The landscape of etiological patterns of hepatocellular carcinoma and intrahepatic cholangiocarcinoma in Thailand.

Thailand is among countries with the highest global incidence and mortality rates of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). While viral hepatitis and liver fluke infections have been associated with HCC and iCCA, respectively, other environmental risk factors, overall risk factor commonality and combinatorial roles, and effects on survival have not been systematically examined. We conducted a TIGER-LC consortium-based population study covering all high-incidence areas of both malignancies across Thailand: 837 HCC, 1474 iCCA, and 1112 controls (2011-2019) were comprehensively queried on lifelong environmental exposures, lifestyle, and medical history. Multivariate logistic regression and Cox proportional hazards analyses were used to evaluate risk factors and associated survival patterns. Our models identified shared risk factors between HCC and iCCA, such as viral hepatitis infection, liver fluke infection, and diabetes, including novel and shared associations of agricultural pesticide exposure (OR range of 1.50; 95% CI: 1.06-2.11 to 2.91; 95% CI: 1.82-4.63) along with vulnerable sources of drinking water. Most patients had multiple risk factors, magnifying their risk considerably. Patients with lower risk levels had better survival in both HCC (HR 0.78; 95% CI: 0.64-0.96) and iCCA (HR 0.84; 95% CI: 0.70-0.99). Risk factor co-exposures and their common associations with HCC and iCCA in Thailand emphasize the importance for future prevention and control measures, especially in its large agricultural sector. The observed mortality patterns suggest ways to stratify patients for anticipated survivorship and develop plans to support medical care of longer-term survivors, including behavioral changes to reduce exposures.

Spontaneous conversion of prenyl halides to acids: application in metal-free preparation of deuterated compounds under mild conditions.

Here we reveal a simple generation of deuterium halide (DX) from common and inexpensive reagents readily available in a synthetic chemistry laboratory, i.e. prenyl-, allyl-, and propargyl halides, under mild conditions. We envisaged that in situ generation of an acid, deuterium halide, would be useful for acid-catalyzed reactions and could be employed for organocatalytic deuteration. The present work reports a metal-free method for deuterium labeling covering a broad range of substrate including phenolic compounds (i.e. flavonoids and stilbenes), indoles, pyrroles, carbonyl compounds, and steroids. This method was also applied for commonly used drugs such as loxoprofen, haloperidol, stanolone, progesterone, androstenedione, donepezil, ketorolac, adrenosterone, cortisone, pregnenolone, and dexamethasone. A gram-scale chromatography-free synthesis of some deuterated compounds is demonstrated in this work. This work provides a simple, clean and by-product-free, site-selective deuteration, and the deuterated products are obtained without chromatographic separation. When applying these initiators for other acid-catalyzed reactions, the deuterium isotope effects of DX may provide products which are different from those obtained from reactions using common acids. Although the mechanism of the spontaneous transformation of prenyl halides to acid is unclear, this overlooked chemistry may be useful for many reactions.

Diterpenoids with Aromatase Inhibitory Activity from the Rhizomes of Kaempferia elegans.

Investigation of bioactive compounds from the rhizomes of Kaempferia elegans led to the isolation and characterization of ten new diterpenoids, namely, five 12,13-seco-diterpenoids named elegansins A-E (1-5) and five new abietanes, elegansols A-E (6-10), together with seven known diterpenoids (11-17). The structure elucidation of the new compounds was achieved by HRESIMS, NMR, and ECD spectroscopic analysis. Compounds (1-5) are the first examples of 12,13-seco-diterpenoid-type compounds representing a decalin fused dihydropyran skeleton. Plausible biosynthetic pathways for compounds 1-5 are proposed. Aromatase inhibitory activities of all compounds were evaluated, and abieta-8,11,13-trien-11-ol (16) was found to be the most potent aromatase inhibitor with an IC50 value of 3.7 µM.

NMR characterization of rearranged staurosporine aglycone analogues from the marine sponge Damiria sp.

The indolocarbazole family of bisindole alkaloids is best known for the natural product staurosporine, a protein kinase C inhibitor that belongs to the indolo[2,3-a]carbazole structural class. A large number of other indolo[2,3-a]carbazoles have subsequently been isolated and identified, but other isomeric forms of indolocarbazole natural products have rarely been reported. An extract of the marine sponge Damiria sp., which represents an understudied genus, provided two novel alkaloids named damirines A (1) and B (2). Their structures were assigned by comprehensive NMR spectroscopic analyses, and for compound 2, this included application of the LR-HSQMBC pulse sequence, a long-range heteronuclear correlation experiment that has particular utility for defining proton-deficient scaffolds. The damirines represent a new hexacyclic carbon-nitrogen framework comprised of an indolo[3,2-a]carbazole fused with either an aminoimidazole or a imidazolone ring. Compound 1 showed selective cytotoxic properties toward six different cell lines in the NCI-60 cancer screen.

Exposure to arsenic in utero is associated with various types of DNA damage and micronuclei in newborns: a birth cohort study.

BACKGROUND: Growing evidence indicates that in utero arsenic exposures in humans may increase the risk of adverse health effects and development of diseases later in life. This study aimed to evaluate potential health risks of in utero arsenic exposure on genetic damage in newborns in relation to maternal arsenic exposure. METHODS: A total of 205 pregnant women residing in arsenic-contaminated areas in Hanam province, Vietnam, were recruited. Prenatal arsenic exposure was determined by arsenic concentration in mother's toenails and urine during pregnancy and in umbilical cord blood collected at delivery. Genetic damage in newborns was assessed by various biomarkers of early genetic effects including oxidative/nitrative DNA damage (8-hydroxydeoxyguanosine, 8-OHdG, and 8-nitroguanine), DNA strand breaks and micronuclei (MN) in cord blood. RESULTS: Maternal arsenic exposure, measured by arsenic levels in toenails and urine, was significantly increased (p <  0.05) in subjects residing in areas with high levels of arsenic contamination in drinking water. Cord blood arsenic level was significantly increased in accordance with maternal arsenic exposure (p <  0.001). Arsenic exposure in utero is associated with genotoxic effects in newborns indicated as increased levels of 8-OHdG, 8-nitroguanine, DNA strand breaks and MN frequency in cord blood with increasing levels of maternal arsenic exposure. Maternal toenail arsenic level was significantly associated with all biomarkers of early genetic effects, while cord blood arsenic levels associated with DNA strand breaks and MN frequency. CONCLUSIONS: In utero arsenic exposure is associated with various types of genetic damage in newborns potentially contributing to the development of diseases, including cancer, later in life.

Correction to: Exposure to arsenic in utero is associated with various types of DNA damage and micronuclei in newborns: a birth cohort study.

Following publication of the original article [1], the author reported that incorrect version of Tables 1, 3, 5 and 6 were published.

Characterization of essential oil from Ocimum gratissimum leaves: Antibacterial and mode of action against selected gastroenteritis pathogens.

Essential oil of fresh leaves of Ocimum gratissimum (OGEO) was water-steam distilled and analyzed by GC-MS. Thirty-seven compounds were identified, with eugenol (55.6%) as the major component followed by cis-ocimene (13.9%), γ-muurolene (11.6%), (Z,E)-α-farnesene (5.6%), α-trans-bergamotene (4.1%), and ß-caryophyllene (2.7%). Antimicrobial activity of OGEO was tested against four gastroenteritis pathogens (Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, and Shigella flexneri). OGEO exhibited antibacterial effect, with MICs of 1-2 mg ml-1, against the tested species. OGEO also displayed rapid killing effect within 5 s at four times of MIC against both E. coli and S. Typhimurium. Various assays were performed to investigate the mode of action of the oil. OGEO increased the permeability of microbial cell membrane as evidenced by LIVE/DEAD BacLight assay. Analyses of the release of absorbing materials at 260 nm, protein leakage, SDS-PAGE, and SEM strongly suggested the disruptive action of the oil on the cytoplasmic membrane of the tested microorganisms. Results revealed that the antibacterial property of OGEO could be due to membrane disruption.

Ultrasound surveillance for cholangiocarcinoma in an endemic area: A prove of survival benefits.

BACKGROUND AND AIM: Cholangiocarcinoma (CCA) is an aggressive malignancy with rapid progression and poor prognosis. Abdominal ultrasound surveillance may detect early-stage malignancy and improve surgical outcome. However, little data exist on the benefits of abdominal ultrasound surveillance in populations at high risk for CCA development in an endemic area. This study compared survival outcomes of CCA patients recruited through abdominal ultrasound surveillance program and those presented to the hospital independent of surveillance. METHODS: The surveillance population-based cohort was 4225 villagers in Northern Thailand, aged 30-60 years, who consented to a 5-year abdominal ultrasound surveillance program, which included interval ultrasound examinations every 6 months. The non-surveillance cohort was hospital-based CCA patients diagnosed during April 2007 to November 2015. Numbers of operable tumors, percentages of R0 resection, and survival analyses were compared between the two cohorts. RESULTS: There were 48 and 192 CCA patients in the surveillance and the non-surveillance cohorts, respectively. Of these, 37/48 (77.1%) and 22/192 (11.5%) were in an operable stage and R0 resections performed in 36/48 (97.3%) and 14/192 (63.6%), respectively. The median survival in each group was 31.8 and 6.7 months, respectively (with correction of lead time bias) (P < 0.0001). By multivariate analysis, abdominal ultrasound surveillance (hazard ratio [HR] = 0.41; P = 0.012), operable stage (HR = 0.11; P < 0.001), and serum albumin ≥ 3.5 g/dL (HR = 0.42; P < 0.001) were significantly associated with decreased mortality, whereas size of CCA (HR = 1.11; P < 0.001), serum alanine aminotransferase > 40 IU/L (HR = 1.71; P = 0.017), and tumor recurrence (HR = 4.86; P = 0.017) were associated with increased mortality. CONCLUSION: Abdominal ultrasound surveillance provided survival benefits and should be considered in areas highly endemic for CCA to reduce mortality.

Cytotoxic Sesterterpenes from Thai Marine Sponge Hyrtios erectus.

Four sesterterpenes, erectusolides B, C, D, and seco-manoalide-25-methyl ether, two 2-furanone derivatives, erectusfuranones A and B, together with thirteen known sesterterpenes, (6Z)-neomanoalide-24-acetate, two diastereomers of 24-O-methylmanoalide, luffariolide B, manoalide, (6E)- and (6Z)-neomanoalide, seco-manoalide, scalarafuran, 12-acetylscalarolide, 12-epi-O-deacetyl-19-deoxyscalarin, 12-epi-scalarin, and 12-O-deacetyl-12-epi-scalarin, three indole alkaloids, 5-hydroxy-1H-indole-3-carbaldehyde, hyrtiosine A, and variabine B, and one norterpene, cavernosine were isolated from the marine sponge Hyrtios erectus. Their structures were determined by means of spectroscopic methods and the absolute configurations of the asymmetric centers were determined using the modified Mosher's method. The cytotoxic activities for the isolated compounds have been reported.

Naphthalene Derivatives and Quinones from Ventilago denticulata and Their Nitric Oxide Radical Scavenging, Antioxidant, Cytotoxic, Antibacterial, and Phosphodiesterase Inhibitory Activities.

New naphthalene derivatives (1 and 2) and a new isomer (3) of ventilagolin, together with known anthraquinones, chrysophanol (4), physcion or emodin 3-methyl ether (5), and emodin (6), were isolated from vines of Ventilago denticulata. The isolated compounds exhibited cytotoxic activity with IC50 values of 1.15 - 40.54 µg/ml. Compounds 1 - 3 selectively exhibited weak antibacterial activity (MIC values of 200.0 - 400.0 µg/ml), while emodin (6) displayed moderate antibacterial activity with MIC value of 25.0 µg/ml. The isolated compounds showed nitric oxide and DPPH radical scavenging activities. Compounds 1 - 3 and 6 exhibited weak xanthine oxidase inhibitory activity, while emodin (6) acted as an aromatase inhibitor with the IC50 value of 10.1 µm. Compounds 1 and 2 exhibited phosphodiesterase 5 inhibitory activity with IC50 values of 8.28 µm and 6.48 µm, respectively.

Metabolite diversification by cultivation of the endophytic fungus Dothideomycete sp. in halogen containing media: Cultivation of terrestrial fungus in seawater.

The endophytic fungus, Dothideomycete sp. CRI7, isolated from the terrestrial plant, Tiliacora triandra, was salt tolerant, capable of growing in the culture medium prepared from seawater; salts in seawater did not have any effects on the fungal growth. Metabolite productions of the fungus CRI7 cultivated in media prepared from seawater (MSW), prepared from deionized water supplemented with potassium bromide (MKBr) or potassium iodide (MKI), and prepared from deionized water (MDW) were investigated. It was found that the cultivation of the fungus CRI7 in MKBr and MSW enabled the fungus to produce nine new metabolites (1-9). The production of an azaphilone, austdiol (10), of the fungus CRI7 grown in MDW was 0.04g/L, which was much lower than that grown in MSW, MKBr, and MKI media which provided the yields of 0.5, 0.9, and 1.2g/L, respectively, indicating that halogen salts significantly enhanced the production of the polyketide 10. The cultivation of terrestrial fungi in media containing halogen salts could therefore be useful for the metabolite diversification by one strain-many compounds (OSMAC) approach. Moreover, the isolated polyketides had significant biosynthetic relationship, suggesting that the cultivation of fungi in halogen containing media could provide the insights into certain polyketide biosynthesis. One of the isolated compounds exhibited antibacterial activity with the MIC value of 100µg/mL.

Efficacy and safety of entecavir versus tenofovir treatment in chronic hepatitis B patients: A randomized controlled trial.

AIM: Entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are considered among the most potent antiviral agents for the treatment of chronic hepatitis B infection. We aimed to compare treatment efficacy and safety of ETV and TDF in nucleoside-naïve chronic hepatitis B patients. METHODS: Inclusion criteria were compensated chronic hepatitis B patients who were either hepatitis B e antigen (HBeAg)-positive or HBeAg-negative. Exclusion criteria were co-infection with hepatitis C virus and/or HIV, concurrent malignancy, and decompensated cirrhosis. Virological, biochemical, and serological end-points at week 96 and 144 were compared. Of 400 patients, 200 patients received ETV and 200 received TDF. RESULTS: There were no significant differences between the two groups in baseline characteristics including age (41.6 ± 11.5 vs. 41.2 ± 11.6, mean baseline hepatitis B virus DNA (5.91 ± 1.79 vs. 5.94 ± 1.68 log10 IU/mL), mean baseline alanine aminotransferase (68.1 ± 64.1 vs. 76.8 ± 79.8 U/L), and cirrhosis (15.5% vs. 14.5%). At week 144 of treatment, 91 and 94% of the ETV and TDF groups, respectively, achieved undetectable hepatitis B virus DNA. In HBeAg-positive patients, HBeAg seroconversion could be achieved in 27.4% and 33.7% at week 144 for ETV and TDF groups, respectively. Quantitative hepatitis B surface antigen dropped significantly over 144 weeks of treatment period but only 1.0 to 1.5% experienced hepatitis B surface antigen loss. Safety profiles were consistent with previous reports of monotherapy. CONCLUSION: Both ETV and TDF showed potent antiviral activity against hepatitis B. Either ETV or TDF can be recommended as a treatment of choice for patients with chronic hepatitis B. Both drugs were safe and well tolerated.

Sterols from Thai Marine Sponge Petrosia (Strongylophora) sp. and Their Cytotoxicity.

Eight new sterols (1-5 and 11-13), together with eight known compounds (6-10 and 14-16) were isolated from marine sponge Petrosia sp. The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis. The cytotoxicity of some compounds against a panel of human cancer cell lines is also reported.

Superbanone, A New 2-Aryl-3-benzofuranone and Other Bioactive Constituents from the Tube Roots of Butea superba.

Phytochemical investigation from the tube roots of Butea superba, led to the isolation and identification of a new 2-aryl-3-benzofuranone named superbanone (1), one benzoin, 2-hydroxy-1-(2-hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)ethanone (2), eight pterocarpans (3 - 10), and eleven isoflavonoids (11 - 21). Compound 2 was identified for the first time as a natural product. The structure of the isolated compounds was elucidated using spectroscopic methods, mainly 1D- and 2D-NMR. The isolated compounds and their derivatives were evaluated for α-glucosidase inhibitory and antimalarial activities. Compounds 3, 7, 8, and 11 showed promising α-glucosidase inhibitory activity (IC50  = 13.71 ± 0.54, 23.54 ± 0.75, 28.83 ± 1.02, and 12.35 ± 0.36 µm, respectively). Compounds 3 and 11 were twofold less active than the standard drug acarbose (IC50  = 6.54 ± 0.04 µm). None of the tested compounds was found to be active against Plasmodium falciparum strain 94. On the basis of biological activity results, structure-activity relationships are discussed.

Ultrasound screening for cholangiocarcinoma could detect premalignant lesions and early-stage diseases with survival benefits: a population-based prospective study of 4,225 subjects in an endemic area.

BACKGROUND: Thailand has a high incidence of cholangiocarcinoma (CCA), particularly in the north and northeastern regions. Most CCA patients come at a late, unresectable stage and presently no optimal screening test for CCA has been established. We determined the prevalence of CCA in a remote northern village and explored if screening could lead to early detection and survival benefits. METHODS: A 5-year population-based study was started in October, 2011 for consented Thai individuals, aged 30-60 years. The screening program comprised blood testing, stool examination and serial ultrasonography every 6 months. RESULTS: During the first 3 years, 4,225 eligible individuals were enrolled. CCA was detected in 32 patients, with a mean age of 51.9 years (41-62 years), and 21/32 cases were at a curative resectable stage. The prevalence rate of CCA was 165.7 per 100,000 and one- and two-year incidence rate was 236.7/100,000 and 520.7/100,000, respectively. One- and 2-year overall survival rates of CCA patients were 90.9 and 61.5 %, respectively. Prognosis was better in resectable cases with 100 % 1-year and 77.8 % 2-year survival rates. Interestingly, premalignant pathological lesions (stage 0) were identified in 11 cases with 100 % 3-year survival rate. Serum biomarkers and alkaline phosphatase were not sufficient to detect early-stage disease. In 22 patients, stool samples were positive for Opistorchis viverrini, based on polymerase chain reaction. CONCLUSION: Detection of premalignant lesions and early-stage resectable CCA by ultrasonography resulted in improved clinical outcome. Ultrasonography should be offered as a first screening tool for CCA in an endemic area until other useful biological markers become available.

High prevalence of advanced colorectal neoplasia in the Thai population: a prospective screening colonoscopy of 1,404 cases.

BACKGROUND: Increasing morbidity and mortality from colorectal cancer is evident in recent years in the developing Asian nations. Particularly in Thailand and most neighbouring low-income countries, screening colonoscopy is not yet recommended nor implemented at the national policy level. METHODS: Screening colonoscopy was offered to 1,500 healthy volunteers aged 50-65 years old who were registered into the program between July 2009 and June 2010. Biopsy and surgery was performed depending on the identified lesions. Fecal immunochemical tests (FIT) were additionally performed for comparison with colonoscopy. RESULTS: There were 1,404 participants who underwent colonoscopy. The mean age of the cohort was 56.9 ± 4.2 years and 69.4 % were females. About 30 % (411 cases) of all colonoscopies had abnormal colonoscopic findings, and of these, 256 cases had adenomatous polyps. High risk adenomas (villous or tubulovillous or high grade dysplasia or size > 1 cm or > 3 adenomatous polyps) were found in 98 cases (7 %), low risk adenoma in 158 cases (11.3 %), and hyperplastic polyps in 119 cases (8.5 %). Eighteen cases (1.3 %) had colorectal cancer and 90 % of them (16 cases) were non-metastatic including five stage 0 cases, seven stage I cases, and four stage IIA cases. Only two cases had metastasis: one to regional lymph nodes (stage IIIB) and another to other organs (stage IVA). The most common cancer site was the distal intestine including rectum (7 cases, 38.9 %) and sigmoid colon (7 cases, 38.9 %). Ten colorectal cancer cases had positive FIT whereas 8 colorectal cancer cases were FIT-negative. The sensitivity and specificity of FIT was 55.6 % and 96.2 %, respectively, while the positive predictive value was 16.4 % and negative predictive value was 99.4 %. The overall survival of colorectal cancer cases at 5-year was 83.3 %. CONCLUSION: High prevalence of colorectal cancer and high-risk adenoma was found in the Thai population aged 50-65 years old by screening colonoscopy. FIT was not sensitive enough to detect colorectal cancer in this asymptomatic cohort. Integration of screening colonoscopy into the national cancer screening program should be implemented to detect early cases of advanced colorectal neoplasia and improve survival of colorectal cancer patients in Thailand.

Cytotoxic Steroidal Glycosides from the Whole Plant of Calamus acanthophyllus.

A new steroidal glycoside, callaphylloside (1), together with seven known glycosides (2-8), was isolated from the whole plant of Calamus acanthophyllus. The structure of the new compound was elucidated by spectral data analyses and chemical transformations. Compounds 5 and 8 exhibited strong cytotoxic activity against four cancer cell lines (0.7 ≤ IC50 ≤ 3.4 µM). Evaluation of the structure-activity relationship among steroidal glycosides revealed that the structure of spirostanol with an α-L-rhamnopyranosyl linked to C-2 of the inner glucopyranosyl residue both play a critical role in the effects of these compounds on the cancer cell lines.

Water-Assisted Nitrile Oxide Cycloadditions: Synthesis of Isoxazoles and Stereoselective Syntheses of Isoxazolines and 1,2,4-Oxadiazoles.

Conventional methods generate nitrile oxides from oxime halides in organic solvents under basic conditions. However, the present work revealed that water-assisted generation of nitrile oxides proceeds under mild acidic conditions (pH 4-5). Cycloadditions of nitrile oxides with alkynes and alkenes easily occurred in water without using catalysts, thus yielding isoxazoles and isoxazolines, respectively, with excellent stereoselectivity toward five- and six-membered cyclic alkenes. A double stereoselective cycloaddition of two units of a nitrile oxide with cyclohexene was also achieved, thus yielding 1,2,4-oxadiazole derivatives having a unique hybrid isoxazoline-oxadiazole skeleton. Enantiomerically pure isoxazolines were prepared from monoterpenes with a ring strain. In one case, the isoxazoline with a butterfly-like structure was simply prepared, and it might be used as a ligand in asymmetric catalysis.

Drimane Sesquiterpene-Conjugated Amino Acids from a Marine Isolate of the Fungus Talaromyces minioluteus (Penicillium Minioluteum).

Four new sesquiterpene lactones (3, 4, 6 and 7) and three known compounds, purpuride (1), berkedrimane B (2) and purpuride B (5), were isolated from the marine fungus, Talaromyces minioluteus (Penicillium minioluteum). New compounds were drimane sesquiterpenes conjugated with N-acetyl-l-valine, and their structures were elucidated by analysis of spectroscopic data, as well as by single crystal X-ray analysis. The isolated compounds could not inhibit the apoptosis-regulating enzyme, caspase-3, while three of the compounds (2, 3 and 7) exhibited weak cytotoxic activity.

Effects of continuous intravenous infusion of morphine and morphine-tramadol on the minimum alveolar concentration of sevoflurane and electroencephalographic entropy indices in dogs.

OBJECTIVE: To compare the effects of continuous rate infusions (CRIs) of intravenous (IV) morphine and morphine-tramadol on the minimum alveolar concentration (MAC) of sevoflurane, and on electroencephalographic entropy indices in dogs. DESIGN: Prospective study. ANIMALS: Eight young, healthy German shepherds, weighing 26.3 ± 3.1 kg (mean ± SD). METHODS: Anaesthesia was induced and maintained with sevoflurane. A standard tail-clamp technique was used for MAC determination. Within one anaesthetic period, MAC was first determined during sevoflurane anaesthesia alone (MACB ); then during morphine infusion (MACM ), (loading dose 0.5 mg kg(-1) IM; CRI, 0.2 mg kg(-1 ) hour(-1)) then finally during morphine-tramadol infusion (tramadol loading dose 1.5 mg kg(-1) IV; CRI, 2.6 mg kg(-1)  hour(-1) ) (MACMT ). At each change, periods of 45 minutes were allowed for equilibration. Stated entropy (SE), response entropy (RE), and RE-SE differences were measured five minutes prior to and during tail clamping. RESULTS: The MACB was 2.1 ± 0.3vol%. The morphine and morphine-tramadol infusions reduced MAC to 1.6 ± 0.3vol% and 1.3 ± 0.3vol%, respectively. MAC was decreased below baseline more during morphine-tramadol than during morphine alone (39 ± 9% versus 25 ± 6%, respectively; p = 0.003). All SE and RE and most RE-SE differences were increased significantly (p < 0.05) over pre-stimulation in all groups when the dogs responded purposefully to noxious stimulation. When no response to noxious stimulation occurred, the entropy indices did not change. CONCLUSION AND CLINICAL RELEVANCE: In dogs, combined morphine-tramadol CRI decreased sevoflurane MAC more than morphine CRI alone. Entropy indices changed during nociceptive responses in anaesthetized animals, suggesting that entropy measurements may be useful in determining anaesthetic depth in dogs.