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Humans are exposed to complex mixtures of mycotoxins through diet. Despite the serious threat they pose, mycotoxin risk assessment often overlooks co-exposure. With the aim of filling this gap, the present study investigates the combined cytotoxicity of sterigmatocystin (STE), ochratoxin A (OTA) and patulin (PAT) in human tumour Neuroblastoma and healthy Mesenchymal Stem Cells three-dimensional (3D) spheroids. The range of concentrations tested (1.56-50 μM for STE, 0.78-25 μM for OTA and 0.15-5 μM for PAT) was selected considering the IC 50 values obtained in previous studies and the estimated dietary exposure of consumers. To ensure appropriate experimental conditions, assessments for single mycotoxins and their combinations were conducted simultaneously. The nature of the toxicological interactions among the mycotoxins was then defined using the isobologram analysis. Our results demonstrated increased cytotoxicity in mycotoxin mixtures compared to individual exposure, with abundance of synergistic interactions. These findings highlight that the co-occurrence of STE, OTA and PAT in food may increase their individual toxic effects and should not be underestimated. Moreover, the use of advanced culture models increased the reliability and physiological relevance of our results which can serve as a groundwork for formulating standardized regulatory approaches towards mycotoxin mixtures in food and feed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: This study examined the cutaneous analgesic effects of lidocaine co-injected with guanfacine and its comparison with dexmedetomidine., Methods: Cutaneous analgesic effects are quantified through the blocking effects of the cutaneous trunci muscle reflex against skin pinpricks in rats. The dose-response curves of guanfacine, dexmedetomidine, and lidocaine were constructed and drug-drug interactions were analyzed by the ED 50 isobologram., Results: Subcutaneous injections of guanfacine, dexmedetomidine, and lidocaine produced dose-dependently nociceptive/sensory blockade. On the ED 50 (50% effective dose) basis, the potency rankings of the drug are dexmedetomidine (0.09 [0.08-0.11] μmol/kg) > guanfacine (3.98 [2.96-5.34] μmol/kg) > lidocaine (25.40 [23.51-27.44] μmol/kg) ( p  < 0.01). On their equipotent doses (ED 25 , ED 50 , and ED 75 ), the duration of sensory blockade induced by guanfacine or dexmedetomidine was longer than lidocaine's ( p  < 0.01). Both guanfacine and dexmedetomidine showed synergistic effects with lidocaine., Conclusions: We showed that guanfacine elicits dose-dependent cutaneous analgesia when administered subcutaneously. Lidocaine is less potent than guanfacine or dexmedetomidine. Both guanfacine and dexmedetomidine enhance the potency and duration of lidocaine. Better synergistic responses we are getting with guanfacine plus lidocaine.

Introduction: Diabetic neuropathies are the most prevalent chronic complications of diabetes, characterized by pain and substantial morbidity. Although many drugs have been approved for the treatment of this type of pain, including gabapentin, tramadol (TMD), and classical opioids, it is common to report short-term results or potentially severe side effects. TMD, recommended as a second-line treatment can lead to unwanted side effects. Cannabidiol (CBD) has been gaining attention recently due to its therapeutic properties, including pain management. This study aimed to characterize the pharmacological interaction between CBD and TMD over the mechanical allodynia associated with experimental diabetes using isobolographic analysis. Materials and Methods: After diabetes induction by streptozotocin (STZ), diabetic rats were systemically treated with CBD or TMD alone or in combination (doses calculated based on linear regression of effective dose 40% [ED 40 ]) and had the mechanical threshold evaluated using the electronic Von Frey apparatus. Both experimental and theoretical additive ED 40 values ( Z mix and Z add , respectively) were determined for the combination of CBD plus TMD in this model. Results: Acute treatment with CBD (3 or 10 mg/kg) or TMD (2.5, 5, 10, or 20 mg/kg) alone or in combination (0.38+1.65 or 1.14+4.95 mg/kg) significantly improved the mechanical allodynia in STZ-diabetic rats. Isobolographic analysis revealed that experimental ED 40 of the combination ( Z mix ) was 1.9 mg/kg (95% confidence interval [CI]=1.2-2.9) and did not differ from the theoretical additive ED 40 2.0 mg/kg (95% CI=1.5-2.8; Z add ), suggesting an additive antinociceptive effect in this model. Conclusions: Using an isobolographic analysis, these results provide evidence of additive pharmacological interaction between CBD and TMD over the neuropathic pain associated with experimental diabetes induced by STZ.

In order to combat various infectious diseases, the utilization of host-directed therapies as an alternative to chemotherapy has gained a lot of attention in the recent past, since it bypasses the existing limitations of conventional therapies. The use of host epigenetic enzymes like histone lysine methyltransferases and lysine demethylases as potential drug targets has successfully been employed for controlling various inflammatory diseases like rheumatoid arthritis and acute leukemia. In our earlier study, we have already shown that the functional knockdown of KDM6B and ASH1L in the experimental model of visceral leishmaniasis has resulted in a significant reduction of organ parasite burden. Herein, we performed a high throughput virtual screening against KDM6B and ASH1L using > 53,000 compounds that were obtained from the Maybridge library and PubChem Database, followed by molecular docking to evaluate their docking score/Glide Gscore. Based on their docking scores, the selected inhibitors were later assessed for their in vitro anti-leishmanial efficacy. Out of all inhibitors designed against KDM6B and ASH1L, HTS09796, GSK-J4 and AS-99 particularly showed promising in vitro activity with IC 50  < 5 µM against both extracellular promastigote and intracellular amastigote forms of L. donovani. In vitro drug interaction studies of these inhibitors further demonstrated their synergistic interaction with amphotericin-B and miltefosine. However, GSK-J4 makes an exception by displaying an in different mode of interaction with miltefosine. Collectively, our in silico and in vitro studies acted as a platform to identify the applicability of these inhibitors targeted against KDM6B and ASH1L for anti-leishmanial therapy., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

In the environment, organisms are exposed to mixtures of different toxicants, which may interact in ways that are difficult to predict when only considering each component individually. Adapting and expanding tools from pharmacology, the toxicology field uses analytical, graphical, and computational methods to identify and quantify interactions in multi-component mixtures. The two general frameworks are concentration addition, where components have similar modes of action and their effects sum together, or independent action, where components have dissimilar modes of action and do not interact. Other interaction behaviors include synergism and antagonism, where the combined effects are more or less than the additive sum of individual effects. This review covers foundational theory, methods, an in-depth survey of original research from the past 20 years, current trends, and future directions. As humans and ecosystems are exposed to increasingly complex mixtures of environmental contaminants, analyzing mixtures interactions will continue to become a more critical aspect of toxicological research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Xylopic acid (XA), a kaurene diterpene from the dried fruits of Xylopia aethiopica, has anxiolytic- and antidepressant-like activity in mice and zebrafish. We aimed to assess the potential synergistic antidepressant-like effects of XA when combined with selected antidepressants in the mouse forced-swim test., Materials and Methods: The antidepressant-like effect of xylopic acid (XA) (10, 30, 100 mgkg -1 ), fluoxetine (Flx) (3, 10, 30 mgkg -1 ), sertraline (Sert) (3, 10, 30 mgkg -1 ), imipramine (Imi) (10, 30, 100 mgkg-1) and ketamine (Ket) (0.1, 0.3, 1.0 mgkg -1 ), was evaluated in forced swim test. The dose (ED 50 ) that achieved a 50% reduction in immobility time was determined from the respective log-dose response curves. XA and the selected antidepressants were co-administered in fixed-dose ratio combinations (1/2:1/2, 1/4:1/4, 1/8:1/8) of the ED 50 to identify the experimental ED 50 (ED 50mix ). The theoretical ED 50 (ED 50add ), of all combinations was determined using isobolograms and compared with the ED 50mix to identify the nature of the interaction. The effect of dose combinations on general locomotor activity was assessed in the open-field test., Results: The interaction index (γ) for the following XA combinations, XA/Flx, XA/Sert, XA/Imi and XA/Ket were 0.42, 0.41, 0.31 and 0.34. An independent sample t- test revealed that the experimental ED 50 (ED 50mix ) was significantly lower than the theoretical ED 50 (ED 50add ) in all combinations of XA, indicative of a synergistic antidepressant-like effect. However, combinations of XA with ketamine significantly reduced general locomotor activity at all dose combinations., Conclusion: The co-administration of xylopic acid and fluoxetine, imipramine, sertraline and ketamine produces a synergistic antidepressant-like effect in mice., Competing Interests: The authors declare no financial or non-financial competing interests., (© 2024 The Authors.)

Background: Cholangiocarcinoma (CCA) exhibits poor response to the present chemotherapeutic agents and frequently develops drug resistance. Finding novel anticancer drugs might enhance patient outcomes. Tiliacorinine, a bisbenzylisoquinoline alkaloid from the Thai medicinal plant Tiliacora triandra, effectively induced apoptosis of human CCA cell lines and inhibited tumor growth in mice. Here, we elucidate further the molecular mechanisms underlining the cytotoxicity of tiliacorinine and its implication in overcoming gemcitabine-resistance of CCA cells., Methods: Cytotoxicity of tiliacorinine against CCA cell lines was assessed using MTT assay. The molecular signaling was determined using Western blot analysis. Molecular docking simulations were applied to predict the binding affinity and orientation of tiliacorinine to the possible binding site(s) of the target proteins., Results: Tiliacorinine induced apoptotic cell death of CCA cells in a dose- and time-dependent manner. Tiliacorinine significantly suppressed the expression of anti-apoptotic proteins, Bcl-xL and XIAP; activated apoptotic machinery proteins, caspase-3, caspase-9, and PARP; and decreased the levels of pAkt and pSTAT3. EGF/EGFR activation model and molecular docking simulations revealed EGFR, Akt, and STAT3 as potent targets of tiliacorinine. Molecular docking simulations indicated a strong binding affinity of tiliacorinine to the ATP-binding pockets of EGFR, PI3K, Akt, JAK2, and SH2 domain of STAT3. Tiliacorinine could synergize with gemcitabine and restore the cytotoxicity of gemcitabine against gemcitabine-resistant CCA cells., Conclusion: Tiliacorinine effectively induced apoptosis via binding and blocking the actions of EGFR, Akt, and STAT3., General Significance: Tiliacorinine is a novel multi-kinase inhibitor and possibly a potent anti-cancer agent, in cancers with high activation of EGFR., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A. Suksamrarn reports financial support was provided by Ministry of Higher Education, Science, Research and Innovation, Thailand., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 LD 50 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Smith, Biancorosso, Camp, Hailu, Johansen, Morris and Carlson.)

The potentiation of pharmacological effects can be achieved through several strategies, such as the association of substances and delivery in nanostructured systems. In practice, potentiation can be measured by the law of mass action and joint evaluation of the combination index (CI) and dose-response curves. In this context, this study aimed to evaluate the anti-inflammatory effect of the association of β-caryophyllene and indomethacin in the free form and delivered in nanoemulsions using the in vitro model of LPS-stimulated murine macrophage. The results indicated potentiation of the anti-inflammatory effect of nanoemulsified substances compared to free substances, as well as synergistic action between the sesquiterpene and the selected NSAID. In comparison, the association of β-caryophyllene and indomethacin in the free form inhibited the production of nitric oxide by 50% at 48.60 µg/mL (CI = 0.21), while the nanoemulsified association of these substances resulted in an IC 50 of 1.45 µg/mL (CI = 0.14). In parallel, cytotoxicity assays on HaCaT and MRC-5 cell lines demonstrated the safety of IC 50 -equivalent concentrations of the anti-inflammatory action, and no irritating effects on the chorioallantoic membrane of embryonated eggs were observed (HET-CAM assay). The results suggest that β-caryophyllene may be an alternative to replace an inert oily core in nanoemulsion systems when anti-inflammatory effects are desirable., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: The antinociceptive pharmacological interaction between N-palmitoylethanolamide (PEA) and morphine (MOR), as well as gabapentin (GBP), was investigated to obtain synergistic antinociception at doses where side effects were minimal. In addition, the possible antinociceptive mechanism of PEA + MOR or PEA + GBP combinations was explored., Methods: Individual dose-response curves (DRCs) of PEA, MOR and GBP were evaluated in female mice in which intraplantar nociception was induced with 2% formalin. Isobolographic method was used to detect the pharmacological interaction in the combination of PEA + MOR or PEA + GBP., Key Findings: The ED50 was calculated from the DRC; the order of potency was MOR > PEA > GBP. The isobolographic analysis was obtained at a 1:1 ratio to determine the pharmacological interaction. The experimental values of flinching (PEA + MOR, Zexp = 2.72 ± 0.2 μg/paw and PEA + GBP Zexp = 2.77 ± 0.19 μg/paw) were significantly lower than those calculated theoretically (PEA + MOR Zadd = 7.78 ± 1.07 and PEA + GBP Zadd = 24.05 ± 1.91 μg/paw), resulting in synergistic antinociception. Pretreatment with GW6471 and naloxone demonstrated that peroxisome proliferator-activated receptor alpha (PPARα) and opioid receptors are involved in both interactions., Conclusions: These results suggest that MOR and GBP synergistically enhance PEA-induced antinociception through PPARα and opioid receptor mechanisms. Furthermore, the results suggest that combinations containing PEA with MOR or GBP could be of interest in aiding the treatment of inflammatory pain., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pain represents one of the leading causes of suffering and disability worldwide. Currently available drugs cannot treat all types of pain and may have adverse effects. Hence, the use of pharmacological combinations is an alternative treatment strategy. Therefore, this study aimed to evaluate the combination of resveratrol and ketorolac through isobolographic analysis. CD1 mice were used to study the antinociceptive effect of this combination using the formalin test and the study was divided into two phases. In the first phase, four individual doses of each drug were evaluated, totaling eight testing groups. From these data, the median effective doses (ED 50 ) of each drug were calculated. In the second phase, four testing groups were used to evaluate the combination of sub-doses of both drugs and obtain the experimental ED 50 . To evaluate gastric damage, five groups were employed, including indomethacin, vehicle, resveratrol, ketorolac, and combined resveratrol and ketorolac groups. Stomach samples from the mice were taken after 5 h of treatment, and the area of the ulcers was determined. Resveratrol plus ketorolac elicited a reduction in nociceptive behavior during both phases of the formalin test, and isobologram analysis revealed that the theoretical and experimental ED 50 values of resveratrol and ketorolac did not differ significantly, implying an additive interaction between the drugs. Additionally, the drug combination did not generate gastric ulcers, thus enhancing the desired effects without increasing the adverse effects. Consequently, these findings substantiate the efficacy of the resveratrol and ketorolac combination in the formalin test, thereby highlighting its potential as a viable alternative for alleviating pain.

Various combination treatments are used for microorganism control in food, medicine, and the environment. Especially in food, combination treatments have been studied using antimicrobial compounds in pasteurization and sterilization but comprehensive quantitative evaluation methods, have not yet been established to evaluate their effectiveness. This review introduces the author's recently published methods for evaluating the effects of combination treatments on the control of harmful microorganisms in food. Particularly important items are 1) the type of action of the control treatment and the mode of the combined method, 2) the choice of endpoint method and growth delay method for analytical evaluation, 3) the construction of extended isobolography that allows the application of conventional isobologram (IBo) for chemicals to various control methods, 4) the extended use of combined index (CI）, and 5) the introduction of synergistic parameter (SP) for quantitative evaluation of synergistic effects. In addition, I describe the characteristics of the action of antimicrobial compounds and disinfectants in their combined effects with heating, and insist on the advantages of using combined treatments and their evaluation methods in the food industry.

To evaluate the synergistic effects of physical and chemical antimicrobial treatments, whether for cidal or static effects, I proposed the extended isobologram (EIBo) analysis method, which is a modification of the isobologram (IBo) analysis commonly used to evaluate synergy between drugs. As the method types for this analysis, the growth delay (GD) assay reported previously by the author was introduced, in addition to the conventional endpoint (EP) assay. The evaluation analysis consists of five stages, analytical procedure establishment, antimicrobial activity assay, dose-effect analysis, IBo analysis, and synergy analysis. In EIBo analysis, the fractional antimicrobial dose (FAD) is introduced for the normalization of the antimicrobial activity of each treatment. For the synergy evaluation, the synergy parameter (SP) is defined as a measure of the degree of the synergistic effect of a combined treatment. This method makes it possible to quantitatively evaluate, predict, and compare various combination treatments as a hurdle technology.

Purpose: Neuropathic pain is sometimes difficult to manage because of limited efficacy of analgesic monotherapy even at high doses. Combination therapy may help address this issue, but there is little evidence for its effectiveness. Therefore, we evaluated the efficacy of combination therapy with pregabalin, an anchor drug for treating neuropathic pain, using the rat L5 spinal nerve ligation model., Methods: Experiments were performed on four-week-old L5 spinal nerve ligated male Sprague-Dawley rats. Mechanical allodynia was assessed using the von Frey test, where the 50% withdrawal threshold was evaluated for five drugs: pregabalin, duloxetine, venlafaxine, tramadol, and celecoxib. The single-drug experiment included 112 rats, where each drug was tested independently. Median effective doses (ED 50 s) were determined. Combinations of pregabalin with each of the other four drugs were tested (n=84). The 50% withdrawal threshold in the von Frey test was evaluated. The ED 50 of each combination was determined experimentally. Isobolographic analyses were conducted to assess the synergistic potential of the drug combinations, excluding pregabalin-celecoxib, since the ED 50 of celecoxib could not be determined., Results: In the single-drug experiment, all drugs except celecoxib resulted in a dose-dependent increase in the 50% withdrawal threshold 2 h after administration, with a maximum possible effect ranging from 4.4% to 79.6%. Similarly, all pregabalin combinations demonstrated a dose-dependent increase in the 50% withdrawal threshold, with pregabalin-tramadol showing the greatest increment. Isobolographic analysis of this combination revealed synergistic effects. Specifically, the combination index was γ=0.4 (<1). Combinations of pregabalin with duloxetine and venlafaxine demonstrated additive (γ=0.9) and antagonistic effects (γ=2.0), respectively., Conclusion: This study demonstrated that combination of pregabalin with tramadol has synergistic antiallodynic effects, while that with duloxetine has additive effects. Moreover, pregabalin combined with venlafaxine was potentially antagonistic. Pregabalin combined with tramadol may serve as a promising drug combination for the effective management of neuropathic pain., Competing Interests: KN and YK are employees of Viatris Pharmaceuticals Japan Inc. YS is an employee of KAC Co., Lt., Japan. TU declares no competing interests in this work., (© 2022 Nozawa et al.)

The bioactive compounds present in the edible products of the olive tree have been extensively studied and their favorable effects on various disease risk factors have been demonstrated. The aim of this study was to perform a comparative analysis of the anti-leishmanial effects of total phenolic fractions (TPFs) derived from extra virgin olive oil with different phenolic contents and diverse quantitative patterns. Moreover, the present study investigated their association with miltefosine, a standard anti-leishmanial drug, against both extracellular promastigotes and intracellular amastigotes of a viscerotropic and a dermotropic Leishmania strain. The chemical compositions of TPFs were determined by high performance liquid chromatography with diode array detection (HPLC-DAD). Analysis of parasite growth kinetics, reactive oxygen species production and apoptotic events were determined by microscopy and flow cytometry. Our results revealed that the presence of oleacein (OLEA) and oleocanthal (OLEO) secoiridoids enhances the anti-leishmanial effect of TPF. The association between TPFs and miltefosine was suggested as being additive in Leishmania infantum and Leishmania major promastigotes, and as antagonistic in intracellular amastigotes, as was evaluated with the modified isobologram method. The obtained data verified that TPFs are bioactive dietary extracts with a strong anti-leishmanial activity and highlighted that fractions that are richer in OLEA and OLEO phenolic compounds possess stronger inhibitory effects against parasites. This study may contribute to improving the therapeutic approaches against leishmaniasis.

The administration of combinations of drugs is a method widely used in the treatment of different pathologies as it can lead to an increase in the therapeutic effect and a reduction in the dose compared to the administration of single drugs. For these reasons, it is of interest to study combinations of drugs and to determine whether a specific combination has a synergistic, antagonistic or additive effect. Various mathematical models have been developed, which use different methods to evaluate the synergy of a combination of drugs. We have developed an open access and easy to use app that allows different models to be explored and the most fitting to be chosen for the specific experimental data: SiCoDEA (Single and Combined Drug Effect Analysis). Despite the existence of other tools for drug combination analysis, SiCoDEA remains the most complete and flexible since it offers options such as outlier removal or the ability to choose between different models for analysis. SiCoDEA is an easy to use tool for analyzing drug combination data and to have a view of the various steps and offer different results based on the model chosen.

Glioblastoma, a type of brain cancer, is one of the most aggressive and lethal types of malignancy. The present study shows that JCI-20679, an originally synthesized mitochondrial complex I inhibitor, enhances the anti-proliferative effects of suboptimal concentrations of the clinically used chemotherapeutic drug temozolomide in glioblastoma cells. Analysis of the effects of temozolomide combined with JCI-20679 using isobologram and combination index methods demonstrated that the combination had synergistic effects in murine and human glioblastoma cells. We found that JCI-20679 inhibited the temozolomide-mediated induction of autophagy that facilitates cellular survival. The autophagy induced by temozolomide increased ATP production, which confers temozolomide resistance in glioblastoma cells. JCI-20679 blocked temozolomide-mediated increases in ATP levels and increased the AMP/ATP ratio. Furthermore, JCI-20679 enhanced the therapeutic effects of temozolomide in an orthotopic transplantation model of glioblastoma. These results indicate that JCI-20679 may be promising as a novel agent for enhancing the efficacy of temozolomide against glioblastoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Emerging artemisinin resistance in Plasmodium falciparum malaria has the potential to become a global public health crisis. In Southeast Asia, this phenomenon clinically manifests in the form of delayed parasite clearance following artemisinin treatment. Reduced artemisinin susceptibility is limited to the early ring stage window, which is sufficient to allow parasites to survive the short half-life of artemisinin exposure. A screen of known clinically-implemented antimalarial drugs was performed to identify a drug capable of enhancing the killing activity of artemisinins during this critical resistance window. As a result, lumefantrine was found to increase the killing activity of artemisinin against an artemisinin-resistant clinical isolate harboring the C580Y kelch13 mutation. Isobologram analysis revealed synergism during the early ring stage resistance window, when lumefantrine was combined with artemether, an artemisinin derivative clinically partnered with lumefantrine. These findings suggest that lumefantrine should be clinically explored as a partner drug in artemisinin-based combination therapies to control emerging artemisinin resistance., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Ursolic acid (UA) is a potent plant-based hepatoprotective agent having poor bioavailability, which hampers its therapeutic efficacy. The present study tries to overcome this limitation by combining it with piperine (PIP), a proven bioenhancer and hepatoprotective agent., Methods: The type of interaction (synergism, addition, or antagonism) resulting between UA and PIP was analyzed and quantified by isobologram and combination index analysis. The hepatoprotective activity of UA and PIP was evaluated by measuring the level of hepatic marker enzymes. Pharmacokinetic analysis was carried out to ascertain the improvement of bioavailability., Results: The combinations significantly decrease the enzyme levels, which indicate better hepatoprotective activity compared to single drugs. The relative oral bioavailability of UA was increased about tenfold (from AUC 0- t =12.78 ± 2.59 µg/h/ml to 125.15 ± 1.84 µg/h/ml) along with the improvement of plasma concentration and elimination half-life., Conclusions: The findings indicated that the combination of PIP and UA is an effective strategy in enhancing the bioavailability and hepatoprotective potential of UA.KEY MESSAGESUrsolic acid in a combination with piperine provides a synergistic hepatoprotective effect in carbon tetrachloride induced liver damage in rats.Piperine improves the pharmacokinetic properties of ursolic acid when given in combination.Piperine improves the relative oral bioavailability of ursolic acid by tenfold when combined together.

Aims: 5-Fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor, has been used as the first-line chemotherapeutic drug for cholangiocarcinoma (CCA). The side effects and drug resistance have developed the limits of the clinical application of 5-FU in CCA treatment. Upregulation of Forkhead box M1 (FOXM1) and TS were shown to play a significant role in 5-FU resistance. In this study, the effect of Siomycin A (SioA), a FOXM1 inhibitor, on enhancing 5-FU cytotoxicity and reversing 5-FU resistance in CCA cell lines were demonstrated., Main Methods: Human CCA cell lines, KKU-100 and KKU-213A were used. Cell viability was determined using MTT assay. Expression of FOXM1 and TS proteins were determined using Western blotting. FOXM1 mRNA expression was quantitated using real-time PCR. The combination and dose reduction (DRI) were analyzed according to the Chou and Talalay method., Key Finding: Single drug treatment of 5-FU and SioA effectively inhibited CCA cell growth in dose and time dependent fashions. The two CCA cell lines had different responses to 5-FU but exhibited similar sensitivity to SioA. FOXM1 and TS expression were increased in the 5-FU treated cells but were suppressed in the SioA treated cells. A direct binding of SioA, to TS and 5,10-methylene-tetrahydrofolate as an inactive ternary complex was simulated. The combined treatment of 5-FU with SioA showed a synergistic effect with a high DRI and restored 5-FU sensitivity in the 5-FU resistant cells., Significance: Targeting FOXM1 using SioA in combination with 5-FU might be a strategy to overcome the 5-FU resistance in CCA., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We evaluated interactions between dopamine D2 receptor and nitric oxide (NO) actions on the regulation of anxiety and memory in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease (PD). A unilateral guide cannula was stereotaxically implanted over the right striatum. Elevated plus-maze test (EPM) test-retest protocol was employed to evaluate anxiety and memory in mice. The results revealed that injection of L-NAME (9 mg/kg) induced anxiolytic and amnesic effects, while L-arginine (9 mg/kg) produced anxiogenic and memory-improvement effects in the 6-OHDA mouse model of PD. Administration of sulpiride (20 mg/kg) induced anxiogenic and memory-improvement effects, whereas quinpirole (20 mg/kg) caused anxiolytic and amnesic effects in PD mice. Co-injection of sulpiride (5, 10, and 20 mg/kg) plus L-NAME (3 mg/kg) induced anxiolytic and amnesic effects. Co-injection of sulpiride (20 mg/kg) plus L-arginine (3 mg/kg) induced anxiogenic and memory-improvement effects. Co-administrations of quinpirole (20 mg/kg) and L-NAME (3 mg/kg) induced anxiolytic effect, but co-administration of quinpirole (20 mg/kg) plus L-arginine (3 mg/kg) caused anxiogenic and memory-improvement effects. The isobologram analysis revealed that there is a synergistic effect between sulpiride and L-arginine as well as quinpirole and L-NAME upon induction of anxiogenic and anxiolytic effects, respectively in PD mice. Our results suggested: (1) NO and dopamine D2 receptor mechanisms affect anxiety and memory in PD mice; (2) L-NAME reversed anxiogenic and memory-improvement effect induced by sulpiride; (3) Anxiolytic and amnesic effects induced by quinpirole reversed by L-arginine; (4) There is a synergistic effect between dopamine D2 receptor and NO systems on the modulation of anxiety and memory., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Several modern drugs, which are derived from traditional herbal medicine are used in contemporary pharmacotherapy. Currently, the study of drug-plant interactions in pain has increased in recent years, looking for greater efficacy of the drug and reduce side effects. The antinociception induced by intragastric co-administration of the combination of pomegranate peel extract (PoPEx) and acetylsalicylic acid (ASA) was assessed using the isobolographic analysis in formalin test (nociceptive and inflammatory pain). The effective dose that produced 30% of antinociception (ED 30 ) was calculated for both drugs from the logarithmic dose-response curves, subsequently generating a curve with the combination on fixed proportions (1:1) of PoPEx and ASA. Through isobolographic analysis, this experimental ED 30 was compared with the calculated theoretical additive ED 30 . The result was a synergistic interaction, the experimental ED 30 was significantly smaller ( p < 0.05) than the theoretical ED 30 . The antinociceptive mechanism of the PoPEx-ASA combination involves the l-Arginine/NO/cGMP pathway, antioxidant capacity, and high content of total phenols. These findings suggest that an interaction between PoPEx and ASA could be a novel treatment for inflammatory and nociceptive pain, also diminish the secondary reactions of ASA.

Noscapine, an opium alkaloid, was discovered to bind tubulin, arrest dividing cells at mitosis, and selectively induce apoptosis to cancer cells. N-3-Br-Benzyl-Noscapine (Br-Bn-Nos), one of the derivatives of noscapine, was demonstrated to have improved anticancer potential compared with noscapine. We approached to evaluate the single and combined effect of Br-Bn-Nos and docetaxel (DOX) based on molecular modeling and cellular study. The individual predicted free energy of binding (∆G bind,pred ) for Br-Bn-Nos and DOX with tubulin was found to be -28.89 and -36.07 kcal/mol based on molecular mechanics generalized Born solvation area (MM-GBSA) as well as -26.21 and -34.65 kcal/mol based on molecular mechanics Poisson Boltzmann solvation area (MM-PBSA), respectively. However, the ∆G bind,pred of Br-Bn-Nos was significantly reduced (-33.02 and -30.24 kcal/mol using MM-GBSA and MM-PBSA) in the presence of DOX on its binding pocket. Parenthetically, the ∆G bind,pred of DOX was significantly reduced (-37.17 and -32.80 kcal/mol using MM-GBSA and MM-PBSA) in the presence of Br-Bn-Nos on its binding pocket. The reduced ∆G bind,pred in the presence of Br-Bn-Nos and DOX together indicated a combination effect of both the ligands. The combined interaction of both the agents onto tubulin dimmer was also determined experimentally using purified tubulin, in which a combination regimen of Br-Bn-Nos and DOX reduced the fluorescence intensity of tubulin to a higher value (68%) compared with the single regimen. Further, isobologram analysis revealed the synergistic effect of Br-Bn-Nos and DOX in antiproliferative activity using MCF-7 cell line at 48 hr (sum FIC = 0.49) and at 72 hr (sum FIC = 0.62). The combination dose regimen of Br-Bn-Nos and DOX blocks the cell cycle progression at the G2/M phase and induces apoptosis to cancer cells more effectively compared with the single regimen. Taken together, our study provides compelling evidence that the anticancer potential of noscapine derivatives may be substantially improved when it is used in a combined application with DOX for breast cancer., (© 2021 John Wiley & Sons A/S.)

Aim/background: The combination of drugs with different mechanisms of action could be more effective due to an enhanced synergistic activity. The pharmacological interactions between rutin and paracetamol, and rutin and non-steroidal anti-inflammatory drugs (NSAIDs), such as naproxen, ketorolac, and diclofenac were investigated using in vitro and in vivo assays., Methods: Individual and drug combination response curves of rutin-NSAIDs, and rutin-paracetamol (each at 0.001-0.1 mmol for in vitro studies and ranging from 1.25-100 mg/kg p.o. for in vivo studies) were evaluated in RAW 264.7 macrophages [inhibition of nitric oxide (NO) using the Griess reagent] and Balb/c mice (acetic acid-induced writhing test). An isobolographic analysis was used to assess the type of interaction between rutin and NSAIDs and rutin-paracetamol in a proportion of 1:1., Results: Rutin alone and in combination with paracetamol and NSAIDs decreased NO production and the number of writhings in a concentration/dose-dependent manner. The isobolograms showed that all in vitro combinations of rutin-NSAIDs and rutin-paracetamol exerted synergistic effects. The combination rutin-diclofenac showed an interaction index of 0.17. In the in vivo assay, the combinations of rutin-diclofenac (interaction index = 0.195) and rutin-ketorolac (interaction index = 0.408) displayed synergistic effects, and the combination rutin-paracetamol exhibited additive effects., Conclusion: Rutin produces synergistic effects with paracetamol and NSAIDs in in vitro and in vivo assays., (Copyright © 2021. Published by Elsevier Inc.)

Interactions between nanoformulations delivering two or more drugs-or a drug and some other therapeutic modality-are often described as synergistic. In fact, synergy is a specific term of art, with important therapeutic implications. The term is often misused in the nanoscience literature. Here we discuss what synergy is and an approach to demonstrating it rigorously.

Plant bioactive compounds have antimicrobial and antioxidant activities that allow them to be used as a substitute for synthetic chemical additives in both food and food packaging. To improve its sensory and bactericidal effects, its use in the form of effective combinations has emerged as an interesting possibility in the food industry. In this study, the antimicrobial activities of essential oils (EOs) of cinnamon bark, cinnamon leaves, and clove and the pure compounds vanillin, eugenol, and cinnamaldehyde were investigated individually and in combination against Listeria monocytogenes and Escherichia coli O157:H7. The possible interactions of combinations of pure compounds and EOs were performed by the two-dimensional checkerboard assay and isobologram methods. Vanillin exhibited the lowest antimicrobial activity (MIC of 3002 ppm against L. monocytogenes and 2795 ppm against E. coli O157:H7), while clove and cinnamon bark EOs exhibited the highest antimicrobial activity (402-404 against L. monocytogenes and 778-721 against E. coli O157:H7). For L. monocytogenes , pure compound eugenol, the main component of cinnamon leaves and clove, showed lower antimicrobial activity than EOs, which was attributed to the influence of the minor components of the EOs. The same was observed with cinnamaldehyde, the main component of cinnamon bark EO. The combinations of vanillin/clove EO and vanillin/cinnamon bark EO showed the most synergistic antimicrobial effect. The combination of the EOs of cinnamon bark/clove and cinnamon bark/cinnamon leaves showed additive effect against L. monocytogenes but indifferent effect against E. coli O157:H7. For L. monocytogenes, the best inhibitory effects were achieved by cinnamon bark EO (85 ppm)/vanillin (910 ppm) and clove EO (121 ppm)/vanillin (691 ppm) combinations. For E. coli , the inhibitory effects of clove EO (104 ppm)/vanillin (1006 ppm) and cinnamon leaves EO (118 ppm)/vanillin (979 ppm) combinations were noteworthy. Some of the tested combinations increased the antimicrobial effect and would allow the effective doses to be reduced, thereby offering possible new applications for food and active food packaging.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are very commonly used, but their adverse effects warrant investigating new therapeutic alternatives. Polyalthic acid, a labdane-type diterpenoid, is known to produce gastroprotection, tracheal smooth muscle relaxation, and antitumoral, antiparasitic and antibacterial activity. This study aimed to evaluate the antinociceptive, antiallodynic, antihyperalgesic and anti-inflammatory effect of polyalthic acid on rats. Moreover, the effectiveness of treating hyperalgesia with a combination of polyalthic acid and naproxen was analyzed, as well as the type of drug-drug interaction involved. Nociception was examined by injecting 1% formalin into the right hind paw and thermal hyperalgesia and inflammation by injecting a 1% carrageenan solution into the left hind paw of rats. Allodynia was assessed on an L5/L6 spinal nerve ligation model. Polyalthic acid generated significant antinociceptive (56-320 mg/kg), antiallodynic (100-562 mg/kg), and antihyperalgesic and anti-inflammatory (10-178 mg/kg) effects. Antinociception mechanisms were explored by pretreating the rats with naltrexone, ODQ and methiothepin, finding the effect blocked by the former two compounds, which indicates the participation of opioid receptors and guanylate cyclase. An isobolographic analysis suggests synergism between polyalthic acid and naproxen in the combined treatment of hyperalgesia.

The present study aimed to evaluate the antileishmanial effect, the mechanisms of action and the association with miltefosine of Vernonia brasiliana essential oil against Leishmania infantum promastigotes. This essential oil was obtained by hydrodistillation and its chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 cells were evaluated by MTT colorimetric assay. Ultrastructural alterations were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential, in the production of reactive oxygen species, and analysis of apoptotic events were determined by flow cytometry. The association between the essential oil and miltefosine was evaluated using the modified isobologram method. The most abundant component of the essential oil was β-caryophyllene (21.47 %). Anti-Leishmania assays indicated an IC 50 of 39.01 ± 1.080 μg/mL for promastigote forms after 72 h of treatment. The cytotoxic concentration for DH82 cells was 63.13 ± 1.211 μg/mL after 24 h of treatment. The effect against L. infantum was proven through the ultrastructural changes caused by the oil, such as kinetoplast and mitochondrial swelling, vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of organelles. It was observed that the oil leads to a decrease in the mitochondrial membrane potential (35.10 %, p = 0.0031), increased reactive oxygen species production, and cell death by late apoptosis (17.60 %, p = 0.020). The combination of the essential oil and miltefosine exhibited an antagonistic effect. This study evidences the antileishmanial action of V. brasiliana essential oil against L. infantum promastigotes., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Patents diagnosed with acute promyelocytic leukemia were treated with Vesanoid ® [all-trans retinoic acid (ATRA)]. ATRA promotes the maturation and differentiation of leukemia cells and is therefore capable of reducing the symptoms of leukemia by preventing aggregation of myeloid cells. However, the clinical applications of ATRA are limited by its side effects, including acute retinoid resistance, hypertriglyceridemia, mucocutaneous dryness, nausea, brief recovery time relapse and drug resistance. Therefore, combinations of ATRA and other anticancer drugs are being investigated to overcome these limitations. In our previous study it was shown that in leukemia cells, (-)-epigallocatechin-3-gallate (EGCG) reduced cell proliferation and induced apoptotic cell death. In the present study, an in vitro evaluation of the effects of the combination of EGCG and ATRA on FLT3 -mutated cell lines was performed using the isobologram method. The results showed that there was an additive effect in leukemic cells when treated with a combination of ATRA and EGCG. Thus, it was concluded that the cytotoxic effects of EGCG were improved by ATRA., (Copyright © 2020, Spandidos Publications.)

Juglone and thymoquinone are cytotoxic to pancreatic cancer cells. The aim of this study was to investigate, using an analysis of isobolograms, the type and degree of interactions between juglone and thymoquinone on MIA PaCa-2 pancreatic cancer cells. Cell viability was evaluated using the MTT assay. Cell death was determined by flow cytometry. The IC 50 value for juglone and TQ in combination was found to be 24.75 μM, which was higher than juglone or TQ alone. Juglone alone killed Mia Paca-2 cells by ferroptosis. At concentrations where 10, 20 or 50% of cells were affected, there existed a moderate antagonistic relationship between juglone and TQ as indicated by the combination index (CI) value determined by the Compusyn software. At concentrations that affected 75% and 90% of cells, there were nearly an additive effect with CI value of 1.09249 and 0.92391, respectively. Moderate synergism was only seen at concentration where 95% of cells were affected, and the corresponding concentration of juglone and TQ at that combination was 40.90 μM and 511.19 μM, respectively., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Bencha-loga-wichian (BLW), a Thai traditional antipyretic formulation, has been reported to have promising antiplasmodial activity, and it was previously revealed that tiliacorinine and yanangcorinine, isolated from Tiliacora triandra, were the active compounds. However, the mechanisms of action of BLW have not been investigated. In addition, these active compounds are bisbenzylisoquinoline alkaloids, many compounds of which have been reported to potentiate the efficacy of chloroquine., Aims of the Study: To investigate the antiplasmodial mechanisms of action of BLW and evaluate the effects of chloroquine combined with tiliacorinine or yanangcorinine., Materials and Methods: Chloroquine-resistant Plasmodium falciparum (PfW2) strains at the ring, trophozoite, and schizont stages were exposed to the extracts or compounds for 2, 4, 6, 8, 10, 12, 24 or 48 h. The percentages of parasitemia were determined by flow cytometry, and their morphologies were examined by Giemsa-stained smear to evaluate the speed of action and stage specificity. For the drug combination assay, a modified fixed-ratio isobologram method was used., Results: The antiplasmodial activity of BLW possessed a slow onset of action and was the most effective against ring-stage parasites. After 48 h of extracts or compounds exposure, most of the treated parasites, at all stages, turned to the pyknotic form and could not recover even after extracts or compounds removal. The results suggested that these extracts and compounds could kill the parasites or possess parasiticidal effects. In addition, the combination of chloroquine with tiliacorinine or yanangcorinine demonstrated a synergistic effect, indicating that these compounds could potentiate chloroquine efficacy against chloroquine-resistant parasites., Conclusion: The antiplasmodial mechanisms of action of BLW appeared to differ from that of chloroquine and other current antimalarial drugs. In addition, tiliacorinine and yanangcorinine, the active compounds of BLW, could potentiate the efficacy of chloroquine. Accordingly, BLW was shown to be a good candidate for development as a new antimalarial and useful for drug combination therapy., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chlorpyrifos (CPF) and aflatoxin B 1 (AFB 1 ) are each known to adversely affect hepatic tissue individually, but their combined hepatic effects have never been previously investigated. HepG2 cell viability, oxidative status, and genetic impairment were examined after exposing HepG2 cells to: (1) CPF alone, (2) AFB 1 alone, and (3) CPF and AFB 1 combined (20:1). CPF exposure decreased cell viability, reduced glutathione (GSH) content, and superoxide dismutase (SOD) activity but increased both glutathione peroxidase (GPx) and paraoxonase 1 activity. AFB 1 exposure decreased cell viability and GSH content but increased reactive oxygen species (ROS) production. CPF and AFB 1 combined exposure decreased GSH content ( p < 0.05) further over individual CPF and AFB 1 exposures. Induction of micronucleus formation was detected in AFB 1 -treated cells but undetected in both CPF and combination-treated cells. In conclusion, cytotoxic effects caused by combined exposure were antagonistic, as shown by a combination index value of 1.67. Although no change in ROS production was observed in CPF groups, the overall results confirmed the occurrence of oxidative stress through the alterations of GSH content, GPx, and SOD activity. Only intracellular GSH was evidently changed upon exposure to CPF and AFB 1 combined. Thus, this study suggested cellular GSH as a potential indicator for detecting the combined effects of CPF and AFB 1 in HepG2 cells, the detection of which could be adapted to estimate the potential toxicity of additional multiple toxicant exposures.

Opioid analgesics represent a critical treatment for chronic pain in the analgesic ladder of the World Health Organization. However, their use can result in a number of unwanted side-effects including incomplete efficacy, constipation, physical dependence, and overdose liability. Cannabinoids enhance the pain-relieving effects of opioids in preclinical studies and dampen unwanted side-effects resulting from excessive opioid intake. We recently reported that a CB 1 positive allosteric modulator (PAM) exhibits antinociceptive efficacy in models of pathological pain and lacks the adverse side effects of direct CB 1 receptor activation. In the present study, we evaluated whether a CB 1 PAM would enhance morphine's therapeutic efficacy in an animal model of chemotherapy-induced neuropathic pain and characterized its impact on unwanted side-effects associated with chronic opioid administration. In paclitaxel-treated mice, both the CB 1 PAM GAT211 and the opioid analgesic morphine reduced paclitaxel-induced behavioral hypersensitivities to mechanical and cold stimulation in a dose-dependent manner. Isobolographic analysis revealed that combinations of GAT211 and morphine resulted in anti-allodynic synergism. In paclitaxel-treated mice, a sub-threshold dose of GAT211 prevented the development of tolerance to the anti-allodynic effects of morphine over 20 days of once daily dosing. However, GAT211 did not reliably alter somatic withdrawal signs (i.e., jumps, paw tremors) in morphine-dependent neuropathic mice challenged with naloxone. In otherwise naïve mice, GAT211 also prolonged antinociceptive efficacy of morphine in the tail-flick test and reduced the overall right-ward shift in the ED 50 for morphine to produce antinociception in the tail-flick test, consistent with attenuation of morphine tolerance. Pretreatment with GAT211 did not alter somatic signs of μ opioid receptor dependence in mice rendered dependent upon morphine via subcutaneous implantation of a morphine pellet. Moreover, GAT211 did not reliably alter μ-opioid receptor-mediated reward as measured by conditioned place preference to morphine. Our results suggest that a CB 1 PAM may be beneficial in enhancing and prolonging the therapeutic properties of opioids while potentially sparing unwanted side-effects (e.g., tolerance) that occur with repeated opioid treatment., (Copyright © 2020 Slivicki, Iyer, Mali, Garai, Thakur, Crystal and Hohmann.)

Opioid analgesics and the α 2 -adrenergic receptor (α 2 AR) agonists are found to produce synergistic antinociception when administered in combination. In this study interactions between butorphanol and dexmedetomidine were investigated in the thermal pain and autonomous locomotor activity. Butorphanol and dexmedetomidine were administered subcutaneously alone and in combination in a fixed-dose ratio (3:1) to assess the antinociceptive and sedative responses. Butorphanol produced antinociception in the hot-plate test via three major opioid receptor subtypes, i.e. MORs, KORs and DORs, while in the tail-immersion test the antinociception was produced by MORs and KORs, whereas dexmedetomidine exhibited antinociception by α 2 ARs in both tests. They exhibited dose- and time-dependent antinociception and inhibition of locomotor activity when administered alone, while their combination displayed enhanced therapeutic effects. Isobolographic analysis revealed that combined butorphanol and dexmedetomidine produced synergistic interactions in the hot-plate, tail-immersion and locomotor activity tests. Furthermore, the analgesic synergy was also approved to be modulated by MORs, KORs, DORs and α 2 ARs. Hence we concluded from this study that combined butorphanol and dexmedetomidine produced synergistic antinociception that may be helpful in facilitating clinical management of acute nociceptive pain., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020 Elsevier B.V. All rights reserved.)

Antimalarial drug resistance in the Plasmodium falciparum parasite poses a constant challenge for drug development. To mitigate this risk, new antimalarial medicines should be developed as fixed-dose combinations. Assessing the pharmacodynamic interactions of potential antimalarial drug combination partners during early phases of development is essential in developing the targeted parasitological and clinical profile of the final drug product. Here, we have studied the combination of M5717, a P. falciparum translation elongation factor 2 inhibitor, and pyronaridine, an inhibitor of hemozoin formation. Our test cascade consisted of in vitro isobolograms as well as in vivo studies in the P. falciparum severe combined immunodeficient (SCID) mouse model. We also analyzed pharmacokinetic and pharmacodynamic parameters, including genomic sequencing of recrudescent parasites. We observed no pharmacokinetic interactions with the combination of M5717 and pyronaridine. M5717 did not negatively impact the rate of kill of the faster-acting pyronaridine, and the latter was able to suppress the selection of M5717-resistant mutants, as well as significantly delay the recrudescence of parasites both with suboptimal and optimal dosing regimens., (Copyright © 2020 Rottmann et al.)

In the past decades, many studies have examined the nature of the interaction between mycotoxins in biological models classifying interaction effects as antagonisms, additive effects, or synergisms based on a comparison of the observed effect with the expected effect of combination. Among several described mathematical models, the arithmetic definition of additivity and factorial analysis of variance were the most commonly used in mycotoxicology. These models are incorrectly based on the assumption that mycotoxin dose-effect curves are linear. More appropriate mathematical models for assessing mycotoxin interactions include Bliss independence, Loewe's additivity law, combination index, and isobologram analysis, Chou-Talalays median-effect approach, response surface, code for the identification of synergism numerically efficient (CISNE) and MixLow method. However, it seems that neither model is ideal. This review discusses the advantages and disadvantages of these mathematical models.

High-grade chloroquine (CQ) resistance has been reported in malaria endemic geographical regions such as Papua New Guinea, northern Papua, and eastern and western provinces of Indonesia, along with low-level resistance in Vietnam, South Korea, Turkey, Burma, South America, and Madagascar. Studies on CQ drug resistance have revealed the association of P. falciparum chloroquine resistance transporter protein. Thus, we are in dire need of alternate chemotherapeutic agents which in combination with artemisinin (or its analogues) are efficacious against chloroquine-resistant strains. Such combinations may thwart the emergence of drug resistant strains, along with reducing the malaria burden. Hypothesizing that newer 4-aminoquinolines, earlier reported by our group, could be part of a combination therapy to efficiently treat malaria, we sought to evaluate these compounds, viz. 1m, 1o, 2c, and 2j against the erythrocytic stages of Plasmodium falciparum, strain 3D7 (chloroquine-sensitive) and strain Dd2 (chloroquine-resistant), in combination with dihydroartemisinin (DHA). Results revealed substantially synergistic interactions between the combination partners, which could be further established by their potential to inhibit hemozoin formation with increased efficiency when combined, as compared to the compounds assessed individually. Furthermore, aminoquinolines and DHA show distinct stage-specific profiles. Our results stand in strong support of the potential of these aminoquinoline derivatives to serve as partner drugs in antimalarial combinations to treat multiple-drug-resistant Plasmodium strains., (Copyright © 2019. Published by Elsevier B.V.)

Background: The suppression of α-glucosidase activity to retard glucose absorption is an important therapy for type-2 diabetes. Corosolic acid (CRA) is a potential antidiabetic component in many plant-based foods and herbs. In this study, the interplay mechanism between α-glucosidase and corosolic acid was investigated by several methods, including three-dimensional fluorescence spectra, circular dichroism spectra, and molecular simulation., Results: Corosolic acid significantly inhibited α-glucosidase reversibly in an uncompetitive manner and its IC 50 value was 1.35 × 10 -5 mol L -1 . A combination of CRA with myricetin exerted a weak synergy against α-glucosidase. The intrinsic fluorescence of α-glucosidase was quenched via a static quenching course and the binding constant was 3.47 × 10 3  L mol -1 at 298 K. The binding of CRA to α-glucosidase was mainly driven by hydrophobic forces and resulted in a partial extension of the protein polypeptide chain with a loss of α-helix content. The molecular simulation illustrated that CRA bound to the entrance part of the active center of α-glucosidase and interacted with the amino acid residues Ser157, Arg442, Phe303, Arg315, Tyr158, and Gln353, which could hinder the release of substrate and catalytic reaction product, eventually suppressing the catalytic activity of α-glucosidase., Conclusions: These results may suggest new insights into corosolic acid from food sources as a potential α-glucosidase inhibitor that could better control diabetes. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Background: Dextrorphan with long-acting local anesthetic effects did not cause system toxicity as fast as bupivacaine, while catecholamines (i.e., epinephrine) with the vasoconstrictive characteristics enhanced the effects of local anesthetic drugs. The objective of the experiment was to examine the synergistic effect of local dopamine (a catecholamine) injection on cutaneous antinociception of dextrorphan., Methods: The panniculus reflex in response to skin stimulation with a needle was used as the primary endpoint when dextrorphan (1.50, 2.61, 5.46, 10.20 and 20.40 μmol) alone, dopamine (16.20, 32.40, 51.60, 60.00 and 81.60 μmol) alone, or dopamine + dextrorphan (a ratio of ED 50 vs. ED 50 ) was injected subcutaneously on the rat's back. We used an isobolographic modelling approach to determine whether a synergistic effect would be observed., Results: We showed that dextrorphan, dopamine, or the mixture of dopamine and dextrorphan produced dose-related skin antinociception. The potency (ED 50 , 50% effective dose) for cutaneous antinociception was dextrorphan [6.02 (5.93-6.14) μmol] greater than dopamine [48.91 (48.80-49.06) μmol] (p < 0.01). The duration of nociceptive inhibition induced by dopamine was longer than that induced by dextrorphan (p <  0.01) based on their equipotent doses (ED 25 , ED 50 , and ED 75 ). Enhancement and prolongation of skin antinociception occurred after co-administration of dopamine with dextrorphan., Conclusions: When compared to dopamine, dextrorphan was more potent and had a shorter duration of skin nociceptive block. Dopamine produced a synergistic effect on dextrorphan-mediated antinociception, and prolonged dextrorphan's antinociceptive duration., (Copyright © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

The aim of this study is to investigate the antimicrobial synergistic effect against Campylobacter jejuni , a leading foodborne pathogen that causes human gastroenteritis, by cinnamon oil, encapsulated curcumin, and zinc oxide nanoparticles (ZnO NPs). We compared three approaches to study the antimicrobial interactions, including the time-killing method, the fractional inhibitory concentration index (FICI) method, and a mathematical concentration-effect model. Isobologram analysis was performed to evaluate the synergy in different combinations, and a median-effect equation was applied to identify the combinations of synergistic effects at median, bacteriostatic, and bactericidal reduction levels. The time-killing method overestimated the synergistic interaction between antimicrobials, while the FICI method failed to detect an existing synergistic phenomenon. This lack of accuracy and sensitivity was mainly due to combining antimicrobials without a deep understanding of their concentration-effect relationships. Our results showed that each antimicrobial had a unique concentration-effect curve. Specifically, encapsulated curcumin showed a sharp sigmoidal curve unlike cinnamon oil and ZnO NPs. A mathematical model was applied to study the interaction between antimicrobials with a different shape of concentration-effect curve. We observed an additive effect of cinnamon oil/ZnO NPs and synergistic interactions of other binary combinations (cinnamon oil/encapsulated curcumin and ZnO NPs/encapsulated curcumin). The tertiary combination of cinnamon oil/ZnO NPs/encapsulated curcumin at IC 25 (additive line <1-log CFU/mL) presented the greatest synergistic effect by reducing the bacterial population over 8-log CFU/mL. This mathematical model provided an alternative strategy to develop a new antimicrobial strategy.

Opioid and neurotensin (NT) receptors are expressed in both central and peripheral nervous systems where they modulate nociceptive responses. Nowadays, opioid analgesics like morphine remain the most prescribed drugs for the treatment of moderate to severe pain. However, despite their daily used, opioids can produce life-threatening side effects, such as constipation or respiratory depression. Besides, NT analogs exert strong opioid-independent analgesia. Here, we thus hypothesized that the combined use of opioid and NT agonists would require lower doses to produce significant analgesic effects, hence decreasing opioid-induced adverse effects. We used isobologram analyses to determine if the combination of a NT brain-penetrant analog, An2-NT(8-13) with morphine results in an inhibitory, synergistic or additive analgesic response. We found that intravenous administration of An2-NT(8-13) reduced by 90% the nocifensive behaviors induced by formalin injection, at the dose of 0.018 mg/kg. Likewise, subcutaneous morphine reduced pain by 90% at 1.8 mg/kg. Importantly, isobologram analyses revealed that the co-injection of An2-NT(8-13) with morphine induced an additive analgesic response. We finally assessed the effects of morphine and An2-NT(8-13) on the gastrointestinal tract motility using the charcoal meal test. As opposed to morphine which significantly reduced the intestinal motility at the analgesic effective dose of 1.8 mg/kg, An2-NT(8-13) did not affect the charcoal meal intestinal transit at 0.018 mg/kg. Interestingly, at the dose providing 90% pain relief, the co-administration of morphine with An2-NT(8-13) had a reduced effect on constipation. Altogether, these results suggest that combining NT agonists with morphine may improve its analgesic/adverse effect ratio., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of the study was to investigate the analgesic effects of adding serotonin to oxybuprocaine or proxymetacaine preparations. We employed a rat model of the cutaneous trunci muscle reflex (CTMR) to conduct the dose-response curves and duration of drugs (oxybuprocaine, proxymetacaine, or serotonin) as an infiltrative anesthetic. The use of isobolographic methods to analyze the drug-drug interactions. We showed that oxybuprocaine and proxymetacaine, as well as serotonin produced dose-dependent skin antinociception. On the basis of 50% effective dose (ED 50 ), the rank order of drug potency was serotonin [7.22 (6.45-8.09) μmol/kg] < oxybuprocaine [1.03 (0.93-1.15) μmol/kg] < proxymetacaine [0.59 (0.53-0.66) μmol/kg] (P < 0.01 for each comparison). The sensory block duration of serotonin was longer (P < 0.01) than that of oxybuprocaine or proxymetacaine at the equipotent doses (ED 25 , ED 50 , and ED 75 ). The mixture of serotonin with oxybuprocaine or proxymetacaine produced a better analgesic effect than the drug itself. We have concluded that oxybuprocaine, proxymetacaine, or serotonin displays dose-related cutaneous analgesia. Oxybuprocaine or proxymetacaine is more potent and has a shorter duration of cutaneous analgesia than serotonin. Serotonin produces a synergistic antinociceptive interaction with oxybuprocaine or proxymetacaine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The current treatment of Chagas disease is based on monopharmacology where the used drugs have limited efficacy and severe side effects. In order to overcome these limitations, some tools have been described including the development or isolation of new drugs, drug repositioning, and polypharmacology. Here, we review the polypharmacology strategy where compounds belonging to different structural chemotypes were combined in order to affect different biochemical pathways of T. cruzi parasite. Therefore ergosterol biosynthesis inhibitors, anti-inflammatory agents, cardiac dysfunction drugs, trypanothione reductase inhibitors, vitamins, between others, were combined looking for new anti-Chagas treatment. Natural products were also used in the application of this strategy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Patulin (PAT), deoxynivalenol (DON) and toxin T-2 (T-2) are mycotoxins distributed worldwide in food and feed. Cytotoxicity of the three mycotoxins individually or in combination in human hepatocellular carcinoma (HepG2) cells was evaluated by MTT assay over 24, 48 and 72 h of exposure. The concentration ranges used were 0.625-15 μM for DON, 1.25-50 nM for T-2 and 0.45-7.5 μM for PAT. The IC 50 values obtained ranged from 9.30 to 2.53 μM, from 33.69 to 44.37 nM and from 2.66 to 1.17 μM for DON, T-2 and PAT, respectively. The most cytotoxic mycotoxin to HepG2 cells was T-2 followed by PAT and DON. The combination ratios used for the mixtures were 1:3 (DON: T-2), 1:5 (DON: PAT), 1:1.7 (T-2: PAT) and 1:3:5 (DON: T-2: PAT). The mixture with the highest cytotoxic effect was T-2+PAT, followed by DON + T-2+PAT, DON + T-2 and DON + PAT respect to the cytotoxic effect of their individuals. In the combinations, at low fa an antagonistic effect was detected, and this effect changes the shape of the combination to additive effect at high fa in the mixtures., (Copyright © 2018. Published by Elsevier Ltd.)

Drug combination for the treatment of pain is common clinical practice. Co-crystal of Tramadol-Celecoxib (CTC) consists of two active pharmaceutical ingredients (APIs), namely the atypical opioid tramadol and the preferential cyclooxygenase-2 inhibitor celecoxib, at a 1:1 molecular ratio. In this study, a non-formulated 'raw' form of CTC administered in suspension (referred to as ctc susp ) was compared with both tramadol and celecoxib alone in a rat plantar incision postoperative pain model. For comparison, the strong opioids morphine and oxycodone, and a tramadol plus acetaminophen combination at a molecular ratio of 1:17 were also tested. Isobolographic analyses showed that ctc susp exerted synergistic mechanical antiallodynic (experimental ED 50 = 2.0 ± 0.5 mg/kg, i.p.; theoretical ED 50 = 3.8 ± 0.4 mg/kg, i.p.) and thermal (experimental ED 50 = 2.3 ± 0.5 mg/kg, i.p.; theoretical ED 50 = 9.8 ± 0.8 mg/kg, i.p.) antihyperalgesic effects in the postoperative pain model. In contrast, the tramadol and acetaminophen combination showed antagonistic effects on both mechanical allodynia and thermal hyperalgesia. No synergies between tramadol and celecoxib on locomotor activity, motor coordination, ulceration potential and gastrointestinal transit were observed after the administration of ctc susp . Overall, rat efficacy and safety data revealed that ctc susp provided synergistic analgesic effects compared with each API alone, without enhancing adverse effects. Moreover, ctc susp showed similar efficacy but improved safety ratio (80, measured as gastrointestinal transit vs postoperative pain ED 50 ratios) compared with the strong opioids morphine (2.5) and oxycodone (5.8). The overall in vivo profile of ctc susp supports the further investigation of CTC in the clinical management of moderate-to-severe acute pain as an alternative to strong opioids., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Preclinical Research & Development Current drugs for obesity treatment have limited efficacy and considerable adverse effects. Combination of drugs with complementary mechanisms of action at lower doses may produce a greater efficacy with a better safety profile. This study was designed to assess the anorectic effect and safety of a diethylpropion + topiramate mixture in rats. The anorectic effect of drugs was measured using a sweetened milk consumption model, and the corresponding interaction was determined by isobolographic analysis, interaction index and confidence intervals. Additionally, blood pressure was measured using a sphygmomanometer in the rat tail. Diethylpropion and topiramate alone or in combination increased the anorectic effect in a dose-dependent fashion in either nondeprived or 12 hr food-deprived rats. All theoretical ED 30 values of diethylpropion + topiramate combinations at 1:1, 1:3, and 3:1 dose ratios were significantly higher than experimental ED 30 values. In addition, interaction indices and confidence intervals confirmed the potentiation between both drugs. Theoretical ED 30 of diethylpropion + topiramate combination did not affect the blood pressure. Data suggests that low doses of the diethylpropion + topiramate combination can potentiate the anorectic effect of individual drugs with a better safety profile, which deserves further investigation in clinical trials., (© 2018 Wiley Periodicals, Inc.)

We studied the combined acute effect (i.e., after 48 h) of deltamethrin (a pyrethroid insecticide) and malathion (an organophosphate insecticide) on Daphnia magna. Two approaches were used to examine the potential interaction effects of eight mixtures of deltamethrin and malathion: (i) calculation of mixture toxicity index (MTI) and safety factor index (SFI) and (ii) response surface methodology coupled with isobole-based statistical model (using generalized linear model). According to the calculation of MTI and SFI, one tested mixture was found additive while the two other tested mixtures were found no additive (MTI) or antagonistic (SFI), but these differences between index responses are only due to differences in terminology related to these two indexes. Through the surface response approach and isobologram analysis, we concluded that there was a significant antagonistic effect of the binary mixtures of deltamethrin and malathion that occurs on D. magna immobilization, after 48 h of exposure. Index approaches and surface response approach with isobologram analysis are complementary. Calculation of mixture toxicity index and safety factor index allows identifying punctually the type of interaction for several tested mixtures, while the surface response approach with isobologram analysis integrates all the data providing a global outcome about the type of interactive effect. Only the surface response approach and isobologram analysis allowed the statistical assessment of the ecotoxicological interaction. Nevertheless, we recommend the use of both approaches (i) to identify the combined effects of contaminants and (ii) to improve risk assessment and environmental management.

Objective: We previously demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate subtype 2 (mGlu 2 ) receptors have potential synergistic interactions with the antiseizure drug levetiracetam (LEV). The present study utilizes isobolographic analysis to evaluate the combined administration of JNJ-46356479, a selective and potent mGlu 2 PAM, with LEV as well as sodium valproate (VPA) and lamotrigine (LTG)., Methods: The anticonvulsant efficacy of JNJ-46356479 was evaluated in the 6-Hz model of psychomotor seizures in mice. JNJ-46356479 was administered in combination with LEV using 3 fixed dose-ratio treatment groups in the mouse 6-Hz (44-mA) seizure test. The combination of JNJ-46356479 with LEV was also evaluated in the mouse corneal kindling model. The potential interactions of JNJ-46356479 with the antiseizure drugs VPA and LTG were also evaluated using fixed dose-ratio combinations. Plasma levels were obtained for analysis of potential pharmacokinetic interactions for each combination studied in the mouse 6-Hz model., Results: JNJ-46356479 was active in the 6-Hz model at both 32-mA and 44-mA stimulus intensities (median effective dose = 2.8 and 10.2 mg/kg, respectively). Using 1:1, 1:3, and 3:1 fixed dose-ratio combinations (LEV:JNJ-46356479), coadministration was significantly more potent than predicted for additive effects, and plasma levels suggest this synergism was not due to pharmacokinetic interactions. Studies in kindled mice further demonstrate the positive pharmacodynamic interaction of LEV with JNJ-46356479. Using 1:1 dose-ratio combinations of JNJ-46356479 with either VPA or LTG, there were no significant differences observed for coadministration., Significance: These studies demonstrate a synergistic interaction of JNJ-46356479 with LEV, whereas no such effect occurred for JNJ-46356479 with either VPA or LTG. The synergy seems therefore to be specific to LEV, and the combination LEV/mGlu 2 PAM has the potential to result in a rational polypharmacy approach to treat patients with refractory epilepsy, once it has been confirmed in clinical studies., (Wiley Periodicals, Inc. © 2018 International League Against Epilepsy.)

In toxicology and related areas, interaction effects between two substances are commonly expressed through a combination index [Formula: see text] evaluated separately at different effect levels and mixture ratios. Often, these indices are combined into a graphical representation, the isobologram. Instead of estimating the combination indices at the experimental mixture ratios only, we propose a simple parametric model for estimating the underlying interaction function. We integrate this approach into a joint model where both the parameters of the dose-response functions of the singular substances and the interaction parameters can be estimated simultaneously. As an additional benefit, this concept allows to determine optimal statistical designs for combination studies optimizing the estimation of the interaction function as a whole. From an optimal design perspective, finding the interaction parameters generally corresponds to a [Formula: see text]-optimality resp. [Formula: see text]-optimality design problem, while estimation of all underlying dose response parameters corresponds to a [Formula: see text]-optimality design problem. We show how optimal designs can be obtained in either case as well as how combination designs providing reasonable performance in regard to both criteria can be determined by putting a constraint on the efficiency in regard to one of the criteria and optimizing for the other. As all designs require prior information about model parameter values, which may be unreliable in practice, the effect of misspecifications is investigated as well.

Aims: To rationalize confusion in the literature concerning the analysis of combined antimicrobials, specifically to see if the combination index (CI) method of analysis was as rigorous as claimed., Methods and Results: Data from previous studies of the inhibition of Staphylococcus aureus by mixed antimicrobials were re-analysed using the CI method and a model which takes account of differences in the concentration exponents of individual antimicrobials., Conclusions: The Chou-Talalay combination index method for the analysis of combined antimicrobials was found to be valid only in the specific cases where concentration exponents were equal. In these cases, the CI method was found to be a function of the residuals of fitting the additive model to the observed data. Where concentration exponents were not equal, the CI method was invalid, whereas the additive model took these differences into account., Significance and Impact of Study: The CI method can be replaced wholly by the additive model described. The model allows simple regression to be used to analyse whole data sets and provides simple graphical output., (© 2017 The Society for Applied Microbiology.)