Dietary serine-microbiota interaction enhances chemotherapeutic toxicity without altering drug conversion.

The gut microbiota metabolizes drugs and alters their efficacy and toxicity. Diet alters drugs, the metabolism of the microbiota, and the host. However, whether diet-triggered metabolic changes in the microbiota can alter drug responses in the host has been largely unexplored. Here we show that dietary thymidine and serine enhance 5-fluoro 2'deoxyuridine (FUdR) toxicity in C. elegans through different microbial mechanisms. Thymidine promotes microbial conversion of the prodrug FUdR into toxic 5-fluorouridine-5'-monophosphate (FUMP), leading to enhanced host death associated with mitochondrial RNA and DNA depletion, and lethal activation of autophagy. By contrast, serine does not alter FUdR metabolism. Instead, serine alters E. coli's 1C-metabolism, reduces the provision of nucleotides to the host, and exacerbates DNA toxicity and host death without mitochondrial RNA or DNA depletion; moreover, autophagy promotes survival in this condition. This work implies that diet-microbe interactions can alter the host response to drugs without altering the drug or the host.

Randomized Controlled Study to Investigate the Effect of Topical Diquafosol Tetrasodium on Corneal Sensitivity in Short Tear Break-Up Time Dry Eye.

INTRODUCTION: Complex mechanisms underlie dry eye (DE) symptom provocation. In particular, corneal hypersensitivity may provoke symptoms in short tear break-up time (BUT) DE characterized by tear film instability. We hypothesized that improved tear film stability may alleviate corneal sensitivity in patients with short tear BUT DE. Therefore, we investigated the effect of topical diquafosol tetrasodium (DQS) on corneal sensitivity in unstable tear film DE. METHODS: This prospective, randomized study included 27 subjects (age: 39.1 ± 8.4 years; range: 25-59 years) with short tear BUT DE, defined based on the presence of DE symptoms and tear film instability. Subjects were randomly divided into DQS (3% DQS, 12 subjects) and artificial tear (AT; preservative-free AT, 15 subjects) groups. Subjects applied the medication 6 times a day for 5 weeks. The perception of touch (S-touch) and pain (S-pain) sensitivity was measured using a Cochet-Bonnet esthesiometer. Tear evaluation, corneal sensitivity, and DE symptoms were compared before and after DQS or AT administration. The correlation between the improvement degrees of corneal sensitivity and DE symptoms following medication was analyzed. RESULTS: DQS significantly improved tear BUT and tear meniscus height (TMH) scores (p < 0.05), while AT significantly improved tear BUT (p < 0.05) but not TMH score. Mean S-pain and DE symptom scores were lower after medication use in the DQS (S-pain and DE symptoms: p  < 0.05) and AT groups (S-pain: p  = 0.05; DE symptoms: p  < 0.05). However, S-touch did not change significantly in either group. A positive correlation was observed between the improvement degrees of S-pain and DE symptoms in the overall subjects studied. CONCLUSION: Both DQS and AT alleviate corneal hypersensitivity and DE symptoms in eyes with short tear BUT DE. However, DQS seems to be more effective to adjust tear environment, leading to the normalization of corneal sensitivity and DE symptoms. TRIAL REGISTRATION: UMIN Clinical Trials Registry Identifier, UMIN000014536.

Immediate Effect of 3% Diquafosol Ophthalmic Solution on Tear MUC5AC Concentration and Corneal Wetting Ability in Normal and Experimental Keratoconjunctivitis Sicca Rat Models.

PURPOSE: To evaluate the immediate effect of 3% diquafosol ophthalmic solution on tear MUC5AC concentration, periodic acid-Schiff (PAS)-positive goblet cells, and tear film stability in normal and keratoconjunctivitis sicca (KCS) rat models. METHODS: Rats were divided into normal and KCS groups. 3% of diquafosol solution was instilled into the right eye and normal saline into the left eye in both groups. To determine the peak time of tear MUC5AC concentration, tears were collected after 3% diquafosol instillation every 5 min up to 20 min. The tear film stability and the numbers of PAS-positive goblet cells were compared in both models. RESULTS: After diquafosol instillation, tear MUC5AC concentration increased steadily for 15 min, at which point the MUC5AC concentration reached its peak. In both normal and KCS groups, the MUC5AC concentration at 15 min was higher after instillation of 3% diquafosol solution (17.77 ± 2.09 ng/ml in the normal group, 9.65 ± 3.51 ng/ml in the KCS group) than that after saline instillation (13.74 ± 2.87 ng/ml in the normal group, 8.19 ± 3.99 ng/ml in the KCS group) (p = 0.018 for both). The corneal wetting ability was significantly longer after instillation of 3% diquafosol solution compared with that after instillation of normal saline in the normal group (p = 0.018). The percentage of PAS-positive goblet cells after the instillation of 3% diquafosol solution was significantly lower than that after instillation of normal saline in both models (p = 0.018 for both). CONCLUSIONS: Diquafosol ophthalmic solution was effective in stimulating mucin secretion in both normal and KCS rat models, and the peak time of tear MUC5AC concentration was 15 min after diquafosol instillation. The increased tear MUC5AC concentration was accompanied by improved tear film stability and a decreased percentage of PAS-positive goblet cells.

Pharmacokinetic evaluation of diquafosol tetrasodium for the treatment of Sjögren's syndrome.

INTRODUCTION: Dry eye is a multifactorial disease of the ocular surface causing ocular discomfort and visual impairment for the patient. A variety of topical and systemic drugs are available to treat dry eye. Conventional treatments are limited to tear supplementation or improvement of ocular surface inflammation by the use of corticosteroids or cyclosporine A. Treatment of severe dry eye associated with Sjögren's syndrome (SS) is even more challenging and is designed to improve the quality and quantity of tear fluid. Diquafosol tetrasodium , a P2Y2 purinergic receptor agonist, acts via a novel mechanism by activating P2Y2 receptors of the ocular surface. AREAS COVERED: The aim of this review is to summarize the pharmacokinetics, and pharmacological and clinical data of 3% diquafosol tetrasodium ophthalmic solution in patients with dry eye, particularly SS. The mechanisms of impaired ocular surface due to severe dry eye, as defined by the International Dry Eye Workshop, are analyzed. EXPERT OPINION: Diquafosol tetrasodium provides a novel mode of action in dry eye syndrome, including SS, by stimulating the quantity and quality of tear fluid secretion via various mechanisms. In clinical trials, 3% Diquafosol tetrasodium ophthalmic solution demonstrated a good safety profile and exhibited efficacy with clinical improvement of the ocular surface in dry eye including SS.

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.

Double-masked, placebo-controlled safety and efficacy trial of diquafosol tetrasodium (INS365) ophthalmic solution for the treatment of dry eye.

PURPOSE: To investigate the safety and efficacy of diquafosol tetrasodium, a P2Y2 receptor agonist that stimulates fluid and mucin secretion on the ocular surface, as a novel topical treatment of dry eye disease. METHODS: Subjects with dry eye (n=527) were evaluated in a randomized, double-masked, parallel-group trial comparing 24 weeks of treatment with 2 concentrations of diquafosol (1% and 2%) versus placebo instilled 4 times daily. Corneal staining, conjunctival staining, Schirmer tests, and subjective symptoms of dry eye were evaluated. Use of artificial tears was permitted as necessary. RESULTS: Subjects treated with 2% diquafosol had significantly lower corneal staining scores compared with placebo at the 6-week, primary efficacy time point (P<0.001), and superiority continued throughout the 24-week study. Reductions in corneal staining were observed as early as after 2 weeks of treatment, were maintained throughout the 24-week study, and were observed to worsen slightly (toward baseline) when diquafosol treatment was discontinued (week 25). Results for conjunctival staining were consistent with those observed for corneal staining. Schirmer scores at week 6 were significantly higher with diquafosol treatment than with placebo (P

Diquafosol tetrasodium: a novel dry eye therapy.

The ophthalmic formulation of diquafosol tetrasodium (INS365), a P2Y2 receptor agonist, is targeted to treat dry eye disease through rehydration of the ocular surface. Existing pharmacological therapies for dry eye disease are limited, therefore, approval of this medication is anticipated. This review summarises key findings during development and in clinical trials including clinical effectiveness and safety. The relevance of P2Y2 receptor technology to dry eye disease and the disease process is discussed.

Diquafosol tetrasodium. Inspire/Allergan/Santen.

Inspire, in collaboration with Allergan and Santen, is developing an eye-drop formulation of diquafosol tetrasodium (INS-365), a second-generation uridine nucleotide analog P2Y, receptor agonist for the potential treatment of dry eye disease. In June 2003, Inspire submitted an NDA for the treatment of dry eye, and in July 2003 the FDA granted the NDA Priority Review status. FDA action is expected in December 2003, and in January 2003 launch was expected in the first half of 2004.

The characteristics of nucleobase transport and metabolism by the perfused sheep choroid plexus.

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (U(max)) for hypoxanthine and adenine, was 35.51+/-1.50% and 30.71+/-0.49% and for guanine, thymine and uracil was 12.00+/-0.53%, 13.07+/-0.48% and 12.30+/-0.55%, respectively with a negligible backflux, except for that of thymine (35.11+/-5.37% of the U(max)). HPLC analysis revealed that the purine nucleobase hypoxanthine and the pyrimidine nucleobase thymine can pass intact through the choroid plexus and enter the cerebrospinal fluid CSF so the lack of backflux for hypoxanthine was not a result of metabolic trapping in the cell. Competition studies revealed that hypoxanthine, adenine and thymine shared the same transport system, while guanine and uracil were transported by a separate mechanism and that nucleosides can partially share the same transporter. HPLC analysis of sheep CSF collected in vivo revealed only two nucleobases were present adenine and hypoxanthine; with an R(CSF/Plasma) 0.19+/-0.02 and 3.43+/-0.20, respectively. Xanthine and urate, the final products of purine catabolism, could not be detected in the CSF even in trace amounts. These results suggest that the activity of xanthine oxidase in the brain of the sheep is very low so the metabolic degradation of purines is carried out only as far as hypoxanthine which then accumulates in the CSF. In conclusion, the presence of saturable transport systems for nucleobases at the basolateral membrane of the choroidal epithelium was demonstrated, which could be important for the distribution of the salvageable nucleobases, adenine and hypoxanthine in the central nervous system.

Liposome suppression of proliferative vitreoretinopathy. Rabbit model using antimetabolite encapsulated liposomes.

The effects of the antimetabolites, cytarabine (Ara-C) and 5-fluorouridine 5'-monophosphate (FUMP), encapsulated in multivesicular liposomes on formation of vitreous fibroproliferative membranes in New Zealand white (NZW) rabbits were studied. In pharmacokinetic studies, the drug half-life in the vitreous cavity was 124 hr after intravitreal administration of 1.0 mg of FUMP in liposomes. By contrast, the drug half-life after a single injection in nonliposome-treated controls was only 4.5 hr. In a heterologous dermal fibroblast model of proliferative vitreoretinopathy (PVR), there was a 92% decrease in frequency of tractional retinal detachments in rabbits receiving a single intravitreal injection of liposome-encapsulated 0.1 mg of FUMP compared with controls receiving liposomes without drug. A dose of 1 mg of Ara-C in liposome-treated rabbits was associated with only a 46% reduction in tractional detachment compared with controls. Multivesicular liposome-encapsulated FUMP may be useful for inhibiting formation of fibroproliferative membranes in the vitreous after vitreoretinal surgery.

The transport and metabolism of the uridine mononucleotides by rat jejunum in vitro.

1. Both uridine 3'-monophosphate (3'-UMP) and uridine 5'-monophosphate (5'-UMP) when perfused through the lumen of isolated rat jejunum gave rise to uracil as the only transported pyrimidine appearing in the serosal medium; neither the nucleotide nor the nucleoside could be detected in the serosal fluid. 2. There was a low level of the nucleoside, uridine, in the luminal fluid after the nucleotide had passed through the jejunal segment. Luminal nucleoside appearance was more marked from the 3' form of the nucleotide. 3. The hydrolysis of the nucleotides to the nucleoside form occurred via a brush-border membrane enzyme, which had the same maximal velocity (Vmax) for the two nucleotides (699 +/- 35 and 747 +/- 10 nmol min-1 (mg protein)-1 for 3'-UMP and 5'-UMP, respectively) but a different Michaelis constant (Km) so that 3'-UMP (Km = 58 +/- 3 microM) hydrolysis is favoured over 5'-UMP hydrolysis (Km = 108 +/- microM) at lower concentrations. 4. At 0.05 mM, luminal 3'-UMP gave rise to a higher rate of serosal uracil appearance than luminal 5'-UMP, but at higher luminal concentrations (0.1-0.2 mM) the rate of serosal uracil appearance was the same from both nucleotides. 5. The transmural transport of uracil from the uridine mononucleotides is discussed with reference to the metabolism and compartmentalization of the small intestine responsible for the appearance of the free pyrimidine in the serosal fluid.

Sustained release of the antimetabolite 5-fluorouridine-5'-monophosphate by multivesicular liposomes.

We have developed a novel liposome for sustained drug delivery to the eye of the antimetabolite 5-fluorouridine-5'-monophosphate (FUMP), a potent metabolite of 5-fluorouracil. The in vitro half-life of FUMP incorporated within these liposomes was 585 hours. Following subconjunctival administration of 1 mg of FUMP to 13 New Zealand white rabbits, the tissue drug level at the injection site was significantly greater in the group treated with liposomes than it was in the group not treated with liposomes (p = .02). These liposomes offer the potential for sustained release of FUMP in the eye. By controlling the rate of drug release, it may be possible to reduce the need for frequent drug administration and its associated ocular toxicity as well as to improve treatment efficacy.