Tisagenlecleucel was approved for the treatment of pediatric and young adult patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (ALL) based on the pivotal ELIANA trial.To comprehensively evaluate the total costs associated with tisagenlecleucel treatment, including costs from pre- to postinfusion periods of tisagenlecleucel in addition to the cost of tisagenlecleucel.An economic model was developed to estimate total costs associated with tisagenlecleucel treatment from the time of leukapheresis to 2 months postinfusion from a U.S. hospital’s perspective. Costs were estimated based on resource use and safety management from the ELIANA trial and were considered during the pretreatment, tisagenlecleucel infusion, and follow-up periods of treatment. Cost components included leukapheresis, lymphodepleting chemotherapy, tisagenlecleucel infusion and hospital administration, inpatient and intensive care unit admissions, medical professional visits, laboratory tests and procedures, and management of major adverse events. Scenario analyses were conducted by varying key assumptions related to adverse events and hospitalization.The total cost associated with tisagenlecleucel treatment among pediatric and young adult patients with r/r ALL was estimated to be $612,779, of which $137,636 (22.5%) was in addition to the list price of tisagenlecleucel  and the associated administration cost of $143.08. The top 3 drivers of the additional cost were adverse event management , inpatient and intensive care unit admissions not attributed to adverse events , and laboratory tests and procedures . The costs incurred during the pretreatment, infusion, and follow-up periods were $29,002, $476,659, and $107,118, respectively. In the scenario analyses, the total costs ranged from $483,169 (tisagenlecleucel treatment in the outpatient setting without adverse events) to $672,373 (tisagenlecleucel treatment in the inpatient setting with grade 3/4 cytokine release syndrome and B-cell aplasia).In this economic model, tisagenlecleucel treatment among pediatric and young adult patients with r/r ALL was estimated to cost $612,779. The cost of care in addition to the price of tisagenlecleucel accounted for 22.5% of the total, with adverse event management and inpatient and intensive care unit admissions being main drivers. Further studies are warranted to assess the cost of tisagenlecleucel treatment in the context of current standards of care in real-world clinical practice. What is already known about this subjectTisagenlecleucel is a CD19-directed chimeric antigen receptor T-cell therapy that is approved for the treatment of pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL).Tisagenlecleucel is priced at $475,000, which does not include the cost of associated resource use and adverse event management.There is limited real-world evidence to inform patterns of health care resource utilization related to tisagenlecleucel treatment and adverse event management; however, comprehensive resource utilization data were collected in the pivotal ELIANA trial.What this study addsIn this economic model, we estimated a total cost of $612,779 associated with tisagenlecleucel treatment from leukapheresis to 2 months postinfusion among pediatric and young adult patients with relapsed/refractory ALL, of which $137,636 (22.5%) was in addition to the list price of tisagenlecleucel  and the associated administration cost ($143.08).The top 3 drivers of the additional cost were adverse event management (51.6%), inpatient and intensive care unit admissions not attributed to adverse events (42.1%), and laboratory tests and procedures (3.8%).2 While the majority of patients with ALL achieve remission after frontline therapy, relapse and refractoriness remain a challenge and are associated with poor clinical outcomes.3Acute lymphoblastic leukemia (ALL) is a hematological malignancy that primarily affects children, adolescents, and young adults, comprising more than 25% of pediatric cancer diagnoses in the United States.6 Conventional treatment options include salvage chemotherapy, such as clofarabine monotherapy or combination therapy with cyclophosphamide or etoposide, and blinatumomab, followed by allogeneic hematopoietic stem cell transplantation (HSCT).7 However, these treatment options have limited efficacy, with median OS of fewer than 10 months and objective response rates of less than 50%.9Historically, patients with refractory disease or those who experience 2 or more relapses after treatment with chemotherapy have especially poor prognoses and dismal survival outcomes, with median overall survival (OS) ranging between 3 and 7.5 months.11 In this global, phase 2, single-arm study, pediatric and young adult patients with r/r ALL treated with a single infusion of tisagenlecleucel achieved promising outcomes compared with current standard therapies, with a median OS of 19.1 months and an overall remission rate of 81% within 3 months.10Recent therapeutic advances within the ALL treatment landscape have led to the introduction of chimeric antigen receptor T-cell (CAR-T) immunotherapy, which involves the genetic modification of a patient’s own T cells to target cancer cells. Tisagenlecleucel, a CD19-directed CAR-T therapy, was recently approved by the U.S. Food and Drug Administration for the treatment of pediatric and young adult patients with relapsed/refractory (r/r) B-cell ALL, based on its efficacy and safety evaluated in the ELIANA clinical trial.11 Subsequently, bridging chemotherapy treatments and lymphodepleting regimens may also be needed to stabilize the patient’s condition in preparation for tisagenlecleucel infusion.12 Moreover, since tisagenlecleucel is designed to use a patient’s own immune system to eliminate cancer cells, treatment is also associated with unique adverse events (AEs), including cytokine release syndrome (CRS) and B-cell aplasia.11 Both of these AEs may need management with expensive therapies , and patients who develop severe CRS may also need to be admitted to the intensive care unit (ICU).As a form of personalized medicine with demonstrated clinical outcomes, tisagenlecleucel has been priced at $475,000 based on wholesale acquisition in the United States. However, because tisagenlecleucel is the first-in-class CAR-T immunotherapy and is individually manufactured for each patient using their own blood cells, the associated resource use could be very different from other cancer treatments. Indeed, before the manufacturing and infusion of tisagenlecleucel, apheresis is required to cultivate the patient’s cells for the manufacturing process.Importantly, there is limited real-world evidence to inform patterns of health care resource utilization (HCRU) related to treatment and AE management, since CAR-T therapy is relatively new to the market. However, comprehensive HCRU data were collected in the pivotal ELIANA trial and included information related to hospitalization and ICU stays, as well as treatment utilization for bridging chemotherapy, lymphodepletion, CRS, and B-cell aplasia.Therefore, this study was conducted to comprehensively evaluate the total costs of tisagenlecleucel treatment, including costs additional to the procedure and from multiple treatment periods, using the latest clinical trial data from the ELIANA study and publicly available data. We also performed scenario analyses to assess the effect on total costs based on varying hospital treatment settings, presence of AEs, cost assumptions, and data input sources. These results aim to provide valuable insight into the cost of care associated with tisagenlecleucel therapy and to fill the current knowledge gaps within the economic literature of CAR-T therapy.13An economic model was developed in Excel  to assess the total costs associated with tisagenlecleucel treatment among pediatric and young adult patients with r/r ALL from a U.S. hospital’s perspective. The total costs were estimated from the time of leukapheresis to 2 months postinfusion, which was consistent with the time frame of collecting HCRU related to the tisagenlecleucel infusion per the trial protocol. The model was developed using a fee-for-service approach, which estimated costs based on the HCRU and safety data from the pivotal, phase 2 ELIANA trial.the pretreatment period, during which T cells were harvested from patients and preparative lymphodepleting chemotherapy was given; (b) the infusion period ; and (c) the follow-up period, during which patients were monitored or treated for disease progression or AEs for up to 2 months (The model considered costs incurred during 3 treatment periods: (a) 2 months . The cos13 Scenario analyses were conducted by varying key inputs and assumptions. Specifically, scenario analyses included: (a) assuming all patients were treated with tisagenlecleucel in the outpatient setting and did not experience any AEs during follow-up; (b) assuming all patients were treated with tisagenlecleucel in the inpatient setting and experienced grade 3/4 CRS and B-cell aplasia during follow-up; (c) considering the variation of individual cost components  within a 95% confidence interval (CI) or ± 25% if 95% CI was not estimable; (d) using Grupp et al. (2019), a study based on real-world use of tisagenlecleucel, to inform grade 3/4 CRS, IVIG use, and tocilizumab use  estimating the daily hospitalization and ICU costs based on an alternative data source—the Healthcare Cost and Utilization Project (HCUP) database.16The base-case model estimated the total costs using the observed hospitalization, ICU, and AE data for all patients receiving tisagenlecleucel infusion in the ELIANA trial.This model considered the period of care starting from cell collection to 2 months post-tisagenlecleucel infusion.13 The model separately estimated costs associated with AEs  and costs associated with inpatient and ICU admissions because of causes other than AEs.This model used the observed hospitalization data based on the ELIANA trial, which indicated that some patients received tisagenlecleucel in the inpatient setting and some in the hospital outpatient setting.13 The remaining 4% of patients did not receive lymphodepleting therapies, as observed in the ELIANA trial.Costs associated with lymphodepleting chemotherapies were considered during the pretreatment period. As estimated from the ELIANA trial data, 96% of the infused population received lymphodepleting therapies (95% received regimen 1 [fludarabine and cyclophosphamide] and 1% received regimen 2 [cytarabine and etoposide]) during the pretreatment period.17 Unit costs for drug administration were obtained from the Centers for Medicare & Medicaid Services (CMS) Physician Fee Schedule.18 Detailed information regarding the drug costs can be found in The default cost inputs for medication and administration were obtained from the public domain and were based on payment from the payers . Specifically, medication costs were estimated using the wholesale acquisition cost (WAC) from RED BOOK.18Tisagenlecleucel drug and administration costs were considered during the infusion period. Similar to the lymphodepleting costs, the cost of the tisagenlecleucel infusion procedure was obtained based on the WAC in the RED BOOK, and the administration cost was derived from the CMS Physician Fee Schedule.13 Only hospitalization not attributed to AEs was considered to avoid double counting with the AE management costs. The unit hospitalization costs were obtained from the literature. The average daily hospitalization cost was obtained from Kaul et al. (2016), and the average daily ICU cost was obtained from Hsu et al. (2015).20 The model inputs are presented in Inpatient and ICU costs were estimated during the entire treatment period. The inpatient and ICU admission proportions and length of stay were estimated from the ELIANA trial data, separately for the pretreatment, infusion, and follow-up periods.18Costs associated with medical professional visits were relevant to the entire treatment period and were thus considered in the model. Resource utilization frequencies were acquired from the ELIANA trial protocol, while the unit costs of medical professional visits were obtained from the CMS Physician Fee Schedule.13 The physician cost associated with leukapheresis and the unit costs of laboratory tests and procedures were derived from the CMS Physician Fee Schedule and Clinical Laboratory Fee Schedule, respectively.21 The laboratory tests and procedures associated with ALL included bone marrow biopsy and/or aspirate, chemistry panel, coagulation panel, echocardiogram, electrocardiogram, hematology panel, influenza A and B test, peripheral blood collection for flow cytometry, pulse oximetry, serum cytokines, serum immunoglobulin levels, urinalysis, and other laboratory tests of special interest.The cost of leukapheresis was considered during the pretreatment period, and other laboratory and procedure costs were considered throughout the tisagenlecleucel treatment period. The frequency inputs of laboratory tests and procedures during each treatment period were based on the ELIANA trial protocol.13 Resource use was modeled separately for the 2 most important tisagenlecleucel-related AEs, grade 3/4 CRS and B-cell aplasia, with observed rates of 48% and 61%, respectively. For patients with grade 3/4 CRS, the proportions of patients with an ICU admission and tocilizumab treatment, as well as the dosage used for CRS management, were estimated based on ELIANA trial data.13 In the ELIANA trial, CRS was graded based on the Penn criteria, which tend to assign a higher grade of CRS compared with the latest criteria recommended by the American Society for Transplantation and Cellular Therapy (ASTCT).22 Similarly, for patients with B-cell aplasia, the proportion of patients with IVIG use, the dosing schedule, and the number of doses was also estimated based on ELIANA trial data.13 Unit costs for other grade 3/4 AEs were derived from the HCUP Kids’ Inpatient Database.23 Of note, grade 3/4 AEs included in the model were AEs regardless of study drug relationship, not restricted to those that were suspected to be study drug-related. The AE inputs are summarized in AE rates during the follow-up period were obtained from the ELIANA trial data, and only grade 3 and 4 AEs with rates ≥ 5% were considered in the model.The overall cost associated with tisagenlecleucel treatment from leukapheresis to 2 months post infusion in patients with r/r ALL was estimated to be $612,779 . ConsideThe costs incurred during the pretreatment period, infusion period, and follow-up period were $29,002, $476,659, and $107,118, respectively. The primary cost drivers were $23,966 for inpatient and ICU admissions (82.6%) during the pretreatment period, $475,143 for the tisagenlecleucel procedure and administration (99.7%) during the infusion period, and $70,968 for AE management (66.3%) during the follow-up period.In the scenario analyses, the total costs ranged from $483,169 (outpatient setting without AEs) to $672,373  for AE management, $83,395 (42.3%) for inpatient and ICU admissions not attributed to AEs, $5,209 (2.6%) for laboratory tests and procedures, $2,177 (1.1%) for medical professional visits, and $1,727 (0.9%) for lymphodepleting drugs and their administration.Under the scenario that each cost component was varied, the greatest effect was seen when AE cost was varied, and the model calculated costs ranging from $109,410 to $165,862 aside from the tisagenlecleucel procedure and administration. The variation in inpatient and ICU admission cost had the second greatest effect; the model calculated costs ranging from $119,118 to $156,180 aside from tisagenlecleucel procedure and administration. The other cost components had more limited effect on the results. The costs aside from tisagenlecleucel procedure and administration changed by less than 1% when these cost components were varied.Under the scenario that used Grupp et al. to inform grade 3/4 CRS, IVIG use, and tocilizumab use, the model calculated a cost of $116,699 aside from the tisagenlecleucel procedure and administration, which included $50,031 (42.8%) for AE management, $57,952 (49.7%) for inpatient and ICU admissions not attributed to AEs, $5,209 (4.5%) for laboratory tests and procedures, $1,780 (1.5%) for medical professional visits, and $1,727 (1.5%) for lymphodepleting drugs and their administration.Under the scenario that used data from the HCUP database to estimate daily hospitalization and ICU costs, the model calculated a cost of $158,168 aside from the tisagenlecleucel procedure and administration, which included $69,460 (43.9%) for AE management, $79,992 (50.6%) for inpatient and ICU admissions not attributed to AEs, $5,209 (3.3%) for laboratory tests and procedures, $1,780 (1.1%) for medical professional visits, and $1,727 (1.1%) for lymphodepleting drugs and their administration.To our knowledge, this is the first study using resource utilization data of tisagenlecleucel treatment from the ELIANA trial to provide total estimated costs and detailed breakdowns by cost component and treatment period of CAR-T immunotherapy for patients with r/r ALL in the United States. In this economic model, we estimated a total cost of $612,779 associated with tisagenlecleucel treatment from leukapheresis to 2 months postinfusion among pediatric and young adult patients with r/r ALL, from a U.S. hospital’s perspective, of which $137,636 (22.5%) was in addition to the list WAC price of tisagenlecleucel  and the associated administration cost ($143.08). The top 3 drivers of the additional cost were AE management (51.6%), inpatient and ICU admissions not attributed to AEs (42.1%), and laboratory tests and procedures (3.8%).This economic modeling study makes several important contributions to the literature. It is the first U.S.-focused study that comprehensively evaluated the health care costs associated with tisagenlecleucel treatment based on HCRU data from a clinical trial. The total estimated costs covered multiple cost components and were summarized within different periods of care from a U.S. hospital’s perspective. Additionally, the model also explored various treatment scenarios to provide a plausible range of the cost estimate by varying key inputs and assumptions. Given the scarcity of comprehensive economic evaluations of novel CAR-T therapies in the literature, the cost estimates provided in the current study shed light on the economic outcomes of tisagenlecleucel for patients with r/r ALL in the context of robust clinical outcomes observed in the clinical trial setting.24 This estimate is lower than that reported in the current study, which may be explained by differing cost components explored in the 2 studies. Hernandez et al. only considered a subset of the cost components evaluated in the current model , and did not account for key resource-use categories related to hospitalization and other important AEs, such as B-cell aplasia. Moreover, Hernandez et al. estimated resource use based on the public data source of the ELIANA trial.24The cost estimates from this study are largely consistent with prior published economic studies of tisagenlecleucel for the treatment of pediatric and young adult patients with r/r ALL. In 1 study that evaluated the total costs of CAR-T immunotherapy, Hernandez et al. (2018) reported a mean expected cost of $510,963 for patients with r/r pediatric ALL treated with tisagenlecleucel in the United States.26In contrast, the current study analyzed the patient-level data from the ELIANA trial to more comprehensively incorporate resource use and costs related to tisagenlecleucel treatment over the entire treatment and postinfusion period, resulting in a slightly higher total cost estimate of $612,779. The total cost of tisagenlecleucel was also evaluated in 2 published cost-effectiveness analysis studies from the U.S. payer’s perspective, though these studies evaluated tisagenlecleucel costs from different time horizons and considered variable cost components and assumptions based on different study focuses.26 Both studies demonstrated the cost-effectiveness of tisagenlecleucel, with incremental cost-effectiveness ratios of $45,871 to $61,000 per quality-adjusted life-year gained compared with standard treatments.The overall cost estimates, ranging from $548,000 to $666,754, were within a similar range of the current study, and in both studies, the majority of the estimated cost was related to the tisagenlecleucel procedure (71%-87%).28 Allogeneic HSCT is currently recommended as part of a treatment option for pediatric patients with r/r ALL receiving conventional treatment, though its role following tisagenlecleucel is unclear.7 No head-to-head comparisons between CAR-T and HSCT have been conducted. The median survival after allogeneic HSCT for pediatric and young adult patients with r/r ALL in first or later relapse ranges from 7.4 months to ~15 months in the literature.31 The median OS of tisagenlecleucel was 19.1 months in the ELIANA trial.10 However, it is important to note that these survival estimates for HSCT were derived from single-arm studies with populations that are not fully comparable to the ELIANA population; in general, there is limited evidence on HSCT efficacy in pediatric and young adult patients in second or later relapse.For the treatment of r/r ALL, the total cost of care associated with tisagenlecleucel was estimated to be $612,779 in this study. This estimated cost is similar to the total reported 100-day cost associated with allogeneic stem cell transplantation , which was the only potentially curative option for pediatric and young adult patients with r/r ALL before the introduction of tisagenlecleucel.33 In cases where allogeneic HSCT is possible, however, the treatment has been associated with high rates of complications, some of which require years to resolve. Indeed, in the study conducted by Crotta et al. (2018), 57% and 35% of patients experienced acute and chronic graft versus host disease, respectively, after the allogeneic HSCT procedure. Additionally, a small fraction of patients developed severe secondary malignancies such as acute myeloid leukemia and lymphoproliferative disease.29 While tisagenlecleucel treatment is associated with unique AEs, most AEs observed in the ELIANA trial were resolved within a few months after infusion.10 However, additional long-term, real-world data are necessary to continue monitoring the situation and to improve our understanding of the safety profile of CAR-T therapy. Additionally, comparative studies on clinical outcomes and costs of tisagenlecleucel and allogeneic HSCT are needed to compare these 2 treatments.Allogeneic HSCT is also not suitable for all patients; poor health or presence of comorbidities may preclude its use, and a suitably matched sibling donor is available in only 30% of cases.26An important component of the overall cost of treatment is the incremental cost of care in addition to the list WAC price and administration cost of tisagenlecleucel. This component was estimated to be $137,636 in the current study, ranging from $8,026 to $197,230 depending on the hospitalization and AE assumptions. In absolute terms, these additional estimated costs are lower compared with the expected medical expenses incurred by pediatric and young adult patients with r/r ALL receiving alternative treatments. Existing modeling studies have estimated the costs of other currently available chemotherapy or targeted treatments, with estimates ranging from $238,000 to $307,000 in addition to the drug costs.34 Finally, the gene therapy voretigene neparvovec accounted for 82% of the total health care costs for patients with inherited retinal disorder in a cost-utility study.35With the continued advancement of innovative cell and gene therapies that offer improved clinical outcomes, the composition of overall cost of patient care may shift with drug/procedure cost becoming the major component of the overall treatment cost. Indeed, the WAC price of tisagenlecleucel and its associated administration cost comprised 77.5% of the total health care cost of tisagenlecleucel treatment in this study. Similarly, based on a cost-effectiveness analysis, the list price of the new gene therapy onasemnogene abeparvovec contributed 57% to 73% of the total cost of care for pediatric patients with spinal muscular atrophy type 1, depending on various pricing assumptions.14 Overall, the study reported lower rates of grade 3 and above CRS (14% vs. 48% from ELIANA) as well as utilization of tocilizumab and IVIG treatment compared with the ELIANA trial.14 This was likely driven by the different grading system and management of CRS in real-world practice from those used in clinical trials as physicians gained more experience with CAR-T treatment. The ELIANA trial used the Penn grading system to classify CRS grade, which tends to assign a higher grade of CRS compared with the ASTCT criteria more commonly adopted in real-world practice.22 However, there is currently a lack of real-world evidence to inform HCRU and costs associated with other cost components  for pediatric patients with tisagenlecleucel infusion.22 With a real-world study currently underway, future analyses may provide updated insight into the HCRU and costs of tisagenlecleucel treatment in real-world clinical practice.14The results of this study should be interpreted in light of some limitations. First, HCRU among patients receiving tisagenlecleucel treatment was estimated based on clinical trial data, which may not represent the real-world HCRU with a more heterogeneous patient population  and practice patterns . A scenario analysis was conducted using CRS rate as well as tocilizumab and IVIG utilization information reported in a real-world study by Grupp et al. with a median follow-up of 6 months.26Second, our analysis was limited to 2 months of follow-up post-tisagenlecleucel infusion, which does not capture long-term safety outcomes associated with the treatment. Therefore, our estimated cost of care in addition to the tisagenlecleucel procedure may be underestimated. However, the total estimated cost of care from the current model was similar to the total costs estimated in the prior cost-effectiveness studies with longer time horizons , which suggests that our current estimates may still capture the majority of the costs associated with tisagenlecleucel treatment.36 Future research with long-term, real-world, follow-up data will be important to provide more accurate and refined cost estimates of tisagenlecleucel treatment in clinical practice.Finally, our estimated costs may not reflect the true costs incurred by hospitals because of the lack of real-world data and use of publicly available databases. For example, we used the WAC in RED BOOK to estimate drug costs, which may not represent the true costs incurred by hospitals or how much hospitals get reimbursed. Indeed, under CMS, hospitals will only receive $242,450 payment in 2020 for CAR-T therapy, which is much lower than the list price of CAR-T therapy, but the payment amount does not necessarily reflect how much the hospital has paid for tisagenlecleucel treatment either.In this economic model, tisagenlecleucel treatment among pediatric and young adult patients with r/r ALL was estimated to cost $612,779. The cost of care in addition to the price of tisagenlecleucel accounted for 22.5% of the total, with AE management and inpatient and ICU admissions being main drivers. While these results provide valuable economic information regarding tisagenlecleucel therapy among pediatric and young adult patients with r/r ALL from a U.S. hospital’s perspective, additional analyses are warranted to assess the cost of CAR-T therapy in the context of current standards of care in real-world clinical practice.


Clostridioides difficile (C. difficile) infections represent a major health risk and a high cost to health-care systems. C. difficile causes pathogenic activity in the colon through the production of two toxins, A and B, which provoke cell death. Several studies conducted on the mechanisms by which these two toxins induce various types of cell death in the intestinal mucosa cells and in the immune system cells failed to identify a common basic event. Since calcium is an important regulator of cellular physiology, here we propose a new point of view in which the alteration in physiological intracellular calcium levels by the two toxins may be the key common event responsible for the activation of cell death. Further exploration of this issue with in vivo studies could be important for possible pharmacological interventions aimed at antagonizing the Ca2+ homeostasis alterations that could tone down the C. difficile pathogenicity until specific antibiotic therapy has brought the infection under control.Clostridioides difficile (C. difficile), responsible for 15–25% of gastrointestinal infections, causes health problems mainly due to the toxic activity of toxins A and B (Tcds). These are responsible for its clinical manifestations, including diarrhea, pseudomembranous colitis, toxic megacolon and death, with a mortality of 5–30% in primary infection, that increase following relapses. Studies on Tcd-induced cell death have highlighted a key role of caspases, calpains, and cathepsins, with involvement of mitochondria and reactive oxygen species (ROS) in a complex signaling pathway network. The complex response in the execution of various types of cell death  depends on the amount of Tcd, cell types, and Tcd receptors involved, and could have as initial/precocious event the alterations in calcium homeostasis. The entities, peculiarities and cell types involved in these alterations will decide the signaling pathways activated and cell death type. Calcium homeostasis alterations can be caused by calcium influx through calcium channel activation, transient intracellular calcium oscillations, and leakage of calcium from intracellular stores. These increases in cytoplasmic calcium have important effects on all calcium-regulated molecules, which may play a direct role in several cell death types and/or activate other cell death effectors, such as caspases, calpains, ROS and proapoptotic Bcl-2 family members. Furthermore, some support for the possible role of the calcium homeostasis alteration in Tcd-induced cell death originates from the similarity with cytotoxic effects that cause pore-forming toxins, based mainly on calcium influx through plasma membrane pores. Clostridioides difficile (C. difficile) [C. difficile infection (CDI) range from mild transient diarrhea to pseudomembranous colitis and toxic megacolon [C. difficile is mainly due to two exotoxins, toxin A (TcdA) and toxin B (TcdB), with TcdB being about 1000 times more potent than TcdA on cytotoxic activity. A minor pathogenic contribution is also due to the binary toxin [fficile) ,2 is a Gfficile) ,4,5. Theegacolon ,7. The pry toxin ,10,11,12C. difficile strains [C. difficile in the anthropized environment, and the increasing ability to colonize humans [At present, CDIs have become a serious health problem because they are not only frequent in subjects undergoing antibiotic therapy or in those hospitalized/institutionalized but also because the incidence is increasing in the community, in both elderly and young subjects, in those with severe illnesses e.g., inflammatory bowel disease (IBD) and also as a consequence of increased feeding tube use and other surgical or therapeutic practices ,15,16,17 strains ,19, the e humans .C. difficile is transmitted person-to-person via the fecal–oral route by spores, and hospitals and community health-care settings may become an important source of infection [C. difficile spores can be found almost ubiquitously in hospitals and nursing home environment, health-care workers’ hands, and several medical devices [C. difficile and hospital outbreaks [nfection ,7,21. Vi devices ,7,20,21. devices ,23. NursC. difficile colonization [C. difficile bacteriophages (phages) [So far, attempts to develop non-antibiotic therapies against CDI based on antibodies ,25, vaccnization , C. diff(phages) ,36 and i(phages)  have giv(phages) ,39,40,41C. difficile toxins (Tcds).Consequently, it is necessary to gain further knowledge of the basic mechanisms underlying the pathogenic activity of TcdA and TcdB to find in the network of activated signaling pathways molecular targets to antagonize in order to inhibit the pathogenic activity of 2+) homeostasis followed by the inhibition of Rho-GTPases. This first phase, still poorly understood, is perhaps decisive in the induction of cell death and for the type of cell death and could be of crucial importance to understand the manner of antagonizing the key pathogenic events of C. difficile.As we discuss below, within the pathogenic mechanism of CDI, there is an early phase of about 40 min duration, characterized by the alteration in calcium . Furthermore, we compare the effects of pore-forming toxins (PFTs) and Tcds, because an additional indirect enforcement of the possible role of the alteration in Ca2+ homeostasis in cell death induced by Tcds comes from the similarity with the effects that PFTs cause during induction of cell death, based mainly on the direct Ca2+ influx by plasma membrane pore formation.In this review, we discuss in depth several significant aspects that indicate, albeit indirectly, that in the mechanism of CDI pathogenicity, there is an early phase characterized by the alteration in Ca2+ with all the consequences due to the profound alteration in intracytoplasmic Ca2+ homeostasis, while the pathogenic activity of Tcds until now has been characterized only as the activation of Rho-GTPase-dependent and Rho-GTPase-independent mechanisms after internalization of the Tcd (see below) [The comparison between PFTs and Tcds takes into account the fact that PFTs form a stable pore in the membrane. This pore is the main responsible for the influx of Cae below) ,10,11,122+ homeostasis alteration could be a key event in the mechanism of pathogenicity of C. difficile is discussed and highlighted. Knowledge of this aspect could thus be of crucial importance to understand an alternative manner to antagonize the pathogenicity of C. difficile.Overall, for the first time, the possibility that CaC. difficile have reached the colon, if the right conditions are present, among which the most relevant is the alteration in the microbiota, they enter the vegetative phase with the dissolution of the spore wall and the production of TcdA and TcdB and, in some strains, also of binary toxin [C. difficile penetrates more deeply into the mucosa [Once the spores of ry toxin ,7,42,43.ry toxin ,10,11,12e mucosa ,9,10.C. difficile may contribute to pathogenicity [Although other molecular components of genicity ,45, the genicity ,11,12,46genicity .2+ as an early key event in the process of cell death.However, little emphasis has been placed on the possible key role of alteration in homeostasis of intracellular CaC. perfrigens and C. novi [TcdA and TcdB belong to the large clostridial toxins family, which also includes the toxins produced by  C. novi . TcdB is C. novi ,10,11,49 C. novi ,9,10,11. C. novi . TcdA co C. novi ,9,10,11.Tcds present both a analogous glucosyltransferase enzymatic activity and a similar structural composition with multi-modular domains, referred as the ABCD model  ,9,10,11.The receptors described for TcdA are two proteins expressed in the cell plasma membrane ,9,51. ThThe receptors described for TcdB number three, and are proteins expressed in cell cytoplasmic membrane ,9,51. ThTcdA and TcdB, after binding to the cell plasma membrane receptors for Tcd, prompt its uptake by different pathways. Uptake of TcdB is mediated by clathrin, while that of TcdA is mediated by PACSIN2/syndapin-II ,59,60. FOf interest, TcdA and TcdB may also induce cell death (cytotoxic effect) independently of autoprocessing or glucosyltransferase activities of the Tcds, which occurs mainly by necrosis, but can also occur by apoptosis, pyknosis, or pyroptosis (see below) ,63,64,65Tcds are able to cause death in several types of cells: colonocytes , enterocNotably, Fettucciari et al. showed that EGCs that survive the TcdB cytotoxic effects go into a senescence state as a survival response to a stressor stimulus prompted by TcdB ,80.Although glucosylation-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question. Recently, Peritore-Galve et al. showed that in a mouse model of CDI, epithelial damage occurs through a glucosylation-independent manner not mediated by immune cell influx. In particular, the authors showed that the glucosylation activity of Tcds causes severe edema and inflammation and recruitment of immune cells. Glucosylation activity of both Tcd was necessary to produce weight loss and severe watery diarrhea, while it was not necessary to cause epithelial damage and pseudomembrane formation. Moreover, glucosylation-independent epithelial damage did not require/elicit recruitment of immune cells .2+ homeostasis [Overall, the cytotoxic activity of Tcds, which are multifunctional proteins with intrinsically disordered regions , is due eostasis ,11,37,82The most evident manifestations of the inactivation by glucosylation of the Rho-GTPase members Rac1, Cdc42 and Rho by Tcds are the cytopathic effects (cell rounding) due to derangements of the actin cytoskeleton with cell cycle arrest and cytotoxic effects that cause cell death ,12,46,61The cytotoxic activity induced by TcdB is manifested in all cell types, while this is not the case for TcdA, probably due to the greater cytotoxic potency of TcdB and the different types of membrane receptors targeted by the two Tcds ,9,10,46.Furthermore, the two Tcds show differences in effects at low and high concentrations. In fact, when TcdB is present at low concentrations (picomolar concentrations) and binds to membrane receptors CSPG4 and FZD, it induces mainly apoptosis in a glucosylation-dependent way, while when TcdB is present at higher concentrations (the nanomolar concentrations) and binds to CPSG4, FZD and PVL3 receptors, it induces mainly necrosis in a glucosylation- and autoprocessing-independent way ,56,57,63Studies on the mechanisms by which TcdA ,85,86,87Effector caspase activation by Tcds is mediated through an extrinsic death pathway dependent on receptors that encompasses caspase-8, or through an intrinsic death pathway dependent on mitochondria that include caspase-9 . TcdA anTcds induce apoptosis also, causing alteration in mitochondrial functionality and a low/moderate increase in reactive oxygen species (ROS) by the ROS/JNK/caspase-3 axis ,103.2+, with the subsequent involvement of caspases and cathepsins [However, it has also been demonstrated that Tcds activated pathways of cell death mediated by calpains, likely due to an early increase in intracellular Cathepsins ,86.TcdB at high concentrations induces cytotoxicity, mainly by necrosis, characterized by early alteration in plasma membrane permeability, ATP depletion, cellular leakage, and condensation of chromatin without activation of caspases ,104,105.TcdB also induces cytotoxicity by pyknotic cell death, characterized by ballooning of the nuclear envelope and condensation of chromatin, both glucosyltransferase domain-dependent and -independent, and ROS-independent ,65,104.Furthermore, TcdB induces a form of cell death called pyroptosis, characterized by a strong inflammation and pore formation mediated by gasdermin-D, which is the substrate of proinflammatory caspases implicated in inflammasome generation ,107,108.2+ mobilization, due to Ca2+ release from both the endoplasmic reticulum and damaged mitochondria. This release plays a critical role in the apoptotic–pyroptosis cascade, which can be mediated by caspase activation triggered by inflammasome activation [2+-mediated calpain activation and subsequent activation of caspases and cathepsins [In this context, the initial key event of assembly and activation of the NLRP3 inflammasome seems to be a rapid Cativation  and by tthepsins ,86,100.2+ flux is activated and/or whether there is mobilization of intracellular Ca2+ stores, events necessary for calpain activation.While the activation of calpains and their primary involvement in the apoptotic cascade induced by TcdB in EGCs has been well documented in vitro , there a2+ in cell death induced by TcdA and TcdB, suggesting possible mechanisms of the increase in intracellular Ca2+ induced by Tcds.However, as discussed in the following paragraphs, several studies seem to provide support for a primary role of Ca2+ homeostasis [2+ can increase due to an influx from the extracellular environment, release from intracellular stores such as the endoplasmic reticulum and lysosomes, or by both mechanisms [2+ induced by bacterial toxins by means of Ca2+ sequestration mechanisms in the endoplasmic reticulum and mitochondria and by extrusion via selective Ca2+ pumps [Most bacterial toxins, either by binding membrane receptors or causing pores on the plasma membrane/membrane of intracellular organelles such as lysosomes and endosomes, can alter cytoplasmic Caeostasis ,115,116.chanisms ,114,115.2+ pumps ,114,115.At the same time, the cells try to repair the damaged plasma membrane by endocytosis or shear and removal of the damaged portion of the membrane (shedding of microvesicles) ,119,120.2+ in cell death and more generally its effects on the mitochondria, lysosomes, and Ca2+-sensitive molecules have scarcely been investigated. In contrast, several studies are available on the family of PFTs [As previously stated, Tcds induce rapid cell cycle arrest, and depending on the Tcd concentration, Tcd receptors, and cell type, can cause death by apoptosis or necrosis ,46,59,63 of PFTs ,115,116,2+ in the toxic activity of Tcds. In fact, it has been demonstrated that when TcdB binds to membrane FZD receptors, which affect cytoplasmic Ca2+ levels, inhibition of calpains strongly inhibits TcdB-induced apoptosis [2+ homeostasis. Additionally, a possible key role of Ca2+ in the activity of Tcds is suggested by several previous observations and studies that in the light of current knowledge can now be better understood and contextualized.Recent studies seem to suggest an important role of Capoptosis ,55,56,58poptosis ,122,123,2+ may be involved in the toxic activity of TcdA and TcdB comes from the studies of Giugliano et al. (1984) through the use of Ca2+ antagonists. In fact, C. difficile filtrates, which cause both the morphological changes and cytotoxicity in Vero cells (monkey kidney), are inhibited when lanthanum chloride  or chlorpromazine hydrochloride  are added to the filtrates. This inhibition is dose-dependent, and when LaCl3 and CRP are added together, the protective effect is greater [3 acts as a Ca2+ antagonist, while CRP, among other properties, also binds calmodulin. Therefore, these results suggest that Ca2+-dependent events are fundamental for the activity of Tcds and that they appear to depend from Ca2+ activation of calmodulin [One of the first demonstrations that Ca greater . LaCl3 almodulin .2+ channel blockers verapamil and LaCl3 protect against the cytopathic effect of TcdB when added before, concomitantly, and up to 30 min after TcdB [2+ and calmodulin activation are required for the toxic activity and internalization process of TcdB. In fact, the functionality of Ca2+ channels is absolutely necessary for TcdB to exert its toxicity [Subsequently, in a more organic study, it was demonstrated in MRC-5 human lung fibroblast cells that the Cater TcdB . Furtherter TcdB . Thus, Ctoxicity .2+ in its toxic activity. In fact, in intestinal epithelial cells, IEC-6, the early alterations in cytoskeleton surface blebbing and nuclear fragmentation (BERN) are not inhibited by Ca2+ channel blockers, inhibitor of Ca2+-activated catabolic enzyme, calmodulin antagonists, or Ca2+ ionophores [2+ homeostasis. A further interesting phenomenon induced by TcdA is the strong formation of surface blebbing in about 50% of the cells [2+ plays no role in TcdA activity on IEC-6 [2+ plays a role inTcdA activity on human granulocytes [However, TcdA appears to be different from TcdB regarding the role of Canophores . Thus, The cells . These ron IEC-6 , are conulocytes ,128,129.2+ by TcdA and TcdB in cytotoxic and cytopathic activity can vary according not only to cell type but also to Tcd dose. Indeed, TcdA in human granulocytes induces a rapid and transient increase in cytosolic Ca2+ that begins within 1–3 s, reaches a peak at 30 s, and returns to baseline values after about 2 min. This transient increase is not due to the Ca2+ influx, but to the mobilization of intracellular Ca2+ from intracellular Ca2+ reserves, partly mediated by guanine nucleotide-binding protein G(i) [However, it is clear that the involvement of Caein G(i) ,129.2+ but only for TcdB internalization. However, it is difficult to exclude that during the internalization process, an influx of Ca2+ or its exit from intracellular reserves does not occur. That Ca2+ may, however, contribute to the toxic activity of TcdB is suggested by the demonstration that the direct introduction of Ca2+ ions into the cell via the Ca2+ ionophore, A23187, enhances the morphological changes induced by TcdB. A further interesting aspect of these studies is represented by the cells that survive TcdB, which increase the cytoplasmic ramifications much more than the controls after 26 days of culture, and further by the appearance of flat epithelioid astrocytes with showy bundles of filaments suggesting the transition to the state of senescence [In primary cultures of astroglia, TcdB induces morphological changes which require extracellular Canescence .2+ in the toxic activity of TcdA is confirmed in a model in which TcdA is given to Chinese hamster ovary (CHO) cells and human colonic tumors cells, T84 [2+ channel blockers or reducing agents, leading to the conclusion that the cytotoxicity of TcdA is not a Ca2+-dependent activity [2+ could depend on the type of receptor involved and the cell type. Thus, some receptors once bound by TcdA have the ability to internalize TcdA and induce toxicity by altering Ca2+ homeostasis, while others internalize TcdA without altering Ca2+ homeostasis [The variability in the role of Calls, T84 ,132. In activity . This coeostasis .2+ due to influx of extracellular Ca2+ [2+ increase follows a bimodal pattern that characterizes two cell populations [2+ concentration (2.5 s) and fast Ca2+ influx followed by a sustained steady state; and (2) 75% of cells characterized by a slow increase in cytosolic Ca2+ concentration, always followed by a steady state. In fast Ca2+ influx, Ca2+ initially localizes in discrete regions close to the membrane, where it reaches higher concentrations, then there is a high and homogeneous diffusion of Ca2+ in the cytosol [2+, the latter diffuses into the cytosol [2+ is exclusively due to its extracellular influx, subsequently the Ca2+ released from intracellular stores contributes to the increase in Ca2+ intracellular concentration [2+ for the toxic activity of TcdB occurs with the sequestration of intracellular Ca2+ by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and Ca2+ clamping, which abolishes the cytotoxicity [Another study demonstrated that the cytotoxic and cytopathic activity of TcdB in human fibroblast cells, NIH-3T3, is due to a sustained increase in intracellular Calar Ca2+ . The celulations : (1) 25% cytosol . Even in cytosol . While tntration . Furthertoxicity . The samtoxicity .2+-independent mechanisms, such as cathepsin activation, may contribute to TcdB toxicity [2+ can activate other Ca2+-dependent protease such as calpains, which can contribute to cytotoxicity [2+ homeostasis, especially if induced from the outside [However, there are other important considerations concerning this phenomenon: (1) Catoxicity ; (2) sustoxicity  and to m outside .2+ and the mobilization of intracellular Ca2+, with the activation of calmodulin. In fact, the association of ethylenediaminetetraacetic acid plus BAPTA completely abrogates TcdA-induced IL-8 secretion, while calpain inhibition abrogates it by about 80% [In human monocytes, TcdA induces an upregulation in the secretion of interleukin-8 (IL-8), one of the cytokines responsible for the inflammatory response that accompanies CDI. In particular, TcdA induces IL-8 upregulation by an influx of Cabout 80% . Furtherbout 80% ,135,136.2+ is required for binding of the cell wall binding domain of TcdA to plasma membrane of CHO cells [2+. Conversely, TcdA at high concentrations has a toxicity independent from Ca2+. Thus, Ca2+ plays a role in TcdA binding to the target cell and renders this more sensitive to TcdA cytotoxicity [Extracellular CaHO cells . In additoxicity .2+ that precedes the final stage of apoptosis [2+ caused by an influx, which is indispensable for altering mitochondrial functionality and the subsequent release of cytochrome C [Most PFTs, in addition to creating pores in cell membranes, creates them in the mitochondrial membranes ,114,115.poptosis . Actuallchrome C .2+. Furthermore, it is possible that the Ca2+ increase induced by TcdB blocks the mitochondrial ATP-sensitive potassium (K+)-channel, thus contributing to apoptosis [TcdB added in vitro to isolated mitochondria causes their swell and releases cytochrome C only in the presence of Capoptosis .2+ also has a very important role in the necrotic response of Hela cells to relatively high concentrations of TcdB (≥100 pM) due to aberrant production of ROS following the assembly of NADPH oxidase (NOX). These high levels of ROS cause mitochondrial damage, lipid peroxidation, and protein oxidation. This phenomenon depends on the activity of L-type Ca2+ channels, because most of their inhibitors block the cytotoxic activity of TcdB [Ca of TcdB ,64.2+ response mechanism to TcdB is characterized by two phases, with an initial peak of Ca2+ increase in 10–15 min, which returns to baseline levels after a further 20 min . This signal is quite small, but it is followed by a second increase at 40–45 min that then persists over time. The increase in intracellular Ca2+ is due to the influx of extracellular Ca2+ [2+ through L-type Ca2+ channels that leads to the rapid activation of PKC with consequent aberrant activation of NOX, which causes the production of a high level of ROS and necrosis as a final event [The Calar Ca2+ ,139. Furlar Ca2+ . Thus, Tal event ,82.2+, also dose-dependent. This increase is due to the upregulation of NOX4, which is mainly responsible for alterations in Ca2+ homeostasis and subsequent endoplasmic reticulum stress. Thus, NOX4 activated by TcdB acting on Ca2+ homeostasis is mainly responsible for cytotoxicity and apoptosis [In another experimental model, at 12 h, TcdB induced a strong increase in intracellular Capoptosis .2+. In fact, in the synaptosomes, where the alpha toxin creates pores allowing the influx of extracellular Ca2+ with the consequent stimulation of phospholipase D, TcdB inhibits the activation of phospholipase D by blocking Rho-GTPase. These results indicate that the Ca2+ influx induced directly by TcdB can be subsequently stopped by TcdB itself through inhibition of Rho-GTPase, which also causes inhibition of phospholipase D, thus preventing the formation of choline and phosphatidic acid [2+ due to extracellular influx and/or mobilization of intracellular Ca2+ stores starts. However, the strongest inhibitory effect is on Ca2+ influx [2+ also depends on the cell type. A relationship between TcdB-induced increase in Ca2 signaling and the phospholipase family is suggested in a macrophage cell model, RAW 264.7, where the phenomenon is independent of Rho-GTPase [Other studies highlight the complexity of the interaction between TcdB and Cadic acid ,141. The+ influx . Of couro-GTPase ,143.2+ alterations.The cytotoxicity of Tcds depends on the cell type because they express different receptors; however, Tcds have the ability to cause cell death in all, further underlining a possible common mechanism of action that could be mediated by Ca2+ homeostasis occur before significant Rho-GTPase inhibition. However, it is possible that after the inhibition of Rho-GTPases, a further alteration in Ca2+ homeostasis occurs, contributing to a stronger activation of Ca2+-dependent molecules, including calpains. This makes the cell death process, which is induced by different molecular mechanisms, irreversible and explains the frequent observation that the inhibition of a single death pathway, for example, that mediated by caspases, does not completely prevent cell death.It has been shown that alterations in CaC. difficile and its Tcds induce activation of pannexin 1 channels, which favor cell death via release of ATP, activation of purinergic receptors and increased intracellular Ca2+ flow, causing the activation of initiator and effector caspases and leading to apoptosis [Loureiro et al.  demonstrpoptosis .2+ mobilization, and PKC activation [Kim et al. demonstrated that TcdA induced colonocytes apoptosis by the release of prostaglandin E2 which by stimulating EP1 receptors induce PLC activation, Cativation .C. difficile by nutrients, including Ca2+, that play a key role in sporulation and germination of C. difficile in the gut [2+ in vitro [2+ uptake or serum Ca2+ levels in CDI in patients or murine models.Loureiro et al.  analyzed the gut , reportiin vitro  and that2+ suggest that in Tcd activity, there is an early phase of about 30–40 min in which Ca2+ homeostasis is altered, with effects that depend on Tcd concentration, cell type and Tcd receptors involved. The increase in intracellular Ca2+ concentration, both distributed throughout the cell and with localized effects on substructures such as vacuoles and vesicular compartments, may be responsible for events crucial for cell survival, such as alteration in mitochondrial polarization, activation of calpains, and alterations in endosome stability, by which the cathepsins are kept under control. There is also the ability of calpains to activate caspases. Then, a series of Ca2+-dependent events fundamental for cell survival takes place, before the occurrence of other events, after about 40 min, resulting from the inhibition by Tcds of Rho-GTPase. These latter are involved in cell cycle arrest and the activation of signaling pathways, which add to the effects already caused by the alteration in Ca2+ homeostasis.Overall, the studies on the relationship between Tcds and Ca2+ homeostasis and the inhibition of the Rho-GTPase family. This in order to be more capable of counteracting the various types of survival pathways in each cell type, including immune cells, the latter with different Tcd susceptibility due to their functional state of activation.All the data above suggest that in their interaction with numerous types of tissue, nervous cells and immune cells, Tcds utilize two important pathways overlapping each other over time, represented by the alteration in Ca2+ has yet to be investigated.It is important to remember that Tcds have a further strategy for neutralizing cells that have resisted toxic activity, i.e., inducing a state of senescence , in whicUnfortunately, there have been no in vivo studies on this issue.2+ that alters its cellular homeostasis, with important consequences on cell fate [2+ homeostasis [2+ homeostasis could have a central role in activating cell death signaling in addition to that derived from Rho-GTPase inhibition [Large clostridial toxins (LCTs) are all PFTs, i.e., toxins that cause pores in the plasma membrane through which occurs an influx of Caell fate ,116,138.eostasis ,138. Althibition ,116.2+ homeostasis  by inositol triphosphate (IP3). This latter is generated through direct or indirect stimulation of G proteins at the plasma membrane level with the subsequent activation of phospholipase A2, favoring the generation of IP3. This in turn stimulates its receptor on the endoplasmic reticulum and final release of Ca2+ [The activation by PFTs of these channels, able to selective release Ca of Ca2+ ,153,154.There are no data available on the possible interaction of Tcds with G proteins; however, TcdA and TcdB are capable of activating phospholipase A2 and therefore of generating IP3 ,156.2+ release from lysosomes through two-pore channels following plasma membrane stimulation of CD38. This activates the pathway involving nicotinic acid adenine dinucleotide phosphate (NAADP), responsible for Ca2+ release [2+ release can contribute to PFT activity [It has been suggested that PFTs might induce Ca release . Damage activity .2+ homeostasis.While there are no data on the activity of Tcds on the CD38-NAADP lysosomal pathway, Tcds are able to act on lysosomes, altering their membrane functionality with release of cathepsins ,123,157,2+-dependent cytoplasmic proteins, if activated, are very sensitive sensors of alterations in Ca2+ homeostasis, and thus of the presence of pores, alterations in the Ca2+ channels of the endoplasmic reticulum, and/or alterations in the Ca2+ channels in lysosomes. In fact, PFTs activate calpains, calmodulin, calcineurin, PKC, and phospholipase [Caholipase ,161,162.2+ in the activity of Tcds, TcdB and to a lesser extent TcdA are able to activate these Ca2+-dependent cytoplasmic proteins. For instance, calpains are activated by TcdB in EGCs [As further confirmation of the role of Ca in EGCs . TcdB is in EGCs , while T in EGCs ,156. Bot in EGCs ,165.2+ homeostasis caused by PFTs activate cell death pathways [2+ levels reached, the capacity of Ca2+ extrusion mechanisms, and the cell type and receptor involved are the elements that decide the prevailing type of cell death.The profound alterations in Capathways ,115,116 2+ are the activation of calpains and calmodulins with consequent activation of caspases. Cathepsins released from lysosomes after functional alteration in their membrane also participate to the necrosis process, and to these events should be added the effects of release of ROS [The main events characterizing cell death by necrosis induced by PFTs and mediated by Cae of ROS ,113,166.e of ROS ,116,167.Even Tcds in certain situations can induce cell death by necrosis ,93,106. 2+-activated calpains and calmodulins [PFT-induced apoptosis is characterized by a mitochondrial pathway with release of AIF and/or cytochrome C from the mitochondria and by a caspase-dependent pathway mainly triggered by Camodulins ,115,116.Tcds are capable of inducing apoptosis ,46,47,602+ mobilization. In fact, it has been reported that Ca2+ mobilization activates NLRP3 inflammasome [2+ signaling/mobilization, which can be promoted by several cellular changes induced by pathogens or their toxins, such as K+ efflux and phagolysosome alteration, thus being responsible for ATP-mediated mitochondrial Ca2+ damage. In fact, it has been demonstrated that Ca2+ signal inhibitors block ATP-mediated mitochondrial Ca2+ damage by evaluating mitochondrial ROS production, loss of mitochondrial membrane potential, and release of mitochondrial DNA into the cytosol. Since these processes are needed for NLRP3 inflammasome activation, the critical role of Ca2+ signaling/mobilization in NLRP3 activation is to mediate mitochondrial damage [2+ homeostasis and calpain activation, effects induced by both PFTs and Tcds.The signal that starts from the mitochondria is mediated by NLRP3 in PFTs and likely also in Tcds and depends on Caammasome . The latl damage . Howeverl damage ,111,112,C. septicum whose activated signal pathways are very analogous to those induced by TcdB and TcdA. The first decisive event is the alteration in Ca2+ homeostasis, with its increase, rapidly followed by the activation of calpains, which act on the stability of the lysosomal membrane, favoring the release of cathepsins acting on various substrates [+/Ca2+ exchange pumps, resulting in further Ca2+ influx. This causes an alteration in MOMP that leads to an increase in ROS and a decreased production of ATP. Finally, DNA damage occurs and activates PARP, which in turn contributes to ATP depletion and thus to cell death [An organic view of cell death mechanisms induced by the most studied PFTs is provided by aerolysin, an orthologue of the alpha toxin of bstrates ,167,176.ll death ,167,176.ll death ,167,176.2+ homeostasis, followed by calpain activation and release of cathepsin from lysosomes [This picture of cell death is very similar to that caused by Tcds, especially by TcdB, as shown from studies demonstrating that the initial rapid event is disruption of Caysosomes ,123,157.ysosomes ,91,102. ysosomes .2+, the cell responds with different types of mechanisms to lower the cytosolic ion concentration and to avoid cell death [2+ extrusion by plasma membrane pumps, Ca2+-ATPase; (2) sequestration of Ca2+ within the endoplasmic reticulum reserves by sarco/endoplasmic reticulum Ca2+-ATPase; (3) and sequestration of Ca2+ in mitochondria by the mitochondrial Ca2+ uniporter. If the sequestration becomes excessive, it can lead to intoxication of the mitochondrion itself.Under increased cytosolic Call death ,116,138:2+ load may be effective whether the influx of Ca2+ is slowed or stopped, acting on the pores to eliminate them [These events aimed at reducing the cytosolic Caate them ,116,138.There are three main mechanisms of pore clearance, as follows.2+—migrate to the plasma membrane where the pores have formed [2+, contributing to the formation of a strong interaction with the plasma membrane. The latter forms membrane folds that are subsequently expelled, detaching from the membrane, which then rapidly reseals the externalized microarea called “microvesicles (MVs)” [Ectocytosis: this is a process of elimination towards the outside of the membrane areas that contain the pores by formation of microvesicles, and it takes place by two modalities ,115,116.e formed . Here, as (MVs)” ,118,119.s (MVs)” ,178.2+ homeostasis, with an increase in the cytoplasmic concentration creating MVs that have two main purposes [2+ by ectocytosis; the second is to surround the cell with MVs derived from the membrane containing the receptors for the PFTs. Therefore, the PFTs are bound before they can reach the cell membrane. Despite the fact that Tcds have been convincingly shown to alter Ca2+ homeostasis, there are no direct studies on MV formation in response to Tcds. However, an interesting study on platelets demonstrated that a pathway that regulates the formation of MVs is blocked by TcdB [2+ increase, at the same time prevent the cell to form MVs as a protective response. In particular, the induction of a Ca2+ influx in platelets leads to the formation of MVs through the Rac1 and p21-activated kinase 1/2 (PAK1/2) activation pathway with the involvement of the activation of calpains, which in turn activate caspases [2+ influx, the cell’s response should be to generate MVs, but this cannot happen because TcdB inhibits Rac1 [Multivesicular bodies: this is one of the membrane repair mechanisms that provokes a profound alteration in Capurposes ,119. The by TcdB . This cocaspases . By inhiits Rac1 .This aspect is relevant for Tcd pathogenesis, because if the MV response also has a protective role , TcdB coEndocytosis: this is a process by which the portion of the membrane that contains the pore is internalized and sent to the lysosome, where it is degraded ,115,116.2+ sequestration and/or the pore repair process.It is also evident that in the cases of PFTs and Tcds, there is always in a given population of cells a percentage that does not undergo any form of cell death, suggesting that in some of them are active effective mechanisms of intracellular CaIJs are one of the primary targets of PFTs, and the main consequence of their destruction is bacterial transmigration across the tissue barrier and the deepening of the infection ,115,180.2+ plays a central role in this process of IJ destruction and on the consequent redistribution of ZO-1 [2+, there is consequent activation of calmodulin and the release of pro-ADAM10, which can be activated by furin. At this point, activated pro-ADAM10 is transferred to the cytoplasmic membrane, where it causes shedding of cadherin and therefore IJ destruction [2+ can alter the permeability of the barrier is mediated by the activation of the myosin light chain kinase pathway [Ca of ZO-1 ,115,180. of ZO-1 ,180,181.truction ,180,181.truction ,180,181. pathway ,180.IJs are an important target of Tcds because their destruction favors the deepening of the infection in the submucosal colonic layers ,174,175.CDIs represent an important and serious widespread health problem, with an increasing incidence in both Western and Eastern communities. This increase is still under investigation, but some data suggest that it might be associated with the appearance of hypervirulent epidemic isolates of ribotype 027  or with The most important event in CDI is represented by cell death caused by TcdA and TcdB, which is then followed by inflammation with immediate and long-term consequences.2+ homeostasis the early key event for the activation of the various types of cell death.Important results have been obtained in studies concerning the complex activation mechanisms and signaling pathways that lead to various types of cell death, apoptosis, necrosis, pyknosis and pyroptosis. Given that Tcd dose, cell type, Tcd receptors involved and their expression in various cell types could account for the complexity of the various signaling pathways activated, perhaps it is possible to identify in the alteration in intracellular Ca2+ is the key player in the activation of cell death, the alterations in its homeostasis in terms of duration, Ca2+ concentration and localization in the various cellular microenvironments can in turn explain the great complexity of the activated signaling pathways involved in the various types of cell death. In particular, the inactivation of Rho-GTPases by Tcds may block the mechanisms that could attempt to counteract the increased intracellular Ca2+ concentration.Of course, if Ca2+ homeostasis are quite similar to those elicited by PFTs, which base most of their toxic action on the direct influx of Ca2+ through membrane pores to alter intracellular Ca2+ homeostasis. Further studies of the key role of Ca2+ in the pathogenetic mechanisms used by Tcds in CDI are needed and could be useful to explore new therapeutic strategies, focused on these early pathogenetic mechanisms, in order to prevent or block them, and to stop the subsequent cascade of events leading to this important inflammatory state of the gut.Additionally, it is interesting that TcdA and TcdB mechanisms and effects on intracellular Ca


To prevent and manage dyslipidemia in young adult men, interventions should be developed separately for the normal, underweight, and obese groups. Effective intervention requires measuring WC and focusing on body fat control. Moreover, regular screening of this population should be performed to ensure early diagnosis and management of dyslipidemia.In recent years, there has been a growing trend of obesity and dyslipidemia among young adult men in South Korea. Therefore, we aimed to identify the obesity-related factors of dyslipidemia among young adult men in Korea using 3-year data (2019–2021) from the 8th Korea National Health and Nutrition Examination Survey. We included 1559 eligible men aged 19–39 years and examined the association between dyslipidemia and participants’ general characteristics, health-related characteristics, and food intake according to obesity status. Statistical analysis was performed using complex sample analysis with weighted household and individual data. The affecting factors of dyslipidemia included age, body image perception, stress, and waist circumference (WC) in the body mass index (BMI) < 25 kg/m Dyslipidemia is characterized by abnormal serum lipid concentrations, including those of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), or their combinations . The incTherefore, weight management and healthy lifestyle modifications are essential for the prevention and appropriate management of dyslipidemia, which can significantly alter CVD incidence and mortality rates . Given tThe Korea National Health and Nutrition Examination Survey (KNHANES)  revealedKNHANES  has alsoWe aimed to identify the obesity-related affecting factors of dyslipidemia in young adult men in Korea using the 8th KNHANES (2019–2021) data and provide evidence that could aid the development of preventive interventions and management strategies for dyslipidemia among young Korean men. The primary objective was to examine the association of dyslipidemia with the participants’ general and health-related characteristics, and food intake according to obesity status. The secondary objective was to identify the risk factors of dyslipidemia according to obesity status.We conducted a secondary analysis of 3-year data from the 8th KNHANES (2019–2021) , a crossAccording to the Korean Society of Lipid and Atherosclerosis criteria , dyslipiParticipants’ general characteristics were recorded, including age, education, and income level, which was based on the monthly household income quartiles presented in the KNHANES . The eduThe assessed health-related characteristics included obesity, waist circumference (WC), body image perception, stress, perceived health, drinking, smoking, walking, muscle training, aerobic physical activity, sleep, work hours, and energy, fat, carbohydrate, and sodium intake. The characteristics were then categorized based on the data collected from the health questionnaire, medical examination, and nutrition survey of the KNHANES .2), nonobese (BMI < 25 kg/m2), and those with a WC of ≥90 and <90 cm [The participants were classified as obese  by the review board of the institution the authors are affiliated with.Statistical analyses were performed using SAS version 9.4 and R version 4.2.3, and household- and individual-weighted values were used for complex sample analysis. Participants’ general characteristics and health-related characteristics were analyzed with complex sample frequency analysis and descriptive statistics (mean and standard error).p < 0.05 was deemed statistically significant. The validity of the predictive model was evaluated by generating calibration plots for each obesity group using the Hosmer–Lemeshow method [Differences in dyslipidemia according to the general and health-related characteristics of the obese and nonobese groups were analyzed with the complex-sample chi-square test. Using a complex-sample multivariate logistic regression analysis that included all risk factors, the factors that affected dyslipidemia in the obese and nonobese groups were analyzed separately. w method , and thew method .2, and 698 (44.8%) had a BMI ≥ 25 kg/m2. The prevalence of dyslipidemia was 20.7% and 44.6% in the BMI < 25 and BMI ≥ 25 kg/m2 groups, respectively. Dyslipidemia prevalence differed significantly according to age, body image perception, stress, aerobic exercise, and WC in the BMI < 25 kg/m2 group and according to age, walking, muscle training, sleep, and WC in the BMI ≥ 25 kg/m2 group  had a BMI < 25 kg/mm2 group .2 group and age and WC in the BMI ≥ 25 kg/m2 group. In both groups, the risk of dyslipidemia increased with increasing age. In the BMI < 25 kg/m2 group, the risk for dyslipidemia was 2.67 times higher among those who perceived themselves to be overweight than among those who perceived themselves to be skinny .We used multivariate logistic regression to separately analyze factors that influenced dyslipidemia in both groups . The sigp = 0.032) and in those with a WC ≥ 90 cm . In the BMI ≥ 25 kg/m2 group, the risk for dyslipidemia was higher in the WC ≥ 90 cm group than in the WC < 90 cm group .The risk of dyslipidemia was higher in participants with stress  [2 and ≥25 kg/m2 groups was 13.7% and 10.1%, respectively. The concordant pair rates were 67.8% and 65.1%, respectively, and accuracy at the same cutoff value of 0.5 was 80.3% and 60.9%, respectively. 2 group (star-shaped) than in the BMI < 25 kg/m2 group (circle-shaped). 2 group (solid line) is further away from the reference line (dashed line) than the curve for the BMI ≥ 25 kg/m2 group (dotted line). The AUC values were 0.68 and 0.65 for the BMI < 25 and ≥25 kg/m2 groups, respectively. To compare the AUCs (or ROC curves) of the obese and non-obese groups, the value obtained with Delong et al.’s D test [p = 0.368), and the value of 2000 bootstraps was 0.93 (p = 0.352). Thus, no statistically significant intergroup difference in the AUC (or ROC curves) was detected.The coefficient of determination (Nagelkerke’s R2)  for the s D test  was 0.902 group and 44.6% in the BMI ≥ 25 kg/m2 group. A previous study [This study was conducted to identify the affecting factors of dyslipidemia in obese and nonobese young adult men in South Korea using the data from the 8th KNHANES (2019–2021)  and to pus study  revealedus study  and Shapus study  reported2 group were age, body image perception, stress, and WC. Other studies [In our study, the affecting factors of dyslipidemia in the BMI < 25 kg/m studies ,15 have  studies  and repo studies . This ma studies ,40. In l studies  in men a studies  in young studies  reported studies . Abdomin studies ,45. TherStress was identified as a predictor of dyslipidemia in the normal and underweight groups. This finding is consistent with that of Tenk et al.’s work , who obs2, both age and WC were identified as affecting factors of dyslipidemia, which is consistent with the findings of many previous studies [2.In the group with a BMI ≥ 25 kg/m studies ,15,41,42 studies , controlChong  reportedBased on the findings of previous studies and the present study, effective management of dyslipidemia requires the identification of relevant factors based on the sociodemographic characteristics of the target population and health interventions based on these factors. However, in reality, the management of dyslipidemia is primarily targeted toward middle-aged and obese populations. Nevertheless, our study reaffirmed the importance of managing dyslipidemia in young adult men and highlighted the need for dyslipidemia management in the normal and underweight BMI groups as well. One key significance of this study is that we identified affecting factors specific to each target group, thus providing insight into the development of interventions for dyslipidemia in young adult men. Finally, to enhance the reliability of the statistical estimations, we merged 3 years of annual data from 2019 to 2021 and converted the year-based weights into combined weights for the analysis.The limitations of this study are as follows: First, although the KNHANES provides a reliable large-scale dataset representative of the Korean population, the cross-sectional survey design prevents the assessment of the long-term effects of factors, such as lifestyle, on the incidence of dyslipidemia in a young adult population. Additionally, causality cannot be determined using these data. Second, the nutrition survey relies on a 24-h dietary recall, which requires the participant to recall the types and amounts of foods consumed in the past 24 h; thus, these data cannot provide objective information on an individual’s usual nutrient intake. Third, our study involved secondary data analysis; therefore, we could not identify other lifestyle-related factors in young adult men. To overcome the limitations of this study, we recommend further replication and longitudinal studies that include lifestyle and health behavior factors and assess nutrient intake using a more accurate nutrition survey. Furthermore, we recommend additional studies for the WHO-based classification of obesity  and further investigation, through diverse study designs and analysis methods, of the underlying factors for obesity in each class while evaluating obesity—a potent risk factor for dyslipidemia—as the mediating variable. We evaluated TC, TG, HDL-C, and LDL-C based on the serum lipid concentration to identify the factors associated with dyslipidemia in our population of interest. However, future studies should also include lipoprotein (a), which was recently identified as an independent causal risk factor for CVD ,49. To cThe prevention and management of dyslipidemia in young adult is imperative for CVD prevention and management, and necessitates interventional strategies that are customized for the young adult male population that has different BMI classifications. In the normal and underweight BMI groups, WC may be a more suitable measure of abdominal obesity and regulating body fat than BMI. Furthermore, interventions for fostering a more positive body image, increasing motivation for body fat management, and managing stress are crucial for these population groups. For patients with obesity, interventions that prioritize weight and body fat control are needed. Furthermore, educational strategies to increase the individual awareness of the importance of early detection and management of dyslipidemia should be undertaken and regular screening should be promoted.


Cannabis sativa’s applications for therapeutical purposes is set apart by the hundreds of known and commercially available strains, the social, cultural and historical context, and the legalization of its use for medical purposes in various jurisdictions around the globe. In an era where targeted therapies are continuously being developed and have become the norm, it is imperative to conduct standardized, controlled studies on strains currently cultivated under Good Manufacturing Practices (GMP) certification, a standard that guarantees the quality requirements for modern medical and therapeutic use. Thus, the aim of our study is to evaluate the acute toxicity of a 15.6% THC: <1% CBD, EU-GMP certified, Cannabis sativa L. in rodents, following the OECD acute oral toxicity guidelines, and to provide an overview of its pharmacokinetic profile. Groups of healthy female Sprague-Dawley rats were treated orally with a stepwise incremental dose, each step using three animals. The absence or presence of plant-induced mortality in rats dosed at one step determined the next step. For the EU GMP-certified Cannabis sativa L. investigated, we determined an oral LD50 value of over 5000 mg/kg in rats and a human equivalent oral dose of ≈806.45 mg/kg. Additionally, no significant clinical signs of toxicity or gross pathological findings were observed. According to our data, the toxicology, safety and pharmacokinetic profile of the tested EU-GMP-certified Cannabis sativa L. support further investigations through efficacy and chronic toxicity studies in preparation for potential future clinical applications and especially for the treatment of chronic pain.The conundrum of  Cannabaceae family, has been cultivated and used for millennia for various medical/therapeutical, religious  or in the experimental groups, regardless of the administrated dose . For allCannabis sativa L. Therefore, based on OECD 423 methodology, the LD50 for oral administration in rats of Cannabixir® Medium Flos was estimated to be equal to or higher than 5000 mg/kg , the maximum time (Tmax), the observed area under the curve (AUC last), the total area under the curve (AVC inf), the half-time (T 1/2), the apparent distribution volume (Vd), and clearance , which necessitates the integrity of the lumbosacral medulla, is characterized by an S-shaped tail erection due to the dopamine release in the central nervous system [In our study, the acute oral toxicity of EU-GMP-certified s system . Generals system . CBGA prs system . CBGA was system . Feigenbs system  identifis system . The neus system . The actThe gross necropsy could not observe any abnormalities in the body, internal cavities or organs. The biochemical analysis results show that the serum creatinine level has a small dose-dependent increase but without statistical significance compared to the control group. In addition, this does not correlate with the levels in urea, which show a downward trend, remaining below control group values, even for the 2000 mg/kg dose, which demonstrates normal or even improved kidney function. Furthermore, this was correlated with the morphological aspects revealed by the histopathological studies. Thus, at the renal level, hyaline areas were observed in the urinary tubules in the paracortical area, represented by uromodulin deposits, at all doses tested, except for the group that received the highest dose, where these changes were less pronounced. Recent studies ,53,54,55There are few studies that attempt to test acute toxicity by oral administration of cannabis plant powder ,26,27,45Cannabis sativa L. plant, higher plasma levels of the acid precursors were detected vs. the THC levels. These precursors can act through direct or indirect mechanisms on the cannabinoid receptors, can have their own actions or lack any pharmacological action, and also, they can reciprocally influence their own pharmacokinetics [After inhalation administration, the THC plasma concentration vs. THCA-A is significantly higher when compared with the oral administration. This is explained by the fact that the acid precursor THCA-A, under the influence of temperature, decomposes to THC. An efficacy study demonstrated that the amount of THC from any standardized THCA-A is in agreement with the decarboxylation kinetics, pointing to the fact that contamination with THC is inevitable . After tkinetics . For sucIn a simulated calculation,For the 300 mg/kg dose, the supraunitary ratio, low Ke, and high Vd could be attributed to the following: the presence of active metabolites, saturation of the metabolization systems, saturation of the transport systems (absorption-elimination), and the presence of cannabinoids that at doses of 5 and 50 mg were in too-low amounts to have biological actions; these parameters may be the result of the synergic or concomitant activities of the cannabinoids present in the phytocomplex. These cumulative tendencies we observed may explain some biological alterations we described in the evaluation of the acute toxicity .In the literature, THCA-A is a weaker agonist of CB1 and CB2 receptors than THC, with a higher affinity for CB1. THCA-A attenuated emesis and vomiting in rats by activating CB1, the effect not being due to the conversion to THC. THCA-A also seems to lack the psychotropic effects of THC. Because THCA-A has a low penetrability of the blood-brain barrier (BBB), it is conceivable that the actions are peripheral. This low penetrability of the THCA-A might be due to the carboxyl group, which reduces the lipid solubility of the molecule. THCA-A can effectively interact with the transient receptor potential melastatin-8 (TRPM8) channels, which are strongly involved in both acute and chronic pain modulation , can inhTHCA-A has also demonstrated a low binding for CB1 and CB2 receptors, at least 62 times smaller than that of THC for CB1 and 125 times smaller than THC for CB2. In efficacy tests, THCA-A slightly inhibited AMPc at CB1, which suggests a weak agonist activity and no efficacy for CB2 .CBGA has antioxidant, antibacterial, neuromodulator and neuroprotective effects  while be® Medium Flos may be harmless to humans. At the same time, the possible favorable pharmacological effects, especially due to the high plasma levels of THCA-A and CBGA after oral administration, warrant a more in-depth exploration of this product in order to identify its potential therapeutic benefits.Considering the reduced toxic effects observed for the dose sequences used in our study, and the calculated LD50, it is most likely that even the accidental ingestion of large doses of CannabixirThe accurate extrapolation of animal data directly to humans may not be fully guaranteed due to interspecies variation in anatomy, physiology, and biochemistry, but they could aid in comprehending acute toxicity mechanisms and health hazards, thereby enhancing the generation of adverse outcome pathways. In addition, appropriate target engagement and safety studies should help define clinically meaningful doses and therapeutic windows. In future studies, we will perform safety screening and chronic toxicity of the phytocomplex. Studies to investigate the product’s efficacy in a broad range of diseases, as well as to establish an interval for the effective dose and a frequency of administration, are additional research directions that can be pursued following the current investigation.® Medium Flos (PZN: 7001905), in the form of a dried inflorescence of Cannabis sativa L. with 15.6% THC and <1% CBD, was sourced from Cansativa GmbH . The completely dried plant material was ground using an electrical mortar grinder RM 200 , and the obtained powder was sieved through a 125 microns strainer (BSS Mesh No. 120).A certified EU GMP batch of CannabixirFormulation: carboxymethylcellulose sodium salt (Na-CMC) was purchased from Alfa Aesar GmbH & Co. .Biochemistry: all the reagents used for biochemical analysis were purchased as in vitro diagnostic ready-to-use reagents from PZ Cormay  and BioSystems S.A.  and used for the automatic analyzer ACCENT-200  according to manufacturer’s instructions.Histopathology: all the reagents used for tissue fixation, processing, embedding and staining were histological grade, purchased from Richard–Allan Scientific , and used according to manufacturer protocol.LC-MS analysis: each natural cannabinoid used in the present study was purchased from Cayman Chemical in the form of a certified reference material supplied at a concentration of 1 mg/mL in either methanol or acetonitrile. Acetonitrile was purchased from VWR Chemicals  and was of HPLC, UHPLC and LC-MS grade. Methanol was purchased from LAB-SCAN Analytical Sciences  and was of HPLC grade. Formic acid 99.7% was from Merck . Ultrapure water (0.055 μS/cm c) was produced by an Arium Mini Plus UV system .Two hundred ninety-four female Sprague–Dawley rats  were housed in the CEMEX animal research facility of the Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania, in a controlled environment , using individually ventilated cages (IVCs), with ad libitum access to food and water, and acclimatized for at least 2 weeks before the experimental protocols. The experimental study was performed in accordance with the European Directive 2010/63/EU and has been approved by the university’s Research Ethics Committee (no. 47/17.02.2021) and authorized by the Romanian National Sanitary Veterinary and Food Safety Authority (no. 34/07.04.2021).The acute toxicity study was carried out following OECD 423: Guideline for testing of chemicals—Acute Oral Toxicity . This meCannabis sativa L. or the possibility of making an informed decision on the dose with a mortality likelihood, we used the minimum starting dose level, 5 mg/kg, accordingly to the OECD 423 [Cannabis sativa L. has been estimated using 4 dose levels, 5, 50, 300, and 2000 mg/kg. A Na-CMC 0.1% aqueous suspension was used as a vehicle to provide uniform doses of Cannabis sativa L. Nine groups have been defined, of 3 animals each, 2 groups for each dose level, and a group for the vehicle (Na-CMC 0.1%), to which the compound suspension was administrated intragastrically using a no. 16 rodent gavage needle.Due to the lack of recent and reliable information regarding the LD50 for OECD 423 . TherefoCannabis sativa L., each individual has been monitored continuously in the first 15 min and then regularly at 30 min, 2 h, 4 h, 8 h, and 24 h, respectively, using a video-tracking system. During the first 24 h, the primary monitoring has been focused on the detection of sedation, convulsions, motor incoordination, loss of balance, reactivity to touch, and tremors and/or stereotypes (head movements). Subsequently, general health parameters such as eating, locomotion, behavior, appearance, and weight loss were monitored and recorded daily for the next 13 days. During the entire monitoring period of 14 days, any abnormalities regarding the behavioral pattern or appearance  have been recorded.The primary monitoring protocol and parameters were adapted from the Primary Observation (Irwin) Test in Rodents , as we dGross necropsy: on the 14th day after oral administration, all the animals were euthanized, and a cardiac puncture was performed to sample 2 mL of terminal blood in 2 3 mL clot activator vacutainer tubes for biochemistry assay. Postmortem, a complete necropsy was performed for all individuals, which consisted of a close examination of the external surface of the body, all the openings and the internal cavities , as well as their contents. Subsequently, 22 internal organs  have been sampled and fixed in 10% formalin for a detailed histopathological examination.Cannabis sativa L. on primary organs responsible for drug metabolism , several biochemical tests were conducted, as follows. After 30 min since harvesting, the vacutainer tubes were centrifuged at 1500× g for 15 min at 4 °C, and the separated serum samples were then subjected to biochemistry analysis using an ACCENT-200 Analyzer . The following parameters have been quantified through the serum biochemical analysis have been quantified the following parameters: glucose, total cholesterol, urea, creatinine, total protein, albumin, alanine transaminase (ALT), AST, alkaline phosphatase and bile acids.Biochemistry assays: to investigate the toxic potential of orally administrated Histopathology: all 22 internal organs harvested in 10% formalin after gross necropsy were let up to 48 h for fixation. Following the fixation step, each organ has been trimmed in organ-specific plans, according to the Revised guides for organ sampling and trimming in rats and mice—Parts 1 to 3 ,78,79. TCannabis sativa L. with 15.6% THC and <1% CBD was assessed using 4 dose levels, 5, 50, 300, and 600 mg/kg. Dose levels were chosen in accordance with OECD 417 guidelines being lower than 1000 mg/kg but still high enough to allow for the identification and measurement of natural cannabinoids in the serum [g for 15 min at 4 °C. The separated serum samples were transferred immediately in microcentrifuge tubes and stored at −20 °C until THC and THCA quantification using LC-MS analysis were performed.The pharmacokinetic profile of the EU-GMP-certified he serum . Each doA method for simultaneous extraction and LC-MS/MS quantitation of 11 cannabinoids was developed and validated for the present study. Cannabinoid extraction from rat serum was performed according to the method described by Wakshlag et al. , with moThe mass spectrometer was operated in positive mode. The source parameters were a capillary voltage of 4000 V, a gas of temperature 325 °C, a gas flow of 8 L/min, and a nebulizer of 35 psi. The parameters of the mass spectrometer for each time and scan segment and the retention time for each compound are presented in For the extraction of natural cannabinoids from rat serum, the following instruments were used: thermocycler for vortexing at temperatures above room temperature model Thermomixer F1.5 , thermocycler for vortexing at temperatures below and above room temperature, model Thermal Mixer with a thermoblock for 24 places for 1.5 mL microtubes , minicentrifuge model MSC-3000  equipped with R-1.5 rotor for 12 1.5 mL microtubes and benchtop centrifuge model SL 8R  equipped with a rotor for 24 1.5 mL microtubes model Microclick 24 × 2. The LC-MS/MS system consisted of an Agilent 1200 liquid chromatograph containing a solvent compartment, a degasser , a binary pump , an autosampler , a thermostatted column compartment , a 10:1 splitter  coupled to a 6410A2k triple quadrupole mass spectrometer  equipped with electrospray source and a Nitrogen generator N118LA, Peak Scientific Instruments Ltd. .The qualitative analysis of the chromatograms and mass spectra was performed by Agilent MassHunter Workstation Software Qualitative Analysis v.B.01.02 and the quantitative analysis by Agilent MassHunter Workstation Software Quantitative Analysis v.B.01.03. Linear calibration curves were obtained for all 11 cannabinoids using the relative response  and the relative concentration . A linear 7-point calibration curve with 1/×2 weighing and a concentration range of 5–1000 ng/mL in rat serum was employed for the quantitation of the serum samples collected in the present study. The analysis of carryover was performed by LC/MS analysis of a blank sample extracted from normal rat serum after LC/MS analysis of a sample extracted from rat serum fortified with 1000 ng/mL natural cannabinoids. According to the results, carryover was absent at this concentration for all tested cannabinoids.Cannabis sativa L. in serum were used for the pharmacokinetic analysis using a non-compartmental model (Linear Up-Log Down method) for extravascular dosing. Pharmacokinetic parameters were estimated using WinNonlin pharmacokinetic software . When performing non-compartmental analysis, the area under the concentration-time curve (AUC) is calculated to determine the total drug exposure over a period of time. Together with the maximum concentration of the drug at a time (Cmax) at the Tmax time point, these parameters are used to evaluate the systemic exposure to the compounds contained by the plant. Also, the half-time (T1/2) and the clearance (Cl) of the compounds have been calculated.The results obtained by LC-MS dosing of major compounds of the EU-GMP-certified Cannabis sativa L. (tested as Cannabixir® Medium Flos), after acute oral administration in rats, is very good, with a determined oral LD50 value human equivalent oral dose ≈ 806.45 mg/kg. The minor-to-moderate effects of the phyto-complex on the behavioral manifestations in a small number of animals do not present particular importance in the overall pharmaco-toxicological picture. The biochemical markers changes corroborated with the macroscopic and microscopic analysis of the target organs are in agreement with earlier studies on cannabinoids reported in the literature and do not raise the suspicion of a potential toxic effect of the tested plant at any of the doses.Our results indicate that the toxicity and safety profile of the EU-GMP certified Cannabis sativa L.  deserves a deeper exploration through future efficacy and chronic use studies, which could support its therapeutic potential in specific targeted pathologies or indications.The favorable pharmacokinetic profile of the EU-GMP certified In this regard, it would be worth testing selective drugs targeting TRPs and/or COX to manage neurodegeneration and treat associated symptoms such as pain and cognitive/motor dysfunction. The evidence suggests that the effectiveness of pharmacological agents regulating TRP channel activity or COX enzyme to treat neuropathological processes and pain deserves further research.