Development of an ELISA for detection of mitragynine and its metabolites in human urine.

An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.

Method validation in quantitative analysis of phase I and phase II metabolites of mitragynine in human urine using liquid chromatography-tandem mass spectrometry.

A method using solid phase extraction and liquid chromatography-tandem mass spectrometry to quantitatively detect mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine samples was developed and validated. The relevant metabolites were identified using multiple reaction monitoring in positive ionization mode using nalorphine as an internal standard. The method was validated for accuracy, precision, recovery, linearity, and lower limit of quantitation. The intra- and inter-day accuracy and precision were found in the range of 83.6-117.5% with coefficient of variation less than 13%. The percentage of recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine was within the range of 80.1-118.9%. The lower limit of quantification was 1 ng/mL for mitragynine, 2 ng/mL for 16-carboxy mitragynine, and 50 ng/mL for 9-O-demethyl mitragynine. The developed method was reproducible, high precision and accuracy with good linearity and recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine.

Isolation and characterization of a novel anti-salbutamol chicken scFv for human doping urinalysis.

Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant ß2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two ß2-agonists were; IC50 salbutamol = ∼0.310 µg/ml, IC50 clenbuterol = ∼0.076 µg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 µg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 µg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.

Ethyl nitrobenzoate: A novel scaffold for cholinesterase inhibition.

A series of novel cholinesterase inhibitors containing nitrobenzoate core structure were synthesized by a facile and efficient method. The structure of the novel compounds were fully characterized and confirmed by analytical as well as spectroscopic methods. Compound indicated as 2f was found to possess the best cholinesterase inhibitory activities of all the evaluated compounds. Results suggest that 2f is a selective acetylcholinesterase inhibitor, although it also inhibits butyrylcholinesterase at higher concentration. Kinetics inhibition result suggest that 2f is a mixed-mode inhibitor of acetylcholinesterase. In addition, it was found to have low cytotoxicity. Molecular docking on compound 2f was carried out to rationalize the observed in vitro enzymatic assay results. Most importantly, the potential of nitrobenzoate derivatives as cholinesterase inhibitor was shown through this study. In summary, we discovered nitrobenzoates as a new scaffold that may eventually yield useful compounds in treatment of Alzheimer's disease.

ABCB1 Polymorphisms and Cold Pressor Pain Responses: Opioid-Dependent Patients on Methadone Maintenance Therapy.

BACKGROUND: Methadone is a substrate of the P-glycoprotein efflux transporter, which is encoded by ABCB1 (MDR1), and thus, ABCB1 polymorphisms may influence the transport of methadone at the blood-brain barrier, affecting its adverse effects. OBJECTIVES: This study investigated the association between ABCB1 polymorphisms and cold pressor pain responses among opioid-dependent patients on methadone maintenance therapy (MMT). METHODS: Malay male opioid-dependent patients receiving MMT (n = 148) were recruited. Cold pressor pain responses (pain threshold, pain tolerance, and pain intensity) were measured at 0, 2, 4, 8, 12, and 24 hours post-methadone dose. DNA was extracted from whole blood and genotyped for ABCB1 polymorphisms including 1236C>T (rs1128503), 2677G>T/A (rs2032582), and 3435C>T (rs1045642) using the allelic discrimination real-time polymerase chain reaction. Repeated-measure analysis of variance between-group analysis was used to compare the three cold pressor pain responses and ABCB1 polymorphisms (1236C>T, 2677G>T/A, and 3435C>T) according to genotypes and allelic additive models, genotype dominant and recessive models, haplotypes, and diplotypes. RESULTS: Patients with 2677 GG or 2677G allele had the lowest pain threshold compared with 2677G>T/A genotypes or alleles (p = .007 and .002, respectively). Haplotype analysis showed a significant association between ABCB1 haplotypes and pain threshold (p = .02). Patients with 2677G allele had the lowest pain tolerance compared to those with 2677T and 2677A alleles (2677G < 2677T < 2677A allele carriers; p = .05). In terms of pain intensity scores, patients with 2677 GG or 2677G allele had the highest scores compared to other 2677G>T/A genotypes or alleles (p = .04 and .008, respectively). Haplotype analysis revealed a significant difference between patients with CGC haplotype and those without this haplotype (p = .02). DISCUSSION: To the best of our knowledge, this study provides the first evidence that ABCB1 polymorphisms are associated with cold pressor pain responses among Malay male patients with opioid dependence on MMT. The results may provide an initial prediction on heightened pain sensitivity or hyperalgesia for individuals who are carriers of the ABCB1 polymorphisms.

Relationship Between ABCB1 Polymorphisms and Cold Pain Sensitivity Among Healthy Opioid-naive Malay Males.

BACKGROUND: Endogenous and exogenous opioids are substrates of the permeability glycoprotein (P-gp) efflux transporter, which is encoded by the ABCB1 (MDR1) gene. Genetic polymorphisms of ABCB1 may contribute to interindividual differences in pain modulation and analgesic responses. We investigated the relationship between ABCB1 polymorphisms and cold pain sensitivity among healthy males. METHODS: Cold pain responses, including pain threshold and pain tolerance, were measured using the cold-pressor test (CPT). DNA was extracted from whole blood and genotyped for ABCB1 polymorphisms, including c.1236C>T (rs1128503), c.2677G>T/A (rs2032582), and c.3435C>T (rs1045642), using the allelic discrimination real-time polymerase chain reaction. RESULTS: A total of 152 participants were recruited in this observational study. Frequencies of mutated allele for c.1236C>T, c.2677G>T/A, and c.3435C>T polymorphisms were 56.6%, 49.7%, and 43.4%, respectively. Our results revealed an association of the CGC/CGC diplotype (c.1236C>T, c.2677G>T/A, and c.3435C>T) with cold pain sensitivity. Participants with the CGC/CGC diplotype had 90% and 72% higher cold pain thresholds (87.62 seconds vs. 46.19 seconds, P = 0.010) and cold pain tolerances (97.24 seconds vs. 56.54 seconds, P = 0.021), respectively, when compared with those without the diplotype. CONCLUSION: The CGC/CGC diplotype of ABCB1 polymorphisms was associated with variability in cold pain threshold and pain tolerance in healthy males.

Comparison of Pain Tolerance between Opioid Dependent Patients on Methadone Maintenance Therapy (MMT) and Opioid Naive Individuals.

PURPOSE: This study compared pain sensitivity among opioid dependent patients on methadone maintenance therapy (MMT) and opioid naive subjects. METHODS: The three hundred participants comprised 152 opioid naive subjects and 148 opioid dependent patients. Opioid naive subjects had not taken any opioids including morphine and methadone to their best knowledge and were presumed so after two consecutive negative urine screenings for drugs. All opioid dependent patients were stabilized in treatment, defined as having been enrolled in the program for more than one month with no change of methadone dosage over the past one month. Excluded from the study were individuals with chronic or ongoing acute pain and individuals with a history of analgesics ingestion within 3 d before the cold pressor test (CPT). Pain tolerance to CPT was evaluated at 0 h, and at 2, 4, 8, 12, and 24 h post-methadone dose. RESULTS: Patients exhibited a significantly shorter mean pain tolerance time of 34.17 s (95% CI 24.86, 43.49) versus 61.36 (52.23, 70.48) [p < 0.001] compared with opioid naive subjects. Time-dependent mean pain tolerance was also significantly different when naive subjects were compared to patients (p = 0.016). CONCLUSIONS: This study revealed hyperalgesia amongst patients on MMT, as manifested by their quicker hand withdrawal. The complaints of pain in this population should not be underestimated and the pain should be evaluated seriously and managed aggressively.

Relationship between ABCB1 polymorphisms and serum methadone concentration in patients undergoing methadone maintenance therapy (MMT).

BACKGROUND: Methadone is a substrate of the permeability glycoprotein (P-gp) efflux transporter, which is encoded by the ABCB1 (MDR1) gene. Large interindividual variability in serum methadone levels for therapeutic response has been reported. Genetic variations in ABCB1 gene may be responsible for the variability in observed methadone concentrations. OBJECTIVE: This study investigated the associations of ABCB1 polymorphisms and serum methadone concentration over the 24-hour dosing interval in opioid-dependent patients on methadone maintenance therapy (MMT). METHODS: One hundred and forty-eight male opioid-dependent patients receiving MMT were recruited. Genomic deoxyribonucleic acid (DNA) was extracted from whole blood and genotyped for ABCB1 polymorphisms [i.e. 1236C>T (dbSNP rs1128503), 2677G>T/A (dbSNP rs2032582), and 3435C>T (dbSNP rs1045642)] using the allelic discrimination real-time polymerase chain reaction (PCR). Blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, and 24 hours after the dose. Serum methadone concentrations were measured using the Methadone ELISA Kit. RESULTS: Our results revealed an association of CGC/TTT diplotype (1236C>T, 2677G>T/A, and 3435C>T) with dose-adjusted serum methadone concentration over the 24-hour dosing interval. Patients with CGC/TTT diplotype had 32.9% higher dose-adjusted serum methadone concentration over the 24-hour dosing interval when compared with those without the diplotype [mean (SD) = 8.12 (0.84) and 6.11 (0.41) ng ml-1 mg-1, respectively; p = 0.033]. CONCLUSION: There was an association between the CGC/TTT diplotype of ABCB1 polymorphisms and serum methadone concentration over the 24-hour dosing interval among patients on MMT. Genotyping of ABCB1 among opioid-dependent patients on MMT may help individualize and optimize methadone substitution treatment.

Understanding the physicochemical properties of mitragynine, a principal alkaloid of Mitragyna speciosa, for preclinical evaluation.

Varied pharmacological responses have been reported for mitragynine in the literature, but no supportive scientific explanations have been given for this. These studies have been undertaken without a sufficient understanding of the physicochemical properties of mitragynine. In this work a UV spectrophotometer approach and HPLC-UV method were employed to ascertain the physicochemical properties of mitragynine. The pKa of mitragynine measured by conventional UV (8.11 ± 0.11) was in agreement with the microplate reader determination (8.08 ± 0.04). Mitragynine is a lipophilic alkaloid, as indicated by a logP value of 1.73. Mitragynine had poor solubility in water and basic media, and conversely in acidic environments, but it is acid labile. In an in vitro dissolution the total drug release was higher for the simulated gastric fluid but was prolonged and incomplete for the simulated intestinal fluid. The hydrophobicity, poor water solubility, high variability of drug release in simulated biological fluids and acid degradable characteristics of mitragynine probably explain the large variability of its pharmacological responses reported in the literature. The determined physicochemical properties of mitragynine will provide a basis for developing a suitable formulation to further improve its solubility, stability and oral absorption for better assessment of this compound in preclinical studies.

3-{5-Eth-oxy-carbonyl-1-[3-(2-oxopyrrol-idin-1-yl)prop-yl]-1H-benzimidazol-2-yl}benzoic acid.

In the title compound, C(24)H(25)N(3)O(5), the eth-oxy group is disordered over two orientations in a 0.853 (14):0.147 (14) ratio. The benzimadazole ring system (r.m.s. deviation = 0.016 Å) makes a dihedral angle of 35.47 (7)° with the attached benzene ring. The pyrrolidine ring adopts an envelope conformation with a methyl-ene C atom as the flap. In the crystal, inversion dimers linked by pairs of O-H⋯N hydrogen bonds generate R(2) (2)(16) loops. C-H⋯O inter-actions link the dimers into a three-dimensional network.

Single-stranded DNA (ssDNA) production in DNA aptamer generation.

The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.

Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti-diabetic mechanism.

BACKGROUND: In the present study, we tested a 50% ethanolic extract of Orthosiphon stamineus plants and its isolated bioactive compound with respect to their α-glucosidase and α-amylase inhibitory activities. METHODS: Bioactive flavonoid sinensetin was isolated from 50% ethanolic extract of Orthosiphon stamineus. The structure of this pure compound was determined on the NMR data and the α-glucosidase and α-amylase inhibitory activities of isolated sinensetin and 50% ethanolic extract of Orthosiphon stamineus were evaluated. RESULTS: In vitro studies of a 50% ethanolic extract of O. stamineus and the isolated sinensetin compound showed inhibitory activity on α-glucosidase (IC50: 4.63 and 0.66 mg/ml, respectively) and α-amylase (IC50: 36.70 mg/ml and 1.13 mg/ml, respectively). Inhibition of these enzymes provides a strong biochemical basis for the management of type 2 diabetes via the control of glucose absorption. CONCLUSION: Alpha-glucosidase and α-amylase inhibition could the mechanisms through which the 50% ethanolic extract of O. stamineus and sinensetin exert their antidiabetic activity, indicating that it could have potential use in the management of non-insulin-dependent diabetes.

A rapid and cost effective method in purifying small RNA.

Purification of RNA fragments from a complex mixture is a very common technique, and requires consideration of the time, cost, purity and yield of the purified RNA fragments. This study describes the fastest method of purifying small RNA with the lowest cost possible, without compromizing the yield and purity. The technique describes the purification of small RNA from polyacrylamide gel, resulting in a good yield of small RNA with minimum experimental steps in avoiding degradation of the RNA, obviating the use of ethidium bromide and phenol-chloroform extraction, as well as siliconized glass wools to remove the polyacrylamide gel particles. The purified small RNA is suitable for a wide variety of applications such as ligation, end labelling with radio isotope, RT-PCR (Reverse Transcriptase-PCR), Northern blotting, experimental RNomics study and also Systematic Evolution of Ligands by Exponential Enrichment (SELEX).

(E)-2-[4-(Trifluoro-meth-oxy)benzyl-idene]indan-1-one.

In the title compound, C(17)H(11)F(3)O(2), the dihydro-indene ring is approximately planar with a maximum deviation of 0.024 (2) Šand makes a dihedral angle of 3.17 (8) Šwith the adjacent benzene ring. In the crystal, mol-ecules are inter-connected by C-H⋯O inter-actions, forming an infinite chain along the c axis.

(2E)-2-Benzyl-idene-5,6-dimethoxy-indan-1-one.

The mol-ecular structure of the title compound, C(18)H(16)O(3), is roughly planar; the maximum deviation of the indanone ring system is 0.027 (1) Šand it makes a dihedral angle of 2.69 (3)° with the phenyl ring. The torsion angles between the two meth-oxy groups and the -indanone ring are -14.67 (11) and -1.11 (12)°. In the crystal, mol-ecules are connected into a ribbon along the a axis via weak inter-molecular C-H⋯O hydrogen bonds. Weak inter-molecular C-H⋯π and π-π [centroid-centroid distance = 3.7086 (6) Å] inter-actions are also observed.

5,6-Dimeth-oxy-4',5'-diphenyl-indane-2-spiro-3'-pyrrolidine-2'-spiro-3''-indoline-1,2''-dione.

In the title compound, C(33)H(28)N(2)O(4), the central pyrrolidine ring adopts a half-chair conformation. Both the indolinone and indanone groups are twisted, with their five-membered rings adopting a half-chair and an envelope conformation, respectively. The two benzene rings and the mean plane of the indolinone and indanone groups make dihedral angles of 71.98 (10), 84.32 (10), 86.26 (9) and 78.50 (9)°, respectively, with the central pyrrolidine ring. Intra-molecular C-H⋯O hydrogen bonds stabilize the mol-ecular conformation. In the crystal, pairs of inter-molecular N-H⋯O hydrogen bonds link the mol-ecules into centrosymmetric dimers. The dimers are inter-connected into ribbons propagating along [110] via weak inter-molecular C-H⋯O hydrogen bonds. Weak inter-molecular C-H⋯π and π-π [centroid-centroid distance = 3.6509 (11) Å] inter-actions are also observed.

(E)-5,6-Dimeth-oxy-2-(pyridin-4-yl-methyl-idene)-2,3-dihydro-1H-inden-1-one.

The mol-ecule of the title compound, C(17)H(15)NO(3), is slightly twisted, with a dihedral angle of 12.12 (3)° between the dihydro-indenone group and the pyridine ring. In the crystal, mol-ecules are connected into layers parallel to the ab plane via inter-molecular C-H⋯O hydrogen bonds. Weak π-π [centroid-centroid distance = 3.5680 (6) Å] inter-actions are also observed.

(E)-2-(3-Chloro-benzyl-idene)-5,6-dimeth-oxy-2,3-dihydro-1H-inden-1-one.

In the title compound, C(18)H(15)ClO(3), the dihydro-indenone group makes a dihedral angle of 8.56 (6)° with the bezene ring. In the crystal, the mol-ecules are inter-connected into a three-dimensional network via inter-molecular C-H⋯O hydrogen bonds. Weak C-H⋯π and π⋯π [centroid-centroid distances 3.6598 (9)-3.6913 (9) Å] inter-actions are also observed.

(E)-2-(3,4-Dimeth-oxy-benzyl-idene)-5,6-dimeth-oxy-2,3-dihydro-1H-inden-1-one.

In the title compound, C(20)H(20)O(5), the 2,3-dihydro-1H-indene ring system is essentially planar [maximum deviation = 0.010 (1) Å] and is inclined at an angle of 4.09 (4)° with respect to the phenyl ring. The C=C bond has an E configuration. In the crystal, the mol-ecules are linked into chains propagating in [102] via inter-molecular C-H⋯O hydrogen bonds. The crystal structure is further consolidated by C-H⋯π inter-actions.

Influence of DRD2 Polymorphisms on the Clinical Outcomes of Opioiddependent Patients on Methadone Maintenance Therapy.

INTRODUCTION: Dopamine receptor D2 (DRD2) is one of the dopamine receptors that have been studied in relation to opioid dependence. It is possible, therefore, that DRD2 gene (DRD2) polymorphisms influence treatment outcomes of patients with opioid dependence. The objective of this study was to investigate the influence of DRD2 polymorphisms on the clinical outcomes of opioid-dependent patients on methadone maintenance therapy (MMT). MATERIALS AND METHODS: Patients with opioid dependence (n = 148) were recruited from MMT clinics. Pain sensitivity, severity of the opiate withdrawal syndrome, and sleep quality were assessed using cold pressor test (CPT), Subjective Opiate Withdrawal Scale (SOWS-M), and Pittsburgh Sleep Quality Index (PSQI)-Malay, respectively. Deoxyribonucleic acid (DNA) was extracted from whole blood, and then was used for genotyping of Val96Ala, Leu141Leu, Val154Ile, Pro310Ser, Ser311Cys, TaqI A, -141C Ins/Del, and A-241G polymorphisms. RESULTS: Among 148 patients, 8.1% (n = 12), 60.8% (n = 90), 27.7% (n = 41), and 29.1% (n = 43) had at least one risk allele for Ser311Cys, TaqI A, -141C Ins/Del, and A-241G polymorphisms, respectively. There were no significant differences in pain responses (pain threshold, tolerance, and intensity), SOWS, and PSQI scores between DRD2 polymorphisms. CONCLUSION: The common DRD2 polymorphisms are not associated with pain sensitivity, severity of the opiate withdrawal syndrome, and sleep quality in patients with opioid dependence on MMT. However, this may be unique for Malays. Additional research should focus on investigating these findings in larger samples and different ethnicity.