Long term effects of denufosol tetrasodium in patients with cystic fibrosis.

RATIONALE: Denufosol stimulates chloride secretion independent of the chloride channel which is dysfunctional in cystic fibrosis (CF) and therefore has the potential to benefit CF patients regardless of genotype. OBJECTIVES: To assess the efficacy of denufosol in CF patients with mild lung function impairment age 5 years and older. METHODS: This multicenter, randomized, parallel group double-blind placebo-controlled trial was conducted at 102 CF care centers in Australia, Canada and the United States (NCT00625612) The active group (n=233) received 60 mg denufosol via inhalation three times daily The primary efficacy endpoint was change in FEV(1) in liters from Day 0 to week 48. MEASUREMENTS AND MAIN RESULTS: 685 patients were screened for the study and 466 patients (233 in each group) were randomized to study treatment. The adjusted mean change in FEV(1)was 40 mL for denufosol and 32 mL for placebo with a resulting treatment effect of 8 mL (95% CI -0.040, 0.056). The average rate of change in FEV(1) percent of predicted over 0 to 48 weeks was -3.04% for placebo vs. -2.30 for denufosol (a difference of 24% relative to placebo) among all patients. The incidence of pulmonary exacerbation was 26% vs. 21% for the placebo and denufosol groups with no differences in the time to first event. The study treatments were well tolerated and there was no evidence of systemic effects in any safety parameter assessed. CONCLUSIONS: In patients with CF treatment with denufosol for 48 weeks did not improve pulmonary function or reduce the incidence of pulmonary exacerbations.

Denufosol: a review of studies with inhaled P2Y(2) agonists that led to Phase 3.

Among the most promising of the new therapies being developed for the treatment of Cystic Fibrosis (CF) are those targeted at increasing mucosal hydration on the surface of the airways. One of these therapies, P2Y(2) receptor agonists, bypasses the defective CFTR chloride channel, and activates an alternative chloride channel. This activation results in an increase in airway surface epithelial hydration, and through these actions and effects on cilia beat frequency, increases mucociliary clearance. The pharmacology of P2Y(2) agonists has been confirmed in several preclinical and clinical studies. Denufosol tetrasodium is a novel second-generation, metabolically stable, selective P2Y(2) receptor agonist currently in Phase 3 clinical development. In radiolabelled deposition studies of P2Y(2) agonists in healthy non-smokers and smokers, approximately 7mg of a 40-mg nebulizer (PARI LC Star) load was deposited in the lungs. In a pharmacokinetic study in healthy volunteers, very limited systemic exposure was observed when doses of 200mg of denufosol were nebulized. Thus, it appears that high concentrations of denufosol can be achieved in the airways with very low systemic absorption. Denufosol has been generally well-tolerated in healthy volunteers and patients with CF. The most common adverse events were in the respiratory system, with cough having the highest frequency. Doses of 20-60mg have been evaluated in Phase 2 trials of up to 28 days duration, and superiority relative to placebo on FEV1 has been observed in patients with relatively normal lung function (FEV1 greater than or equal to 75% of predicted). The first Phase 3 trial is a comparison of denufosol 60mg and placebo in 350 patients with CF with FEV1 at study entry greater than or equal to 75% of predicted.

Effect of mycophenolate mofetil on the pharmacokinetics of antiretroviral drugs and on intracellular nucleoside triphosphate pools.

OBJECTIVE: To study the effect of mycophenolate mofetil therapy on the pharmacokinetic parameters of a number of antiretroviral drugs, on intracellular pools of deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP), and on intracellular concentrations of the triphosphate of lamivudine (3TCTP). DESIGN: Randomised pharmacokinetic study. PARTICIPANTS: Nineteen HIV-1-infected patients. METHODS: Antiretroviral-naive men starting treatment with didanosine 400 mg once daily, lamivudine 150 mg twice daily, abacavir 300 mg twice daily, indinavir 800 mg twice daily, ritonavir 100 mg twice daily and nevirapine 200 mg twice daily were randomised to a group with or without mycophenolate mofetil 500 mg twice daily. After 8 weeks of therapy, the plasma pharmacokinetic profiles of mycophenolic acid (the active metabolite of mycophenolate mofetil), abacavir, indinavir and nevirapine, and triphosphate concentrations (dCTP, dGTP and 3TCTP) in peripheral blood mononuclear cells, were determined. RESULTS: Nine of the 19 patients received mycophenolate mofetil. There was no difference in plasma clearance of indinavir or abacavir between the two groups. The clearance of nevirapine was higher in patients using mycophenolate mofetil (p = 0.04). In 12 patients, of whom five also received mycophenolate mofetil, intracellular triphosphates were measured. There was no significant difference in intracellular dCTP, dGTP or 3TCTP concentrations between the two groups. CONCLUSION: In this small cohort of patients, mycophenolate mofetil therapy reduced the plasma concentration of nevirapine but had no effect on plasma concentrations of indinavir and abacavir. There were no consistent effects of mycophenolic acid on the intracellular concentrations of dCTP, dGTP or 3TCTP.

The effect of nicotine on DNA repair in adult myocytes.

Recent findings have demonstrated that terminally differentiated adult ventricular myocytes are capable of repairing DNA that has been damaged by exposure to oxygen free radicals. Despite the potential importance of DNA repair in cells that may survive many decades after injury, little is known about the mechanisms or regulation of repair. Since tobacco use has a well-defined role in the epidemiology and pathophysiology of heart disease, we tested the effects of nicotine on repair of free radical damaged plasmids by whole-cell protein extracts from adult myocytes. Exposure to a concentration of 25 microM nicotine increased incorporation of (32P)dCTP into damaged plasmids by 16%, and 50 or 100 microM nicotine increased incorporation by 32%. Nicotine did not alter the rate or amount of poly (ADP-ribose) on the major protein acceptor of molecular weight 113-116 kDa. Inhibition of DNA polymerase activity with pyridoxal 5'-phosphate revealed greater plasmid degradation in the presence of nicotine. We conclude that nicotine enhances DNA degradation and the increased repair is a consequence of this greater degradation.

Targeting antiviral nucleotide analogues to macrophages.

Macrophages are important target cells for human immunodeficiency virus type 1 (HIV-1) infection. We have developed a drug targeting system for the selective delivery of phosphorylated nucleoside analogues to these phagocytosing cells. This system is based on the possibility of encapsulating the phosphorylated drugs into autologous erythrocytes and on the subsequent selective modification of their membranes to promote macrophage recognition and phagocytosis. Targeted delivery of phosphorylated nucleoside analogues to human, feline, and murine macrophages inhibits the infectivity of HIV-1, feline immunodeficiency virus, and LP-BM5 viruses more efficiently than the administration of the corresponding nucleoside analogues. In vivo administration of 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) encapsulated into autologous erythrocytes to LP-BM5-infected mice was found to reduce infectivity and disease progression. Furthermore, the simultaneous administration of AZT or ddC produced additive antiviral effects. The possibility of using red cells as drug targeting systems was useful for the design, synthesis, and delivery of new antiviral nucleoside analogues. As a prototype of these new drugs, di-(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophospha te (AZTp2AZT) was prepared. Although this drug in solution has the same antiviral activity as AZT, when administered encapsulated into erythrocytes it was several times more efficient in inhibiting the infectivity of human, feline, and murine immunodeficiency viruses. Thus, the availability of a drug targeting system for the selective delivery of antivirals to macrophages offers an additional possibility for the development of new drugs and of new combination antiviral therapies.

Synthesis and antileukemic activity of chymotrypsin-activated derivatives of 3'-amino-2',3'-dideoxycytidine. (Synthetic nucleosides and nucleotides. XXXIII.

3'-Amino-2',3'-dideoxycytidine (8) was directly synthesized from 2'-deoxycytidine. 2',3'-Dideoxy-3'-(N-acyl-L-phenylalanylamino)cytidines (acyl = butoxycarbonyl (9a), acetyl (9b), benzoyl (9c), and n-hexanoyl (9d)) were synthesized as chymotrypsin-activated prodrugs of 8. This N-protection was required for activation by chymotrypsin to 8. In vitro, compound 8 showed high cytotoxic activity against P388 cells, but the prodrugs 9a-d were ineffective. In vivo, however, these prodrugs showed much higher activity than 8 in mice bearing P388 cells.

Cellular pharmacology of phosphorothioate homooligodeoxynucleotides in human cells.

The uptake and distribution of phosphorothioate oligodeoxynucleotides by human cells were studied using 35S-labeled 28-mer phosphorothioate oligodeoxycytidine [S-(dC)28]. Accumulation of intracellular S-(dC)28 was found to be higher in the carcinoma cells (grown in monolayers) than in the leukemia cells (grown in suspension culture). A hepatoma cell line transfected with hepatitis B virus, 2215, was chosen for further studies. The uptake of S-(dC)28 was partially dependent on temperature and energy. The intracellular concentration was significantly higher than that in the medium and the amount accumulated was dependent on the extracellular concentration. It appears that the uptake of S-(dC)28 involves mechanisms of both fluid-phase pinocytosis and adsorptive endocytosis. Neither oligonucleotides nor 5'-phosphorylated nucleotides inhibited S-(dC)28 uptake. Unlike horseradish peroxidase, which was primarily associated with endosomes once it was taken into the cell, S-(dC)28 was found to be present in both nuclear and cytoplasmic fractions. Efflux of S-(dC)28 from the cell was multiphasic; a trapping mechanism that could be due to a potent interaction of S-(dC)28 with cellular proteins was implicated. This trapping mechanism could be responsible for the lack of biological activity such as cytotoxicity and antisense activity of phosphorothioate oligodeoxynucleotides in some human cells.