Synthesis and biological activity of phosphonoacetate- and thiophosphonoacetate-modified 2'-O-methyl oligoribonucleotides.

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.

Molecular docking of alpha-enolase to elucidate the promising candidates against Streptococcus pneumoniae infection.

PURPOSE: To predict potential inhibitors of alpha-enolase to reduce plasminogen binding of Streptococcus pneumoniae (S. pneumoniae) that may lead as an orally active drug. S. pneumoniae remains dominant in causing invasive diseases. Fibrinolytic pathway is a critical factor of S. pneumoniae to invade and progression of disease in the host body. Besides the low mass on the cell surface, alpha-enolase possesses significant plasminogen binding among all exposed proteins. METHODS: In-silico based drug designing approach was implemented for evaluating potential inhibitors against alpha-enolase based on their binding affinities, energy score and pharmacokinetics. Lipinski's rule of five (LRo5) and Egan's (Brain Or IntestinaL EstimateD) BOILED-Egg methods were executed to predict the best ligand for biological systems. RESULTS: Molecular docking analysis revealed, Sodium (1,5-dihydroxy-2-oxopyrrolidin-3-yl)-hydroxy-dioxidophosphanium (SF-2312) as a promising inhibitor that fabricates finest attractive charges and conventional hydrogen bonds with S. pneumoniae alpha-enolase. Moreover, the pharmacokinetics of SF-2312 predict it as a therapeutic inhibitor for clinical trials. Like SF-2312, phosphono-acetohydroxamate (PhAH) also constructed adequate interactions at the active site of alpha-enolase, but it predicted less favourable than SF-2312 based on binding affinity. CONCLUSION: Briefly, SF-2312 and PhAH ligands could inhibit the role of alpha-enolase to restrain plasminogen binding, invasion and progression of S. pneumoniae. As per our investigation and analysis, SF-2312 is the most potent naturally existing inhibitor of S. pneumoniae alpha-enolase in current time.

Antitumor activity of the weekly intravenous push schedule of 5-fluoro-2'-deoxyuridine +/- N-phosphonacetyl-L-aspartate in mice bearing advanced colon carcinoma 26.

We have investigated the effects of N-(phosphonacetyl)-L-aspartate (PALA) administered i.v. as a single dose (100 mg/kg) on the antitumor activity of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluorouracil (FUra), on the pharmacokinetic parameters of FdUrd and FUra, and on the tumor pyrimidine ribonucleotide triphosphate pools in mice bearing advanced colon carcinoma 26 and leukemia 1210. The antitumor activity was evaluated with PALA administered i.v. 24 h prior to the maximum tolerated dose of FUra and FdUrd administered by: (a) 4 days of continuous infusion (schedule 1, c.i. days 1-4); (b) daily for 4 days by i.v. push (schedule 2, i.v. days 1-4); and (c) weekly for 3 weeks (schedule 3, i.v. weekly for 3 weeks). The maximum tolerated doses of FdUrd were 20, 150, and 400 mg/kg/day and for FUra were 25, 50, and 80 mg/kg/day for schedule 1, 2, and 3, respectively. At the maximum tolerated doses, the antitumor activity in mice bearing advanced colon carcinoma can be summarized as follows: (a) FdUrd is significantly more active than FUra; (b) for both drugs the weekly for 3 weeks i.v. push schedule is superior to the c.i. or i.v. push daily for 4 days schedules; (c) pretreatment with PALA enhances the antitumor activity of FdUrd and FUra and resulted in 95 and 13% complete responses, respectively; (d) long-term survivors with FUra could only be achieved in the presence of PALA; in mice bearing leukemia 1210 cells, FdUrd or FUra with or without PALA exhibited no significant antitumor activity when PALA was administered in a single dose 24 h prior to fluoropyrimidine treatment; and (e) in C-26 and L1210, PALA reduced the pools of CTP and UTP equally, to about 10% of controls with significant difference in their rates of recovery.

The role of low-dose PALA in biochemical modulation.

Phosphonacetyl-L-aspartate (PALA) is a rationally-synthesized analog of the transition-state intermediate in the formation of carbamyl aspartate from carbamyl phosphate and aspartic acid by aspartate carbamyl transferase (ACTase). PALA is thus a potent inhibitor of the enzyme (Ki about 10(-8) M for ACTases of various origins), which in whole cells blocks the de novo synthesis of pyrimidines. In vivo, low doses of PALA inhibit whole body pyrimidine synthesis. While this action is cytotoxic in vitro, extensive human testing demonstrates that PALA alone is devoid of selective antitumor activity. Recent interest in the therapeutic action of PALA derives from the demonstration that its action potentiates the cytotoxicity of several cytotoxic drugs, notably 5-fluorouracil (5-FU). Results from clinical trials of PALA and 5-FU in combination in colorectal cancer suggest that biochemical modulation with regimens which follow the principles determined in preclinical studies may enhance the efficacy of current therapy.

Pharmacokinetics and absorption of foscarnet after intravenous and oral administration to patients with human immunodeficiency virus.

Six patients with human immunodeficiency virus were given foscarnet in oral solution, 4000 mg every 6 hours for 3 days, followed by a washout period for 2 days and continuous intravenous infusion of 16,000 mg/24 hr over 72 hours. After oral foscarnet, plasma concentrations were less than 33 mumol/L in four patients; two had occasional concentrations of 35 to 50 mumol/L. The extent of absorption varied between 12% and 22%. During intravenous infusion, plasma concentrations ranged between 75 and 265 mumol/L. The disposition of foscarnet was triphasic, with mean half-lives of 0.45, 3.3, and 18 hours. Excretion data suggested elimination was by tubular secretion and glomerular filtration. Renal clearance was 176 ml/min 1.73 m2. The apparent nonrenal clearance, 40 ml/min 1.73 m2, probably reflects sequestration of foscarnet into bone. Ten percent to 28% of the cumulative dose may have been deposited in bone 2 days after infusion. A slight increase in serum calcium levels and changes in serum phosphate values may reflect the uptake of foscarnet in bone. Five patients had diarrhea (oral) and two had thrombophlebitis (intravenous).

Pharmacokinetics of potential anti-AIDS agents thiofoscarnet and foscarnet in the cat.

The pharmacokinetics of thiophosphonoformate (TPFA) and phosphonoformate (foscarnet, PFA) were studied in normal adult cats, a species susceptible to feline immunodeficiency virus (FIV) infection. Parent drugs and metabolites were quantitated by high-performance liquid chromatography (HPLC). TPFA had a mean terminal plasma half-life of 42 min, a total clearance of 4.58 ml/min/kg, and a renal clearance of 1.24 ml/min/kg (N = 4). TPFA underwent in vivo metabolism to PFA and thiophosphonic acid (TPA); the latter was inactive against HIV reverse transcriptase. The 6-h cumulative urinary excretion was 42.3% of the intravenous administered dose of TPFA, consisting of 23.5% unchanged TPFA, 13.8% PFA, and 5.0% TPA. In comparison, PFA had a mean (N = 5) terminal half-life of 172 min and a total clearance of 1.88 ml/min/kg, approximating its renal clearance. There was no evidence of PFA metabolism. Oral doses of TPFA were administered either in enteric-coated capsules or in solution by gavage. The mean oral bioavailability of encapsulated TPFA and PFA was 22 and 8%, respectively. When given by gavage, TPFA had a higher mean bioavailability (33%), but with a greater variability. Based on the 6-h cumulative urinary excretion of TPFA, the mean oral bioavailability of TPFA was 44%, similar to that based on plasma data. The TPFA appears to be superior to PFA because of its greater oral bioavailability and its ability to deliver an active metabolite, PFA, to the systemic circulation after oral dosing.

Approaches to the treatment of cytomegalovirus retinitis: ganciclovir and foscarnet.

Both ganciclovir, a nucleoside analogue, and foscarnet, a pyrophosphate analogue, specifically bind cytomegalovirus (CMV) DNA polymerase and inhibit CMV replication at plasma concentrations achievable with intravenous administration. The agents have similar plasma half-lives, and both are cleared solely by the kidneys. Foscarnet has a low solubility and a high degree of ionization at physiologic pH, requiring it to be administered in higher doses and larger volumes. Both drugs are administered as an initial induction regimen followed by a long-term maintenance regimen. Among patients with the acquired immune deficiency syndrome (AIDS) who have CMV retinitis, the efficacy of long-term maintenance therapy, as measured by median time to retinitis progression, appears to be similar for the two drugs. The major toxicity of ganciclovir is myelosuppression, with dose-limiting neutropenia occurring in approximately 16% and thrombocytopenia in 5% of AIDS patients. The major toxicity of foscarnet is nephrotoxicity, with dose-limiting toxicity occurring in approximately 10-23% of patients; other effects of foscarnet include hypocalcemia, which may be associated with seizure and arrhythmia. Studies in vitro indicate an additive or synergistic inhibitory effect on CMV when these two drugs are combined, suggesting that lower-dose combination regimens or higher-dose alternating regimens may result in greater efficacy with less toxicity than with either drug alone.

Clinical pharmacology: foscarnet.

Foscarnet exerts its antiviral effects via reversible inhibition of viral polymerases. Pharmacodynamic data indicate that herpesvirus and human immunodeficiency virus replication is inhibited by therapeutically achievable concentrations of foscarnet; however, the concentrations of foscarnet required for such inhibition have been found to vary widely. Pharmacokinetic data indicate that foscarnet is eliminated via the renal route, undergoes negligible metabolism, and appears to be distributed widely from the circulation. However, the available data indicate that the pharmacokinetics of the drug varies among patients and within the individual patient, particularly with regard to plasma drug levels; furthermore, such factors as the intracellular kinetics of the drug have yet to be well characterized. It is thus difficult to formulate optimal dosing regimens on the basis of what is known of foscarnet pharmacodynamics and pharmacokinetics. Nevertheless, dosages that produce clear-cut therapeutic benefits without unacceptable toxicity have been identified in clinical trials of foscarnet in acquired immunodeficiency syndrome (AIDS) patients with cytomegalovirus (CMV) retinitis.

Foscarnet. A review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis.

The pyrophosphate analogue, foscarnet, selectively inhibits the DNA polymerase of human herpes viruses, including cytomegalovirus, and the reverse transcriptase of HIV. Viral replication is therefore prevented, but resumes when the drug is cleared from infected cells. In vitro, the combination of foscarnet and zidovudine (azidothymidine) has an additive effect against cytomegalovirus and acts synergistically against HIV. An improvement in cytomegalovirus retinitis is obtained in over 85% of affected AIDS patients during foscarnet induction therapy, but relapse usually occurs within a month of ceasing treatment. There is a similar duration of remission during maintenance therapy given for 5 days each week, but this can be extended 4- to 5-fold with daily administration of higher doses. In allograft recipients, progression of retinitis can be halted by foscarnet until immune function recovers and eradicates the virus. The incidence of acute renal failure, which is common during foscarnet therapy, may be reduced by dosage adjustment and adequate prehydration. Anaemia, phlebitis, nausea and vomiting, and disturbances in serum calcium and phosphate levels, perhaps resulting from uptake of foscarnet into bone or chelation with ionised calcium, have also been associated with administration of the drug. Cytomegalovirus retinitis is difficult to treat, with few therapeutic options available. Although treatment with foscarnet produces some severe adverse effects, with care these can be minimised, and the drug produces clinical improvement in a large proportion of patients; this is a highly encouraging finding at this stage in its development. Preliminary comparative data indicate that foscarnet and ganciclovir are similarly effective, but foscarnet may have some theoretical advantages in AIDS patients since it can be used in combination with zidovudine without potentiating myelosuppression.

"Body burden" of phosphonoformic acid after topical and vaginal administration to rabbits and beagle dogs.

Trisodium phosphonoformate (PFA), an antiviral compound, was studied in two animal species, dogs and rabbits, for systemic absorption after vaginal and topical application. Antiviral agents to be possibly used against genital and skin herpes should penetrate deeply into the skin and, hopefully, should remain there as long as possible with no or little systemic uptake. In the rabbit the absolute bioavailability after vaginal and topical administration is 14% and 12%, respectively, and in the dog 34% and 3%, respectively. Because PFA has a low toxicity the body burden may be acceptable if the compound is found to be high clinical benefit.

Influence of dimethylacetamide, N-N-diethyl-m-toluamide and 1-dodecylazacycloheptan-2-one on ex vivo permeation of phosphonoformic acid through rat skin.

Transdermal permeation of trisodium phosphonoformate (PFA) alone and in presence of sorption promoters, dimethylacetamide (DMAC), N-N-diethyl-m-toluamide (NNDEMT) and 1-dodecylazacycloheptan-2-one (Azone) was studied using excised rat skin. DMAC in concentrations between 0.05% to 10% had no effect on amount of PFA in the skin or permeated across skin, flux or lag time. 10% NNDEMT doubled the amount of PFA in the skin, increased fourfold the amount permeated across the skin, and increased the flux fivefold. There was no influence on lag time. Azone doubled the amount of PFA in the skin, more than tripled the amount permeated across the skin, and increased the flux fourfold. There was no influence on lag time.

Age-related differences in pharmacokinetics of phosphonoformate in cats.

Phosphonoformate (PFA) is a simple PPi analog which inhibits the activities of a variety of viral DNA polymerase, RNA polymerase, and reverse transcriptase enzymes. PFA is a topical and parenteral treatment for human herpesvirus infections and is currently in phase I trials for treatment of acquired immunodeficiency syndrome. Pharmacokinetic properties of PFA in young (growing) and adult specific-pathogen-free cats were compared. Mean PFA clearance from plasma was twofold higher in young cats (7.52 ml/min per kg of body weight) than in adult cats (3.70 ml/min per kg). Higher PFA clearance from plasma observed in young cats may result from higher renal clearance or enhanced accumulation of PFA in bone tissue of young versus adult cats. No plasma protein binding of PFA was observed. Mean oral bioavailability was 35% in young cats. These data indicate that age-related differences in PFA clearance from plasma occur in cats.

Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: implications for allosteric regulation.

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For Escherichia coli aspartate transcarbamoylase (ATCase; EC) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-A resolution crystal structure of the C trimer-PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer-PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.

Carrot cells detoxify N-phosphonoacetyl-L-aspartate by esterification.

Unlike bacterial and mammalian cells, carrot cells are able to tolerate N-phosphonoacetyl-L-aspartate (PALA), a potential inhibitor of pyrimidine biosynthesis, by detoxifying the compound. Anion-exchange chromatography showed that detoxified PALA was less negatively charged than PALA, and allowed detoxified PALA to be isolated. Incubation of detoxified PALA with a low-specificity carboxylic-ester hydrolase fully restored the ability to inhibit aspartate transcarbamoylase, the target enzyme, indicating that the detoxification involves the formation of carboxylic ester. G.1.c. analysis of the alcohol products of enzymic hydrolysis, and of their ratio to PALA, showed that the detoxification produced a mixture of mono- and di-carboxylic esters and of methyl and ethyl esters. The detoxification mechanism showed considerable specificity towards PALA, since the analogous carboxy groups of succinate were not modified in the same way. Succinate was depleted much more slowly, no succinate esters could be detected, and the presence of a 10-fold excess of succinate did not inhibit the esterification rate of PALA. The possible significance of these results is discussed.

Pharmacokinetics of foscarnet and distribution to cerebrospinal fluid after intravenous infusion in patients with human immunodeficiency virus infection.

To investigate the pharmacokinetics and effects of intravenous foscarnet, 13 relatively healthy male patients with human immunodeficiency virus infection and a mean CD4+ lymphocyte value of 0.45 x 10(-9) cells per liter were given a continuous intravenous infusion of foscarnet (0.14 to 0.19 mg/kg per min) for 8 to 21 days. Blood and urine samples were taken during and after drug administration to monitor foscarnet concentrations. Lumbar puncture was performed during the infusion in five patients. The concentrations in plasma showed large variations both within and between patients. The disposition of foscarnet could be explained by a triexponential equation (t1/2 lambda 1, 0.40 to 2.52 h; t1/2 lambda 2, 3.20 to 16.7 h; t1/2 lambda 3, 36 to 196 h). Renal clearance accounted for most of the plasma clearance, the difference probably reflecting the passage of foscarnet into bone. Up to 20% of the cumulative dose may have been deposited in bone 7 days postinfusion. Foscarnet was distributed to the cerebrospinal fluid in a concentration varying from 13 to 68% of the simultaneous concentration in plasma. Polyuria and polydipsia were recorded in all patients. There appears to be an association between the degree of malaise, including symptoms such as nausea, vomiting, fatigue, and headache, and concentrations in plasma above 350 mumol/liter.

Phase I-II trial of foscarnet for prevention of cytomegalovirus infection in autologous and allogeneic marrow transplant recipients.

The safety and efficacy of foscarnet for prevention of cytomegalovirus (CMV) infection was evaluated in 19 CMV-seropositive bone marrow transplant (BMT) recipients. All patients received intermittent intravenous (iv) foscarnet: 40 mg/kg every 8 h from 7 days before to day 30 after BMT, then 60 mg/kg once a day until day 75. The main toxicity was transient renal dysfunction, with a greater than 50 mumol/L increase in serum creatinine above baseline in 5 of the 7 autograft recipients and in 6 of the 12 allograft recipients. Only 4 allograft recipients developed CMV infection during foscarnet prophylaxis, and no patient showed evidence of CMV disease. Because 3 allograft recipients receiving concomitant iv amphotericin B showed rapid impairment of renal function, foscarnet prophylaxis should not be given to allograft recipients requiring amphotericin B; otherwise, foscarnet prophylaxis at this dose appears safe after BMT.

Foscarnet in the treatment of cytomegalovirus retinitis in acquired immune deficiency syndrome.

Cytomegalovirus (CMV) retinitis is the major cause of visual loss in acquired immune deficiency syndrome (AIDS). Thirty-one patients with active CMV retinitis were treated with the new antiviral drug, Foscarnet (trisodium phosphonoformate). After a 3-week course of induction therapy, the retinitis improved in 29 of 31 patients (93.5%). Complete resolution of the retinitis was seen in 19 cases (61.3%). Ten patients had partial resolution (32.2%) and two (6.5%) failed to respond. After induction therapy, six patients were put on a low-dose maintenance regimen. All patients without maintenance therapy relapsed within 3 weeks after discontinuation of Foscarnet. The rate of relapse on maintenance therapy was 50% (3/6) within the first 5 weeks. The three other patients of Foscarnet maintenance did not relapse after a follow-up period of 12 weeks. In contrast to ganciclovir, Foscarnet did not induce neutropenia but it produced kidney toxicity that led to reversible renal insufficiency in three cases. Thus, Foscarnet appears to be a useful alternative to ganciclovir, particularly when combined with bone marrow toxic drugs, such as zidovudine (azidothymidine).

Foscarnet sodium.

Cytomegalovirus (CMV), a major opportunistic viral pathogen frequently causing disease in immunocompromised patients such as organ transplant recipients and people with AIDS, may present as pneumonitis, gastrointestinal disease, or encephalitis. Its most common manifestation in patients with AIDS is retinitis which, if left untreated, invariably progresses to extensive retinal necrosis and ultimately to blindness. Ganciclovir sodium, currently the only licensed antiviral agent for the treatment of CMV retinitis, effectively controls this infection in a majority of AIDS patients, but significant granulocytopenia or thrombocytopenia related to ganciclovir therapy often limit its clinical application. Myelosuppression may be further exacerbated in AIDS patients by such other agents as zidovudine or trimethoprim/sulfamethoxazole, often necessitating dosage reductions or discontinuation of these agents in patients receiving ganciclovir. Foscarnet sodium, a pyrophosphate analog active against both cytomegalovirus and the human immunodeficiency virus type 1 (HIV), may be an effective alternative to ganciclovir in the management of CMV retinitis. Trials with intravenous foscarnet in CMV retinitis have reported favorable results using initial daily doses of 180-230 mg/kg/d given as intermittent infusions every eight hours, followed by maintenance regimens of 60-90 mg/kg/d given as single daily one- or two-hour infusions. Foscarnet therapy may result in renal impairment, and indefinite intravenous maintenance therapy may be required to prevent recurrence of CMV infection. Despite these drawbacks, foscarnet's lack of major myelosuppressive toxicity, and its activity in suppressing HIV replication, make this a potentially safe and effective alternative agent for the management of CMV infection, especially in AIDS patients.