3-amino-3-demethoxyfortimicin A and the C-2 epimeric-2-amino-3-O-demethyl-2-deoxyfortimicins A

Selective reactions of 3-O-demethyl-3-O-methanesulfonyl-4-N, 5-O-methylenefortimicin derivatives have been used as the key steps in the syntheses of 3-amino-3-demethoxyfortimicin A and the C-2 epimeric 2-amino-3-O-demethyl-2-deoxyfortimicins A. In vitro antibacterial activities of the new fortimicin derivatives are reported.     

6-O-demethyl-5-deoxyfusarubin and its anhydro derivative produced by a mutant of the fungus Nectria haematococca blocked in fusarubin biosynthesis

6-O-Demethyl-5-deoxyfursarubin and 6-O-demethyl-5-deoxyanhydrofusarubin have been isolated from the mutant redD169.yelY9 of the fungus Nectria haematococca blocked in fusarubin biosynthesis. These products were identified on the basis of physico-chemical data by comparison with known substances.     

Influence of aglycone modifications on the binding of anthracycline drugs to DNA: the molecular structure of idarubicin and 4-O-demethyl-11-deoxydoxorubicin complexed to d(CGATCG)

X-ray diffraction analyses of the complexes between two anthracycline antitumor compounds, idarubicin (IDR) and 4-O-demethyl-11-deoxydoxorubicin (ddDOX), with the DNA hexamer d(CGATCG) provided the detailed three-dimensional molecular structures at 1.7 A and 1.8 A resolution, respectively. Their structures have been refined with the constrained refinement procedure to final R-factors of 0.188 (1724 reflections for IDR) and 0.179 (1247 reflections for ddDOX). The overall structures of both complexes are similar to those of the previously studied DAU- and DOX-DNA complexes. In both complexes, two IDR (and ddDOX) molecules bind to the DNA hexamer double helix with the elongated aglycone chromophore intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans the GC Watson-Crick base pairs with its amino sugar lying in the minor groove where little structural difference is seen, compared with the daunorubicin-d(CGATCG) and doxorubicin-d(CGATCG) complexes. In contrast, the missing C4 methoxy of IDR and the missing methyl group at the O4 position of ddDOX result in a different binding surface in the major groove. The O4 hydroxyl group is capable of receiving and/or donating a hydrogen bond to proteins that bind to the drug-DNA complex. The missing O11 hydroxyl group in ring B creates an empty space in the intercalation cavity between the two GC base pairs, which appears to affect the stacking interactions between the aglycone and the DNA base pairs. Those structural changes in the major groove of the drug-DNA complexes due to the modifications of the aglycone chromophore may be responsible in part for the difference in their biological activities.     

Low toxic derivatives of istamycin B: synthesis and preliminary evaluation.

3-O-Demethylistamycin B derived from istamycin B was one of the most potent aminoglycoside antibiotics against various bacteria. 3-O-Demethylistamycin B, however, showed considerable acute toxicity in mice. The authors attempted to prepare the derivatives of istamycin B having high potency and low toxicity. The selective N-acylation or N-amidination at the C-2 position of istamycin B could not improve the acute toxicity. The replacement of the amino group at the C-2 position of istamycin B by a hydroxyl group markedly decreased the acute toxicity. Among 2'-deamino-2'-hydroxyistamycins, 4-N-(beta-alanyl)-2'-deamino-3-O-demethyl-2'-hydroxyistamycin B0 (9d) showed good antibacterial activity against Gram-positive and Gram-negative bacteria and a low acute toxicity in mice.     

Preparation and biological evaluation of 4-O-demethyldaunorubicin (carminomycin I) and of its 13-dihydro derivative

A mutant strain of Streptomyces peucetius produced an anthracycline antibiotic whose structure has been established to be 4-O-demethyl-13-dihydrodaunorubicin (4), by application of spectroscopic methods and chemical degradation. A new synthesis of 4-O-demethyl-daunorubicin (carminomycin I, 2) starting from daunomycinone, together with the comparison of the antitumor activity of the anthracycline glycosides 2 and 4 are also reported.     

Pharmacogenomics as molecular autopsy for forensic toxicology: genotyping cytochrome P450 3A4*1B and 3A5*3 for 25 fentanyl cases

Pharmacogenomics, the study on genetic contributions to drug action may help in certifying fentanyl toxicity. Fentanyl is used clinically as an adjunct to surgical anesthesia and for chronic pain management. Its toxicity may be partially due to cytochrome P450 (CYP) 3A4*1B and 3A5*3 variant alleles, resulting in variable fentanyl metabolism. In this study, we examined 25 fentanyl-related deaths (22 Caucasians, 1 African-American, and 2 Native-Americans) from the Milwaukee County Medical Examiner's Office and referral cases. Fentanyl and norfentanyl in postmortem blood samples were analyzed by radioimmunoassay and liquid chromatography-mass spectrometry-mass spectrometry. The samples were then genotyped for CYP3A4*1B and 3A5*3 using Pyrosequencing. Genotyping showed: 1 CYP3A4*1B homozygous and CYP3A5*3 heterozygous, 1 compound CYP3A4*1B and CYP3A5*3 heterozygous, 22 CYP3A4*1B wild type and CYP3A5*3 homozygous, and 1 CYP3A5*3 and CYP3A4*1B wild type. CYP variant allelic frequencies of the 25 cases were 6% for CYP3A4*1B and 92% for CYP3A5*3, compared with normal Caucasian CYP3A4*1B, 3-8%, and CYP3A5*3, 85-95%. The mean fentanyl concentration and metabolic ratio of fentanyl to norfentanyl of the 2 cases with CYP3A4*1B and CYP3A5*3 variants were 12.8 and 1.4 microg/L, respectively, lower than those of 22 cases with wild type CYP3A4*1B and CYP3A5*3 homozygous variants, 16.7 and 7.3 microg/L, respectively. The postmortem/in vivo data provided the first scientific evidence that CYP3A5 is involved in the fentanyl metabolism, and homozygous CYP3A5 *3 causes impaired metabolism of fentanyl, and genotyping CYP3A4*1B and 3A5*3 variants may help to certify the fentanyl toxicity.     

CYP3A5~*3和CYP3A4~*18B基因多态性对肾移植患者环孢素药代动力学的影响

目的回顾性研究肾脏移植后1mon,CYP3A5*3和CYP3A4*18B基因多态性对CsA药代动力学参数的影响。方法采用PCR-RFLP方法分析了63名肾脏移植患者CYP3A5*3和CYP3A4*18B基因型;荧光偏正免疫法用于检测肾移植患者静脉全血中的CsA浓度。结果在63名肾移植患者中,CYP3A5*3和CYP3A4*18B突变等位基因发生频率分别为0.770(95CI:0.767～0.773),0.235(95CI:0.235～0.241),而且这些等位基因表现出完全连锁不平衡。在移植术后1mon内,携带CYP3A4*1/*1野生型纯合子患者的C0以及剂量校正谷血浓度(C0/D)均明显高于携带CYP3A4*1/*18B杂合子或CYP3A4*18B/*18B突变型纯合子患者(P<0.05,Mann-WhitneyUtest);CYP3A5*1/*1基因型组的给药剂量明显高于CYP3A5*1/*3或CYP3A5*3/*3基因型组(P=0.004<0.01,Kruakal-Wallistest);CYP34*18B和CYP3A5*3联合考虑,对于CYP3A5表达组,同样发现C0、C0/D在CYP3A4*1/*1组C0以及C0/D均明显高于CYP3A4*1/*18B或CYP3A4*18B/*18B组(P<0.05,Mann-WhitneyUtest);而其他药动学参数在CYP3A5*3及CYP3A4*18B各组间相比差异则没有统计学意义。结论CYP3A5*3和(或)CYP3A4*18B基因多态性对肾移植后1monCsA药代动力学有一定影响,移植前CYP3A5*3基因型的分析仍需进一步研究。     

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors α3β2 and α4β2 stably expressed in HEK293 cells

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations α3β2 or α4β2. [³H]-(±)Epibatidine ([³H]-(±)EPI) bound to membranes from A3B2 (α3β2) and A4B2.2 (α4β2) cells with K_d values of 7.5 and 33.4 pM and B_max values of 497 and 1564 fmol/mg protein, respectively.Concentration-dependent increases in intracellular free Ca²⁺ concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-β-erythroidine (DHβE) in A3B2 cells and MEC=DHβE>d-Tubo in A4B2.2 cells.Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in α3β2 and α4β2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca²⁺, respectively.Our results indicate that stably expressed α3β2 and α4β2 human nAChRs are pharmacologically and functionally distinct.     

Pharmacological comparison of human homomeric 5-HT3A receptors versus heteromeric 5-HT3A/3B receptors

The present study determined the detailed pharmacological profile of heterologously expressed human (h) homomeric 5-HT3A receptors in direct comparison to heteromeric h5-HT3A/3B receptors. The very minor differences in their respective pharmacological profiles indicates that the 5-HT3B receptor subunit alters, predominantly, the biophysical rather than the pharmacological properties of the 5-HT3 receptor.     

Expression of cyclic GMP-inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: Differential subcellular localization and regulated expression by cyclic AMP

1. A combination of pharmacological, molecular biological and biochemical approaches were used to investigate the differential expression of two cyclic GMP-inhibited cyclic nucleotide phosphodiesterase genes (PDE3A and PDE3B) in the rat.
2. RT–PCR using PDE3A- or PDE3B-specific oligonucleotide primers allowed amplification of products encoding PDE3A (508 bp) or PDE3B (499 bp) sequences from several rat tissues (heart, aorta, liver, kidney and epididymal fat), from primary cultures of aortic vascular smooth muscle cells (VSMC) as well as from an SV40 large T-antigen immortalized aortic VSMC line.
3. Immunoblotting experiments with PDE3-selective antisera allowed the detection of both PDE3A and PDE3B immunoreactive proteins in several rat tissues, including tissues of the cardiovascular system, in primary cultures of aortic VSMC and in an SV40 large T-antigen immortalized aortic VSMC line. In all cases, PDE3A was expressed as a 120 kDa protein which was only detected in the cytosolic fraction. PDE3B was expressed as a 135 kDa protein and its expression was limited to the particulate fraction of all tissues and cells studied.
4. Prolonged incubation of cultured aortic VSMC with agents that increase VSMC cyclic AMP (forskolin or 8-bromo-cyclic AMP) produced marked time-dependent increases in PDE3 activity which correlated with increases in PDE3A and PDE3B RT–PCR signals and a marked increase in particulate PDE3 activity and PDE3B protein.
5. The physiological, pharmacological and biochemical implications of these findings are discussed based on previous reports of the effects of PDE3 inhibitors in the cardiovascular system and the relevance of our findings are presented in the context of the development of PDE3A and/or PDE3B-selective pharmacological agents.

     

Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8)

OBJECTIVE: Hepatocellular uptake transporters are involved in the hepatobiliary elimination of endogenous and xenobiotic substances. Mutations in genes encoding these uptake transporters may be key determinants of interindividual variability in hepatobiliary elimination and drug disposition. Our aim was to investigate the functional consequences of mutations in the SLCO1B3 gene encoding the hepatic uptake transporter for organic anions OATP1B3, formerly termed OATP8. METHODS: Mutations occurring in Caucasian Europeans and observed in databases were introduced into the SLCO1B3 cDNA and the consequences were analyzed in stably transfected canine MDCKII cells and human HEK293 cells. The functional consequences were examined for two frequent polymorphisms SLCO1B3-334T>G, encoding OATP1B3-S112A (allelic frequency of 74%) and SLCO1B3-699G>A, encoding OATP1B3-M233I (allelic frequency of 71%) and one rare polymorphism SLCO1B3-1564G>T, encoding OATP1B3-G522C (allelic frequency of 1.9%) and one artificial mutation SLCO1B3-1748G>A, encoding OATP1B3-G583E. RESULTS: OATP1B3-S112A, OATP1B3-M233I, and the OATP1B3 protein corresponding to the reference sequence (accession NM_019844), showed a comparable lateral localization in stably transfected MDCKII cells, whereas OATP1B3-G522C and OATP1B3-G583E proteins were retained intracellularly. Both latter amino acid substitutions abolished the transport of bile acids mediated by OATP1B3, whereas other substrates, like bromosulfophthalein, were transported by all polymorphic variants of the protein. CONCLUSIONS: The functional consequences of three polymorphisms and one artificial mutation include differences in the localization and in transport characteristics of several OATP1B3 proteins. This study demonstrates the importance of the analysis of genetic variations in genes encoding transport proteins for the understanding of individual variations in the hepatobiliary elimination of substances.     

CYP3A5*3 and CYP3A4*1B allele distribution and genotype combinations: differences between Spaniards and Central Americans

The aim of this study was to detect genotypic differences between three populations of healthy volunteers from Northern Spain (204 subjects), Nicaragua (120 subjects), and El Salvador (112 subjects) regarding CYP3A4*1B and CYP3A5*3 polymorphisms. No significant differences were found by comparing allelic frequencies between the two Central American populations. The CYP3A5*3 allele frequency was significantly different (P < 0.01) between Central Americans (76%) and Spaniards (91%). By contrast, CYP3A4*1B allele was more prevalent among Central Americans (12.5%) than among North Spaniards (4%) (P < 0.01). Analysis of CYP3A4-3A5 genotype combinations revealed that individuals carrying CYP3A4*1B/CYP3A5*1 were more represented in Central Americans (16.9%) than in Spaniards (5.4%), suggesting a marked linkage disequilibrium. These data are compatible with a higher CYP3A enzyme activity in Central Americans as opposed to Spaniards and other white groups, which could imply differences in dose requirements for drugs metabolized by CYP3A and should be considered in allele-disease association studies.     

Naturally occurring variants in the HTR3B gene significantly alter properties of human heteromeric 5-hydroxytryptamine-3A/B receptors

OBJECTIVES: The 5-hydroxytryptamine-3 (5-HT3) receptor, a ligand-gated ion channel, is known to be involved in gut motility and peristalsis, the mediation of pain and psychiatric diseases. 5-HT3 receptor antagonists are effectively used to treat chemotherapy-induced emesis and irritable bowel syndrome. We have characterized the impact of four naturally occurring variants in the HTR3B gene leading to amino acid exchanges within the respective subunit of heteromeric 5-HT3A/B receptors on a functional and expressional level. METHODS AND RESULTS: For functional characterization, a Ca influx assay based on aequorin bioluminescence was used. Radioligand-binding studies with the 5-HT3 receptor antagonist [H]GR65630 were carried out to determine expression levels of heteromeric 5-HT3A/B receptors. Transiently transfected human embryonic kidney 293 cells using 5-HT3A and 5-HT3B complementary DNA constructs were shown to coexpress homopentameric 5-HT3A next to heteromeric 5-HT3A/B receptors. The variant p.V183I decreased surface expression, whereas p.Y129S and p.S156R led to pronounced increases of 5-HT maximum responses, despite nearly unaltered surface expression levels of heteromeric 5-HT3A/B receptors. CONCLUSION: These results may help to explain earlier reported association findings of the frequent p.Y129S and p.V183I variants with psychiatric diseases. Replication studies with larger sample pools, especially regarding the rare p.S156R variant would be useful, to obtain an idea about the predisposing role of these single nucleotide polymorphisms as susceptibility variants.     

Substituted tetrahydroisoquinoline compound B3 inhibited P-glycoprotein-mediated multidrug resistance in-vitro and in-vivo

P-glycoprotein (P-gp) mediated multidrug resistance (MDR) is one of the main obstacles in tumour chemotherapy. A promising approach to reverse MDR is the combined use of nontoxic and potent P-gp inhibitor with conventional anticancer drugs. We have examined the potential of a newly synthesized tetrahydroisoquinoline derivative B3 as a MDR-reversing agent. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to examine the effect of B3 on the cytotoxicity in K562/A02 and MCF-7/ADM cells caused by doxorubicin (adriamycin). Accumulation and efflux of P-gp substrate rhodamine123 in K562/A02 and primary cultured rat brain microvessel endothelial cells (RBMECs) were measured to evaluate the inhibitory effect of B3 on P-gp. The K562/A02 xenograft model in nude mice was established to examine MDR-reversing efficacy of B3 in-vivo. The results indicated that co-administration of B3 resulted in an increase on chemosensitivity of K562/A02 and MCF-7/ADM cells to doxorubicin in a dose-dependent manner. Rhodamine123 accumulation in K562/A02 cells and RBMECs were significantly enhanced after the incubation with various concentrations of B3. Furthermore, B3 inhibited the efflux of rhodamine123 from RBMECs. Co-administration of B3 with doxorubicin significantly decreased weight and volume of tumour in nude mice. In conclusion, B3 is a novel and potent MDR reversal agent with the potential to be an adjunctive agent for tumour chemotherapy.     

Introduction of the 5-HT3B subunit alters the functional properties of 5-HT3 receptors native to neuroblastoma cells

The identification of a second 5-HT(3) (5-HT(3B)) subunit provides an explanation for 5-HT(3) receptor heterogeneity. We investigated whether introduction of recombinant 5-HT(3B) subunits would alter the functional properties of mouse neuroblastoma 5-HT(3) receptors. RT-PCR analysis revealed that NB41A3 cells contain mRNAs encoding 5-HT(3A) and 5-HT(3B) subunits. 5-HT increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and caused the concentration-dependent activation of inward currents recorded at -60 mV. Both actions of 5-HT were antagonized by ondansetron. The 5-HT concentration-response relationship of NB41A3 cells was indistinguishable from that of the related NG108-15 cell line. The selective 5-HT(3)-receptor agonist mCPBG also elevated [Ca(2+)](i) and activated inward currents. 2-M-5HT was less efficacious than 5-HT as an activator of 5-HT(3) receptors in NB41A3 cells and did not significantly increase [Ca(2+)](i). The 5-HT induced increase in [Ca(2+)](i) did not involve caffeine- or thapsigargin-sensitive intracellular Ca(2+) stores. The introduction of the 5-HT(3B) subunit by transient transfection of NB41A3 cells caused 5-HT to become less potent as an activator of 5-HT(3) receptors and altered the kinetics of 5-HT activated currents so that they resembled currents mediated by 5-HT(3AB) receptors. The 5-HT(3B) subunit also abolished the 5-HT induced [Ca(2+)](i) increase seen in untransfected NB41A3 cells. These data are consistent with the hypothesis that NB41A3 cells predominantly express homomeric 5-HT(3A) receptors that become heteromeric 5-HT(3AB) receptors upon introduction of the recombinant 5-HT(3B) subunit.     

Enzymes in addition to CYP3A4 and 3A5 mediate N-demethylation of dextromethorphan in human liver microsomes

Both indinavir and troleandomycin (CYP3A inhibitors) are incapable of completely inhibiting dextromethorphan metabolism to 3-methoxymorphinan in human liver microsomes. It is hypothesized that CYPs in addition to CYP3A4 and 3A5 contribute to this biotransformation. The effect of CYP-selective inhibitors on the residual 3-methoxymorphinan activity in human liver microsomes (i.e. in the presence of 30 microM indinavir, a selective CYP3A4 and 3A5 inhibitor) was measured to identify these enzymes. At this concentration, indinavir completely inhibited the formation of 3-methoxymorphinan by rCYP3A4 and rCYP3A5. In addition, the formation kinetics of 3-methoxymorphinan in rCYPs was measured. Only CYP2B6, 2C8 and 2C18 were considered likely candidates as contributors to residual 3-methoxymorphinan activity. The residual 3-methoxymorphinan activity was highly correlated with CYP2B6 activity as measured by CYP2B6 antibody (r(2)=0.90, p<0.001) and by orphenadrine (r(2)=0.97, p<0.001), but was not correlated (r(2)=0.12, p>0.05) with CYP2C8 activity. Collectively, these findings suggest that CYP2B6 is a major contributor towards residual 3-methoxymorphinan activity, while CYP2C8 and 2C18 are either minor contributors or do not contribute to this metabolic process.     

Effect of CYP3A4 genetic polymorphisms on pharmacokinetics of tinidazole

In the present study, we aimed to investigate the frequency of CYP3A4*18B genetic polymorphism in Han Chinese populations, and to assess the effect of the CYP3A4*18B genetic polymorphism on the pharmacokinetics of tinidazole. A total of 100 healthy volunteers from Han nationalities in China were recruited. DNA was extracted from peripheral leukocytes using a standard protocol. A PCR-RFLP method was developed to detect the alleles of CYP3A4*18B. A pharmacokinetic study of tinidazole was then carried out in two groups with CYP3A4*1/*1 (n = 10) and CYP3A4*1/*18B (n = 9) genotypes. Concentrations of tinidazole were determined using high-performance liquid chromatography in plasma samples that were collected up to 72 h after drug intake. In this study, 88 healthy volunteers were found with CYP3A4*1/*1 genotype, and 12 were found with CYP3A4*1/*18Bgenotype. CYP3A4*18B/*18B were absent from all subjects. The allele frequencies of CYP3A4*18B were 6%. The pharmacokinetic parameters of CYP3A4*1/*1 genotype and CYP3A4*1/*18B genotype in healthy subjects were as follows: t_1/2: (15.92±1.62), (15.77±1.67) h; C_max: (18.72±3.10), (20.25±3.42) mg/L; t_max: (1.50±0.66), (1.45±0.69) h; V_d/F: (55.73±10.66), (51.30±7.75) L; CL/F: (2.44±0.47), (2.26±0.30) L·h; AUC_0–∞: (424.40±82.38), (450.53±69.48) mg·h/L. Collectively, the CYP3A4*18B genetic polymorphism did not affect pharmacokinetics of tinidazolein healthy volunteers.     

Interaction of 3′-Azido-3′-Deoxythymidine With the Organic Base Transporter in a Cultured Renal Epithelium

In humans and various animal species, 3′-azido-3′-deoxythymidine (AZT) is in part eliminated by the kidneys, where it undergoes significant tubular secretion. The goal of this project was to develop, in a continuous renal epithelial cell line (LLCPK₁), a model of AZT transport in which mechanisms of drug interactions could be investigated. Transport properties of H³-AZT were studied in LLCPK₁ cells grown as monolayers on permeable filters. This system provides access to the basolateral and apical surfaces of the epithelium and allows the determination of substrate transepithelial flux from the basolateral side to the apical side (B → A/secretory direction) and apical to basolateral side (A → B/reabsorptive direction). The B → A flux of AZT was significantly greater than B → A flux of mannitol (a nontransported substrate) and was temperature dependent (37°C > 4°C). The AZT A → B flux was significantly smaller than the B → A flux, indicating that the drug is predominantly secreted in this renal epithelium. The B → A flux was significantly inhibited by the organic bases cimetidine, quinine, quinidine, and trimethoprim. Log concentration dose studies indicate that quinine is a weak inhibitor (IC₅₀ = 9.61 mM) of AZT B → A flux, and that AZT is a moderate inhibitor (IC₅₀ = 0.69 mM) of the organic base cimetidine. These results suggest that AZT may share the organic base transporter in the renal epithelium, and that this model can be used successfully to study transport properties and renal drug-drug interactions of AZT.