A Case of Phenibut Directed Detoxification Leading to Toxicity During the COVID-19 Pandemic.

BACKGROUND: Phenibut is a non-Food and Drug Administration-approved gamma-aminobutyric acid analog marketed in the United States as an anxiolytic, cognitive enhancer, and alcohol withdrawal treatment through online supplement vendors. In this case report, we describe a woman's self-directed detoxification with phenibut used to manage withdrawal symptoms from fentanyl and benzodiazepines in March 2020 during the height of the COVID-19 pandemic. CASE: A 38-year-old woman with severe opioid, benzodiazepine, gabapentin, stimulant use disorders developed altered mental status after oral phenibut ingestion intended to help self-manage opioid and benzodiazepine withdrawal. She chose self-directed detoxification as she feared COVID-19 exposure in detoxification facilities. Her altered mental status drove her to jump out a third-story window causing multiple spinal fractures. After a long hospitalization, she self-directed her discharge home due to concerns about COVID-19. Her premature discharge disrupted opioid and benzodiazepine use disorder treatment plans. CONCLUSION: This case highlights the risks of phenibut use for selfdirected detoxification. With COVID-19 related changes in the drug supply, people may be more likely to use online pharmaceuticals, therefore, substance use assessments should inquire about the online acquisition of new psychoactive drugs. Public health messaging regarding the risks of infectious disease transmission in addiction care settings is needed to guide addiction treatment choices among people who use substances.

The scavenging capacity of γ-aminobutyric acid for acrolein and the cytotoxicity of the formed adduct.

Acrolein is a notorious aldehyde with hazardous impacts on humans. γ-Aminobutyric acid (GABA) is a functional amino acid present widely in foods. This study aimed at investigating the protective mechanism of GABA against acrolein. In simulated physiological and thermal processing models, GABA effectively scavenged acrolein by adduct formation. The cytotoxicity of the formed adduct was evaluated in human bronchial epithelial cell line HBE and normal colonic epithelial cell line NCM460. It tremendously decreased acrolein toxicity and exerted protective effects by ROS reduction. Apoptotic staining and signaling analysis showed that it also interfered with apoptosis via extrinsic and intrinsic pathways. Our findings provide the basic knowledge that GABA is an effective acrolein scavenger and it has potential detoxifying capacity for both exogenous and endogenous acrolein sourced cellular damage.

F-phenibut (ß-(4-Fluorophenyl)-GABA), a potent GABAB receptor agonist, activates an outward-rectifying K+ current and suppresses the generation of action potentials in mouse cerebellar Purkinje cells.

The GABA analog phenibut (ß-Phenyl-GABA) is a GABAB receptor agonist that has been licensed for various uses in Russia. Phenibut is also available as a dietary supplement from online vendors worldwide, and previous studies have indicated that phenibut overdose results in intoxication, withdrawal symptoms, and addiction. F-phenibut (ß-(4-Fluorophenyl)-GABA), a derivative of phenibut, has not been approved for clinical use. However, it is also available as a nootropic supplement from online suppliers. F-phenibut binds to GABAB with a higher affinity than phenibut; therefore, F-phenibut may lead to more serious intoxication than phenibut. However, the mechanisms by which F-phenibut acts on GABAB receptors and influences neuronal function remain unknown. In the present study, we compared the potency of F-phenibut, phenibut, and the GABAB agonist (±)-baclofen (baclofen) using in vitro patch-clamp recordings obtained from mouse cerebellar Purkinje cells slice preparations Our findings indicate that F-phenibut acted as a potent GABAB agonist. EC50 of outward current density evoked by the three GABAB agonists decreased in the following order: phenibut (1362 µM) > F-phenibut (23.3 µM) > baclofen (6.0 µM). The outward current induced by GABAB agonists was an outward-rectifying K+ current, in contrast to the previous finding that GABAB agonists activates an inward-rectifying K+ current. The K+ current recorded in the present study was insensitive to extracellular Ba2+, intra- or extracellular Cs+, and intra- or extracellular tetraethylammonium-Cl. Moreover, F-phenibut suppressed action potential generation in Purkinje cells. Thus, abuse of F-phenibut may lead to severe damage by inhibiting the excitability of GABAB-expressing neurons.

Pre-clinical evidences for the efficacy of tryptanthrin as a potent suppressor of skin cancer.

OBJECTIVE: Clinical trials have demonstrated the efficacy of indigo naturalis, a traditional Chinese medicine ingredient, against psoriasis, a skin disease characterized by keratinocyte hyperproliferation and inflammation. The present study investigates the efficacy of tryptanthrin, a bioactive compound in indigo naturalis, against non-melanoma skin cancer (NMSC) and the signalling events involved. METHODS: Efficacy of tryptanthrin against NMSC was assessed using DMBA/PMA-induced skin carcinogenesis model in Swiss albino mice. Immunostaining for PCNA and ki-67 was used to mark proliferating cells in tissues. Haematoxylin and eosin staining and toluidine staining were employed to assess inflammation, and TUNEL assay was used to detect apoptosis in tissues. The signalling events were evaluated using Western blot, imunohistochemistry and immunofluorescence staining. MTT assay and clonogenic assay were performed to assess the viability and proliferation of cancer cells, in vitro. RESULTS: In mice, topical application of tryptanthrin suppressed skin carcinogenesis. It attenuated inflammation, impeded the proliferation of hair follicle (HF) cells and suppressed the activation of ß-catenin, a major driver of HF cell proliferation. Additionally tryptanthrin suppressed the activation of ERK1/2 and p38, both of which promote ß-catenin activation and lowered the expression of c-Myc and cyclin-D1. Tryptanthrin suppressed the proliferation of the human NMSC cell line, A431 and abrogated EGF-induced activation of ß-catenin and subsequent cytoskeletal rearrangement. CONCLUSION: The study demonstrates with molecular evidence that tryptanthrin is an effective suppressor of NMSC.

γ-Aminobutyric acid-modified graphene oxide as a highly selective and low-toxic fluorescent nanoprobe for relay recognition of copper(II) and cysteine.

A sensitive and selective graphene oxide (GO)-based fluorescent nanoprobe has been developed for the relay recognition of Cu2+ and cysteine (Cys) by covalently grafting γ-aminobutyric acid (GABA) onto GO. The fluorescence of the probe (with excitation/emission maxima at 360/445 nm) is selectively quenched by Cu2+ via static fluorescence quenching. Fluorescence drops linearly as the concentration of Cu2+ is increased from 50 nM to 1.0 µM, and the detection limit for Cu2+ is calculated as 15 nM. By virtue of the strong interaction between Cys and Cu2+, the GO-GABA/Cu2+ complex can further sensitively recognize Cys in a "switch-on" mode. The linear range for Cys detection is from 50 nM to 1.0 µM, and the detection limit is 38 nM. The probe has low cytotoxicity, and it works well inside living cells, which is verified by the successful application in imaging of LLC-PK1 cells. Graphical abstract Gamma-Aminobutyric Acid (GABA) modified graphene oxide (GO) is a highly selective nanoprobe for the fluorometric relay recognition of Cu2+ and Cys.

The flavonoid 6-methoxyflavone allays cisplatin-induced neuropathic allodynia and hypoalgesia.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting side-effect of several commonly used chemotherapeutic agents (such as cisplatin) that profoundly impairs patient quality of life. Unfortunately, neither prophylactic strategies nor symptomatic treatments have proven useful in this condition. Flavonoids are found ubiquitously in fruits and vegetables and exert a multiplicity of beneficial effects. In this study, the antinociceptive activity of 6-methoxyflavone (6-MF) was investigated and evaluated in comparison with gabapentin in a rat model of CIPN. The effect on motor balance was also assessed using the rotarod and footprint analysis paradigms. 6-MF possessed both peripheral and central antinociceptive activities against tonic and phasic nociceptive stimuli. Cisplatin administration (3.0mg/kg/week, i.p.) for four consecutive weeks generated temporal mechanical allodynia (decreased paw withdrawal threshold; PWT) and thermal hypoalgesia (increased paw thermal threshold; PTT) in the bilateral hindpaws. Daily treatment with 6-MF (25, 50 and 75mg/kg/day, i.p) for four weeks attenuated the cisplatin-induced expression of nocifensive behaviors observed as a significant increase in PWT and alleviation of PTT during the third and fourth weeks of cisplatin administration. Accordingly, daily gabapentin (75mg/kg, i.p) suppressed the expression of CIPN by normalizing the PWT and hotplate response latency. However, these antinociceptive actions were associated with motor impairment exemplified by a significant decrease in rotarod endurance latency and a deficit in the uniformity of step alternation. In contrast, 6-MF was devoid of these adverse side-effects. These findings suggested that 6-MF afforded desirable neuropathic pain alleviating effects in CIPN and it was devoid of gabapentin-like unwanted motor side-effects.

Comparison of Toxicity of Taurine and GABA in Combination with Alcohol in 7-Day-Old Mice.

Previously, we described the combined toxicity of taurine and alcohol, and assumed hypoglycemia to be one reason of this toxicity. To understand whether taurine-ethanol combined toxicity is exclusively connected to taurine or whether other inhibitory amino acids may have similar effects when combined with ethanol, we tested different doses of gamma-aminobutyric acid (GABA) in combination with ethanol in 7-day-old mice. The minimal dose of GABA in combination with 5 g/kg ethanol which could kill a mouse was 2 g/kg. GABA combined with ethanol at doses of 3 g/kg, 4 g/kg, 6 g/kg induced lethality of 30%, 90% and 100%, correspondingly. Taurine at the doses of 4 and 6 g/kg combined with ethanol induced death in 60 and 100% of mice. Ethanol (5 g/kg), taurine (6 g/kg), GABA (4 g/kg) administered alone and the combination of ethanol (5 g/kg) with taurine (3 g/kg) have no lethal effects. GABA (6 g/kg) applied alone induced 90% lethality. Taurine or GABA alone decreased blood glucose in a dose-depending manner. Ethanol potentiated GABA- and taurine-induced decrease in blood glucose and in some animals it dropped from 8.8 (intact) to a hypoglycemic level 3.1-3.3 mmol/L (GABA 4 g/kg, taurine 6 g/kg), but this may not be considered a single reason of death. We conclude that the combination of GABA and ethanol has a lethal effect and this is stronger than the combined toxicity of ethanol and taurine.

Effects of GABA microinjection into dorsal raphe nucleus on behavior and activity of lateral habenular neurons in mice.

The dorsal raphe nucleus (DRN) is a key site for 5-hydroxytryptamine (5-HT) synthesis and release. DRN dysfunction has been implicated in several stress-related disorders, including depression and anxiety. The lateral habenular nucleus (LHb) has been shown to inhibit the activity of DRN 5-HT neurons, and thus the LHb-DRN pathway plays an important role in the pathogenesis of depression. Although it is known that the LHb also receives the projection from the 5-HT neuron in the DRN, whether 5-HT neurons in the DRN can influence activity of the LHb in vivo and whether this effect is related to the induced behavioral changes have not been investigated. In the current study, we determined how injecting γ-aminobutyric acid (GABA) into the DRN to inhibit 5-HT neurons affected behavior and the changes in the activity of LHb neurons in mice. We found that GABA injection into the DRN induced depression-like behavior in mice, as indicated by increased immobility time, and decreased climbing time in the forced swimming test and the tail suspension test, decreased time spent in the center and total distance moved in the open field test. Using extracellular single unit recording, we showed that the firing rate of LHb neurons decreased after GABA microinjection into the DRN. Further, c-Fos expression in LHb neurons was inhibited. Together our results indicate that inhibition of DRN 5-HT neurons can cause decreased LHb activity and depression-like behavior in mice, however this depression-like behavior could be independent of the LHb activity. The observed decrease in LHb activity is probably due to the presence of a negative feedback loop between the DRN and the LHb, which may play a role in maintaining emotional homeostasis.

Effect of topical neuromodulatory medications on oral and skin keratinocytes.

BACKGROUND: Neuromodulatory medications (NMs), such as amitriptyline, carbamazepine and gabapentin, are used as topical preparations for the management of neuropathic orofacial pain (NOP) and have produced promising preliminary results. The aim of this study was to investigate the effects of three aforementioned NMs on cell lines relevant to the orofacial tissues in vitro as no published studies have examined the effect of these topical NMs. METHODS: Cellular viability was measured using alamarBlue® , testing cumulative and specific time point effects of NMs on human skin keratinocytes and oral keratinocytes. Effects of the NMs on cell counts were investigated by CCK-8 assay. Drug concentrations released from NM orabase pastes after 30-min incubation were measured by high-performance liquid chromatography. Using these clinical concentrations, morphological changes and cytokine expression were investigated using scanning electron microscopy (SEM) and human inflammatory antibody array (AAH), respectively. RESULTS: Cumulative and specific time point viability and cell count methods revealed that amitriptyline caused a significant decrease in cellular viability and counts in both cell lines. Carbamazepine also had significant effects after long-term exposure and at higher concentrations, whilst gabapentin had little demonstrable effect. SEM confirmed the cytotoxicity of amitriptyline, whilst AAH revealed no significant changes in cytokine expression following amitriptyline, carbamazepine or gabapentin exposure compared with control. CONCLUSIONS: The results raise concerns about the safety of topical amitriptyline as it was cytotoxic to skin and oral keratinocytes in both exposure times and concentrations, whilst carbamazepine was cytotoxic only at high concentrations and after longer exposure times and gabapentin had no demonstrable effects.

Identification of phototransformation products of the antiepileptic drug gabapentin: Biodegradability and initial assessment of toxicity.

The anticonvulsant drug Gabapentin (GAB) is used for the treatment of various diseases (e.g. epilepsy, bipolar disorder, neuropathic pain) and is being consumed in high amounts. As GAB is not metabolized and shows a weak elimination in sewage treatment plants (STPs), it has been detected in surface water and even in raw potable water. Moreover, the confirmed teratogenic effects of GAB indicate the need for further investigations regarding options for the elimination of GAB in the water cycle. Little is known about the behavior of GAB during treatment with UV light, which is normally used for the disinfection of potable water and discussed for advanced wastewater treatment. In this study, GAB was exposed to polychromatic UV irradiation at different initial concentrations in aqueous solution. Afterwards the structures of the resulting phototransformation products (PTPs) were identified and elucidated by means of high-resolution mass spectrometry. GAB and photolytic mixtures were submitted to the Closed Bottle Test (CBT; OECD 301 D) to assess biodegradability. Furthermore, the toxicity of GAB and its photolytic mixtures was initially addressed on screening level using a modified luminescent bacteria test (LBT) and the umu-test (ISO/FDIS 13829). Environmentally realistic concentrations of GAB were disclosed by predicting STP influent concentrations (24.3 and 23.2 µg L(-1)). GAB with initial concentration of 100 mg L(-1) was eliminated by 80% after 128 min of direct UV irradiation, but just 9% of non-purgeable organic carbon (NPOC) was removed indicating the formation of dead-end transformation products (TPs). Structures of different PTPs were elucidated and several identical PTPs could also be identified at lower initial treatment concentrations (20 mg L(-1), 5 mg L(-1), 1 mg L(-1) and 0.1 mg L(-1)). GAB was classified as not readily biodegradable. Moreover, photo treatment did not result in better biodegradable PTPs. With increasing UV treatment duration, photolytic mixtures of GAB showed an increased inhibition of both, the bacterial luminescence emission as well as the growth in the modified LBT. In the umu-test no significant induction of the umuC gene as an indicator of genotoxicity was observed. Our results show that UV irradiation of GAB containing water would lead to the formation of recalcitrant PTPs. Considering that GAB was found in raw drinking water, the formation of toxic PTPs during drinking water treatment with UV light might be possible. Therefore, further studies should be conducted regarding the fate and effects on human health and the environment of GAB and the PTPs identified within this study.

Second generation anti-epileptic drugs adversely affect reproductive functions in young non-epileptic female rats.

Reproductive endocrine disturbances are a major health concern in women with epilepsy due to their long term use of antiepileptic drugs (AEDs). Second generation AEDs such as topiramate (TPM) and gabapentin are frequently used for the treatment of epilepsy as well as migraine, bipolar disorder etc. Despite the widespread clinical complications, however the definitive mechanism(s) mediating the side effects of TPM and gabapentin remain obscure. The present study was aimed to evaluate the long term effects of TPM and gabapentin on reproductive functions in young female Wistar rats. Estrous cyclicity, ovarian histology as well as estradiol, LH, leptin and insulin hormones level were studied to elucidate the long-term effect of these AEDs monotherapy on reproductive functions in non-epileptic animals. Further to explore the effects on gonadotropin releasing hormone (GnRH) neuroendocrine plasticity, the expression of GnRH, gamma-amino butyric acid (GABA), glutamic acid decarboxylase (GAD), glial fibrilliary acidic protein (GFAP) and polysialylated form of neural cell adhesion molecule (PSA-NCAM) was studied in median eminence (ME) region of these animals by immunohistochemistry, Western blot hybridization and RT-PCR. Our results demonstrate that TPM and gabapentin treatment for 8 weeks cause reproductive dysfunction as ascertained by disturbed hormonal levels and estrous cyclicity as well as alterations in GABAergic system and GnRH neuronal-glial plasticity. Our findings suggest that treatment with TPM and gabapentin disrupts the complete hypothalamo-hypophyseal-gonadal axis (HPG) through GnRH pulse generator in hypothalamus.

Subchronic toxicity evaluation of γ-aminobutyric acid (GABA) in rats.

γ-Aminobutyric acid (GABA) is an amino acid compound contained in vegetables such as tomatoes and also widely distributed in mammals. GABA acts as an inhibitory neurotransmitter and promotes parasympathetic activity to provide several beneficial effects, for instance, relaxation, anti-stress, and insomnia. GABA, produced via a fermentation process, has been available as a functional food ingredient. As part of a program to assess its safety, GABA was administered by oral gavage at doses of 500, 1250, and 2500mg/kg body weight to groups of 10 male and 10 female Sprague-Dawley rats for 13weeks. Treatment was not associated with the test substance-related mortality and appeared to be well tolerated. There were no toxicologically and statistically significant changes in urinalysis, hematology, clinical chemistry parameters, and in necropsy findings. A few statistically significant changes in food consumption and body weights were noted in the male groups while any significant changes were not noted in female groups. There was no effect of treatment on organ weights or on the results of the histopathological examinations. The results of toxicity evaluation support the safety use of GABA and the potential use as a functional food ingredient.

Effect of combined treatment with diuretics and gabapentin on convulsive threshold in mice.

Research data show that diuretics can have anticonvulsant properties. This study examined effects of ethacrynic acid, a loop diuretic, and hydrochlorothiazide, a thiazide-type diuretic, on the anticonvulsant activity of gabapentin, a newer antiepileptic drug, in the maximal electroshock seizure threshold test in mice. Diuretics were administered intraperitoneally (ip.) both acutely (single dose) and chronically (once daily for seven days). Electroconvulsions were produced by an alternating current (50 Hz, 500 V, 0.2 s stimulus duration) delivered via ear-clip electrodes by a generator. Additionally, the influence of combined treatment with the diuretics and gabapentin on motor performance in the chimney test has been assessed. In the current study, ethacrynic acid at the chronic dose of 12.5 mg/kg and the single dose of 100 mg/kg did not affect the anticonvulsant activity of gabapentin. Similarly, hydrochlorothiazide (100 mg/kg), both in acute and chronic experiments, had no effect on the gabapentin action. On the other hand, in the chimney test, the combined treatment with ethacrynic acid (100 mg/kg) and gabapentin (50 mg/kg) significantly impaired motor performance in mice. Based on the current preclinical findings, it can be suggested that the diuretics should not affect the anticonvulsant action of gabapentin in epileptic patients. However, the combination of ethacrynic acid with gabapentin may cause neurotoxicity.

Pregabalin induces hepatic hypoxia and increases endothelial cell proliferation in mice, a process inhibited by dietary vitamin E supplementation.

The preceding article identified key components of pregabalin's mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.

Hemangiosarcoma in mice administered pregabalin: analysis of genotoxicity, tumor incidence, and tumor genetics.

Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, binds with high affinity to the α(2)δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Two-year carcinogenicity studies were completed in B6C3F1 and CD-1 mice and two separate studies in Wistar rats. Doses in mice were 200, 1000, and 5000 mg/kg/day, with systemic exposures (AUC(0-24 h)) up to 31 times the mean exposure in humans, given the maximum recommended clinical dose. In rats, doses were 50, 150, and 450 mg/kg/day in males and 100, 300, and 900 mg/kg/day in females; systemic exposures up to 24 times were achieved in clinical trials. In both strains of mice, pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and bone marrow. The incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Thus, pregabalin is a single-species, single tumor-type, nongenotoxic mouse carcinogen. Hemangiosarcomas occurring in mice treated with pregabalin were genotypically distinct from hemangiosarcomas induced by genotoxic carcinogens in humans with respect to ras and p53 mutation patterns and were similar to spontaneous tumors. Furthermore, there was a strong association between pregabalin treatment and bone marrow changes in these studies in mice, suggesting a possible link between the effects observed in bone marrow and the increase in tumor incidence in pregabalin-treated mice.

Mode of action associated with development of hemangiosarcoma in mice given pregabalin and assessment of human relevance.

Pregabalin increased the incidence of hemangiosarcomas in carcinogenicity studies of 2-year mice but was not tumorigenic in rats. Serum bicarbonate increased within 24 h of pregabalin administration in mice and rats. Rats compensated appropriately, but mice developed metabolic alkalosis and increased blood pH. Local tissue hypoxia and increased endothelial cell proliferation were also confirmed in mice alone. The combination of hypoxia and sustained increases in endothelial cell proliferation, angiogenic growth factors, dysregulated erythropoiesis, and macrophage activation is proposed as the key event in the mode of action (MOA) for hemangiosarcoma formation. Hemangiosarcomas occur spontaneously in untreated control mice but occur only rarely in humans. The International Programme on Chemical Safety and International Life Sciences Institute developed a Human Relevance Framework (HRF) analysis whereby presence or absence of key events can be used to assess human relevance. The HRF combines the MOA with an assessment of biologic plausibility in humans to assess human relevance. This manuscript compares the proposed MOA with Hill criteria, a component of the HRF, for strength, consistency, specificity, temporality, and dose response, with an assessment of key biomarkers in humans, species differences in response to disease conditions, and spontaneous incidence of hemangiosarcoma to evaluate human relevance. Lack of key biomarker events in the MOA in rats, monkeys, and humans supports a species-specific process and demonstrates that the tumor findings in mice are not relevant to humans at the clinical dose of pregabalin. Based on this collective dataset, clinical use of pregabalin would not pose an increased risk for hemangiosarcoma to humans.

Key components of the mode of action for hemangiosarcoma induction in pregabalin-treated mice: evidence of increased bicarbonate, dysregulated erythropoiesis, macrophage activation, and increased angiogenic growth factors in mice but not in rats.

In carcinogenicity studies, pregabalin increased hemangiosarcoma incidence in mice but not in rats. Investigative studies, ranging in length from 24 h to 12 months, were conducted in mice (1000 or 5000 mg/kg) and rats (900 mg/kg) to evaluate a potential mode-of-action scheme for tumor formation. Three areas were evaluated: (1) hematopoiesis (because endothelial and hematopoietic cells arise from the same precursor and hemangiosarcomas are primarily located in mouse hematopoietic tissues), (2) angiogenic growth factors (because increased angiogenic growth factors may stimulate vascular tumors), and (3) pulmonary/blood gas parameters (because hypoxia is a known driver for endothelial cell proliferation). In mice, pregabalin rapidly increased platelet and megakaryocyte counts, activated platelets and bone marrow erythrophages, decreased the myeloid-to-erythroid (M:E) ratio (49%), and produced bone marrow and splenic congestion and extramedullary hematopoiesis (EMH). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor immunohistochemical staining were also increased in mouse bone marrow and spleen and vascular endothelial growth factor receptor 2 immunolabeling was increased in liver. Serum bicarbonate was increased within 24 h of pregabalin administration, persisted over time, and was accompanied by decreased respiratory rate (up to 34%) and increased partial pressure of carbon dioxide (pCO(2)), resulting in sustained metabolic alkalosis and elevated blood pH in mice. In contrast, in rats, pregabalin decreased overall bone marrow cellularity, including decreased number of megakaryocytes (24%) with no evidence of erythrophages, no change in M:E ratio, no EMH, and no increase in angiogenic growth factors or blood pH. Persistent alterations in serum bicarbonate, respiratory function, and blood gas parameters in mice, without adequate compensatory mechanisms, has the potential to create chronic tissue hypoxia, an accepted driver of endothelial cell proliferation.

GABAergic excitotoxicity injury of the immature hippocampal pyramidal neurons' exposure to isoflurane.

BACKGROUND: Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. γ-Aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, depolarizing the membrane potential and inducing a cytosolic Ca2+ increase ([Ca2+]i), because of a reversed transmembrane chloride gradient. Recent experimental data from several rodent studies have demonstrated that exposure to isoflurane during an initial phase causes neuronal excitotoxicity and apoptosis. GABAA receptor-mediated synaptic voltage-dependent calcium channels' (VDCCs) overactivation and Ca2+ influx are involved in these neural changes. METHODS: We monitored [Ca2+]i using Fluo-4 AM fluorescence imaging. Using whole-cell patch clamp techniques, IVDCC (voltage-dependent calcium channel currents) were recorded from primary cultures of rat hippocampal neurons (5-day culture) exposed to isoflurane. To further investigate the neurotoxicity of high cytosolic-free calcium after isoflurane in a dose- and time-dependent manner, the possibility of increased caspase-3 levels was evaluated by Western blot and quantitative real-time polymerase chain reaction. Statistical significance was assessed using the Student t test or 1-way analysis of variance followed by the Tukey post hoc test. RESULTS: Under control conditions, isoflurane enhanced the GABA-induced [Ca2+]i increase in a dose-dependent manner. Dantrolene and nicardipine markedly inhibited this enhancement mediated by isoflurane. Moreover, in Ca2+-free media, pretreatment with isoflurane did not show any influence on the caffeine-induced increase of [Ca2+]i. Similarly, using whole-cell recording, isoflurane increased the peak amplitude of IVDCC in the cultured neurons from rat hippocampus by depolarization pulses. Isoflurane (0.25, 0.5, 0.75, and 1 minimum alveolar concentration [MAC]) potentiated IVDCC peak current amplitude by 109.11%±9.03%, 120.56%±11.46%, 141.33%±13.87%, and 146.78%±15.87%, respectively. To analyze variation in protein levels, the effect of treatments with isoflurane on caspase-3 activity was dose- and time-dependent, reaching a maximal caspase-3 activity after exposure to 1 MAC for 6 hours (P<0.001). However, in the mRNA levels, hippocampal caspase-3 mRNA levels began to be significantly increased in isoflurane-treated developing rat hippocampal neurons after 6 hours of exposure to 0.25 MAC isoflurane (P<0.001). CONCLUSIONS: Isoflurane-mediated enhancement of GABA-triggered [Ca2+]i release results from membrane depolarization with subsequent activation of VDCCs and further Ca2+-induced Ca2+ release from the ryanodine-sensitizing Ca2+ store. An increase in [Ca2+]i, caused by activation of the GABAA receptor and opening of VDCCs, is necessary for isoflurane-induced calcium overload of immature rat hippocampal neurons, which may be involved in the mechanism of an isoflurane-induced neurotoxic effect in the developing rodent brain.