Fracture healing failure remains a major complication in trauma and orthopedic surgery. The transplantation of autologous cancellous bone grafts represents the gold standard for the treatment of atrophic non-unions. However, during revision surgery the grafts can be exposed to a significant period of intraoperative ischemia, which may have detrimental effects on their quality and functionality. Therefore, we analyzed the effects of different periods of ischemia (0, 30, 60 and 90 min) on cellular stress, gene expression and viability of the bone grafts, to determine a critical ischemia time window for transplantation. Graft samples were harvested from 24 patients undergoing revision surgery due to bone healing failure. Analyses included mRNA profiler arrays, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Ischemia lasting 60 min or longer induced the expression of stress-induced genes, such as JUN and DUSP1. This was associated with early cellular stress within the grafts, as indicated by the presence of hypoxia-inducible factor (HIF)-1α-positive cells and an increased number of senescent p16-positive cells at early time points of ischemia. Additional analyses revealed a significantly higher number of apoptotic cleaved caspase-3-positive cells at 60 and 90 min of ischemia, demonstrating a compromised viability of the grafts. Moreover, RT-PCR analyses revealed a shift from a pro-osteogenic towards a pro-chondrogenic extracellular matrix (ECM) gene expression profile. Taken together, periods of ischemia of 60 min or longer after tissue harvesting should be avoided during cancellous bone graft transplantation to preserve graft viability.

Autologous fat grafting is increasingly used in plastic, reconstructive, and esthetic surgery. Cryopreservation offers a promising solution for the long-term storage of adipose tissue, enabling multiple grafting sessions while minimizing patient discomfort associated with repeated liposuction for fat harvesting. This systematic review aims to analyze the current literature focusing on factors that influence the outcome of cryopreservation, including the use of cryoprotectants, the cooling and warming rate, the storage temperature, and the enrichment of cryopreserved fat grafts. A systematic search of the PubMed/MEDLINE database up to November 2024 was performed, including original preclinical and clinical studies written in English describing the cryopreservation of unprocessed or mechanically processed adipose tissue (macrofat, microfat, or nanofat). Eligible articles needed to describe the applied cryopreservation protocol, at least the storage temperature. Studies on cryopreservation of adipose-derived stem cells (ASCs), stromal vascular fraction, microvascular fragments, and other isolated components of adipose tissue were excluded. Data on cryoprotectants, cooling and warming rates, storage temperature, and eventual supplementation or enrichment of frozen fat were collected. Of the 679 records identified, 59 met the inclusion criteria. Adipose tissue cryopreservation at -80°C with a cryoprotectant, controlled slow cooling, and fast warming represented the most often applied protocol with encouraging outcomes in maintaining tissue survival and histological structure. Several studies indicated that the supplementation of frozen adipose tissue with ASCs improves tissue survival. Taken together, existing studies present diverse, and to some extent, conflicting results regarding cryopreservation protocols and their effects on adipose tissue viability. Hence, the ideal cryopreservation protocol for autologous fat remains to be established. Moreover, tailored protocols may be necessary for the cryopreservation of fat derivatives, such as nanofat.

Vasculogenic mimicry (VM) contributes significantly to tumor aggressiveness and resistance to anti-angiogenic therapies. Simultaneous inhibition of both angiogenesis and VM represents a promising strategy to improve therapeutic outcomes in aggressive cancers, such as triple-negative breast cancer (TNBC), which responds poorly to anti-angiogenic therapies. In this study, we identified carvacrol, a natural monoterpenoid phenol widely used as a food additive, as a dual inhibitor of angiogenesis and VM in TNBC. Carvacrol preferentially inhibited angiogenesis in endothelial cells (ECs) and VM in TNBC cells at concentrations that had minimal effects on TNBC cell proliferation. Mechanistically, carvacrol directly bound to the vanilloid-like (VL) site of transient receptor potential melastatin 7 (TRPM7), thereby inhibiting channel activity and attenuating Zn(2+ )influx. This triggered dephosphorylation of the mammalian target of rapamycin (mTOR) and subsequent proteasomal and lysosomal degradation of key receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptor 2 (VEGFR2), Tie2, fibroblast growth factor receptor 1 (FGFR1), and insulin-like growth factor 1 receptor (IGF1R) in ECs, as well as FGFR1 and IGF1R in TNBC cells. Genetic knockdown of TRPM7 recapitulated the anti-vascular effects and signaling alterations induced by carvacrol. In vivo, carvacrol effectively suppressed TNBC vascularization and growth in a mouse dorsal skinfold chamber model and an orthotopic xenograft model. Together, these findings suggest that carvacrol preferentially targets angiogenesis and VM in TNBC by suppressing the TRPM7/Zn2+/mTOR/RTKs axis, highlighting it as a promising therapeutic candidate for TNBC and potentially other tumors resistant to anti-angiogenic therapies, while positioning the TRPM7 channel as a novel anti-vascular target for TNBC treatment.

Aims In the therapy of type 1 diabetes mellitus, the subcutaneous space has been suggested to be a clinically preferable transplantation site for pancreatic islets due to its easy accessibility. However, its poor vascularisation capacity and, thus, challenging environment typically result in islet engraftment failure. In the present proof-of-principle study, we demonstrate that this problem can be overcome by nanofat, an emulsified fat derivative already used in clinical practice.Materials and Methods The cellular composition of nanofat was assessed by immunohistochemistry. The angiogenic activity of the soluble and cellular nanofat fraction was analyzed by an angiogenic protein array, tube formation and spheroid sprouting assays. The viability and endocrine function of islets exposed to the nanofat fractions was investigated by flow cytometry, qRT-PCR and ELISA. In vivo, islets and nanofat were co-transplanted under the kidney capsule as well as into the subcutaneous space of diabetic animals.Results In a panel of in vitro assays, we showed that the soluble and cellular nanofat fraction improve the viability, hormone release, and angiogenic activity of islets. The beneficial effects of these two fractions were validated in vivo in the murine diabetic kidney capsule model, as indicated by an accelerated restoration of normoglycaemia. The co-transplantation of islets with nanofat resulted in successful islet engraftment within the subcutaneous space of diabetic mice.Conclusions These findings demonstrate that nanofat markedly boosts the vascularisation and endocrine function of islet grafts. Hence, its co-transplantation with pancreatic islets represents a simple, clinically feasible approach to make the subcutaneous space available for future islet transplantation.

The surgical treatment of geriatric patients represents a major challenge in traumatology. It is well known that aging affects fracture healing. However, the exact pathophysiology of age-related changes in angiogenesis, inflammation and bone remodeling remains still elusive. Therefore, we herein studied the differences of femoral fracture healing in young adult (3–4 months) and aged (16–18 months) CD-1 mice by using a stable closed femoral fracture model with intramedullary screw fixation. The callus tissue was analyzed by means of X-ray, micro-computed tomography (µCT), histology and immunohistochemistry. We found a deteriorated trabecular architecture and a reduced bone formation within the callus tissue of aged mice. Moreover, aged animals showed an increased number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts at an early healing time point, whereas the fraction of mature α-smooth muscle actin (SMA)-positive microvessels was significantly reduced. Furthermore, the numbers of macrophages and granulocytes were higher in the callus tissue of aged animals at the end of the healing process. Taken together, these results demonstrate a delayed femoral fracture healing in aged CD-1 mice. This is most likely caused by an early overshooting osteoclast response, a decelerated maturation of the callus microvasculature and a late increased recruitment of pro-inflammatory cells. Targeting these alterations may contribute to the development of novel treatment approaches for the stimulation of bone regeneration in geriatric patients.

Metamizole is a commonly used analgesic drug in clinical fracture management, which does not affect the healing process under physiological conditions. However, many fracture patients suffer from co-morbidities resulting in ischemic conditions with impaired bone healing. The effect of metamizole on fracture healing under ischemic conditions has not been analyzed so far. Accordingly, in this study 44 CD-1 mice underwent ligation of the deep femoral artery to induce mild ischemia in the right hind limb. The femur was then fractured and stabilized with an intramedullary lag screw and the animals were daily treated per os with 50 mg/kg metamizole (n = 23) or vehicle (control; n = 21). Serum concentrations of the active metamizole metabolites, 4-methyl-amino-antipyrine (4-MAA) and 4-amino-antipyrine (4-AA), were determined 30, 60 and 90 min after administration. Bone healing was analyzed by biomechanical, radiological, histomorphometrical and Western blot analysis at 2 and 5 weeks postoperatively. The plasma level of 4-MAA was high at all time points, whereas 4-AA peaked at 90 min after administration. Biomechanical, radiological and histomorphometrical analyses revealed no differences between metamizole-treated and control mice, while both groups showed a delayed fracture healing. Of interest, Western blot analyses of callus tissue showed an increased expression of the pro-angiogenic factor Cyr61 and the osteoanabolic runt-related transcription factor 2 (RUNX2) as well as the osteocatabolic receptor activator of NF-κB ligand (RANKL) in metamizole-treated animals when compared to controls. Taken together, these findings indicate that the application of metamizole does not affect fracture healing under ischemic conditions. Therefore, treatment with this analgesic drug may be also recommended in fracture patients suffering from co-morbidities resulting in tissue ischemia.

Islet-resident macrophages contribute to hypoxia-induced islet cell death during pancreatic islet transplantation. However, their specific role during this process remains elusive. Here, we report that interleukin-1α (IL-1α) and IL-1β are released by islet-resident macrophages, resulting in the suppression of insulin secretion. This may be due to a decreased inflammation-driven expression of pancreatic and duodenal homeobox 1 (PDX-1) and MafA in β cells. Islet-resident macrophages release significantly less IL-1α when compared to IL-1β. However, both cytokines inhibit insulin expression and secretion to a comparable extent. We identified heparan sulfate on the islet surface, which acts as a "molecular glue" potentiating the inhibitory action of IL-1α on insulin expression via specific binding to IL-1 receptor (IL-1R). In vivo analyses revealed that the loss of IL-1 signaling in isolated islets accelerates their revascularization and, thus, enhances their endocrine function. These findings indicate that heparan sulfate fine-tuned IL-1 signaling crucially determines the outcome of islet transplantation.

Radiotherapy, while effective in cancer treatment, can lead to side effects, such as radiodermatitis with potential long-term consequences including telangiectasias, ulceration and fibrosis of the skin, eventually resulting in impaired wound healing. In this study, we analyzed whether the healing of such challenging wounds can be improved by nanofat (NF). NF is generated by mechanical emulsification and filtration of fat samples and, thus, is a random mixture of adipose-derived stem cells, microvascular fragments, extracellular matrix components and growth factors. Two months after localized ionizing radiation of the skin with a total dose of 20 Gy, full-thickness wounds were created in dorsal skinfold chambers of mice, which were filled with platelet-rich plasma (PRP; control, n = 8) or NF fixed in PRP (PRP + NF, n = 8). The healing process was assessed by means of stereomicroscopy, intravital fluorescence microscopy, histology and immunohistochemistry over 14 days. The closure of PRP + NF-treated wounds was accelerated, as indicated by significantly smaller wound areas on day 14 when compared to controls. This was associated with a higher density of blood-perfused microvessels inside the wounds. Moreover, PRP + NF-treated wounds showed a tendency towards an improved granulation tissue formation, lymphatic drainage and M2/M1 macrophage ratio. Taken together, these findings suggest that the application of NF represents a promising therapeutic strategy for the management of complex wounds in irradiated skin.

Background/Objectives: Patients suffering from fractures are often treated with clopidogrel during the phase of bone healing due to multiple comorbidities. Studies indicate that clopidogrel suppresses osteoblast proliferation and the formation of trabecular bone. However, it is unknown whether clopidogrel also affects fracture healing under ischemic conditions, as they may occur in multimorbid patients. Methods: To test this in the present study, a murine ischemia model was performed in CD-1 mice by ligating the right deep femoral artery to induce mild ischemia of the right lower limb. A closed fracture of the femur was then stabilized by inserting an intramedullary lag screw. The animals received either 3 mg/kg body weight clopidogrel daily per os or vehicle (control). Bone healing was assessed by biomechanical, radiological, histomorphometrical and Western blot analyses 2 and 5 weeks postoperatively. Results: The fractured femurs in the clopidogrel group exhibited no increase in biomechanical stiffness throughout the observation period in contrast to controls. While the radiological analysis showed no differences between both groups, histomorphometric analyses demonstrated a significantly reduced bridging score, less bone and more connective tissue within the callus of clopidogrel-treated animals. Western blot analyses revealed a significantly reduced expression of the osteogenic marker bone morphogenetic protein (BMP)-4 and an increased expression of the blood vessel marker CD31. Conclusions: These results show that clopidogrel may impair fracture healing under challenging ischemic conditions, which is associated with a shift in angiogenic and osteogenic expression markers in the callus tissue. Therefore, clopidogrel treatment may not be recommended in fracture patients with tissue ischemia.

Heat preconditioning has been shown to promote nutritive perfusion and tissue survival in autologous fat grafting as well as in flap and breast surgery. However, its impact on the vascularization properties of nanofat has not been investigated so far. Therefore, we exposed nanofat from donor mice to a temperature of 43 °C for 1 h and assessed the effects of this heat stress on cell viability and the expression of heat shock proteins (HSPs) and angiogenesis-related factors. Moreover, dermal substitutes seeded with heat-preconditioned and non-preconditioned control nanofat were implanted into dorsal skinfold chambers of recipient mice to study their vascularization and tissue integration in vivo by means of repeated intravital fluorescence microscopy, histology and immunohistochemistry. Heat preconditioning upregulated the expression of HSPs in nanofat without affecting cell viability. Moreover, it resulted in the downregulation of many pro-angiogenic factors and the increased expression of anti-angiogenic factors, indicating a shift towards an anti-angiogenic phenotype. Accordingly, implanted dermal substitutes seeded with heat-preconditioned nanofat exhibited a reduced vascularization and were not better integrated into the host tissue when compared to controls. These findings indicate that heat preconditioning cannot be recommended for enhancing the vascularization capacity of nanofat.

Background:Chloride intracellular channel 1 (CLIC1) is expressed in endothelial cells and platelets. Loss-of-function studies suggest that CLIC1 is involved in adhesive interactions in either cell type, but the exact mechanism of CLIC1 action is still a matter of debate. Methods:Cultured endothelial cells and platelets were probed for CLIC1 function as well as subcellular location using fluorescence microscopy, flow cytometry, and light transmission aggregometry. CLIC1 function in vivo was tested using a mouse dorsal skin fold chamber model to assess thrombus formation. Results:Knocking down CLIC1 in endothelial cells is associated with the inability of cells to spread after attachment to the extracellular matrix. Critical to this process is the endothelial integrin αvβ3, which mediates the recruitment of CLIC1 into newly formed lamellipodia and subsequent colocalization with F-actin. Inhibiting CLIC1 with siRNA or the synthetic CLIC1 inhibitor IAA94, on the other hand, reduced F-actin formation in nascent adhesions, indicating that CLIC1 supports integrin β3-mediated cytoskeletal dynamics during endothelial cell attachment. In addition to endothelial cells, colocalization of CLIC1 with F-actin was detected in lamellipodia of platelets, which relocate CLIC1 to their cell surface in an integrin-dependent manner. Treatment with the CLIC1 inhibitor IAA94 hindered CLIC1 relocation to the platelet membrane, diminished platelet aggregation, and reduced integrin αIIbβ3 activation. Injecting mice with IAA94 delayed vaso-occlusion in a mouse model of photochemical thrombus formation in vivo. Conclusion:CLIC1 is regulated by adhesive interactions with integrin ligands that cause CLIC1 to relocate to the cell membrane of endothelial cells and platelets. This process in turn appears to be relevant for integrin-mediated functions involved in platelet thrombus formation in vitro and in vivo.

Cilostazol has previously been shown to reduce liver steatosis and enhance hepatic perfusion. We investigated the effects of cilostazol after major hepatectomy in a steatotic rat model. Six weeks prior to surgery, Sprague–Dawley rats were fed with a high-fructose diet. The treatment group received daily 5 mg/kg cilostazol. Seven days following the cilostazol treatment, all animals underwent 70% liver resection (PHX). Analysis of hepatic blood flow and microcirculation and immunohistochemical examinations were conducted 30 min after PHX (postoperative day [POD] 0) as well as on POD 1, POD 3 and POD 7. The weight of cilostazol-treated animals was significantly reduced compared to untreated controls after completion of the 6-week high-FRC diet. Furthermore, 41% macrovesicular steatosis was found in the control group compared to 8% in the cilostazol group. Hepatic arterial and portal venous perfusion were increased in the cilostazol group on POD 7. Lower liver enzyme release was found postoperatively in cilostazol-treated animals. Moreover, apoptosis and neutrophil infiltration were reduced after cilostazol treatment. Proliferation of hepatocytes and liver regeneration after PHX were significantly increased in the cilostazol group. Consequently, cilostazol should be evaluated as a novel strategy to reduce the rate of liver failure after PHX in steatotic liver.

Inhibition of angiogenesis, either as monotherapy or in conjunction with other treatments, holds significant promise in cancer treatment. However, the limited efficacy of clinically approved anti-angiogenic agents underscores the urgent need for the development of novel drugs and therapeutic strategies. In this study, we demonstrate the highly selective inhibitory effects of clioquinol, a topical antifungal and antibiotic agent, on the angiogenic activity of endothelial cells (ECs) in a series of in vitro angiogenesis assays. Moreover, clioquinol effectively suppressed blood vessel formation in ex vivo aortic ring and in vivo Matrigel plug assays. Mechanistic studies revealed that clioquinol directly binds to the ATP-binding site of vascular endothelial growth factor receptor 2 (VEGFR2), promoting its degradation through both proteasome and lysosome pathways. This led to the down-regulation of the downstream extracellular signal-regulated kinase (ERK) pathway. In addition, the combination with the AKT inhibitor MK-2206 synergistically boosted the anti-angiogenic efficacy of clioquinol in vitro and in an in vivo dorsal skinfold chamber model of triple-negative breast cancer (TNBC), leading to the suppression of TNBC growth. Accordingly, clioquinol, either alone or in combination with AKT inhibitors, represents a promising therapeutic agent for future anti-angiogenic cancer treatment.

Background Despite growing insights into the pathophysiology of non-union formation, failed fracture healing remains a major complication in trauma and orthopedic surgery. The transplantation of cancellous bone grafts represents the gold standard for the treatment of atrophic non-unions and large-scaled bone defects. Depending on the type of procedure and the available personnel, the bone grafts may be exposed to a significant period of intraoperative ischemia before the transplantation to the defect site. This ischemia may have detrimental effects on the quality and functionality of the grafts. Methods Therefore, we analyzed in this study the effects of different periods of ischemia (0, 30, 60 and 90 minutes) on oxidative stress, gene expression and viability of autologous bone grafts, to determine a critical ischemia time window for cancellous bone graft transplantation. Graft samples were harvested from 24 patients undergoing revision surgery due to bone healing failure. The samples were analyzed by mRNA profiler arrays, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Results An ischemia of 60 minutes or longer induced the expression of pro-inflammatory and stress-induced genes, such as CXCL8 , JUN and DUSP1 . This was associated with early cell stress within the grafts, as indicated by the presence of hypoxia-inducible factor (HIF)-1α-positive cells and an increased number of senescent p16-positive cells. Additional immunohistochemical analyses revealed a significantly higher number of apoptotic cleaved caspase-3-positive cells at 60 and 90 minutes of ischemia, demonstrating a compromised viability of the grafts. RT-PCR analyses revealed a shift from a pro-osteogenic towards a pro-chondrogenic extracellular matrix (ECM) gene expression profile, along with evidence for potentially compromised angiogenesis and hematoma formation at the later transplantation site. Conclusion Taken together, these findings indicate that periods of ischemia of 60 minutes or longer should be avoided during cancellous bone graft transplantation to preserve graft function and regenerative capacity.

The elderly exhibit a reduced healing capacity after fracture, which is often associated with delayed or failed bone healing. This is due to a plethora of factors, such as an impaired bone vascular system and delayed angiogenesis. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil exerts pro-angiogenic and pro-osteogenic effects. Hence, we herein investigated in aged mice whether sildenafil can improve fracture healing. For this purpose, 40 aged CD-1 mice (16–18 months) were daily treated with 5 mg/kg body weight sildenafil (n = 20) or vehicle (control, n = 20) by oral gavage. The callus tissue of their femora was analyzed at 2 and 5 weeks after fracture by X-ray, biomechanics, micro-computed tomography (µCT), histology, immunohistochemistry as well as Western blotting. These analyses revealed a significantly increased bone volume and higher ratio of callus to femoral bone diameter in sildenafil-treated mice at 5 weeks after fracture when compared to controls. This was associated with a reduced number and activity of osteoclasts at 2 weeks after fracture, most likely caused by an increased expression of osteoprotegerin (OPG). Taken together, these findings indicate that sildenafil does not improve fracture healing in the elderly but delays the process of bone remodeling most likely by reducing the number and activity of osteoclasts within the callus tissue.

ObjectiveTo investigate whether tibiofemoral alignment influences early knee osteoarthritis (OA). We hypothesized that varus overload exacerbates early degenerative osteochondral changes, and that valgus underload diminishes early OA.MethodNormal, over- and underload were induced by altering alignment via high tibial osteotomy in adult sheep (n = 8 each). Simultaneously, OA was induced by partial medial anterior meniscectomy. At 6 weeks postoperatively, OA was examined in five individual subregions of the medial tibial plateau using Kellgren-Lawrence grading, quantification of macroscopic OA, semiquantitative histopathological OA and immunohistochemical type-II collagen, ADAMTS-5, and MMP-13 scoring, biochemical determination of DNA and proteoglycan contents, and micro-computed tomographic evaluation of the subchondral bone.ResultsMultivariate analyses revealed that OA cartilaginous changes had a temporal priority over subchondral bone changes. Underload inhibited early cartilage degeneration in a characteristic topographic pattern (P ≥ 0.0983 vs. normal), in particular below the meniscal damage, avoided alterations of the subarticular spongiosa (P ≥ 0.162 vs. normal), and prevented the disturbance of otherwise normal osteochondral correlations. Overload induced early alterations of the subchondral bone plate microstructure towards osteopenia, including significantly decreased percent bone volume and increased bone surface–to–volume ratio (all P ≤ 0.0359 vs. normal).ConclusionThe data provide high-resolution evidence that tibiofemoral alignment modulates early OA induced by a medial meniscus injury in adult sheep. Since underload inhibits early OA, these data also support the clinical value of strategies to reduce the load in an affected knee compartment to possibly decelerate structural OA progression.

Alternative matrices, especially exhaled breath (EB), have gained increasing attention for a few years. To interpret toxicological findings, knowledge on the toxicokinetic (TK) properties of a substance in EB is indispensable. While such data are already accessible for various drugs (e.g. Delta 9-tetrahydrocannabinol), they are still not available for new psychoactive substances, particularly synthetic cannabinoids (SCs). As SCs raise a high public health concern, the aim of this study was to assess these data in future TK studies in pigs. For this purpose, an in vitro sampling technique of EB was initially developed, which is prospectively applied to anesthetized and ventilated pigs for the detection of SCs in a controlled and reproducible manner as exemplified by cumyl-5F-P7AICA. Furthermore, a method for the qualitative and quantitative detection of cumyl-5F-P7AICA in EB using glass fiber filters (GFFs) was established and fully validated. Therefore, cumyl-5F-P7AICA (0.5 mg/mL in ethanol absolute) was initially nebulized using a ventilation machine and a breathing tube, as they are also used in surgeries. The aerosol was delivered into a simulated pig lung. To collect EB, a pump was connected to that part of the breathing tube, which contains EB (expiratory limb), and sampling was performed repeatedly (n = 6) for 15 min (2 l EB/min) each using GFF. For extraction of the substance, the GFFs were macerated with acetone and the remaining experimental components were rinsed with ethanol. After sample preparation, the extracts were analyzed by liquid chromatography tandem mass spectrometry. In the complete experimental setup, about 40% of the initially nebulized cumyl-5F-P7AICA dose was found, with 3.6 +/- 1.3% being detected in the GFF. Regarding the comparably high loss of substance, the open ventilation system and a conceivable adsorption of the SC in the ventilator have to be considered. However, the herein introduced approach is promising to determine the TK properties of cumyl-5F-P7AICA in EB.

Many fatal intoxications have been reported in connection with the consumption of newer, highly potent synthetic cannabinoids. Yet, a possible postmortem redistribution (PMR) might complicate reliable interpretation of analytical results. Thus, it is necessary to investigate the PMR-potential of new synthetic cannabinoids. The pig model has already proven to be suitable for this purpose. Hence, the aim of this study was to study the PMR of the synthetic cannabinoid 5F-MDMB-P7AICA and its main metabolite 5F-MDMB-P7AICA-dimethylbutanoic acid (DBA). 5F-MDMB-P7AICA (200 µg/kg body weight) was administered by inhalation to anesthetized and ventilated pigs. At the end of the experiment, the animals were euthanized and stored at room temperature for 3 days. Tissue and body fluid samples were taken daily. Specimens were analyzed after solid phase extraction using a standard addition method and LC–MS/MS, blood was quantified after protein precipitation using a validated method. In perimortem samples, 5F-MDMB-P7AICA was found mainly in adipose tissue, bile fluid, and duodenum contents. Small amounts of 5F-MDMB-P7AICA were found in blood, muscle, brain, liver, and lung. High concentrations of DBA were found primarily in bile fluid, duodenum contents, urine, and kidney/perirenal fat tissue. In the remaining tissues, rather low amounts could be found. In comparison to older synthetic cannabinoids, PMR of 5F-MDMB-P7AICA was less pronounced. Concentrations in blood also appear to remain relatively stable at a low level postmortem. Muscle, kidney, fat, and duodenum content are suitable alternative matrices for the detection of 5F-MDMB-P7AICA and DBA, if blood specimens are not available. In conclusion, concentrations of 5F-MDMB-P7AICA and its main metabolite DBA are not relevantly affected by PMR.

Synthetic cannabinoids (SCs) are consumed as an alternative to cannabis. Novel compounds are developed by minor modifications in their chemical structure, e.g. insertion of a carboxamide moiety as a linker, which can potentially lead to altered toxicokinetics (TK). Knowledge on the TK data of SCs, especially structural modified substances, is scarce. Hence, interpretation of toxicological results is challenging. Therefore, the aim of the present study was to evaluate the TK of cumyl-5F-P7AICA in a pig model, which was shown to be suitable for TK studies of SCs. A 200 µg/kg body weight dose of cumyl-5F-P7AICA was administered intravenously (n = 6) or inhalatively (n = 10) via an ultrasonic nebulizer to pigs. Blood specimens were repeatedly drawn over 6 h and the concentrations of cumyl-5F-P7AICA as well as its N-pentanoic acid (NPA) metabolite were determined using a fully validated LC–MS/MS method. Based on the concentration–time profiles, a population TK analysis yielded a three-compartment model for the TK of cumyl-5F-P7AICA, whilst a two-compartment model described the NPA best. The incorporation of transit compartments accounts for the time delay between the appearance of cumyl-5F-P7AICA and NPA in serum. Finally, the model was upscaled to humans using allometric scaling. In comparison to older SCs, a higher volume of distribution was determined for cumyl-5F-P7AICA. No further relevant differences of the TK properties were observed. Insertion of a carboxamide moiety into the chemical structure of SCs does not appear to have only minor influence on the TK.