesic-encapsulated nanoemulsion formulations for CNS delivery and therapeutic efficacy.

Preliminary safety evaluation of a taurocholate‐conjugated low‐molecular‐weight heparin derivative (LHT7): a potent angiogenesis inhibitor

In our previous studies, taurocholic acid (TA)-conjugated low-molecular-weight heparin derivative (LHT7) has been proven to be a potent anti-angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism-related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg(-1) doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg(-1) doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism-related studies showed that blood pressure at 50 mg kg(-1) did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg(-1) repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material.

The performance of a fly-larva shell-derived chitosan sponge as an absorbable surgical hemostatic agent

Chitosan is a versatile biomaterial lately used as a new generation of local hemostatic agent approved to date for external use only. Here we introduced a fly-larva shell-derived chitosan sponge (CS) and its feasibility for internal use as an absorbable surgical hemostatic agent was evaluated in a rat hepatic hemorrhage model. CS was a better implantable hemostatic material than gelatin sponge (GS) or oxidized cellulose (OC) in both the acute and chronic bleeding models. The better efficacy of CS may be due to its greater ability to enhance platelet activation, erythrocyte aggregation and morphological alteration, and thrombin generation at sites it is applied than GS or OC. Moreover, preliminary safety evaluations have demonstrated excellent blood and cell compatibility in hemorheological measurements, blood coagulation analysis, histological evaluations and hepatocytes culture experiments. None of CS, GS, or OC induced acute inflammation or other adverse effects while normal tissue growth and vascularization were observed in each case 4 weeks after each hemostatic agent had been implanted. Thus, CS has promising properties as an absorbable, implantable agent for promoting perioperative hemostasis and this material warrants further study.

Toxicological Evaluation of Polyphenol Extract from Acerola (Malpighia emarginata DC.) Fruit

Acerola (Malpighia emarginata DC.) is known to be one of the best fruits rich in ascorbic acid and polyphenols. Acerola polyphenols (APs) were found to have radical scavenging activities and inhibitory effects on both alpha-glucosidase and advanced glycation endproducts (AGEs) production. While polyphenols from several foodstuffs have been shown to be safe, APs, which differ from those of other foodstuffs, have not been evaluated for their toxicological properties. Here, we report preliminary toxicological and safety evaluations of crude APs (C-AP), which were obtained by eluting an XAD7HP column-adsorbed fraction of APs with 70% ethanol containing malic acid. The total polyphenol content of C-AP was 57.7% with the main polyphenols being proanthocyanidin and cyanidin-3-alpha-O-rhamnoside. For toxicological evaluations, C-AP was administered orally to rats at doses of 2000 mg/kg body weight (acute) or 100, 300, and 1000 mg/kg body weight/d for 28 (subacute) and 90 (subchronic) d. In the acute oral toxicological test, no deaths or abnormalities at necropsy on day 14 were observed, confirming that the minimum fatal dose of C-AP is greater than 2000 mg/kg body weight. In both subacute and subchronic toxicological tests, no death was recorded and the body weights and food intakes of the rats did not differ significantly from the control groups. Besides, there were no abnormal clinical signs related to administration of C-AP in any of the experimental animals. These results provide an important reference for the safety of APs as a food supplement for human consumption.

Prediction of Icing Effects on the Dynamic Response of Light Airplanes

The accumulation of ice on an airplane in flight is one of the leading contributing factors to general aviation accidents. To date, only relatively sophisticated methods based on detailed empirical data and flight data exist for its analysis. This paper develops a methodology and simulation tool for preliminary safety and performance evaluations of airplane dynamic response and climb performance in icing conditions. The important aspect of dynamic response sensitivity to pilot control input with the autopilot disengaged is also highlighted. Using only basic mass properties, configuration, propulsion data, and known icing data from a similar configuration, icing effects are applied to the dynamics of a non-real-time, six degree-of-freedom simulation model of a different, but similar, light airplane. Besides evaluating various levels of icing severity, the paper addresses distributed icing which consists of wing alone, horizontal tail alone, and unequal distributions of combined wing and horizontal tail icing. Results presented in the paper for a series of simulated climb maneuvers with various levels and distributions of ice accretion show that the methodology captures the basic effects of ice accretion on pitch response and climb performance, and the sensitivity of the dynamic response to pilot control inputs.

Nubac Disc Arthroplasty: Preclinical Studies and Preliminary Safety and Efficacy Evaluations

Background Disc arthroplasty is gaining popularity for treatment of low-back pain caused by degenerative disc disease (DDD). It can involve total disc replacement or partial disc or nucleus replacement (or augmentation). Compared with total disc replacement, nucleus replacement is less invasive, has less surgical risk, has faster postoperative recovery, and doesn’t “burn bridges” should further surgery be required. However, nucleus replacement has a high risk of implant expulsion because the device is not fixed to the vertebrae. Nubac is the first polyetheretherketone (PEEK)-on-PEEK articulated disc arthroplasty device designed to optimally restore the lumbar anatomy and biomechanics. Methods ISO 10993 standards were used to evaluate the biocompatibility of the PEEK material. Chemical and thermal–mechanical tests and in vivo study assessed PEEK's biostability after exposure to high g irradiation and harsh oxidative conditions. Biomechanical tests to evaluate kinematic properties and anatomical restoration of the implanted lumbar motion segments and implant expulsion risk assessments were performed with a human cadaveric model. Because of the novelty of PEEK-on-PEEK as a self-mating articulating material, extensive wear tests were conducted with unidirectional and coupled motions. Static and fatigue strength also were tested. Animal study with a baboon model was conducted with gross, radiographic, biomechanical, and histological evaluations at 6 and 12 months postoperatively. Preliminary clinical data were collected through a prospective multicenter cohort study. Results PEEK demonstrated exceptional biocompatibility and biodurability. Nubac restored disc height and motion segment range of motion. The unique articulating design of the Nubac demonstrated low risk of implant expulsion in a human cadaveric model. Wear tests showed that the Nubac has minimal wear and compares favorably to other disc arthroplasty materials. The Nubac also had excellent static and fatigue properties for the intended application. The animal study showed that the Nubac caused no adverse local or systematic tissue reaction and there was no detectable wear debris. The preliminary clinical data showed no major intraoperative vascular and neurological complications. There was significant Visual Analog Scale and Oswestry Disability Index score improvement. Conclusions The preclinical data supported the design rationale, and the preliminary clinical data (level II evidence) on safety and efficacy were encouraging. Clinical Relevance The Nubac could be a viable first surgical option for patients with back pain caused by DDD.

Nubac disc arthroplasty: preclinical studies and preliminary safety and efficacy evaluations.

BackgroundDisc arthroplasty is gaining popularity for treatment of low-back pain caused by degenerative disc disease (DDD). It can involve total disc replacement or partial disc or nucleus replacement (or augmentation). Compared with total disc replacement, nucleus replacement is less invasive, has less surgical risk, has faster postoperative recovery, and doesn't “burn bridges” should further surgery be required. However, nucleus replacement has a high risk of implant expulsion because the device is not fixed to the vertebrae. Nubac is the first polyetheretherketone (PEEK)-on-PEEK articulated disc arthroplasty device designed to optimally restore the lumbar anatomy and biomechanics.MethodsISO 10993 standards were used to evaluate the biocompatibility of the PEEK material. Chemical and thermal–mechanical tests and in vivo study assessed PEEK's biostability after exposure to high g irradiation and harsh oxidative conditions. Biomechanical tests to evaluate kinematic properties and anatomical restoration of the implanted lumbar motion segments and implant expulsion risk assessments were performed with a human cadaveric model. Because of the novelty of PEEK-on-PEEK as a self-mating articulating material, extensive wear tests were conducted with unidirectional and coupled motions. Static and fatigue strength also were tested. Animal study with a baboon model was conducted with gross, radiographic, biomechanical, and histological evaluations at 6 and 12 months postoperatively. Preliminary clinical data were collected through a prospective multicenter cohort study.ResultsPEEK demonstrated exceptional biocompatibility and biodurability. Nubac restored disc height and motion segment range of motion. The unique articulating design of the Nubac demonstrated low risk of implant expulsion in a human cadaveric model. Wear tests showed that the Nubac has minimal wear and compares favorably to other disc arthroplasty materials. The Nubac also had excellent static and fatigue properties for the intended application. The animal study showed that the Nubac caused no adverse local or systematic tissue reaction and there was no detectable wear debris. The preliminary clinical data showed no major intraoperative vascular and neurological complications. There was significant Visual Analog Scale and Oswestry Disability Index score improvement.ConclusionsThe preclinical data supported the design rationale, and the preliminary clinical data (level II evidence) on safety and efficacy were encouraging.Clinical RelevanceThe Nubac could be a viable first surgical option for patients with back pain caused by DDD.

Nonlinear response of dams founded on alluvial deposits in narrow canyons

This paper presents a simplified model for the response of earth dams founded on alluvial deposits at the base of canyons. The goal of the model is to capture the effect of the most important factors influencing the response and to provide a realistic and efficient tool for preliminary safety evaluations. The developed theoretical closed-form solution combines the effects on the response of: (a) the foundation layer, (b) the canyon narrowness, (c) the variation of soil stiffness within the dam versus depth, and (d) soil nonlinearity. The latter is incorporated in a simple piece-wise linear scheme, in which the soil shear moduli and damping ratios within the dam and the layer are continuously updated at small time intervals during the analysis. The presence of a soft foundation layer is found to have a dramatic effect on the natural periods of the dam. A series of parametric studies is undertaken to investigate the effect on the response of the fundamental geometric and material characteristics of the dam-layer-canyon system subjected to transient and harmonic motion. The results show that a foundation layer with an average S -wave velocity similar or less than that of the dam under moderate or strong base excitation may experience significant shear strains. Such strains are capable of inducing high excess pore water pressures and perhaps liquefaction within contractive saturated cohesionless soils in the foundation. The cost of the simplified analysis is about three orders of magnitude less than that of an elastoplastic FE analysis.