Pharmacokinetics and Tissue Distribution of Pilocarpine After Application to Eyelid Skin of Rats.

Extending the delivery of drugs into the eyes while reducing systemic bioavailability is of utmost importance in the management of chronic ocular diseases. Topical application onto the lower eyelid skin, as an alternative to eye drops, is seen to be a valuable strategy in the treatment of chronic eye diseases. To elucidate the critical value of delivering drugs in solution onto the eyeball through the eyelid skin, pharmacokinetic studies of pilocarpine were conducted, and the results were verified using a direct pharmacodynamic study in rats. The mean residence time of pilocarpine after topical eyelid application to the eyelid skin, conjunctiva, eyeball, and plasma were 14.9, 8.50, 6.29, and 8.11 h, respectively. Conjunctiva and eyeball concentrations of pilocarpine at 8 h were 80-fold and 8-fold higher after topical eyelid application, respectively, than those for eye drops. Pupillary constriction was sustained over 8 h after topical eyelid application. Topical eyelid skin application exhibited a localized drug absorption and specific drug accumulation in the ocular tissues. Hence, it is rational to prepare topical formulations directed onto the eyelid skin, which is suitable for drugs required for long-term treatment.

Estimation of skin concentrations of topically applied lidocaine at each depth profile.

Skin concentrations of topically administered compounds need to be considered in order to evaluate their efficacies and toxicities. This study investigated the relationship between the skin permeation and concentrations of compounds, and also predicted the skin concentrations of these compounds using their permeation parameters. Full-thickness skin or stripped skin from pig ears was set on a vertical-type diffusion cell, and lidocaine (LID) solution was applied to the stratum corneum (SC) in order to determine in vitro skin permeability. Permeation parameters were obtained based on Fick's second law of diffusion. LID concentrations at each depth of the SC were measured using tape-stripping. Concentration-depth profiles were obtained from viable epidermis and dermis (VED) by analyzing horizontal sections. The corresponding skin concentration at each depth was calculated based on Fick's law using permeation parameters and then compared with the observed value. The steady state LID concentrations decreased linearly as the site became deeper in SC or VED. The calculated concentration-depth profiles of the SC and VED were almost identical to the observed profiles. The compound concentration at each depth could be easily predicted in the skin using diffusion equations and skin permeation data. Thus, this method was considered to be useful for promoting the efficient preparation of topically applied drugs and cosmetics.

Proteomics accelerating the identification of the target molecule of bioactive small molecules.

Failures in many drug development programs in the past decades were related to unspecified mechanism of action and poor pharmacokinetic (PK) properties. Recent developments are focused on well defined targets, improved PK profiles, however, not much is known about off-target effects, especially those responsible for diminishing drug activity. Steadily increasing application of proteomics in drug development should expose clinically relevant proteins for the analysis of drug effects, to show what group of patients will respond and who should not be treated with an agent.

Changes to the dihydropyrimidine dehydrogenase gene copy number influence the susceptibility of cancers to 5-FU-based drugs: Data mining of the NCI-DTP data sets and validation with human tumour xenografts.

Patient response to the anti-tumour drug 5-fluorouracil (5-FU) is variable, but predicting the response rate is important for the selection of effective chemotherapy. Our aim was to identify alterations in DNA copy number that influence susceptibility of cancer cells to 5-FU-based drugs. The NCI public database was used to identify chromosome loci associated with drug sensitivity and DNA copy number. One of the 11 candidates, the cytogenetic band 1p21.3, harbours the dihydropyrimidine dehydrogenase (DPD) gene. To validate this finding, the DPD copy number and in vivo sensitivity to 5-FU-based drugs were determined in 31 human tumour xenografts. Those xenografts demonstrating low sensitivity had significantly higher DPD copy numbers than highly sensitive tumours (P<0.002). Moreover, DPD mRNA expression levels were significantly correlated with DPD copy numbers (P<0.046). An assessment of copy number may be a more precise method of predicting the sensitivity of cancer patients to 5-FU related drugs.

Synthesis and cytostatic activity of 4,7-dihydroxythioaurone derivatives. Effect of B ring substitution on the activity.

Biological activity of thioaurones was not tested so far and the group constitute completely unexplored source of new molecules of pharmacological interest. We report synthesis and evaluation of cytotoxic activity of thioaurone derivatives bearing p-hydroquinone system in ring A. Their activity was found to depend strongly on substitution pattern, so eventually both the activity and pharmacokinetic parameters of the molecules could be tailored by further structural modifications.

A novel cinnamic acid derivative that inhibits Cdc25 dual-specificity phosphatase activity.

The Cdc25 dual-specificity phosphatases are key regulators of cell cycle progression through activation of cyclin-dependent kinases (Cdk). Three homologs exist in humans: Cdc25A, Cdc25B, and Cdc25C. Cdc25A and Cdc25B have oncogenic properties and are overexpressed in some types of tumors. Compounds that inhibit Cdc25 dual-specificity phosphatase activity might thus be potent anticancer agents. We screened several hundred compounds in a library using an in vitro phosphatase assay, with colorimetric measurement of the conversion of p-nitrophenyl phosphate (pNPP) to p-nitrophenol by the catalytic domain of recombinant human Cdc25, and discovered TPY-835, which inhibits Cdc25A and Cdc25B activity (IC50 = 5.1 and 5.7 microM, respectively). TPY-835 had mixed inhibition kinetics for Cdc25A and Cdc25B. TPY-835 caused cell cycle arrest in the G1 phase in human lung cancer cells (A549 and SBC-5) but not cell cycle arrest in the G2/M phase. After treatment with TPY-835, the activation of Cdk2 was suppressed and phosphorylation of the retinoblastoma (Rb) protein was decreased in SBC-5 cells. In addition, TPY-835 induced an increase of the sub-G1 phase cell population after 48-72 h treatment. The growth inhibitory effects of TPY-835 against cisplatin (CDDP)-, camptothecin- and 5-FU-resistant cell lines are comparable to the growth inhibitory effect on their parental lines, thus indicating that TPY-835 did not show cross-resistance to these cell lines. These results suggest that TPY-835 is a promising candidate for constructing a novel class of antitumor agents that can control the cell cycle progression of cancer cells.

Napthalimidobenzamide DB-51630: a novel DNA binding agent inducing p300 gene expression and exerting a potent anti-cancer activity.

Control of gene expression by small molecule compounds is a novel therapeutic strategy for cancer and usually it requires the presence of specific molecular recognition. The development of the compounds preferentially binding to the specific DNA sequence is one of the potential but very difficult approaches in this strategy. We designed and synthesized novel napthalimidobenzamide derivatives and analyzed their binding preferences to oligonucleotides by EtBr-displacement assay with DNA sequences, being essential fragments of the genes. To test whether these compounds modify the expression of specific genes, we analyzed the effect on the gene expression in AZ521 cells by differential display analysis using the compounds showing different characteristics in the recognition of specific DNA sequence. Among them, DB-51630, which showed approximately 350 times higher preferential binding to GC-repeats than to the AT and AA-repeating oligomers, caused the induction of a specific mRNA. The genetic sequence was identified to be the p300 gene by sequencing of the cloned cDNA. The p300 is a transcriptional co-activator protein that acts with other nuclear proteins in various cell differentiation and signal transduction pathways. This protein has intrinsic histone acetyltransferase activity and may act on chromatin directly to facilitate transcription. The increase of the amount of p300 mRNA increased after DB-51630 treatment by real time RT-PCR and Northern blot analysis. DB-51630 inhibited cell growth in various cancer cell lines at nanomolar range of concentrations, whereas p300 mRNA induction was observed at sub-nanomolar concentrations and the maximal induction occurred 8h after DB-51630 treatment. In contrast, anti-cancer drugs such as doxorubicin, vincristine, cisplatin, etoposide, and actinomycin D did not increase p300 transcription. DB-51630 revealed potent anti-cancer activity against human solid tumor xenografts. Thus, we demonstrated the anti-cancer activity of DB-51630, which interacts with a specific DNA sequence, thereby inducing p300 gene expression and exhibited significant anti-cancer activity in human tumor xenografts. Furthermore, such compounds that bind to specific DNA sequences may not only control the expression of specific genes but also exert other mechanisms in the anti-cancer effect than those of classical DNA binding drugs.

TAS-108, a novel oral steroidal antiestrogenic agent, is a pure antagonist on estrogen receptor alpha and a partial agonist on estrogen receptor beta with low uterotrophic effect.

PURPOSE: Investigators are currently conducting phase II trials on TAS-108, a novel oral steroidal antiestrogenic agent. The purpose of this study is to investigate the molecular and pharmacologic properties of TAS-108 compared with other antiestrogenic agents such as tamoxifen,raloxifene, and fulvestrant. EXPERIMENTAL DESIGN: The antagonistic or agonistic activities of these agents against both estrogen receptors (ER) alpha and beta were compared in the reporter assay systems. Their effects on the uterus were evaluated in ovariectomized rat models. The antitumor activity of TAS-108 given p.o. was evaluated in both dimethylbenzanthracene-induced mammary tumor model and human breast cancer MCF-7 cell line xenografts. RESULTS: TAS-108 inhibited the transactivation of ERalpha under the presence of 17beta-estradiol (E2) and did not induce the transactivation of ERalpha in the absence of E2, unlike the agonistic activity of tamoxifen. On the other hand, it exhibited the most agonistic activity on ERbeta among the antiestrogenic agents tested. When given p.o. in the ovariectomized rat, TAS-108 showed a much weaker estrogenic effect on utterine weight compared to tamoxifen, or with similar levels of raloxifene, a selective estrogen receptor modulator. Also, TAS-108 strongly inhibited tumor growth in dimethylbenzanthracene-induced mammary carcinomain the rat, the endogenous E2 model, at a dosage of 1 to 3 mg/kg/day. It also inhibited high exogenous E2, inducing tumor growth against MCF-7 xenografts at a dosage of 1 mg/kg/day without any toxic manifestation. CONCLUSIONS: Taken together, p.o. treatment with TAS-108 has a novel mode of action on ERs and inhibits E2-dependent tumor growth with little uterotrophic effect.

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 1: discovery of novel orally active inhibitors of human thymidine phosphorylase.

A series of novel 6-methylene-bridged uracil derivatives have been prepared as inhibitors of human thymidine phosphorylase (TP). To enhance the in vivo antitumor activity of fluorinated pyrimidine 2'-deoxyribonucleosides such as 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd), a potent TP inhibitor preventing their degradation to an inactive compound, has become a target of medicinal chemistry. We present here the synthesis and evaluation of novel human TP inhibitors. Introduction of an N-substituted aminomethyl side chain at the 6-position of 5-chlorouracil has improved water solubility and enhanced inhibitory activity compared with the known TP inhibitor, 6-amino-5-chlorouracil. Compound 42 was reasonably well absorbed in mice after oral administration. When combined with F(3)dThd, compound 42 exerted its TP inhibitory potency by increasing the maximum plasma concentrations of the former as evidenced in experiments with monkeys. Positive changes in pharmacokinetic profile were accompanied by the enhanced in vivo antitumor activity of this combination when compared to F(3)dThd alone, in mice bearing human tumor xenografts. Both biochemical and pharmacological effects appeared to fit the concept as anticipated.

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase.

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.

Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex.

Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.