Isobolographic Analysis Research Articles

Experimental study of the combined effect of benzene exposure and physical load in rats

Introduction. The working process in many industries is often associated not only with chemical exposures but also with heavy physical workload, which can be potent of toxic effects of the former differently. The purpose of the study was to establish body responses to effects of benzene exposure combined with exercise in an experiment on rats. Materials and methods. The study was conducted on mature male Wistar rats, ten animals in each of the following groups: “Control”, “Running”, “Benzene exposure”, and “Benzene exposure + running”. Subchronic toxicity was modelled by intragastric administration of benzene thrice a week for 4 weeks in a cumulative dose of 100 mg/kg body weight. Exercise was modelled by means of 10-minute forced running sessions at a speed of 25 m/min 5 times a week for 4 weeks. We then measured post-exposure biochemical and hematological indices and the proportion of different types of cells in imprint smears of rat internal organs. Statistical data analysis was carried out using the Student’s t-test with p<0.05. For mathematical modelling of the combined effect of factors, we applied a Response Surface Method (RSM) with the construction of Loewe isobols on its basis. Results. Almost no statistically significant changes were registered in the rodents who exercised while statistical ones were sporadic and diagnostically insignificant in most cases. Benzene exposure induced changes in blood indices indicating blood clotting disorders, typical of chronic exposure to benzene, and in the antioxidant system. The impact of running on health effects of benzene exposure was ambiguous but isobolographic analysis showed possible additivity and even synergy, both detrimental to health, in 15.6 % of cases. Limitations. Extrapolation of the study results to human should be done with caution, since the combined effect has been examined only for subchronic exposure using a single species and sex of laboratory animals. Conclusion. Additivity proven by a number of indices of toxic effect gives important information for understanding and predicting health effects of exposure to organic pollutants combined with physical activity.

D1 dopamine / mu opioid receptor interactions in operant conditioning assays of pain-depressed responding and drug-induced rate suppression, and a conditioned place preference procedure: assessment of therapeutic index in male Sprague Dawley rats.

In vivo receptor interactions vary as a function of behavioral endpoint, with key differences between reflexive and non-reflexive measures that assess the motivational aspects of pain and pain relief. There have been no assessments of D1 dopamine agonist / mu opioid receptor (MOR) agonist interactions in non-reflexive behavioral measures of pain. We examined the hypothesis that D1/MOR mixtures show enhanced effectiveness in blocking pain depressed behaviors while showing decreased side effects such as sedation and drug reward. SKF82958 and methadone were used as selective/high efficacy D1 and mu agonists, respectively. An FR10 operant schedule was utilized in the presence and absence of a lactic acid inflammatory pain-like manipulation, to measure antinociceptive and operant-rate-suppressing effects, respectively. Rewarding properties of the drug combinations were determined using a conditioned place preference procedure. Methadone alone, but not SKF82958 alone, produced dose-dependent restoration of pain-depressed responding. Both SKF82958 and methadone produced dose-dependent response rate suppression. Three fixed proportion mixtures, based on the relative potencies of the drugs in the rate suppression assay, produced dose-dependent antinociception and sedation. Isobolographic analysis indicated that the 0.17:1 mixture produced supra-additive antinociception and additive sedation. The 0.055:1 mixture produced additive antinociception with sub-additive sedation, and the 0.018:1 mixture had the highest therapeutic index (TI) relative to other mixtures and drugs alone. The antinociceptive doses and component doses for the 0.018:1 mixture did not produce conditioned place preference. These results suggest that D1-selective dopamine agonists may have utility as candidate opioid-sparing analgesics.

The role of mitochondrial dysfunction in the cytotoxic synergistic effect of gemcitabine and arsenic on breast cancer.

Breast cancer is the most common type of cancer in women worldwide. A common approach to cancer treatment in clinical practice is to use a combination of drugs to enhance the anticancer activity of drugs while reducing their side effects. In this regard, we evaluated the effectiveness of combined treatment with gemcitabine (GCB) and arsenic (ATO) and how they affect the cell death pathway in cancer cells. Cytotoxic activity of drugs individually or combined against MDA-MB-231 and MCF-7 was performed by MTT method and isobolographic analysis was used to determine the interaction between these factors. The combination of ATO and GCB showed synergistic anti-cancer activity (CI < 1) in both cancer cell lines. The combination of ATO and GCB induced sub-G1 phase arrest, apoptosis and death rates in MCF-7 and MDA-MB-231 cells. The apoptotic response induced by the combination of GCB and ATO was dependent on caspase 3/7. Combined treatment with mitochondrial membrane potential (MMP) reduction and increased reactive oxygen species (ROS) production caused mitochondrial dysfunction. Co-treatment significantly reduced catalase (CAT) activity in both cancer cells compared to the control group and cells treated with each monotherapy. A significant decrease in cellular GSH was observed in cancer cells treated with ATO and GCB. In addition, migration and invasion were significantly reduced in breast cancer cells treated with the combination of ATO and GCB compared to cells treated with ATO and GCB. In conclusion, the combined treatment of ATO and GCB synergistically increased the anti-cancer activity, and these findings provide an effective approach for the treatment of breast cancer. To the best of our knowledge, this is the first study showing promising results for combination therapy with ATO and GCB in breast cancer.

Isobolographic interactions of cannabidiol and AM 1172 with cisplatin in human neuroblastoma and glioblastoma cell lines: An in vitro study.

Glioblastoma is the most aggressive brain cancer in humans with very poor prognosis and high mortality rate. Despite advances in treatment, glioblastoma almost always recurs and new therapeutic methods are urgently needed. This study aimed at assessing the cytotoxic and antiproliferative effects of AM 1172 and cannabidiol (two cannabinoid receptor ligands) in vitro, when used alone and in combination with cisplatin (a standard cytotoxic drug), in various human neuroblastoma (CHP-134, KELLY), human glioblastoma (U-87MG and T98G) and rat glioblastoma (C6) cell lines. Our experiments showed that AM 1172 and cannabidiol inhibited cell proliferation with IC50 values in the range of 2.29-17.21μM (for AM 1172) and 11.61-20.35μM (for cannabidiol), respectively. The selectivity index for AM 1172 ranged from 0.61 to 4.60 and that for cannabidiol ranged from 1.45 to 2.55 in the studied glioblastoma and neuroblastoma cell lines. With isobolographic analysis, it was found that AM 1172 combined with cisplatin exerted a synergistic interaction in the CHP-134cell line (p<0.01). In contrast, AM 1172 when combined with cisplatin produced an antagonistic interaction in the C6 cell line (p<0.01). The remaining combinations of AM 1172 with cisplatin in the U-87MG, KELLY and T98G cell lines were additive. In case of cannabidiol, its combination with cisplatin produced an antagonistic interaction in the T98G cell line (p<0.0001), whereas the combinations of cannabidiol with cisplatin in the CHP-134, U-87MG, KELLY, and C6 cell lines were additive in nature. The synergistic and additive interactions for the combination of AM 1172 and cannabidiol with cisplatin seem to be a promising direction in glioblastoma therapy. Unfortunately, the combinations producing antagonistic interactions (AM 1172+cisplatin in C6, and cannabidiol+cisplatin in T98G cell lines) should be avoided due to the antagonistic antiproliferative effect of two-drug mixtures.

Anti-allodynic effect of intrathecal antibodies against macrophage-inducible C-type lectin in spinal nerve ligation model in rat

IntroductionMacrophage-inducible C-type lectin (Mincle) has emerged as a potential contributor to neuropathic pain induction and neuroinflammatory responses within the spinal cord. Moreover, evidence suggests a close association between toll-like receptor (TLR) and Mincle expression in myeloid cells. This study evaluated the effectiveness of Mincle antibodies in neuropathic pain and identified the epitope of these antibodies. In addition, the mode of interaction between Mincle and TLR inhibition was explored using isobolographic analysis. MethodsThree different Mincle antibodies and a specific TLR4 inhibitor (TAK-242) were intrathecally administered, and mechanical allodynia was evaluated using the von Frey test in a rat model of spinal nerve ligation (SNL). Isobolographic analysis was conducted on the effect of combination of TAK-242 and Mincle Ab. Microarray analysis examined the specific region of Mincle targeted by the antibodies. ResultsAll Mincle antibodies and TAK-242 significantly alleviated mechanical allodynia in a dose-dependent manner. However, the maximal possible effects (MPE) produced by the antibodies ranged widely from 37.1% to 91.8%, comparable to that of TAK-242 (88.7%). The combination of TAK-242 and the antibody with the highest MPE resulted in an additive interaction for their anti-allodynic effects. Epitope mapping revealed that each antibody targeted the extracellular domain, with epitope lengths ranging from 5 to 15 amino acids. ConclusionsThe current study demonstrates the anti-allodynic effect of Mincle antibodies and additive interaction with TLR4 inhibition in spinal nerve ligation model, suggesting the potential of blocking of Mincle signaling with its antibodies as a novel treatment strategy for neuropathic pain.

The Effect of Metoclopramide on the Antinociceptive, Locomotor and Neurobehavioral Effects of Metamizole in Mice

Abstract Combining analgesic medications with distinct pharmacodynamics may result in effective analgesia and reduced adverse effects by lowering dosages of one or both medications. The objective was to investigate the nature of interactions of metoclopramide and metamizole by measuring the antinociceptive and neurobehavioral effects in mice. The drugs were administered intraperitoneally (ip) in mice. The antinociceptive effect was determined by the up-and-down method. Isobolographic analysis was performed by simultaneous injection of the two drugs at ED50 ratios of 1:1 and 0.5:0.5. The treated mice were also subjected to an open-field behavioral test as well as dorsal immobility and negative geotaxis behavioral paradigms. The ED50 of metoclopramide and metamizole were 33.71 and 43 mg/kg ip, respectively. Lower ED50 was established for metoclopramide and metamizole by 48.44% and 47.49%, respectively when given at a ratio of 0.5:0.5, and by 35.81% and 29.88%, respectively at a ratio of 1:1. The combined dose of metoclopramide and metamizole (8 and 10 mg/kg, ip, respectively) significantly decreased the open field-activity test in mice. This was observed by lower number of squares crossing compared to the control. Additionally, the drug combination resulted in a substantial increase in the duration of dorsal immobility response and delayed the negative geotaxis task when compared with the control group. The results indicated that the interaction between metoclopramide and metamizole in lower dose ratio with ED50 50:50, was synergistic in producing analgesia in mice with reduced risk for adverse reaction but with decreased behavioral and motor neuronal activity.

Antiproliferative effects of LY-2183240 combined with various chemotherapeutic drugs in an isobolographic in vitro model of malignant melanoma

Despite a great progress in identifying treatment options for patients with malignant melanoma, novel therapies tend to be costly and, in some cases, produce adverse effects forcing the melanoma patients to withdraw drugs. There is a strong need for less expensive drugs with a more favorable spectrum of anticancer actions. This study was designed to assess whether LY-2183240 (a potent inhibitor of both, anandamide cellular reuptake and fatty acid amide hydrolase (FAAH), an enzyme that degrades anandamide) has antiproliferative and cytotoxic effects on various human malignant melanoma cell lines (primary A375 and FM55P, metastatic SK-MEL28 and FM55M2) when administered alone or in combination with docetaxel, paclitaxel, mitoxantrone and cisplatin via the MTT assay. The MTT, LDH and BrdU assays were used to evaluate the potency and safety of LY-2183240, whereas isobolographic analysis of interactions was applied to characterize the interactions of LY-2183240 with the studied chemotherapeutics (docetaxel, paclitaxel, mitoxantrone and cisplatin). The isobolography confirmed that the combinations of LY-2183240 with docetaxel, paclitaxel and mitoxantrone produced additive interactions in all the tested melanoma cell lines. Only two antagonistic interactions for LY-2183240 combined with cisplatin in the A375 and FM55P cell lines were observed by the MTT assay. In conclusion, LY-2183240 can be considered an add-on drug for the treatment of melanoma, when combined with docetaxel, paclitaxel, or mitoxantrone, but not with cisplatin.

Combined antiallodynic effects of Neurotropin®–tramadol and Neurotropin®–mirogabalin in rats with L5-spinal nerve ligation

We aimed to examine the efficacy of combination therapies of Neurotropin® with tramadol and Neurotropin with mirogabalin for neuropathic pain management. A neuropathic pain model (L5 spinal nerve ligation model: L5-SNL) using male Wistar rats was generated through tight ligation of the left fifth lumbar nerve using silk sutures. Mechanical allodynia was assessed using the 50% paw withdrawal threshold. The combined antiallodynic effects were evaluated using isobolographic analyses. Small intestinal transit was evaluated using the charcoal meal test, and motor coordination using the rota-rod test. Neurotropin (50–200 NU/kg, p.o.), tramadol (7.5–60 mg/kg, p.o.), and mirogabalin (3–30 mg/kg, p.o.) showed a dose-dependent antiallodynic effect in L5-SNL rats. The combined antiallodynic effects of Neurotropin and tramadol were additive or synergistic, whereas those of Neurotropin and mirogabalin were additive. Neurotropin (100–400 NU/kg, p.o.) did not affect the small intestinal transit, whereas tramadol (30–100 mg/kg, p.o.) significantly inhibited it. Neurotropin (100–400 NU/kg, p.o.) did not affect the walking time, whereas mirogabalin (10–100 mg/kg, p.o.) significantly decreased it. Neurotropin dose-dependently ameliorated mechanical allodynia in rats, and combination therapy with Neurotropin–tramadol or Neurotropin–mirogabalin may alleviate neuropathic pain without aggravating the adverse effects of tramadol and mirogabalin.

The synergistic anti-nociceptive effects of nefopam and gabapentinoids in inflammatory, osteoarthritis, and neuropathic pain mouse models

Pain is a common public health problem and remains as an unmet medical need. Currently available analgesics usually have limited efficacy or are accompanied by many adverse side effects. To achieve satisfactory pain relief by multimodal analgesia, new combinations of nefopam and gabapentinoids (pregabalin/gabapentin) were designed and assessed in inflammatory, osteoarthritis and neuropathic pain. Isobolographic analysis was performed to analyze the interactions between nefopam and gabapentinoids in carrageenan-induced inflammatory pain, mono-iodoacetate-induced osteoarthritis pain and paclitaxel-induced peripheral neuropathic pain in mice. The anti-inflammatory effect and motor performance of monotherapy or their combinations were evaluated in the carrageenan-induced inflammatory responses and rotarod test, respectively. Nefopam (1, 3, 5, 10, 30 mg/kg, p.o.), pregabalin (3, 6, 12, 24 mg/kg, p.o.) or gabapentin (25, 50, 75, 100 mg/kg, p.o.) dose-dependently reversed mechanical allodynia in three pain models. Isobolographic analysis indicated that the combinations of nefopam and gabapentinoids exerted synergistic anti-nociceptive effects in inflammatory, osteoarthritis, and neuropathic pain mouse models, as evidenced by the experimental ED50 (median effective dose) falling below the predicted additive line. Moreover, the combination of nefopam–pregabalin/gabapentin alleviated carrageenan-induced inflammation and edema, and also prevented gabapentinoids-related sedation or ataxia by lowering their effective doses. Collectively, the co-administration of nefopam and gabapentinoids showed synergistic analgesic effects and may result in improved therapeutic benefits for treating pain.

Isobolographic Analysis of the Cytoprotective Effect of Dapsone and Cannabidiol Alone or Combination upon Oxygen-Glucose Deprivation/Reoxygenation Model in SH-SY5Y Cells.

Oxidative stress and apoptosis cell death are critical secondary damage mechanisms that lead to losing neighboring healthy tissue after cerebral ischemia. This study aims to characterize the type of interaction between dapsone (DDS) and cannabidiol (CBD) and its cytoprotective effect in an in vitro model of oxygen and glucose deprivation for 6 h followed by 24 h of reoxygenation (OGD/R), using the SH-SY5Y cell line. For the combined concentrations, an isobolographic study was designed to determine the optimal concentration-response combinations. Cell viability was evaluated by measuring the lactate dehydrogenase (LDH) release and 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assays. Also, the reactive oxygen species (ROS) and reduced glutathione (GSH) levels were analyzed as oxidative stress markers. Finally, caspase-3 activity was evaluated as a marker cell death by apoptosis. The results showed a decrease in cell viability, an increase in oxidant stress, and the activity of caspase-3 by the effect of OGD/R. Meanwhile, both DDS and CBD demonstrated antioxidant, antiapoptotic, and cytoprotective effects in a concentration-response manner. The isobolographic study indicated that the concentration of 2.5 µM of DDS plus 0.05 µM of CBD presented a synergistic effect so that in treatment, cell death due to OGD/R decreased. The findings indicate that DDS-CBD combined treatment may be a helpful therapy in cerebral ischemia with reperfusion.

Peripherally restricted cannabinoid and mu-opioid receptor agonists synergistically attenuate neuropathic mechanical hypersensitivity in mice.

Many medications commonly used to treat neuropathic pain are associated with significant, dose-limiting adverse effects, including sedation, dizziness, and fatigue. These adverse effects are due to the activity of these medications within the central nervous system. The objective of this work was to investigate the interactions between peripherally restricted cannabinoid receptor and mu-opioid receptor (MOR) agonists on ongoing and evoked neuropathic pain behaviors in mouse models. RNAscope analysis of cannabinoid receptor type 1 (CB1R) and MOR mRNA demonstrated that the mRNA of both receptors is colocalized in both mouse and human dorsal root ganglion. Single-cell RNAseq of dorsal root ganglion from chronic constriction injury mice showed that the mRNA of both receptors ( Cnr1 and Oprm1 ) is coexpressed across different neuron clusters. Myc-CB1R and FLAG-MOR were cotransfected into immortalized HEK-293T cells and were found to interact at a subcellular level. We also find that CB-13 (a peripherally restricted dual CB1R and cannabinoid receptor type 2 agonist) and DALDA (a peripherally restricted MOR agonist) both attenuate mechanical hypersensitivity in a murine model of neuropathic pain. Using isobolographic analysis, we demonstrate that when coadministered, these agents synergistically attenuate mechanical hypersensitivity. Importantly, combination dosing of these agents does not cause any detectable preferential behaviors or motor impairment. However, repeated dosing of these agents is associated with the development of tolerance to these drugs. Collectively, these findings suggest that leveraging synergistic pain inhibition between cannabinoid receptor and MOR agonists in peripheral sensory neurons may be worth examining in patients with neuropathic pain.

Investigation of Herb-Drug Interactions between Xylopia aethiopica, Its Principal Constituent Xylopic Acid, and Antidepressants.

Depression affects an estimated 350 million people worldwide and is implicated in up to 60% of suicides. Only about 60-70% of patients respond to antidepressant therapy. One of the factors causing patients to not attain therapeutic goals is herb-drug interactions. To investigate any potential herb-drug interaction that might exist between Xylopia aethiopica extract (XAE) or xylopic acid (XA) and selected conventional antidepressants (imipramine, fluoxetine, and venlafaxine) in mice. Dried, powdered fruits of Xylopia aethiopica were cold macerated in 70% ethanol to obtain XAE. XA was isolated by cold macerating dried fruits of Xylopia aethiopica in petroleum ether, crystallising impure XA with ethyl acetate, and purifying XA crystals with 96% ethanol. Pharmacodynamic interaction was assessed via isobolographic analysis of tail suspension tests of the agents individually and in their respective combinations. Pharmacokinetic interaction was assessed by monitoring the effect of coadministrations on the plasma concentration of antidepressants and xylopic acid via HPLC analysis. XAE and XA in mice showed significant antidepressant-like activity in the tail suspension test. With interaction indices less than one, synergism of antidepressant effect was observed in the Xylopia aethiopica extract/fluoxetine (γXAE/FL = 0.502), Xylopia aethiopica extract/imipramine (γXAE/IP = 0.322), Xylopia aethiopica extract/venlafaxine (γXAE/VL = 0.601), xylopic acid/imipramine (γXA/IP = 0.556), xylopic acid/venlafaxine (γXA/VL = 0.451), and xylopic acid/fluoxetine (γXA/FL = 0.298) combinations, which may be potentially due to elevation of serotonergic neurotransmission via varying mechanisms. The AUC of imipramine (AUCIP = 1966 ± 58.98 µg/ml.h) was significantly (P < 0.0001) reduced by Xylopia aethiopica extract (AUCIP = 1228 ± 67.40 µg/ml.h) and xylopic acid (AUCIP = 1250 ± 55.95 µg/ml.h), while the AUC of xylopic acid (AUCXA = 968.10 ± 61.22 µg/ml.h) was significantly (P < 0.0001) reduced by venlafaxine (AUCXA = 285.90 ± 51.92 µg/ml.h) and fluoxetine (AUCXA = 510.60 ± 44.74 µg/ml.h), possibly due to the effect of interfering agents on gastric emptying hence reducing oral absorption. Xylopia aethiopica extract and xylopic acid interacted synergistically with imipramine, fluoxetine, and venlafaxine and reduced the systemic circulation of imipramine.

Synergistic Antinociceptive Effect of β-Caryophyllene Oxide in Combination with Paracetamol, and the Corresponding Gastroprotective Activity.

Pain is the most frequent symptom of disease. In treating pain, a lower incidence of adverse effects is found for paracetamol versus other non-steroidal anti-inflammatory drugs. Nevertheless, paracetamol can trigger side effects when taken regularly. Combined therapy is a common way of lowering the dose of a drug and thus of reducing adverse reactions. Since β-caryophyllene oxide (a natural bicyclic sesquiterpene) is known to produce an analgesic effect, this study aimed to determine the anti-nociceptive and gastroprotective activity of administering the combination of paracetamol plus β-caryophyllene oxide to CD1 mice. Anti-nociception was evaluated with the formalin model and gastroprotection with the model of ethanol-induced gastric lesions. According to the isobolographic analysis, the anti-nociceptive interaction of paracetamol and β-caryophyllene oxide was synergistic. Various pain-related pathways were explored for their possible participation in the mechanism of action of the anti-nociceptive effect of β-caryophyllene oxide, finding that NO, opioid receptors, serotonin receptors, and K+ATP channels are not involved. The combined treatment showed gastroprotective activity against ethanol-induced gastric damage. Hence, the synergistic anti-nociceptive effect of combining paracetamol with β-caryophyllene oxide could be advantageous for the management of inflammatory pain, and the gastroprotective activity should help to protect against the adverse effects of chronic use.

Synergistic effect of combining dual enkephalinase inhibitor PL37 and sumatriptan in a preclinical model of migraine.

The aim of this study was to test whether a combination of sumatriptan with dual enkephalinase inhibitor PL37 would result in an additive or a synergistic effect. Combination treatment is frequently used to improve the therapeutic efficacy of drugs. The co-administration of two drugs may result in efficacy at lower doses than those needed for either drug alone, thus minimizing side effects. Here, we tested the effect of the co-administration of two drugs on cutaneous mechanical hypersensitivity (MH), a symptom often affecting cephalic regions in patients with migraine: dual enkephalinase inhibitor PL37, a small molecule that protects enkephalins from rapid degradation, and sumatriptan, a serotonin 5-HT1B/1D receptor agonist. We investigated the effects of oral administrations of sumatriptan, PL37, or their combination on changes in cutaneous mechanical sensitivity induced by a single intraperitoneal administration of the nitric oxide donor, isosorbide dinitrate (ISDN) in male rats. Mechanical sensitivity was assessed using von Frey filaments applied to the face of animals to determine pain thresholds. Isobolographic analysis was performed to determine the nature of the interaction between sumatriptan and PL37. Sumatriptan as well as PL37 each produced a dose-dependent inhibition of ISDN-induced cephalic MH. Median effective dose (ED50 ) values were 0.3 and 1.1 mg/kg for sumatriptan and PL37, respectively. An isobolographic analysis of the effect of combined doses of sumatriptan and PL37 based on their calculated ED50 values demonstrated a synergistic effect of the combination on cephalic MH, with an interaction index of 0.14 ± 0.04. These results suggest that PL37 acts synergistically with sumatriptan to produce an anti-allodynic effect in a rat model of migraine. Thus, combining PL37 and sumatriptan may be a useful therapeutic strategy in the management of migraine. There have been many advances in migraine treatment, but we still need more options that are effective and have few side effects. Sumatriptan is one available drug for acute treatment of migraine, but it does not work for every patient and is not suitable for some people. We tested a new drug called PL37 (that blocks enkephalinases) together with sumatriptan and the combination minimized side effects and allowed lower doses of the drugs for effective migraine treatment in an animal model.

Methylcobalamin Acts as a Synergistic Effector of Cadmium Chloride (Cdcl2) and Enhances Cadmium Induced Stress in Human A549 Cells through Methylation of MT2A Promoter

Methylcobalamin (MeB12) a vitamin B12 analogue is used in the treatment of several neurological disorders was used in this study to check for its role in cadmium toxicity. Cadmium is a highly toxic heavy metal can cause considerable damage to airway epithelium which can lead to many pulmonary disorders and even lung cancer. Human A549 cells were grown in medium supplemented with MeB12 for 48 hours, followed by 24 hours exposure to increasing concentrations of CdCl2 . MeB12 enhanced cadmium induced cells death. Isobolographic analysis revealed a synergistic effect of these two compounds. Studies on the revealed while MT1A promoter is already heavily methylated in A549 cell line, MT2A appeared to be methylated only in the presence of CdCl2 and MeB12. Our results suggest that the epigenetic modification of MT2A gene may be one of the contributing factors towards the enhanced cadmium sensitivity in A549 cells in the presence of MeB12.

Isobolographic analysis of interactions – a pre-clinical perspective

Isobolographic analysis of interactions – a pre-clinical perspective

Combining Cisplatin with Different Radiation Qualities-Interpretation of Cytotoxic Effects In Vitro by Isobolographic Analysis.

The combination of platinum-containing cytostatic drugs with different radiation qualities has been studied for years. Despite their massive side effects, these drugs still belong to the therapeutic portfolio in cancer treatment. To overcome the disadvantages of cisplatin, our study investigated the cytotoxic effects of combining radionuclides with cisplatin. FaDu cells were treated with cisplatin (concentration ≈ 2 µM) and additionally irradiated after two hours with the alpha-emitter 223Ra, the beta-emitter 188Re as well as external X-rays using dose ranges of 2-6 Gy. Cell survival was followed by colony formation assays and plotted against cisplatin concentration and radiation dose. The results were interpreted by isobolograms. Isobolographic analyses revealed a supra-additive cytotoxic effect for the combination of cisplatin and 223Ra. A sub-additive effect was observed for the combination of cisplatin and 188Re, whereas a protective effect was found for the combination with X-rays. The combination of cisplatin and 223Ra may have the potential to create a successfully working therapy scheme for various therapy approaches, whereas the combination with 188Re as well as single-dose X-ray treatment did not lead to a detectable radiosensitizing effect. Thus, the combination with alpha-emitters might be advantageous and, therefore, should be followed in future studies when combined with cytostatic drugs.

Antinociceptive Effect of the Combination of a Novel α4β2* Agonist with Donepezil in a Chronic Pain Model.

Chronic pain presents a major challenge in contemporary medicine, given the limited effectiveness and numerous adverse effects linked to available treatments. Recognizing the potential of the cholinergic pathway as a therapeutic target, the present work evaluates the antinociceptive activity of a combination of Cris-104, a novel α4β2* receptor agonist, and donepezil, a central anticholinesterase agent. Isobolographic analysis revealed that equimolar combination was approximately 10 times more potent than theoretically calculated equipotent additive dose. Administration of Cris-104 and donepezil combination (3 μmol/kg) successfully reversed hyperalgesia and mechanical allodynia observed in rats subjected to spinal nerve ligation (SNL). The combination also modulated neuroinflammation by reducing astrocyte activation, evident in the decreased expression of glial fibrillary acidic protein (GFAP) in the spinal cord. The observed synergism in combining a nicotinic receptor agonist with an anticholinesterase agent underscores its potential for treating chronic pain. This alternative therapeutic distinct advantage, including dose reduction and high selectivity for the receptor, contribute to a more favorable profile with minimized adverse effects.

AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) reduces the viability of the various melanoma cells, but it exerts significant cytotoxic effects on healthy cells: an in vitro study based on isobolographic analysis.

Despite great advances in our understanding of the impact of cannabinoids on human organism, many of their properties still remain undetermined, including their potential antineoplastic effects. This study was designed to assess the anti-proliferative and cytotoxic effects of AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) administered alone and in combinations with docetaxel (DOCX), paclitaxel (PACX), mitoxantrone (MTX) and cisplatin (CDDP) on various human malignant melanoma A375, FM55P, SK-MEL 28 and FM55M2 cell lines. In the MTT, LDH, and BrdU assays, the potency and safety of AM1172 when administered alone and in combinations with DOCX, PACX, MTX, and CDDP were determined. The isobolographic analysis revealed that combinations of AM1172 with PACX, DOCX, MTX, and CDDP exerted additive interactions, except for a combination of AM1172 with PACX in primary melanoma A375 cell line, for which synergy was observed (*p<0.05). Nevertheless, AM1172 when administered alone produced cytotoxic effects on healthy human melanocytes (HEMa-LP) and human keratinocytes (HaCaT), which unfortunately limits its potential therapeutic utility. AM1172 cannot be used separately as a chemotherapeutic drug, but it can be combined with PACX, DOCX, MTX, and CDDP, offering additive interactions in terms of the anti-proliferative effects in various malignant melanoma cell lines.

"Tranq-dope" overdose and mortality: lethality induced by fentanyl and xylazine.

Introduction: The recreational use of fentanyl in combination with xylazine (i.e., "tranq-dope") represents a rapidly emerging public health threat characterized by significant toxicity and mortality. This study quantified the interactions between these drugs on lethality and examined the effectiveness of potential rescue medications to prevent a lethal overdose. Methods: Male and female mice were administered acute doses of fentanyl, xylazine, or their combination via intraperitoneal injection, and lethality was determined 0.5, 1.0, 1.5, 2.0, and 24 h after administration. Both fentanyl and xylazine produced dose-dependent increases in lethality when administered alone. Results: A nonlethal dose of fentanyl (56 mg/kg) produced an approximately 5-fold decrease in the estimated LD50 for xylazine (i.e., the dose estimated to produce lethality in 50% of the population). Notably, a nonlethal dose of xylazine (100 mg/kg) produced an approximately 100-fold decrease in the estimated LD50 for fentanyl. Both drug combinations produced a synergistic interaction as determined via isobolographic analysis. The opioid receptor antagonist, naloxone (3 mg/kg), but not the alpha-2 adrenergic receptor antagonist, yohimbine (3 mg/kg), significantly decreased the lethality of a fentanyl-xylazine combination. Lethality was rapid, with death occurring within 10 min after a high dose combination and generally within 30 min at lower dose combinations. Males were more sensitive to the lethal effects of fentanyl-xylazine combinations under some conditions suggesting biologically relevant sex differences in sensitivity to fentanyl-xylazine lethality. Discussion: These data provide the first quantification of the lethal effects of "tranq-dope" and suggest that rapid administration of naloxone may be effective at preventing death following overdose.