Biologically active oligodeoxyribonucleotides. 5. 5'-End-substituted d(TGGGAG) possesses anti-human immunodeficiency virus type 1 activity by forming a G-quadruplex structure.

A series of hexadeoxyribonucleotides (6-mers), d(TGGGAG), substituted with a variety of aromatic groups at the 5'-end were synthesized and tested for anti-human immunodeficiency virus type 1 (HIV-1) activity. While unmodified d(TGGGAG) (31) had no anti-HIV-1 activity, compound 23 with a 3,4-di(benzyloxy)benzyl (DBB) group at the 5'-end potently inhibited the HIV-1IIIB-induced cytopathicity of MT-4 cells in vitro (IC50 = 0.37 microM) without cytotoxicity up to 40 microM. A thermal denaturation study on the 5'-end-substituted 6-mers by means of the circular dichroism (CD) spectra demonstrated that the aromatic substituent attached at the 5'-end of the 6-mer strongly enhanced the formation of a parallel helical structure consisting of four strands (quadruplex). On the contrary, compound 36, in which one of the guanosines of 23 was replaced by a thymidine, did not form a quadruplex, thus exhibiting no anti-HIV-1 activity. Moreover, both compound 15, with a tert-butyldiphenylsilyl group solely at its 3'-end, and compound 21, with a relatively small substituent, a benzyl group, at the 5'-end, formed quadruplexes but had no anti-HIV-1 activity. These findings led us to the conclusion that both the quadruplex structure and the aromatic substituent with adequate size at the 5'-end are crucial for the interaction of the 5'-end-substituted 6-mers with the V3 loop as well as the CD4 binding site on viral gp120, resulting in anti-HIV-1 activity.

Genetic analysis of HIV-1 during rapid progression to AIDS in an apparently healthy man.

We encountered a case of HIV-1 infection in a previously healthy man, which was characterized by rapid progression to AIDS and death within 7 months in association with high levels of antigenemia throughout the clinical course and no humoral immune response for at least 6 months. Genetic changes of the third variable domain (V3) of the envelope gene of HIV-1 in serum samples were analyzed at four time points during his rapid clinical course. The nucleotide changes were confined to a maximum of three substitutions among 105 nucleotides of the V3 region. A major population of the viral clones in this patient showed one amino acid substitution from aspartic acid (a negatively charged amino acid) to lysine (a positively charged amino acid) at position 30 from the first cysteine of the V3 loop. This substitution was thought to be associated with phenotypic changes, and viruses with this sequence in the V3 region had a strong syncytium-inducing ability in MT-4 cells. It appears that the lack of a humoral immune response accelerated disease progression in our patient and a genetic change that appeared to produce a phenotypic change occurred at an early stage of the disease.

Establishment of lymphoid cell lines from persons with different P blood group phenotypes and the biosynthesis of the antigenic glycosphingolipids.

Human lymphoid cell lines were established from the peripheral lymphocytes of persons with different P blood group phenotypes by in vitro transformation with EB virus. The glycosphingolipid compositions of these cell lines were examined by TLC. The results show that P1k and P2k phenotype cells lack globoside (P antigen) and that two cell lines established from persons with the p phenotype lack both globoside and CTH (Pk antigen), while both the glycosphingolipids were detected in two cell lines established from persons with usual phenotypes (P1 or P2 phenotype). CTH synthetase activity was detected at decreased levels in two p phenotype cell lines, however, globoside synthetase activities could not be significantly detected in all the P phenotype cells.

Absorption, distribution and excretion of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate in rats, dogs and monkeys.

Absorption, distribution and excretion of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate (MN-1695) were studied in rats, dogs and monkeys after administration of [14C]-MN-1695. MN-1695 was found to be well absorbed from the small intestine after oral administration in all species examined. Plasma level of unchanged MN-1695 reached a maximum at 1 to 4 h after oral administration of [14C]-MN-1695 in rats, dogs and monkeys. The mean elimination half-life of unchanged MN-1695 from plasma was about 3, 4 and 50 h in rats, dogs and monkeys, respectively. Tissue levels of radioactivity after oral administration of [14C]-MN-1695 in rats indicated that [14C]-MN-1695 was distributed throughout the body and the radioactivity in tissues disappeared with a rate similar to that in plasma. A stomach autoradiogram after intravenous administration of [14C]-MN-1695 in the rat revealed the radioactivity localized in the gastric mucosa where MN-1695 was assumed to exert its pharmacological activity. In pregnant rats, [14C]-MN-1695 was distributed to the fetus with levels similar to maternal blood levels. After oral administration of [14C]-MN-1695 in rats, 39 to 46% of the dose was excreted into the urine and 50 to 63% of the dose into the feces, within 96 h. In dogs, about 40% of the dose was excreted into the urine and about 50% of the dose into the feces, within 6 days after oral administration. In monkeys, within 14 days after oral administration, about 60 and 30% of the dose were excreted into the urine and feces, respectively, and the main excretion route was the urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of prulifloxacin. 1st communication: absorption, distribution and excretion in rats, dogs and monkeys after a single administration.

The pharmacokinetics of prulifloxacin ((+/-)-6-fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4-yl) methyl-1-piperazinyl]-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-car boxylic acid. CAS 123447-62-1, NM441), a quinolone antibacterial prodrug, was investigated after i.v. (14C-NM394, CAS 112984-60-8) or oral (14C-NM441) administration to rats, dogs and monkeys. 1. 14C-NM441 was absorbed mainly from the upper small intestine and then metabolized to NM394 partly in the intestinal membrane but mainly in the portal blood and liver. Thus NM441 was not detected in the systemic circulation. 2. After i.v. administration of 14C-NM394 (5 mg/kg), the plasma concentration of radioactivity decreased biexponentially, and the elimination half-life in rats, dogs and monkeys was 4.2, 5.8 and 7.0 h, respectively. After oral administration of 14C-NM441 (20 mg/kg), the plasma concentration of radioactivity reached a maximum at 0.7-3.3 h, and thereafter decreased as observed after i.v. administration of 14C-NM394. An effect of food on the absorption of NM441 was found. No clear sex-related differences were observed in the plasma concentration profiles of rats. 3. The concentration of radioactivity in most tissues of rats reached a maximum within 1 h after oral administration of 14C-NM441 and thereafter decreased along with the plasma concentration. At 0.5 h, the radioactivity concentrations were highest in the liver and kidney, moderately high in the spleen, pancreas, lung and mandibular gland and extremely low in the cerebrum and cerebellum. 4. The radioactivity in the excreta collected over a 96-h period was 96-98% of the oral dose (urine, 22-32%; feces, 64-75%) in rats, dogs and monkeys, 35% of the radioactivity administered was excreted in the bile of rats during a 48-h period after oral administration, and only a small portion of the biliary radioactivity was reabsorbed. 5. The proportion of 14C-NM394 that bound to serum proteins in vitro in rats, dogs, monkeys and humans was 41-59% in a concentration range of 0.1-10 micrograms/ml.

Pharmacokinetics of prulifloxacin. 2nd communication: pharmacokinetics and effect on hepatic drug-metabolizing enzyme activities after repeated administration and transfer into fetus and milk after a single administration in rats.

Prulifloxacin ((+/-)-6-fluoro-1-methyl-7-[4-(5-methyl-2-oxo-1,3-dioxolen-4-yl) methyl-1-piperazinyl]-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-car boxylic acid, CAS 123447-62-1, NM441) is a prodrug of a new quinolone carboxylic acid antibacterial agent, NM394 (CAS 112984-60-8). The pharmacokinetics of radioactivity after repeated oral administration of 14C-NM441, the effects of NM441 on hepatic drug-metabolizing enzyme activities after repeated oral administration of NM441, and the transfer of radioactivity into the fetus and milk after a single oral administration of 14C-NM441 were investigated in rats. 1. The plasma concentration of radioactivity 6 h after each oral dose of 14C-NM441 (20 mg/kg) to male rats once a day for 21 days was almost constant. There was no marked difference in the plasma concentration-time curves for radioactivity after the single, 7th, 14th or 21st administration. The averaged cumulative urinary and fecal excretion of radioactivity during repeated administration did not differ from the corresponding values after a single administration. The concentration of radioactivity 8 h after each dose had reached a plateau in most tissues by the 14th administration. After the 21st dose, the radioactivity concentration in most tissues decreased along with the plasma concentration, whereas a slower elimination was observed in the skin and bone. 2. Repeated oral administration of 20 or 200 mg/kg of NM441 to male rats did not affect hepatic drug-metabolizing enzyme activities. 3. In pregnant rats, the maximum concentration of radioactivity in the fetus was lower than that in the maternal plasma. Furthermore, the total amount of radioactivity in the fetus was only 0.01% of the dose at 0.5 h. 4. In lactating rats, the concentration of radioactivity in the milk was substantially higher than in the plasma. 5. In conclusion, repeated administration of NM441 did not alter its pharmacokinetics, and no evidence was found that it accumulated in the body. Furthermore, there was little placental transfer. These characteristics add to the suitability of NM441 as an effective prodrug of NM394.

Inhibition of human immunodeficiency virus type-1-induced syncytium formation and cytopathicity by complestatin.

Complestatin, an anti-complement agent, was shown to be a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) infection in vitro. It inhibited HIV-1-induced cytopathicity and HIV-1 antigen expression in MT-4 cells; the 50% effective doses for these effects were 2.2 and 1.5 micrograms/ml, respectively. No toxicity for MT-4 cells was observed at concentrations up to 400 micrograms/ml. In addition, the agent inhibited the focus formation in HT4-6C cells (CD4-positive HeLa cells); the concentration for 50% focus reduction was 0.9 microgram/ml. HIV-1-induced cell fusion in cocultures of MOLT-4 cells and MOLT-4/HTLV-IIIB were also blocked by complestatin (the concentration for 50% cell fusion inhibition, 0.9 microgram/ml). Complestatin had no ability to inhibit HIV-1 reverse transcriptase activity. When MT-4 cells were pretreated with complestatin for 2 hrs prior to the exposure to HIV-1, the HIV-1-induced cytopathicity was markedly inhibited, while pretreatment of HIV-1 with the agent did not affect the infection. These results suggest that complestatin primarily interacts with cells and inhibits viral adsorption to the cell surface as well as adsorption of infected cells to adjacent cells.

Biologically active oligodeoxyribonucleotides--I: Syntheses and anti-HIV-1 activities of 5'-modified pentadecadeoxyribonucleotides.

5'-Modified pentadecadeoxyribonucleotides with a sequence (5'-TGGGAGGTGGGTCTG-3') (15mer) complimentary (antisense) to the tat 2nd splicing acceptor region of human immunodeficiency virus type 1 (HIV-1) were prepared and evaluated for anti-HIV-1 activity. The modified antisense oligodeoxyribonucleotides (AS-ODNs) were synthesized using 5'-modified thymidine 3'-phosphoramidites as the 5'-terminal residues. The structures of the 5'-modified 15mers were confirmed by negative ion LSI mass spectroscopy, and the anti-HIV-1 activities were evaluated in vitro by the MTT assay using MT-4 cells. While the unmodified 15mer had no activity in our assay system, the 15mers bearing modifications with trityl-type substituents at the 5'-end showed high anti-HIV-1 activities.

Mechanism of inhibition of HIV-1 infection in vitro by guanine-rich oligonucleotides modified at the 5' terminal by dimethoxytrityl residue.

Oligodeoxyribonucleotides (ODN) linked at their 5'-end with dimethoxytrityl (DmTr) residue were examined for antiviral activities against human immunodeficiency virus type 1 (HIV-1). We found that guanine-rich oligonucleotides exhibit anti-HIV activity upon 5'-end modification with DmTr. One oligonucleotide, DmTr-TGGGAGGTGGGTCTG (SA-1042), showed potent anti-HIV activity in vitro. A greater than 95% reduction of infectivity was observed if the cells were treated with 10 micrograms/ml of SA-1042 at the time of viral infection, PCR analysis confirmed that there was a significant reduction of provirus in the cells exposed to virus in the presence of SA-1042. By contrast, no inhibition was observed if the cells were treated with the oligomer 1 h after virus adsorption. SA-1042 prevented syncytium formation between chronically infected cells and CD4 positive uninfected cells. Furthermore, the oligomer interfered the interaction of purified gp120 to the CD4 receptor. By contrast, SA-1042 had no inhibitory effect on chronically HIV-infected cells. These results strongly suggest that the DMTr-ODNs with appropriate base sequences antagonize HIV-1 infection during the stage of virus-cell interaction.

Thyroid function in children with long-term anticonvulsant treatment.

Thyroid functions were studied in 287 children given long-term treatment with anticonvulsants. Of these 287 patients, 26 were treated with carbamazepine (CBZ) alone, 63 with phenobarbital (PB) alone, 66 with sodium valproate (VPA) alone and 132 with combination therapy. Serum triiodothyronine (T3) and thyroxine (T4) concentrations were decreased after more than 3 years of treatment with CBZ. The regression line of serum T4 concentrations against serum CBZ concentrations indicated a negative correlation. Serum T3 and T4 concentrations were decreased after more than 6 years of treatment with PB, although there was no statistical significance in the relationship between serum T4 and PB concentrations. There was no significant decrease in serum T3 or T4 concentration in patients treated with VPA. Serum-free T4 and thyroxine-binding globulin were decreased and serum thyrotropic hormone concentration was increased in patients with low serum T4 concentrations. We conclude that thyroid functions should be examined in children given long-term treatment with anticonvulsants.

Identification of a phosphodiester hexanucleotide that inhibits HIV-1 infection in vitro on covalent linkage of its 5'-end with a dimethoxytrityl residue.

It has been shown in previous reports that a guanine-rich phosphodiester oligonucleotide bearing a dimethoxytrityl (DmTr) residue on its 5'-terminal. DmTr-TGGGAGGTGGGTCTG (SA-1042), is an inhibitor of HIV-1 infection in vitro. SA-1042 interfered with the attachment of gp120 to the CD4 receptor and the subsequent entry stage of viral infection. We investigated the structure-activity relationship of the DmTr-conjugated oligomer by using 15-mer oligonucleotides with various nucleotide sequences. Results show that location of guanine nucleosides at the 5'-terminal and modification of the 5'-terminal with DmTr are essential for anti-HIV-1 activity. First, substitution of the guanine nucleoside close to the 5'-terminal of SA-1042 with another nucleotide prevented antiviral activity. Second, the existence of at least three consecutive guanine nucleosides adjacent to the 5'-terminal was required for the activity. Finally, modification of the 5'-terminal was essential for the activity. Based on these findings, the hexanucleotide, DmTr-TGGGAG, was identified as a potent inhibitor of HIV-1 infection. The hexamer was found to be capable of inhibiting the binding of gp120 to its receptor CD4 molecule, and it was also capable of inhibiting accessibility of anti-V3 monoclonal antibody to its ligand V3 peptide.

Joint-preserving operation for osteoarthrosis of the hip in adult cerebral palsy.

BACKGROUND: A joint-preserving operation was performed on 15 hips with osteoarthrosis, involving 12 patients who had adult cerebral palsy. METHODS: Eleven hips underwent Chiari pelvic osteotomy only; three hips underwent Chiari pelvic osteotomy with femoral osteotomy and the other one hip underwent femoral varus osteotomy only. The mean follow-up period after surgery was 6 years and 2 months (with follow-up range of 2 years and 3 months to 10 years and 6 months). RESULTS: Good results were achieved in 13 of the 15 hips (86.6%). Two patients with athetotic tetraplegia treated with Chiari pelvic osteotomy had pelvic obliquity. Progressive osteoarthrotic change continued in bilateral hips in one case treated with Chiari pelvic osteotomy. CONCLUSION: We confirm that usual treatment for osteoarthrosis of the hip was also applicable for osteoarthrosis of the hip in cases of adult cerebral palsy, provided sufficient attention is given to the complications accompanying spastic paralysis.

Supplementary thyroxine therapy in patients with hypothyroidism induced by long-term anticonvulsant therapy.

Of 287 patients under long-term therapy with anticonvulsants, 24 with low serum thyroxine and free thyroxine concentrations were prescribed supplementary thyroxine in the present study. In addition, the basal metabolic rate (BMR) was measured in 13 out of 24 patients and in eight of them it was low (under -15%). Serum thyroid hormone concentrations improved after administration of thyroxine. However, improvement of the BMR was not obtained after one month of supplementary therapy. EEG after administration showed an increase in the power spectra of the occipital alpha 2 band (10.0-12.8 Hz) and beta 1 band (13.0-19.8 Hz), and a decrease in that of the theta band (4.0-7.8 Hz). The interpeak latency from wave I to wave V in the ABR was normalized after administration. These results suggest that supplementary therapy with thyroxine may be necessary in patients with hypothyroidism induced by anticonvulsants.

Synthesis and anti-HIV activity of arylpiperazinyl fluoroquinolones: a new class of anti-HIV agents.

Synthesis and anti-HIV activity of a series of novel arylpiperazinyl fluoroquinolones are reported. In the SAR study, the aryl substituents on the piperazine nitrogen were found to play an important role for the anti-HIV-1 activity. A few of the compounds exhibited potent anti-HIV activity: IC50=0.06 microM in chronically infected cells.

A new fluoroquinolone derivative exhibits inhibitory activity against human immunodeficiency virus type 1 replication.

The inhibitory activity of several fluoroquinolone antibiotics against human immunodeficiency virus type 1 (HIV-1) replication was investigated. R-71762, (+/-) 9-fluoro-3-fluoromethyl-2, 3-dihydro-10-[4-(2-pyridyl)-1-piperazinyl]-7-oxo-7H-pyrido[1,2, 3-de][1,4]benzoxazine-6-carboxylic acid, protected MT-4 cells from HIV-1-induced cytopathic effects. Furthermore, the compound inhibited virus replication both in acutely and in chronically HIV-1-infected cells. On the other hand, ofloxacin, levofloxacin, ciprofloxacin, norfloxacin and enoxacin, that were previously reported to be protective against HIV-1-induced cytopathic effects, did not show any protective activity in our assay system. These results indicate that R-71762 is a novel inhibitor of HIV-1 replication and is effective even in HIV-1 chronically infected cells.

Biologically active oligodeoxyribonucleotides--IX. Synthesis and anti-HIV-1 activity of hexadeoxyribonucleotides, TGGGAG, bearing 3'- and 5'-end-modification.

We have determined that hexadeoxyribonucleotides (5'TGGGAG3'), with modified aromatic groups such as a trityl group at the 5'-end, have anti-HIV-1 activity in vitro. The 6-mer bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5'-end had the most potent activity and the least cytotoxicity. When the 3'-end of the 5'-(3,4-DBB)-modified 6-mer was substituted with a 2-hydroxyethylphosphate, a 2-hydroxyethylthiophosphate, or a methylphosphate group at the 3'-end, anti-HIV-1 activity increased. Moreover, among various 3'- and 5'-end-modified 6-mers that were tested, the 6-mer (R-95288) bearing a 3,4-DBB group at the 5'-end and a 2-hydroxyethylphosphate group at the 3'-end was the most stable, when incubated with mouse, rat, or human plasma. Therefore, R-95288 was chosen as the best candidate for possible use in therapy on the basis of its anti-HIV-1 activity.