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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. [ABSTRACT FROM AUTHOR]

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.

Introduction. Isobolographic analysis is the preferred method of assessment of pharmacodynamic interactions occurring among drugs administered in mixture in both pre-clinical and clinical studies. Despite its mathematical complexity, rigorous preliminary conditions and various prerequisites to be met, it assesses the pharmacodynamic interactions, classifying them as additive, antagonistic, synergistic or indifferent in nature. These interactions are usually plotted in the Cartesian system of coordinates forming isobolograms. The strength (power) of interactions is calculated and presented as an interaction index. Conclusion. This report provides basic information on the isobolographic analysis used experimentally in preclinical conditions indicating the underestimation of this valuable method in pharmacology and toxicology. [ABSTRACT FROM AUTHOR]

(1) The treatment of metastatic or drug-resistant melanoma is still a significant therapeutic problem. The aim of this study was to evaluate the anticancer potential of daphnetin (7,8-dihydroxycoumarin) and its combinations with five different cytostatic drugs (mitoxantrone, docetaxel, vemurafenib, epirubicin and cisplatin). (2) The viability, proliferation and cytotoxicity of daphnetin against four human malignant melanoma cell lines were evaluated. The interactions were assessed using isobolographic analysis for the combinations of daphnetin with each of the five cytostatic drugs. (3) Daphnetin showed anticancer activity against malignant melanoma, with IC50 values ranging from 40.48 ± 10.90 µM to 183.97 ± 18.82 µM, depending on the cell line. The combination of daphnetin with either vemurafenib or epirubicin showed an antagonistic interaction. Moreover, additive interactions were observed for the combinations of daphnetin with cisplatin and docetaxel. The most desirable synergistic interactions for human melanoma metastatic cell lines were observed for the combination of daphnetin with mitoxantrone. (4) The obtained results suggest that daphnetin should not be combined with vemurafenib or epirubicin in the treatment of malignant melanoma due to the abolition of their anticancer effects. The combination of daphnetin with mitoxantrone is beneficial in the treatment of metastatic melanoma due to their synergistic interaction. [ABSTRACT FROM AUTHOR]

The study aimed to reveal pregabalin’s median effective analgesic dose (ED50) and determine the type of analgesic interaction with tramadol, diclofenac, and paracetamol in chicks. The electrical stimulator device was used to detect pain before and after treatment, and through ascending and descending in doses and depending on the up and down method, the median effective analgesic doses were determined for all drugs used in the study, and then the interaction experiment was conducted at a fixed ratio 0.5:0.5 of pregabalin with each of tramadol, diclofenac and paracetamol of their individual ED50 values, the results were subjected to the isobolographic analysis to determine the type of interaction. Results showed that ED50s for pregabalin, tramadol, diclofenac, and paracetamol in chicks were 156.5, 0.82, 5.65, and 10.74 mg/kg, respectively. Concomitant administration of drugs pregabalin: tramadol, pregabalin: diclofenac and pregabalin: paracetamol at a fixed ratio 0.5:0.5 of their individual ED50 values reduced their ED50s to 36.2:0.18, 64.3:2.3 and 64.3:4.3 mg/kg respectively. Isobolographic analysis showed synergistic analgesic effects of both drugs interaction. The calculated interaction indexes were 0.45, 0.81, and 0.81, respectively. We conclude from the outcomes that the analgesic interaction was synergistic between pregabalin and tramadol significantly, while the analgesic interaction of pregabalin with both diclofenac and paracetamol was also synergistic, but to a lesser extent.

(1) Malignant melanomas are dangerous skin cancers, and the treatment of melanomas with various cytostatic drugs often causes side effects and after their prolonged use resistance to these drugs appears. The aim of this study was to evaluate the anticancer effects of esculetin (a simple coumarin) and to assess pharmacodynamic interactions between esculetin and six commonly used cytostatic drugs (cisplatin, epirubicin, docetaxel, paclitaxel, mitoxantrone and vemurafenib) using an isobolographic analysis. (2) The experiments were carried out on four human malignant melanoma cell lines (FM55P, A375, FM55M2 and SK-MEL28). The effects of esculetin on viability, cell proliferation and cytotoxicity were verified in the range of concentrations of 2–200 μM. (3) Esculetin inhibited, in a dose-dependent manner, malignant melanoma cell viability and proliferation. The IC50 for esculetin ranged from 18.20 ± 2.93 to 120.64 ± 30.39 μM depending on the melanoma cell lines used. The combinations of esculetin with epirubicin and vemurafenib showed antagonistic interactions, the combinations of esculetin with cisplatin, docetaxel and paclitaxel showed additive interactions. For the combinations of esculetin with mitoxantrone, the isobolographic analysis displayed synergy. (4) In the treatment of malignant melanoma, esculetin should not be combined with epirubicin or vemurafenib, due to the reduction of their anticancer effects, while the synergistic interactions (esculetin + mitoxantrone) deserve a preclinical recommendation as a beneficial combination during anticancer therapy. [ABSTRACT FROM AUTHOR]

Rationale: Drug combinations are commonly used in pain management, which can produce potent analgesic effects with reduced dosage and adverse effects. Objective: This study was designed to evaluate the anti-nociceptive effects and adverse effects of new combinations of flupirtine (a Kv7 potassium channel opener) and antihistamines (promethazine, fexofenadine) on acute and chronic pain in mice, and the possible mechanisms behind the synergistic analgesic effects were preliminarily investigated. Methods: In acetic acid writhing test, carrageenan-induced inflammatory pain model, and paclitaxel-induced neuropathic pain model, the interaction indexes (γ) between flupirtine and antihistamines were determined by isobolographic analysis. Furthermore, the Kv7 channel blocker XE991 was used to determine whether the effects of single agents and drug combinations on paclitaxel- and carrageenan-induced mechanical allodynia were mediated by Kv7 channels. Finally, hepatotoxicity markers, liver histopathology, and the rotarod test were used to investigate the adverse effects of drugs in combination doses. Results: The interaction indexes of flupirtine-promethazine and flupirtine-fexofenadine in all the above three pain models were lower than 1. The analgesic effects of flupirtine (13 mg/kg), promethazine (5 mg/kg), fexofenadine (20 mg/kg), and their combinations were antagonized significantly by XE991 (3 mg/kg). And the adverse effects of flupirtine and antihistamines in combination doses were not significantly different from the vehicle group. Conclusions: Flupirtine and antihistamines produced synergistic analgesic effects in all the above pain models. The analgesic effects of antihistamines were partially mediated by Kv7/M channels, and the activation of Kv7/M channels may be partly responsible for the synergistic analgesic effects between flupirtine and antihistamines. [ABSTRACT FROM AUTHOR]

Ranolazine, an antianginal and antiarrhythmic drug blocking slow inactivating persistent sodium currents, is described as a compound with anticonvulsant potential. Since arrhythmia often accompanies seizures, patients suffering from epilepsy are frequently co-treated with antiepileptic and antiarrhythmic drugs. The aim of this study was to evaluate the effect of ranolazine on maximal-electroshock (MES)-induced seizures in mice as well as interactions between ranolazine and classical antiepileptic drugs in this model of epilepsy. Types of pharmacodynamic interactions were established by isobolographic analysis of obtained data. The main findings of the study were that ranolazine behaves like an antiseizure drug in the MES test. Moreover, ranolazine interacted antagonistically with carbamazepine, phenytoin, and phenobarbital in the proportions of 1:3 and 1:1. These interactions occurred pharmacodynamic, since ranolazine did not change the brain levels of antiepileptic drugs measured in the fluorescence polarization immunoassay. Ranolazine and its combinations with carbamazepine, phenytoin, and phenobarbital did not impair motor coordination evaluated in the chimney test. Unfortunately, an attempt to conduct a passive avoidance task (evaluating long-term memory) resulted in ranolazine-induced delayed lethality. In conclusion, ranolazine exhibits clear-cut anticonvulsant properties in the MES test but interacts antagonistically with some antiepileptic drugs. The obtained results need confirmation in clinical studies. The mechanisms of ranolazine-induced toxicity require specific explanation. [ABSTRACT FROM AUTHOR]

The study aimed to investigate the analgesic as well as anti-inflammatory effects of diclofenac and silymarin in chicks. The up and down procedure was used to assess the effective median analgesic dosages (ED50s) of diclofenac and silymarin administered intraperitoneally either alone or at the same time in chicks. Also, Analgesic and anti-inflammatory effects were measured by using the formalin test. Isobolographically, ED50s of drugs were assessed for the manner of interaction between both. Formalin testing also supervised analgesic and anti-inflammatory coadministration impact of diclofenac and silymarin at doses 5 and 40 mg/kg and 2.5 and 20 mg/kg respectively. Analgesic ED50s for diclofenac and silymarin in chicks were 9.3 and 76.6 mg/kg separately. Concomitant administration of drugs at a fixed ratio 0.5:0.5 and 0.25:0.25 of their individual ED50 values reduced their ED50s to 2.3:18.6 mg/kg and 2.2:16.5 mg/kg separately. ED50s isobolographic analysis showed synergistic analgesic effects of both drugs. Additionally, coadministration of both drugs had effective analgesic and anti-inflammatory effect, as seen by formalin test, led to a significant rise in latency to lift right foot beside a significant decline in foot lifting frequency when compared with control value, the anti-inflammatory reaction was demonstrated by a significant decrease in foot thickness compared to control value. In conclusion, the data indicate that diclofenac and silymarin coadministration controls acute pain synergistically, and suppress inflammatory reaction.

The objective of our research was to estimate the therapeutic index and assess the interaction of alfaxalone (IP) with ketamine or xylazine (IM) in chicks by using isobolographic analysis. The up-and-down technique was involved to calculate the median effective anesthetic dosages (ED50) of alfaxalone, xylazine, and ketamine given separately or at the same time in young chicks. Then the up-and-down technique was involved to estimate the median lethal dosage (LD50) of alfaxalone (IP) to determine the safety profile. The ED50 of all anesthetics was evaluated isobolographically to assess the type of interaction between alfaxalone and xylazine or alfaxalone and ketamine. The alfaxalone ED50 was 32.88 mg/kg (IP), whereas the LD50 was 102.40 mg/kg (IP). The ED50 values for alfaxalone, ketamine, and xylazine were 32.88, 12.24, and 2.45 mg/kg, respectively. The ED50 values of alfaxalone with ketamine or xylazine (25:25 ED50 values) were: 7.39+2.35, and 8.61+0.63 mg/kg, respectively. ED50 values were decreased when the combinations of alfaxalone/ketamine or alfaxalone/xylazine were administered by 22-21% and 26-25%, respectively. The anesthesia of chicks with alfaxalone is safe, produces a surgical stage of anesthesia, and can be used for minor surgical procedures. The use of alfaxalone with ketamine or xylazine has been shown to have a synergistic effect and these findings may be of clinical relevance in poultry or may be extended to mammals following further clinical trials.

Introduction Aspergillus fumigatus is the most common pathogen causing allergic bronchopulmonary mycosis. The pathogenic capacity of Aspergillus fumigatus is related to its thermal tolerance and the small size of the spores which enables transfer to the respiratory tract. In the case of fungal diseases, their treatment is based on fungicidal antibiotics, such as fluconazole. Due to the growing problem of drug resistance, new therapeutic solutions are sought, especially of natural origin. Essential oils, due to their anti-bacterial, anti-fungal, anti-inflammatory and immunostimulatory properties, constitute interesting research material in the fight against mould. Objective The aim of the study was to assess the type of pharmacodynamic interactions between fluconazole and selected essential oils: lemon, orange, tangerine, and grapefruit in an in vitro study against Aspergillus fumigatus . Isobolographic analysis of the results allowed determining the type of interactions between fluconazole and the tested essential oils. Results According to the research results, a IC50 dose of fluconazole versus Aspergillus fumigatus IC50=1.87±0.88 mg/ml. The most active essential oil was lemon oil, which at the concentration of 4% in medium completely inhibited the growth of Aspergillus fumigatus. Tangerine essential oil is the least active against A. fumigatus . Isobolographic analysis of the interactions between fluconazole and essential oils showed additive interactions for the combination of fluconazole with lemon, orange and grapefruit ols, and an additive interaction with a tendency to synergism for the combination of fluconazole with tangerine oil. Conclusions Isobographic analysis can contribute to the introduction of natural substances into the therapy of many diseases.

Abstract Background Evidence of Plasmodium resistance to some of the current anti-malarial agents makes it imperative to search for newer and effective drugs to combat malaria. Therefore, this study evaluated whether the co-administrations of xylopic acid-amodiaquine and xylopic acid-artesunate combinations will produce a synergistic anti-malarial effect. Methods Antiplasmodial effect of xylopic acid (XA: 3, 10, 30, 100, 150 mg kg−1), artesunate (ART: 1, 2, 4, 8, 16 mg kg−1), and amodiaquine (AQ: 1.25, 2.5, 5, 10, 20 mg kg−1) were evaluated in Plasmodium berghei (strain ANKA)-infected mice to determine respective ED50s. Artemether/lumefantrine was used as the positive control. XA/ART and XA/AQ were subsequently administered in a fixed-dose combination of their ED50s (1:1) and the combination fractions of their ED50s (1/2, 1/4, 1/8, 1/16, and 1/32) to determine the experimental ED50s (Zexp). An isobologram was constructed to determine the nature of the interaction between XA/ART, and XA/AQ combinations by comparing Zexp with the theoretical ED50 (Zadd). Bodyweight and 30-day survival post-treatment were additionally recorded. Results ED50s for XA, ART, and AQ were 9.0 ± 3.2, 1.61 ± 0.6, and 3.1 ± 0.8 mg kg−1, respectively. The Zadd, Zexp, and interaction index for XA/ART co-administration was 5.3 ± 2.61, 1.98 ± 0.25, and 0.37, respectively while that of XA/AQ were 6.05 ± 2.0, 1.69 ± 0.42, and 0.28, respectively. The Zexp for both combination therapies lay significantly (p

Introduction Cladosporium is one of the most abundant genera of environmental fungi worldwide and a very common respiratory allergen. To-date, many C. cladosporoides infections have been identified. Risk factors for C. cladosporioides infection are primarily injuries, metabolic disorders, organ transplantation and autoimmune diseases, among others. Objective The aim of the study was to assess the type of pharmacodynamic interactions between fluconazole and some selected essential oils: orange, mandarin, lemon and grapefruit, in an in vitro study against C. cladosporioides. Material and methods Experiments were carried out using the plate cultivation method. Fluconazole was tested against C. cladosporioides at concentrations ranging from 0.05–3.3 mg / ml, and the activity of essential oils added to PDA medium at concentrations ranging from 1–30%. The dose-effect curves for the collected results were determined with by the log-probit method. Isobolographic analysis of the results allowed determining the type of interactions between fluconazole and the tested essential oils. Results Lemon essential oil was the most active, and in a concentration of 1% it inhibited the growth of C. cladosporioides by 21%. Isobolographic analysis showed that the combination of fluconazole with orange and grapefruit essential oil had an additive interaction, and with mandarin and lemon – an additive interaction with a tendency to synergy in the plate culture test for C. cladosporioides. Conclusions The use of isobolographic analysis can contribute to the introduction of natural substances with the desired activities into the pharmacotherapy of many infections and diseases. The use of natural substances can also help to reduce the number of side-effects caused by conventional and standard therapies

This study aimed to investigate the possibility of utilizing oat by-products for fiber preparation. Oat husk (OH) and oat bran (OB) were micronized and used to prepare a novel product rich in fiber and with enhanced antioxidant properties. The basic chemical composition and phenolic acid profile were determined in OH and OB. The antioxidant properties of OH and OB were also analyzed. The type and strength of interactions between the biologically active compounds from their mixtures were characterized by an isobolographic analysis. The analyses showed that the sum of phenolic acids was higher in OH than in OB. Ferulic acid was dominant in both OH and OB; however, its content in OH was over sixfold higher than that in OB. The results also suggested that both OH and OB can be used for preparing fiber with enhanced antioxidant properties. The optimal composition of the preparation, with 60–70% of OH and 30–40% of OB, allows for obtaining a product with 60–70% fiber and enhanced antioxidant activity due to bioactive substances and their synergistic effect. The resulting product can be a valuable additive to various food and dietary supplements. [ABSTRACT FROM AUTHOR]

Background: Diverse studies suggest that non-steroidal anti-inflammatory drugs (NSAIDs) induce antinociception through the inhibition of cyclooxygenases. Objectives: This study evaluated the effect of NSAIDs in inducing antinociception either alone or in combination in mice formalin orofacial pain. Methods: Male mice were injected intraperitoneally with dexibuprofen, dexketoprofen, diclofenac meloxicam, metamizole and piroxicam. Then from a dose-response curve the ED50 (dose that produce 50% of maximum effect) was obtained from each drug. Results: The administration of NSAIDs produced a dose-dependent antinociception in both phases of the assay with different potency. Then, combinations of the cited NSAIDs were tested and analyzed by isobolographic analysis. The results demonstrate that the nocifensive response induced when dexketoprofen (DEX), the dextrorotatory enantiomer of the S (+) configuration of ketoprofen, was combined with piroxicam, diclofenac, dexibuprofen, metamizole, and meloxicam, was synergistic, either in Phase I or Phase II of the formalin orofacial mice assay. Conclusion: The data demonstrated that the NSAIDs administered alone or in combination produce antinociception. These effects need to be explained by other mechanisms of action of NSAIDs other than the simple inhibition of COXs. The findings may be relevant for the relief of acute or chronic pain such as migraine, post‐herpetic neuralgia and tooth pain.

Introduction Triple-therapy with antiepileptic drugs (AEDs) is usually prescribed for epilepsy patients, whose seizures are not fully controlled with standard medications. Although 25 various AEDs are currently licensed for treating epilepsy, no algorithms allowing for the proper combination of AEDs are available. Objective The aim of the study is to isobolographically assess the type of interaction among three AEDs (lacosamide [LCM], phenobarbital [PB] and valproate [VPA]), in the model of tonic-clonic seizures in mice. Material and methods The electrically-evoked (25 mA, 500 V, 50 Hz, 0.2 s of stimulus duration) tonic-clonic seizures in male albino Swiss mice allowed determination of the anticonvulsant action of the three-drug mixture of LCM, PB and VPA combined in a dose ratio of 1:1:1 by means of type I isobolographic analysis of interaction. Results The experimentally-determined ED50 exp value for the three-drug mixture was 112.04 mg/kg and did not differ from the theoretically calculated ED50 add value, which was 112.36 mg/kg. Lack of statistical significance confirmed that the mixture of LCM, PB and VPA in a dose-ratio of 1:1:1 exerted additive interaction in the mouse tonic-clonic seizure model. Conclusions Although the three-drug combination of LCM, PB and VPA produced additive interaction in the mouse tonic-clonic seizure model, the three-drug combination could be recommended for epilepsy patients whose seizures are refractory to the standard medication.

Breast cancer is the most common cancer that affects women globally and is among the leading cause of women’s death. Triple-negative breast cancer is more difficult to treat because hormone therapy is not available for this subset of cancer. The well-established therapy against triple-negative breast cancer is mainly based on surgery, chemotherapy, and immunotherapy. Among the drugs used in the therapy are cisplatin and carboplatin. However, they cause severe toxicity to the kidneys and brain and cause nausea. Therefore, it is urgent to propose new chemotherapy techniques that provide new treatment options to patients affected by this disease. Nowadays, peptide drugs are emerging as a class of promising new anticancer agents due to their lytic nature and, apparently, a minor drug resistance compared to other conventional drugs (reviewed in Jafari et al., 2022). We have recently reported the cytotoxic effect of the antimicrobial peptide LyeTx I-b against glioblastoma cells (Abdel-Salam et al., 2019). In this research, we demonstrated the cytotoxic effect of the peptide LyeTx I-b, alone and combined with cisplatin, against triple-negative cell lines (MDA-MD-231). LyeTx-I-b showed a selectivity index 70-fold higher than cisplatin. The peptide:cisplatin combination (P:C) 1:1 presented a synergistic effect on the cell death and a selective index value 16 times greater than the cisplatin alone treatment. Therefore, an equi-effective reduction of cisplatin can be reached in the presence of LyeTx I-b. Cells treated with P:C combinations were arrested in the G2/M cell cycle phase and showed positive staining for acridine orange, which was inhibited by bafilomycin A1, indicating autophagic cell death (ACD) as a probable cell death mechanism. Furthermore, Western blot experiments indicated a decrease in P21 expression and AKT phosphorylation. The decrease in AKT phosphorylation is indicative of ACD. However, other studies are still necessary to better elucidate the pathways involved in the cell death mechanism induced by the peptide and the drug combinations. These findings confirmed that the peptide LyeTx I-b seems to be a good candidate for combined chemotherapy to treat breast cancer. In addition, in vivo studies are essential to validate the use of LyeTx I-b as a therapeutic drug candidate, alone and/or combined with cisplatin.

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• Novel mathematical models propose curved isoboles for mixtures of partial agonists. • Hypothesis testing of curvature of isoboles by experiments. • A mixture of drugs with different maximal effects leads to curved isoboles. • Nonlinear isoboles may change the null model for mixture toxicology. Concentration addition as a classic null model for toxicology and pharmacology is based on Loewe's mathematical formulation and the linearity of the isoboles. Novel mathematical models, however, propose curved isoboles in certain conditions. This article aims to test the hypothesis of the curvature of isoboles in experimental measurements. With the assumption of linear isoboles, a partial agonist acts as an antagonist above its maximal effect level. The isoboles automatically convert to a positive slope. For curved isoboles, a partial agonist acts as an antagonist at higher effect levels than its maximal effect alone. The discrepancies between effect levels were studied with an estrogen receptor binding assay (BMAEREluc/ERα) using a mixture of 17β-estradiol and fulvestrant as a partial agonist. A mixture of 17β-estradiol and fulvestrant acts as a partial agonist and causes the diminishing of the effect level of 17β-estradiol at a significantly higher level than the maximal effect of their partial-agonistic dose-response curve. Measured, elevated effect levels were well predicted by the mathematical model. Nonlinear isoboles may change our understanding and definition of synergism or antagonism and prompt further attention in receptor theory. [ABSTRACT FROM AUTHOR]

Abstract Background: Phoneutria nigriventer venom contains Phα1β. This toxin and its recombinant form have a remarkable analgesic potential that is associated with blockage of voltage-gated calcium channels and TRPA1 receptors. Although morphine is a mainstay drug to treat moderate and severe pain related to cancer, it has serious and dose-limiting side effects. Combining recombinant Phα1β and morphine to treat pain is an interesting approach that has been gaining attention. Therefore, a quantitative and reliable method to establish the strength of the antinociceptive interaction between these two substances is necessary. The present study was designed to investigate the nature of the functional antinociceptive (analgesic) interaction between Phα1β recombinant toxin and morphine in a model of cancer pain. Methods: Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw of C57BL/6J mice. Von Frey filaments measured the paw-withdrawal threshold after intrathecal administration of morphine, recombinant Phα1β, and their combination. Thermal hyperalgesia was assessed using Hargreaves apparatus. The degree of interaction was evaluated using isobolographic analysis. Spontaneous and forced motor performance was assessed with the open-field and rotarod tests, respectively. Results: Co-administration of recombinant Phα1β and morphine synergistically reverses the melanoma-induced mechanical hyperalgesia. The potency of the mixture, measured as the effective dose to reach 50% of maximum possible effect (MPE) in ameliorating mechanical hyperalgesia, was about twice fold higher than expected if the interaction between morphine and recombinant Phα1β was merely additive. Treatment with the combination at doses necessary to reach 50% of MPE caused no spontaneous nor forced motor alterations. Conclusion: The combinatorial use of recombinant Phα1β and morphine allows significant and effective dose reduction of both agents, which has translational potential for opioid-sparing approaches in pain management related to cancer.

The objective of our research was to estimate the therapeutic index and assess the interaction of alfaxalone (IP) with ketamine or xylazine (IM) in chicks by using isobolographic analysis. The up-and-down technique was involved to calculate the median effective anesthetic dosages (ED50) of alfaxalone, xylazine, and ketamine given separately or at the same time in young chicks. Then the up-and-down technique was involved to estimate the median lethal dosage (LD50) of alfaxalone (IP) to determine the safety profile. The ED50 of all anesthetics was evaluated isobolographically to assess the type of interaction between alfaxalone and xylazine or alfaxalone and ketamine. The alfaxalone ED50 was 32.88 mg/kg (IP), whereas the LD50 was 102.40 mg/kg (IP). The ED50 values for alfaxalone, ketamine, and xylazine were 32.88, 12.24, and 2.45 mg/kg, respectively. The ED50 values of alfaxalone with ketamine or xylazine (25:25 ED50 values) were: 7.39+2.35, and 8.61+0.63 mg/kg, respectively. ED50 values were decreased when the combinations of alfaxalone/ketamine or alfaxalone/xylazine were administered by 22-21% and 26-25%, respectively. The anesthesia of chicks with alfaxalone is safe, produces a surgical stage of anesthesia, and can be used for minor surgical procedures. The use of alfaxalone with ketamine or xylazine has been shown to have a synergistic effect and these findings may be of clinical relevance in poultry or may be extended to mammals following further clinical trials. [ABSTRACT FROM AUTHOR]

Introduction. Aspergillus fumigatus is the most common pathogen causing allergic bronchopulmonary mycosis. The pathogenic capacity of Aspergillus fumigatus is related to its thermal tolerance and the small size of the spores which enables transfer to the respiratory tract. In the case of fungal diseases, their treatment is based on fungicidal antibiotics, such as fluconazole. Due to the growing problem of drug resistance, new therapeutic solutions are sought, especially of natural origin. Essential oils, due to their anti-bacterial, anti-fungal, anti-inflammatory and immunostimulatory properties, constitute interesting research material in the fight against mould. Objective. The aim of the study was to assess the type of pharmacodynamic interactions between fluconazole and selected essential oils: lemon, orange, tangerine, and grapefruit in an in vitro study against Aspergillus fumigatus. Isobolographic analysis of the results allowed determining the type of interactions between fluconazole and the tested essential oils. Results. According to the research results, a IC50 dose of fluconazole versus Aspergillus fumigatus IC50 = 1.87±0.88 mg/ml. The most active essential oil was lemon oil, which at the concentration of 4% in medium completely inhibited the growth of Aspergillus fumigatus. Tangerine essential oil is the least active against A. fumigatus. Isobolographic analysis of the interactions between fluconazole and essential oils showed additive interactions for the combination of fluconazole with lemon, orange and grapefruit ols, and an additive interaction with a tendency to synergism for the combination of fluconazole with tangerine oil. Conclusions. Isobographic analysis can contribute to the introduction of natural substances into the therapy of many diseases. [ABSTRACT FROM AUTHOR]

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are the most commonly used drugs due to their antipyretic, anti‐inflammatory, and analgesic properties. However, NSAIDs can cause adverse reactions, mainly gastrointestinal damage. Omeprazole (OMP) exhibits gastroprotective activity, but its protection is limited at the intestinal level. For this reason, it is essential to utilize a combination of therapies that provide fewer adverse effects, such as the combined treatment of OMP and docosahexaenoic acid (DHA), an omega‐3 polyunsaturated fatty acid with anti‐inflammatory, analgesic, and gastroprotective activities. The objective of this study was to evaluate the pharmacological interaction between DHA and OMP in a murine model of indomethacin‐induced gastrointestinal damage. The gastroprotective and enteroprotective effects of DHA (0.3–10 mg/kg, p.o.), OMP (1–30 mg/kg, p.o.), or the combination treatment of both compounds (3–56.23 mg/kg, p.o.) were evaluated in the indomethacin‐induced gastrointestinal damage model (30 mg/kg, p.o.). Since DHA and OMP exhibited a protective effect in a dose‐responsive fashion, the ED30 for each individual compound was determined and a 1:1 combination of DHA and OMP was tested. Isobolographic analysis was used to determine any pharmacodynamic interactions. Since the effective experimental dose ED30 (Zexp) of the combined treatment of DHA and OMP was lower than the theoretical additive dose (Zadd; p <.05) in both the stomach and small intestine their protective effects were considered synergistic. These results indicate that the synergistic protective effects from combined treatment of DHA and OMP could be ideal for mitigating damage generated by NSAIDs at the gastrointestinal level. [ABSTRACT FROM AUTHOR]

Rationale: Opioid and GABAA receptors are both located in central nociceptive pathways, and compounds that activate these receptors have pain-relieving properties. To date, the interactive effects of concurrent administration of these compounds in preclinical models of pain-like behaviors have not been assessed. Objective: The purpose of this study was to examine the interactive effects of the μ-opioid agonist morphine and the α2GABAA and α3GABAA receptor positive allosteric modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024) in preclinical models of mechanical hyperalgesia and thermal nociception. Methods: The antihyperalgesic and antinociceptive effects of morphine and MP-III-024 administered alone were assessed initially, followed by fixed-ratio mixtures of MP-III-024/morphine combinations. Drug interaction data were analyzed using isobolographic and dose-addition analyses. All studies were conducted in male CD-1 mice. Results: In the assay of mechanical hyperalgesia, each compound produced dose-dependent antihyperalgesic effects, whereas only morphine was effective on thermal nociception. Fixed-ratio mixtures of MP-III-024/morphine were also dose-dependently effective in both procedures. These drug combination studies revealed that morphine and MP-III-024 produced supra-additive (synergistic) effects in both assays, depending on their relative proportions. Conclusions: These results demonstrate an interaction between α2GABAA and α3GABAA receptor- and μ-opioid receptor-mediated signals and suggest that combination therapy may be useful for the treatment of pain-related disorders. [ABSTRACT FROM AUTHOR]

Evidence suggests that the antinociceptive activity of various drugs can be increased when administered in combination with B vitamins (BVs). The aim of this study was to examine the potential interaction between statins and BVs to counter nociception, the latter measured by the formalin test. Rats were orally administered atorvastatin (1, 3, 10 and 30 mg/kg), pravastatin (1, 3, 10 and 30 mg/kg), rosuvastatin (1, 3, 10 and 30 mg/kg), BVs (31, 56, 100 and 180 mg/kg) or calculated combinations of BVs with each drug. The effective dose 30 (ED30) was calculated for each statin and BVs and subjected to isobolographic analysis, thus finding the ED30 of the combinations. The antinociceptive experimental ED30 values for BVs administered with atorvastatin, pravastatin or rosuvastatin were 1.53 ± 0.38, 6.74 ± 0.04 and 4.26 ± 0.39, respectively, being lower (p <.05) than the corresponding theoretical ED30: 28.02 ± 2.20, 28.17 ± 2.20 and 29.86 ± 2.21. Since BVs likely boost the antinociceptive effect of statins, these combinations could possibly be advantageous in pain management. [ABSTRACT FROM AUTHOR]

Metamizole is used to relieve the visceral pain but its adverse effects limit its use. An alternative to improve its efficacy with lower doses is to combine it with a natural product as hesperidin. Aim of the study. The aim of this study was to evaluate the antinociceptive interaction between metamizole and hesperidin in a visceral pain model using an isobolographic analysis. Antinociception was evaluated in the writhing model using acetic acid (1%) to induce writhes in mice. Metamizole (1–316 mg/kg), hesperidin (3–300 mg/kg), or combinations with a fixed-dose ratio of 1:1 were administered intraperitoneally 30 min before the acetic acid and the number of writhes was counted for 30 min. Isobolographic analysis was employed to define the nature of the compound interaction. Metamizole and hesperidin in individual administration induced dose-dependent antinociceptive effects, reached an efficacy of 84.2 ± 5.9% and 66.3 ± 7.4%, respectively. The ED 50 values calculated from their dose-response curves were 84.5 ± 22.7 and 108.9 ± 17.9 mg/kg, respectively. The analysis of DRC for the metamizole + hesperidin combination, in a ratio 1:1 showed a ED 50 COMB value lower than the ED 50 ADD estimated from the additivity line from the isobologram (46.7 ± 6.3 vs. 96.7 ± 11.9 mg/kg, respectively). In addition, the pharmacological interaction calculated was of 0.48. These results suggest a synergistic interaction for the antinociceptive activity of metamizole + hesperidin combination. These data suggest that metamizole + hesperidin combination could be useful in treating visceral pain as it can interact synergistically using low dose of both drugs with the possibility of reducing the risk of adverse effects. [Display omitted] [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the antinociceptive interaction between dexketoprofen and tapentadol in three different dose ratios, as well as the ulcerogenic activity of this combination. Dose–response curves were carried out for dexketoprofen, tapentadol, and dexketoprofen–tapentadol combinations in the acetic acid‐induced writhing test in mice. On the other hand, the gastric damage of all treatments was assessed after the surgical extraction of the stomachs. Intraperitoneal administration of dexketoprofen and tapentadol induced a dose‐dependent antinociceptive effect, reaching a maximal effect of about 58% and 99%, respectively. Isobolographic analysis and the interaction index showed that the three proportions produced an analgesic potentiation (synergistic interaction). Interestingly, the 1:1 and 1:3 ratios of the drugs combination produced minor gastric injury in comparison with the 3:1 proportion. Our data suggest that all proportions of the dexketoprofen–tapentadol combination produced a synergistic interaction in the acetic acid‐induced visceral pain model in mice with a low incidence of gastric injury. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the nature of the antinociceptive interaction of systemic administration of a combination of the anticonvulsant gabapentin with the antidepressant nortriptyline, by isobolographic analysis in the formalin orofacial pain test of mice. Methods: The study was carried out in 168 male CF-1 mice weighing 30 g, and the protocol was to test each drug (at dosages of 1, 3, 10, 30, and 100 mg/kg of gabapentin and 0.1, 1, 3, 10, and 30 mg/kg of nortriptyline; ip) alone and in combination. The isobolographic assay has two phases: phase 1 corresponds to the 5-minute period starting immediately after the formalin injection and reflects a tonic acute pain due to peripheral nociceptor sensitization; phase 2 is recorded as the 10-minute period starting 20 minutes after the formalin injection and reflects an inflammatory pain state. Results were analyzed by Student t test for independent means. Results: Gabapentin was 1.61 times more potent in phase 2 than in phase 1, and nortriptyline 1.37 times more potent in phase 2 than in phase 1. The combination of both drugs was synergic, with an index of interaction of 0.134 and 0.148 for phase 1 and phase 2, respectively. Differences in the pharmacological profiles of gabapentin and nortriptyline could underlie the synergism of the two drugs. Conclusion: The findings of this study are important, because they are concordant with some clinical studies and also raise the possibility of potential clinical advantages of combining gabapentin and nortriptyline in pain management, since the low doses of the components may potentially have a lower incidence of adverse reactions. [ABSTRACT FROM AUTHOR]

Background: Evidence of Plasmodium resistance to some of the current anti-malarial agents makes it imperative to search for newer and effective drugs to combat malaria. Therefore, this study evaluated whether the co-administrations of xylopic acid-amodiaquine and xylopic acid-artesunate combinations will produce a synergistic anti-malarial effect. Methods: Antiplasmodial effect of xylopic acid (XA: 3, 10, 30, 100, 150 mg kg−1), artesunate (ART: 1, 2, 4, 8, 16 mg kg−1), and amodiaquine (AQ: 1.25, 2.5, 5, 10, 20 mg kg−1) were evaluated in Plasmodium berghei (strain ANKA)-infected mice to determine respective ED50s. Artemether/lumefantrine was used as the positive control. XA/ART and XA/AQ were subsequently administered in a fixed-dose combination of their ED50s (1:1) and the combination fractions of their ED50s (1/2, 1/4, 1/8, 1/16, and 1/32) to determine the experimental ED50s (Zexp). An isobologram was constructed to determine the nature of the interaction between XA/ART, and XA/AQ combinations by comparing Zexp with the theoretical ED50 (Zadd). Bodyweight and 30-day survival post-treatment were additionally recorded. Results: ED50s for XA, ART, and AQ were 9.0 ± 3.2, 1.61 ± 0.6, and 3.1 ± 0.8 mg kg−1, respectively. The Zadd, Zexp, and interaction index for XA/ART co-administration was 5.3 ± 2.61, 1.98 ± 0.25, and 0.37, respectively while that of XA/AQ were 6.05 ± 2.0, 1.69 ± 0.42, and 0.28, respectively. The Zexp for both combination therapies lay significantly (p < 0.001) below the additive isoboles showing XA acts synergistically with both ART and AQ in clearing the parasites. High doses of XA/ART combination significantly (p < 0.05) increased the survival days of infected mice with a mean hazard ratio of 0.40 while all the XA/AQ combination doses showed a significant (p < 0.05) increase in the survival days of infected mice with a mean hazard ratio of 0.27 similar to AL. Both XA/ART and XA/AQ combined treatments significantly (p < 0.05) reduced weight loss. Conclusion: Xylopic acid co-administration with either artesunate or amodiaquine produces a synergistic anti-plasmodial effect in mice infected with P. berghei. [ABSTRACT FROM AUTHOR]

Introduction. Cladosporium is one of the most abundant genera of environmental fungi worldwide and a very common respiratory allergen. To-date, many C. cladosporoides infections have been identified. Risk factors for C. cladosporioides infection are primarily injuries, metabolic disorders, organ transplantation and autoimmune diseases, among others. Objective. The aim of the study was to assess the type of pharmacodynamic interactions between fluconazole and some selected essential oils: orange, mandarin, lemon and grapefruit, in an in vitro study against C. cladosporioides. Materials and method. Experiments were carried out using the plate cultivation method. Fluconazole was tested against C. cladosporioides at concentrations ranging from 0.05-3.3 mg / ml, and the activity of essential oils added to PDA medium at concentrations ranging from 1-30%. The dose-effect curves for the collected results were determined with by the logprobit method. Isobolographic analysis of the results allowed determining the type of interactions between fluconazole and the tested essential oils. Results. Lemon essential oil was the most active, and in a concentration of 1% it inhibited the growth of C. cladosporioides by 21%. Isobolographic analysis showed that the combination of fluconazole with orange and grapefruit essential oil had an additive interaction, and with mandarin and lemon - an additive interaction with a tendency to synergy in the plate culture test for C. cladosporioides. Conclusions. The use of isobolographic analysis can contribute to the introduction of natural substances with the desired activities into the pharmacotherapy of many infections and diseases. The use of natural substances can also help to reduce the number of side-effects caused by conventional and standard therapies. [ABSTRACT FROM AUTHOR]

Clinical and preclinical research that contributes pain palliation has suggested that drugs favor the expected effects and minimize the adverse effects. Among the most widely used strategies is the combination of analgesic drugs among those in the same group, with those in another group of analgesics or with co‐adjuvants (nonanalgesic drugs or elements of traditional medicine). This work aims to evaluate the interaction between eugenol (EUG) and diclofenac (DFC) on nociception in the presence of a noxious stimulus through the formalin test and isobolographic analysis. The results indicate that EUG, DFC, or the combination of both produce an antinociceptive effect in rodents (p ≤ 0.05). Local co‐administration of EUG and DFC gave a theoretical effective dose (Zadd) 2,936.27 ± 155.33 μg/kg (p ≤ 0.05) significantly higher as compared to the effective experimental doses (Zmix) of 866.89 ± 0.02 μg/kg in phase 1 and 292.88 ± 0.05 μg/kg in phase 2, with an interaction index of 0.29 and 0.09, respectively. These data allow concluding that the interaction derived from the joint administration of EUG and DFC, in the rodent at a local level, is synergistic. [ABSTRACT FROM AUTHOR]

Hydrophilic interaction liquid chromatography coupled to mass spectrometry was employed for simultaneous determination of vitamin B6 and catechins in dietary supplements. The obtained results clearly shows the potential of the application of alcohol eluent (instead mainly used acetonitrile) as a component of an with zwitterionic stationary phase. The limits of detection on ZIC-HILIC column were 0.01 mg/L for catechins and vitamins B (only for pyridoxal phosphate was 0.10 mg/L). The investigations between green tea extract (GTE) and vitamin B were also evaluated using isobolographic analysis as well as the interaction indexes. Antioxidant activities of single components and their mixtures were determined by DPPH assay. It was found that the mixtures of GTE and vitamin B acted synergistically. In comparison to GTE alone, faster DPPH radical bleaching of the mixtures was observed in the presence of different forms of vitamin B6 (pyridoxine, pyridoxal, or pyridoxal phosphate), particularly for pyridoxal. [ABSTRACT FROM AUTHOR]

Introduction: Triple-therapy with antiepileptic drugs (AEDs) is usually prescribed for epilepsy patients, whose seizures are not fully controlled with standard medications. Although 25 various AEDs are currently licensed for treating epilepsy, no algorithms allowing for the proper combination of AEDs are available. Objective: The aim of the study is to isobolographically assess the type of interaction among three AEDs (lacosamide [LCM], phenobarbital [PB] and valproate [VPA]), in the model of tonic-clonic seizures in mice. Materials and Method: The electrically-evoked (25 mA, 500 V, 50 Hz, 0.2 s of stimulus duration) tonic-clonic seizures in male albino Swiss mice allowed determination of the anticonvulsant action of the three-drug mixture of LCM, PB and VPA combined in a dose ratio of 1:1:1 by means of type I isobolographic analysis of interaction. Results: The experimentally-determined ED50 exp value for the three-drug mixture was 112.04 mg/kg and did not differ from the theoretically calculated ED50 add value, which was 112.36 mg/kg. Lack of statistical significance confirmed that the mixture of LCM, PB and VPA in a dose-ratio of 1:1:1 exerted additive interaction in the mouse tonic-clonic seizure model. Conclusions: Although the three-drug combination of LCM, PB and VPA produced additive interaction in the mouse tonic-clonic seizure model, the three-drug combination could be recommended for epilepsy patients whose seizures are refractory to the standard medication. [ABSTRACT FROM AUTHOR]

Rationale: Acute pain states in the trigeminal region (headaches, dental pain) fall into the most prevalent painful conditions. Standard analgesics (paracetamol/NSAIDs) represent the cornerstone of their treatment, whereas triptans are primarily used in migraine attacks. Due to limited efficacy and/or side effects of current treatments, identifying favorable combinations of available drugs is justified. Objectives: Eslicarbazepine acetate (ESL) is a novel antiepileptic drug whose effectiveness against trigeminal pain was recently demonstrated. Here, we examined the interactions between ESL and several standard/alternative analgesics (paracetamol, propyphenazone, naproxen, zolmitriptan, and metoclopramide) in a model of trigeminal pain. Methods: The antinociceptive effects of orally administered ESL, standard/alternative analgesics, and two-drug ESL-analgesic combinations were examined in the orofacial formalin test in mice. The type of interaction between drugs was determined by isobolographic analysis. Results: ESL, analgesics, and two-drug ESL-analgesic combinations significantly and dose-dependently reduced nociceptive behaviour in the second, inflammatory phase of the test. Isobolographic analysis revealed that ESL interacted additively with paracetamol/propyphenazone/zolmitriptan and synergistically with naproxen/metoclopramide (with about a 4-fold and 3-fold reduction of doses in the ESL-naproxen and ESL-metoclopramide combination, respectively). Conclusions: ESL interacted in a beneficial manner with several analgesics that are used for trigeminal pain treatment, producing synergistic interactions with naproxen/metoclopramide and additive interactions with paracetamol/propyphenazone/zolmitriptan. Our results suggest that combining ESL with analgesics could theoretically enable the use of lower doses of individual drugs for achieving pain relief. [ABSTRACT FROM AUTHOR]

Palmatine (PLT) is a natural isoquinoline alkaloid that belongs to the class of protoberberines and exhibits a wide spectrum of pharmacological and biological properties, including anti-cancer activity. The aim of our study was to isolate PLT from the roots of Berberis cretica and investigate its cytotoxic and anti-proliferative effects in vitro alone and in combination with doxorubicine (DOX) using human ER+/HER2− breast cancer cell lines. The alkaloid was purified by column chromatography filled with silica gel NP and Sephadex LH-20 resin developed in the mixture of methanol: water (50:50 v/v) that provided high-purity alkaloid for bioactivity studies. The purity of the alkaloid was confirmed by high resolution mass measurement and MS/MS fragmentation analysis in the HPLC-ESI-QTOF-MS/MS-based analysis. It was found that PLT treatment inhibited the viability and proliferation of breast cancer cells in a dose-dependent manner as demonstrated by MTT and BrdU assays. PLT showed a quite similar growth inhibition on breast cancer cells with IC50 values ranging from 5.126 to 5.805 µg/mL. In contrast, growth of normal human breast epithelial cells was not affected by PLT. The growth inhibitory activity of PLT was related to the induction of apoptosis, as determined by Annexin V/PI staining. Moreover, PLT sensitized breast cancer cells to DOX. Isobolographic analysis revealed synergistic and additive interactions between studied agents. Our studies suggest that PLT can be a potential candidate agent for preventing and treating breast cancer.

Usnic acid (UA) is a chiral lichen metabolite with an interesting pharmacological profile. The aim of this study was to compare the anti-melanoma effect of (+)-UA and (−)-UA in an in vitro model by studying their impact on the cells as well as the processes associated with cancer progression. The effect of UA enantiomers on the viability, proliferation, and invasive potential of three melanoma cell lines (HTB140, A375, WM793) was evaluated. Their interaction with a chemotherapeutic drug—doxorubicin was assessed by isobolographic analysis. Anti-inflammatory and anti-tyrosinase properties of (+)-UA and (−)-UA were also examined. Both UA enantiomers dose- and time-dependently decreased the viability of all three melanoma cell lines. Their synergistic effect with doxorubicin was observed on A375 cells. (+)-Usnic acid at a sub-cytotoxic dose strongly inhibited melanoma cells migration. Both UA enantiomers decreased the release of pro-inflammatory mediators. The cytotoxic effect of (+)-UA and (−)-UA depends greatly on the melanoma cell type; however, the overall anti-melanoma potential is perspective. Our results indicate that the strategy of combining usnic acid enantiomers with cytostatic drugs may be an interesting option to consider in combating melanoma; however, further studies are required.

Hyperbaric oxygen (HBO2) is acutely toxic to the central nervous system, culminating in EEG spikes and tonic-clonic convulsions. GABA enhancers and sodium channel antagonists improve seizure latencies in HBO2 when administered individually, while combining antiepileptic drugs from different functional classes can provide greater seizure latency. We examined the combined effectiveness of GABA enhancers (tiagabine and gabapentin) with sodium channel antagonists (carbamazepine and lamotrigine) in delaying HBO2-induced seizures. A series of experiments in C57BL/6 mice exposed to 100% oxygen at 5 atmospheres absolute (ATA) were performed. We predicted equally effective doses from individual drug-dose response curves, and the combinations of tiagabine + carbamazepine or lamotrigine were tested to determine the maximally effective combined doses to be used in subsequent experiments designed to identify the type of pharmacodynamic interaction for three fixed-ratio combinations (1:3, 1:1, and 3:1) using isobolographic analysis. For both combinations, the maximally effective combined doses increased seizure latency over controls > 5-fold and were determined to interact synergistically for fixed ratios 1:1 and 3:1, additive for 1:3. These results led us to explore whether the benefits of these drug combinations could be extended to the lungs, since a centrally mediated mechanism is believed to mediate hyperoxic-induced cardiogenic lung injury. Indeed, both combinations attenuated bronchoalveolar lavage protein content by ~ 50%. Combining tiagabine with carbamazepine or lamotrigine not only affords greater antiseizure protection in HBO2 but also allows for lower doses to be used, minimizing side effects, and attenuating acute lung injury. [ABSTRACT FROM AUTHOR]

The aims of the study were to evaluate the pharmacodynamic interaction between 3α‐hydroxymasticadienonic acid and diligustilide (DLG), isolated from the plants Amphiptherygium adstringens and Ligusticum porteri, respectively, using the indomethacin‐induced gastric injury model, as well as their individual gastroprotective efficacy in this model. Male Wistar rats were orally administered with 3α‐hydroxymasticadienonic acid, DLG or the mixture of 3α‐hydroxymasticadienonic acid‐DLG (at a fixed‐ratio combination of 1:1, 1:3, and 3:1). Thirty minutes later, the gastric damage was induced by a single oral dose of indomethacin (30 mg/kg). Three hours later, the gastric injury (mm2) was determined. 3α‐hydroxymasticadienonic acid and DLG as individual compounds showed a gastroprotective effect against indomethacin‐induced gastric damage (p < .05). The effective dose (ED50) values for each compound were 6.96 ± 1.25 mg/kg for 3α‐hydroxymasticadienonic acid and 2.63 ± 0.37 mg/kg for DLG. The isobolographic analysis performed showed that the combination exhibited super‐additive interaction as the experimental ED50 values (Zexp) were lower than theoretical additive dose values (Zadd; p < .05). Our results identify the super‐additive (synergist) interaction between 3α‐hydroxymasticadienonic acid and DLG and the gastric safety of both compounds in the indomethacin—induced gastric injury model, suggesting their potential in the future as a strategy to decrease the gastric damage associated to the chronic use of nonsteroidal anti‐inflammatory drugs (NSAIDs). [ABSTRACT FROM AUTHOR]

Introduction. Polytherapy with three antiepileptic drugs (AEDs) is used in patients who have seizure episodes classified by physicians and neurologists as refractory or drug-resistant. Although doctors can prescribe these patients 25 various AEDs, no algorithms are available which allow them to choose the most efficacious combinations of AED. Objective. The aim of this study was to isobolographically classify interaction among three classical AEDs (carbamazepine, phenobarbital and valproate), applied in the fixed-ratio combination of 1:1:1, in the maximal electroshock-induced seizures in mice. Materials and method. The anti-seizure activity of the mixture of carbamazepine, phenobarbital and valproate (in the fixed-ratio of 1:1:1) was determined in maximal electroshock-induced seizures - an experimental model of tonic-clonic seizures in mice using type I isobolographic analysis. Results. The mixture of carbamazepine, phenobarbital and valproate (in the fixed-ratio of 1:1:1) produced additive interaction in the maximal electroshock-induced seizure model in mice. The experimentally-derived median effective dose (ED50 exp value) for the mixture was 94.35 mg/kg, whereas the theoretically additive median effective dose (ED50 add value) amounted to 116.77 mg/kg. Conclusions. Although the combination of carbamazepine, phenobarbital and valproate exerted additivity in the mouse maximal electroshock-induced seizure model, it could be recommended for the treatment of patients, if the results of this study would be directly transposed to clinical settings. [ABSTRACT FROM AUTHOR]

Although nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the main types of drugs used to treat pain, they have several adverse effects, and such effects can be reduced by combining two analgesic drugs. The aim of this study was to evaluate the nociceptive activity of methyleugenol combined with either diclofenac or ketorolac, and determine certain parameters of pharmacokinetics. For the isobolographic analysis, the experimental effective dose 30 (ED30) was calculated for the drugs applied individually. With these effective doses, the peak plasma concentration (Cmax) was found and the other parameters of pharmacokinetics were established. Methyleugenol plus diclofenac and methyleugenol plus ketorolac decreased licking behavior in a dose-dependent manner in phase II, with an efficacy of 32.9 ± 9.3 and 39.8 ± 9.6%, respectively. According to the isobolographic analysis, the experimental and theoretical ED30 values were similar for methyleugenol plus diclofenac, suggesting an additive effect, but significantly different for methyleugenol plus ketorolac (3.6 ± 0.5 vs. 7.7 ± 0.6 mg/kg, respectively), indicating a probable synergistic interaction. Regarding pharmacokinetics, the only parameter showing a significant difference was Cmax for the methyleugenol plus diclofenac combination. Even with this difference, the combinations studied may be advantageous for treating inflammatory pain, especially for the combination methyleugenol plus ketorolac.

This study was aimed at characterizing the anticonvulsant effects of levetiracetam in combination with gabapentin, in the mouse 6 Hz psychomotor seizure model. Herein, psychomotor seizures were evoked in male albino Swiss mice by a current (32 mA, 6 Hz, 3 s stimulus duration) delivered via ocular electrodes. Type II isobolographic analysis was used to characterize the anticonvulsant interactions between the drugs in combination, for fixed-ratios of 1:1, 1:2, 1:5 and 1:10. The type II isobolographic analysis revealed that the combinations of levetiracetam with gabapentin for the fixed-ratios of 1:5 and 1:10 were supra-additive (synergistic; P

Study Question: Does the chemosensory activation of CatSper Ca2+ channels in human sperm give rise to additive, sub-additive or even synergistic actions among agonists?Summary Answer: We show that oviductal ligands and endocrine disrupting chemicals (EDCs) activate human CatSper highly synergistically.What Is Known Already: In human sperm, the sperm-specific CatSper channel controls the intracellular Ca2+ concentration and, thereby, several crucial stages toward fertilization. CatSper is activated by oviductal ligands and structurally diverse EDCs. The chemicals mimic the action of the physiological ligands, which might interfere with the precisely coordinated sequence of events underlying fertilization.Study Design, Size, Duration: For both oviductal ligands and EDCs, we examined in quantitative terms whether stimulation of human sperm in vitro with mixtures results in additive, sub-additive or synergistic actions.Participants/materials, Setting, Methods: We studied activation of CatSper in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. The combined action of progesterone and prostaglandins and of the EDCs benzylidene camphor sulfonic acid (BCSA) and α-Zearalenol was evaluated by curve-shift analysis, curvilinear isobolographic analysis and the combination-index method.Main Results and the Role Of Chance: Analysis of the action of progesterone/prostaglandin and BCSA/α-Zearalenol mixtures in human sperm by fluorimetry revealed that the oviductal ligands and EDCs both evoke Ca2+ influx via CatSper in a highly synergistic fashion. Patch-clamp recordings of CatSper currents in human sperm corroborated the synergistic ligand-activation of the channel.Limitations, Reasons For Caution: This is an in vitro study. Future studies have to assess the physiological relevance in vivo.Wider Implications Of the Findings: These findings indicate that the fertilization process is orchestrated by multiple oviductal CatSper agonists that act in concert to control the behavior of sperm. Moreover, our results substantiate the concerns regarding the negative impact of EDCs on male reproductive health. So far, safety thresholds like the "No Observed Adverse Effect Level (NOAEL)" or "No Observed Effect Concentration (NOEC)" are set for individual EDCs. Our finding that EDCs act synergistically in human sperm challenges the validity of this procedure.Study Funding/competing Interest(s): This work was supported by the German Research Foundation (SFB 645; CRU326), the Cells-in-Motion (CiM) Cluster of Excellence, Münster, (FF-2016-17), the 'Innovative Medical Research' of the University of Münster Medical School (BR121507), an EDMaRC research grant from the Kirsten and Freddy Johansen's Foundation, and the Innovation Fund Denmark (InnovationsFonden; 14-2013-4). The authors have no competing financial interests. [ABSTRACT FROM AUTHOR]

Introduction. Assessment of interactions among antiepileptic drugs (AEDs) during polytherapy is still a challenging issue for physicians and epileptologists worldwide. In spite of 25 currently licensed AEDs, there are no algorithms allowing a proper choice of these drugs to create combinations which would offer epileptic patients an efficacious therapy in the case of seizures refractory to monotherapeutic use of the AEDs. To characterize a type of interaction for a three-drug mixture of oxcarbazepine (OXC), pregabalin (PGB) and topiramate (TPM) in an experimental model of tonic-clonic seizures, an isobolographic analysis of interaction was applied. Materials and Method. The anticonvulsant effects of the three-drug mixture of OXC, PGB and TPM with respect to suppression of tonic-clonic seizures in mice were assessed in the mouse maximal electroshock-induced seizure model. Type I isobolographic analysis was used to characterize the type of interactions among three AEDs. Potential acute adverse effects were evaluated in the chimney, passive avoidance and grip-strength tests. Results. The three-drug mixture of OXC, PGB and TPM exerted supra-additive (synergistic) interaction in the mouse maximal electroshock-induced seizure model. The combination of OXC, PGB and TPM did not produce any acute adverse effects in mice in the chimney, passive avoidance and grip-strength tests. Conclusions. The isobolographic synergy observed experimentally for the combination of OXC, PGB and TPM could be recommended to patients with drug-resistant epilepsy, if the results of this study were translated to clinical settings. [ABSTRACT FROM AUTHOR]

The antioxidant interactions between acetaminophen (APAP) and green tea polyphenols were investigated using 1,1-diphenyl- 2-picryl-hydrazyl (DPPH) radicals and cupric reducing antioxidant capacity (CUPRAC). The results obtained experimentally for the mixtures were compared with theoretical values calculated by adding up the effects of both individual components analyzed separately. The potential antagonistic, additive or synergistic effects were also evaluated using the isobolographic analysis and interaction indexes. The results obtained for DPPH assays suggest the antagonistic type of interaction, while the results from CUPRAC method showed synergism. The observed differences in the type of interactions were probably a result of different reaction mechanisms between two used assays. [ABSTRACT FROM AUTHOR]

Aims Despite many antiepileptic drugs (AEDs) are available to treat epilepsy, there is still about 30% of epilepsy patients inadequately treated with these AEDs. For these patients, polytherapy with two or three AEDs to fully control their seizure attacks is recommended. Unfortunately, polytherapy is always associated with drug interactions, whose nature may be beneficial, neutral or unfavorable. To determine a type of interaction for the combination of three AEDs (i.e., phenobarbital [PB], phenytoin [PHT] and pregabalin [PGB]) at the fixed-ratio of 1:1:1, we used a model of tonic-clonic seizures in male albino Swiss mice. Materials and method Tonic-clonic seizures in mice were evoked by a current (sine-wave, 25 mA, 500 V, 0.2 s stimulus duration) delivered via auricular electrodes. The anticonvulsant effects of the three-drug combination (PB, PHT and PGB) in terms of suppression of tonic-clonic seizures in mice were assessed with type I isobolographic analysis. Potential acute side effects for the mixture of PB, PHT and PGB along with total brain concentrations of the AEDs were determined to confirm pharmacodynamic nature of observed interaction. Results The three-drug combination of PB, PHT and PGB (at the fixed-ratio of 1:1:1) exerted synergistic interaction (at P < 0.01) in the mouse model of tonic-clonic seizures. The combination of PB, PHT and PGB did not produce any side effects in experimental animals, when measuring long-term memory, muscular strength and motor coordination. The measurement of total brain concentrations of PB, PHT and PGB was conducted to confirm that none of the three AEDs significantly influenced total brain concentrations (pharmacokinetic profiles) of the other co-administered AEDs in mice. Conclusions The synergistic pharmacodynamic interaction for the combination of PB, PHT and PGB observed in this preclinical study can be translated into clinical settings and this favorable AED combination is worthy of being recommended to some patients with refractory epilepsy. [ABSTRACT FROM AUTHOR]

Background. Epilepsy is a serious neurological disease affecting about 1% of people worldwide (65 million). Seizures are controllable with antiepileptic drugs (AEDs) in about 70% of epilepsy patients, however, there remains about 30% of patients inadequately medicated with these AEDs, who need a satisfactory control of their seizure attacks. For these patients, one of the treatment options is administration of 2 or 3 AEDs in combination. Objectives. To determine the anticonvulsant effects of a combination of 3 selected AEDs (i.e., lacosamide - LCM, lamotrigine - LTG and phenobarbital - PB) at the fixed-ratio of 1:1:1 in a mouse maximal electroshock-induced (tonic-clonic) seizure model by using isobolographic analysis. Material and methods. Seizure activity was evoked in adult male albino Swiss mice by a current (sinewave, 25 mA, 500 V, 50 Hz, 0.2 s stimulus duration) delivered via auricular electrodes. Type I isobolographic analysis was used to detect interaction for the 3-drug combination. Results. With type I isobolographic analysis, the combination of LCM, LTG and PB (at the fixed-ratio of 1:1:1) exerted additive interaction in the mouse maximal electroshock-induced (tonic-clonic) seizure model. Conclusions. The combination of LCM with LTG and PB produced additive interaction in the mouse tonicclonic seizure model, despite various molecular mechanisms of action of the tested AEDs. [ABSTRACT FROM AUTHOR]

Receptor ligands in mixtures may produce effects that are greater than the effect predicted from their individual dose-response curves. The historical basis for predicting the mixture effect is based on Loewe’s concept and its mathematical formulation. This concept considers compounds with constant relative potencies (parallel dose-response curves) and leads to linear additive isoboles. These lines serve as references for distinguishing additive from nonadditive interactions according to the positions of the experimental data on or outside of the lines. In this paper, we applied a highly relevant two-state model for a description of the receptor-ligand interaction in the construction of the isobologram. In our model we consider partial agonists that have dose-response curve slopes differing from one. With this theoretical basis, we demonstrated that a combination of compounds with different efficacies leads to curved isoboles. This model should overwrite Tallarida’s flawed assumption about isobolographic analysis of partial agonists and enhance our understanding of how the partial agonists contribute to the overall mixture effect. [ABSTRACT FROM AUTHOR]

The antagonistic effects of water hardness have led to a decrease in herbicides' efficacy, especially in regions where the bedrock is mainly composed of lime and dolomite. In such conditions, tank mixtures of herbicides may improve herbicides' efficacy. Our study evaluated how different agents of water hardness, such as calcium chloride (CaCl 2), magnesium chloride (MgCl 2), iron(III) chloride (FeCl 3), and deionized water (without water hardness), affect the tank mixture of 2,4-D amine and sulfosulfuron in various ratios (0:100, 25:75, 50:50, 75:25, and 100:0). The results showed that the efficacy of both herbicides in decreasing the biomass and survival of flixweed [ Descurainia sophia (L.) Webb ex Prantl] decreased when exposed to water hardness agents. In the case of 2,4-D amine and sulfosulfuron, the presence of water hardness caused by iron(III) chloride resulted in a 45% and 52% increase in the ED 50 of biomass, respectively, compared to the condition without water hardness. The lowest ED 50 values for survival were obtained with deionized water (10.06 g a.i. ha−1), CaCl 2 (39.21 g a.i. ha−1), MgCl 2 (12.08 g a.i. ha−1), and FeCl 3 (36.60 g a.i. ha−1) in different ratios of 2,4-D amine and sulfosulfuron (50:50, 25:75, 50:50, and 25:75). Isobolographic analysis was used to confirm the mixture effects of these herbicides, which corroborated the dose-response findings of this study. Based on the concentration addition and Hewlett models, herbicides' efficacy was higher when used in tank mixing, especially in a 50:50 ratio, compared to using them alone. This study proved that tank mixing these herbicides prevents them from reducing their efficacy in controlling broadleaf weeds such as flixweed in areas with water hardness. In addition, using these herbicides in a tank mixture may be beneficial in delaying the development of herbicide resistance and the shift in weed flora that often occurs when a single herbicide is used. • Water hardness decreased the efficacy of both herbicides in decreasing the biomass and survival of flixweed. • Iron(III) chloride caused the most significant decrease in efficacy. • Tank mixing 75% sulfosulfuron and 25% 2,4-D amine with iron(III) chloride is the most effective in reducing survival by 50%. • The synergistic or antagonistic effects of herbicide mixtures can vary depending on the specific conditions and herbicides used. • The isobole diagrams show the different ratios of the two herbicides and their effects on flixweed biomass. [ABSTRACT FROM AUTHOR]