Synthesis of a novel L-nucleoside, beta-L-D4A and its inhibition on the replication of hepatitis B virus in vitro / 药学学报

<p><b>AIM</b>Nucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, beta-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus (HBV) in 2. 2. 15 cells derived from HepG2 cells transfected with HBV genome.</p><p><b>METHODS</b>beta-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, 1H NMR and MS. 2. 2. 15 Cells were placed at a density of 5 x 10(4) per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a 32P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC50 was calculated. 2. 2. 15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC50 was calculated.</p><p><b>RESULTS</b>The synthesized compound structure conformed with beta-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC50 0.2 micromol x L(-1). The experiment of cytotoxicity gained IC50 200 micromol x L(-10.</p><p><b>CONCLUSION</b>beta-L-D4A has been synthesized successfully. beta-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.</p>

Effect and mechanism of beta-L-D4A (a novel nucleoside analog) against hepatitis B virus / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effect and the molecular targets of anti-hepatitis B virus (HBV) by beta-L-D4A in vitro.</p><p><b>METHODS</b>2.2.15 cells were cultured and treated with various concentrations of beta-L-D4A for 6 hours, then the effect of anti-HBV was examined by Southern blot and the replicating core particles from the cells were isolated. The endogenous polymerase reaction and activity gel experiment were performed to monitor the activities of the DNA polymerase and reverse transcriptase.</p><p><b>RESULTS</b>The replication of HBV DNA was inhibited in a dose-dependent manner. The endogenous polymerase reaction showed both the two enzymatic activities were irreversibly inactivated in a concentration -dependent manner, with IC50 at 0.51 micromol/L and 0.55 micromol/L, respectively. But the activities of DNA polymerase and reverse transcriptase were found to remain active by activity gel with exogenous templates.</p><p><b>CONCLUSIONS</b>The mechanism of inhibiting HBV replication by beta-L-D4A may be in that either the DNA replication priming is blocked or the elongation of DNA chain is terminated irreversibly.</p>

Toxicidade dose e tempo dependente da 2', 3' - didesoxiinosina em pâncreas, linfonodo e timo de rato / Toxicity dose-and time-dependent 2 ', 3'- dideoxyinosine in pancreas, lymph node and thymus mouse

O anti-retroviral análogo de nucleosídeo 2',3' - didesoxiinosina (ddl) tem uma ampla utilização clínica no tratamento de pacientes com AIDS iu HIV positivos. Apesar da toxicidade reduzida, alguns problemas tem sido levantados com a utilização da ddl em tratamento a longo prazo. Vários estudo clínicos tem associado a pancreatite com a terapia prolongada de ddl em pacientes infectados com o HIV...Nenhum tratamento com ddl modificou a amilase sérica determinada na época do sacrifício dos animais. Nos ratos tratados com ddl, 600 mg kg -1 dia -1, por mais de 28 dias foi observada uma marcada hipoglicemia (i.p. = 34,7 +- 7,4 mgdL-1 p,,0,05, s.c.= 45,0 +- 7,5 mg dL -1, p,,0,05). Conclui-se que a ddl induz um efeito tóxico direto dose e tempo dependente nos tecidos pancreático e linfóide de rato. Os resultados suportam o envolvimento da ddl na pancreatite relatada em pacientes com AIDS tratados com ddl e são compatíveis com a hipótese de que esse anti-retroviral pode afetar a resposta imune humoral. No conjunto, este estudo indica que a toxicidade potencial do tratamento crônico com ddl deve ser mais profundamente examinada.

Augmentation of Regional Cerebral Blood Flow Response by Repeated Administration of Methamphetamine in Rat / 대한정신약물학회지

OBJECTIVES: It was aimed to observe the regional cerebral blood flow (rCBF) response on methamphetamine challenge test in rats which were subjected to repeated administration of methamphetamine, and to investigate the mechanism(s) of changes in rCBF response in relation to the dopaminergic receptors and cyclic AMP. METHODS: Male Sprague-Dawley rats received daily injections of methamphetamine (0.3 mg/kg, i.p.) for 10 days, and were then allowed a 4-day drug-free period. Naive and methamphetamine-pretreated rats were challenged with topical application of methamphetamine on the surface of parietal cortex through a cranial window. The changes in rCBF were measured by laser-Doppler flowmetry. RESULTS: Acute topical application of methamphetamine dose-dependently increased rCBF with little effect on mean arterial blood pressure. The methamphetamine-induced increases in rCBF were significantly blocked by SCH23390, a D1-like receptor antagonist, but not by sulpiride, a D2-like receptor antagonist. Repeated administration of methamphetamine induced progressive augmentation of rCBF in response to the challenge of methamphetamine. Repeated administration of methamphetamine in combination with SKF38393, a D1-like receptor agonist, as well as with SCH23390 significantly attenuated the development of augmentation of rCBF response to methamphetamine. The augmentation of rCBF response was markedly inhibited by pretreatment with 2',3'-dideoxyadenosine, a specific adenylyl cyclase inhibitor, and Rp-cAMPS, a protein kinase A inhibitor, respectively. CONCLUSION: Based on these results, it is suggested that repeated administration of methamphetamine induces an augmentation of rCBF in response to the challenge of methamphetamine, and that D1-like receptor-mediated cyclic AMP plays a critical role in the development of augmentation of methamphetamine-induced rCBF response.

Effect of adenylate cyclase inhibitor and protein kinase C inhibitor on GnRH-induced LH release and LH beta subunit biosynthesis in rat anterior pituitary cells

According to our previous studies together with others, GnRH, a hypothalamic decapeptide, has been known to be a major regulator for LH release and its subunit biosynthesis in anterior pituitary gonadotropes. But the precise mechanisms by which GnRH exerts stimulatory effects on LH release and its subunit biosynthesis have not been clearly understood. In the present study we examined the effect of GnRH on protein kinase C (PKC) activity and intracellular cAMP content in cultured anterior pituitary cells of rat to clarify whether PKC or cAMP are involved in GnRH action. Moreover, we examined the effects of staurosporine (ST), a PKC inhibitor and 2',3'-dideoxyadenosine (2',3'-DDA), an adenylate cyclase inhibitor, on LH release and steady state LH beta subunit mRNA levels in cultured anterior pituitary cells of rat. PKC activity was rapidly increased within 30 min after GnRH treatment whereas intracellular cAMP level was elevated 18 h after GnRH treatment. ST significantly inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner, showing an half maximal response at 50 nM ST. 2',3'-DDA inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner in pituitary cells. From these results, it is suggested that GnRH stimulates LH beta subunit mRNA level as well as LH release in anterior pituitary cells and this GnRH action might be mediated by PKC activation and cAMP stimulation.

Farmacología de las drogas empleadas en el tratamiento del SIDA / Pharmacology of the drugs employee in the treatment of AIDS

En esta revisión (transcripción de la presentación realizada en el Simposio SIDA, Avellaneda 1990) se enumeran las drogas empleadas para: 1)tratar las complicaciones del SIDA (solo algunos ejemplos, incluyendo foscarnet y glanciclovir); 2)realizar la inmunoterapia del SIDA; y 3)inhibir la replicación del HIV (quimioterápicos anti-HIV). En la segunda clase se enfatiza la necesidad de actuar precozmente (por ej., con valores no muy bajos de CD4), y se analizan evidencias preliminares sobre isoprinosina, timopentina y ditiocarb. En la tercera se analiza en detalle el mecanismo de acción de los dideoxinucleósidos, ejemplificando con la zidovudina, sus efectos adversos, sus limitaciones, en particular para tratamiento precoz, y el desarrrollo de resistencia en el HIV tanto in vitro como in vivo. Se analiza, también, el efecto del interferón * sobre la replicación viral y se esbozan los tratamientos más experimentales en farmacología clínica como el CD4 recombinante soluble) e in vitro (como los oligonucleótidos antisense). Pese a disponer de drogas efectivas en cada una de las tres categorías enunciadas, ninguna hace más que retrasar el curso de la infección hacia la destrucción del sistema inmune y muerte del paciente