Establishing a Pharmacy-Based Patient Registry System: A Pilot Study for Evaluating Pharmacist Intervention for Patients with Long-Term Medication Use.

Background: In Japan, an increasing number of patients are prescribed a large amount of long-term medications by large hospitals that are then dispensed by a community pharmacy. This practice often leads to considerable wastage of medicine. As part of their professional role, community pharmacists are expected to contribute more to the appropriate use of medication by patients. Using a prospective cohort, we aimed to evaluate pharmacists' role in the community. Methods: We created a patient registry system for community pharmacies to monitor long-term medication use by patients with chronic conditions. Patient drug adherence and potential problems were monitored through regular home visits or telephone calls by the pharmacist at least once a month between patient hospital visits. Patient data were collected and stored in an internet-based system. Results: Over a one-year follow-up, 28 out of 37 registered patients from 14 community pharmacies were continuously monitored. In total, we extracted 19 problems relating to medication use, 17 to physical complaints, eight to patient concerns, and two others. Conclusion: The registry system was useful for identifying medication-related problems as well as patient concerns and changes in their condition. Pharmacists might play a key role in improving patient care in the community.

Analysis of the Mechanism of Prolonged Persistence of Drug Interaction between Terbinafine and Amitriptyline or Nortriptyline.

The purpose of the study was to quantitatively estimate and predict drug interactions between terbinafine and tricyclic antidepressants (TCAs), amitriptyline or nortriptyline, based on in vitro studies. Inhibition of TCA-metabolizing activity by terbinafine was investigated using human liver microsomes. Based on the unbound Ki values obtained in vitro and reported pharmacokinetic parameters, a pharmacokinetic model of drug interaction was fitted to the reported plasma concentration profiles of TCAs administered concomitantly with terbinafine to obtain the drug-drug interaction parameters. Then, the model was used to predict nortriptyline plasma concentration with concomitant administration of terbinafine and changes of area under the curve (AUC) of nortriptyline after cessation of terbinafine. The CYP2D6 inhibitory potency of terbinafine was unaffected by preincubation, so the inhibition seems to be reversible. Terbinafine competitively inhibited amitriptyline or nortriptyline E-10-hydroxylation, with unbound Ki values of 13.7 and 12.4 nM, respectively. Observed plasma concentrations of TCAs administered concomitantly with terbinafine were successfully simulated with the drug interaction model using the in vitro parameters. Model-predicted nortriptyline plasma concentration after concomitant nortriptylene/terbinafine administration for two weeks exceeded the toxic level, and drug interaction was predicted to be prolonged; the AUC of nortriptyline was predicted to be increased by 2.5- or 2.0- and 1.5-fold at 0, 3 and 6 months after cessation of terbinafine, respectively. The developed model enables us to quantitatively predict the prolonged drug interaction between terbinafine and TCAs. The model should be helpful for clinical management of terbinafine-CYP2D6 substrate drug interactions, which are difficult to predict due to their time-dependency.

Pharmacokinetic model incorporating mechanism-based inactivation of CYP2D6 can explain both non-linear kinetics and drug interactions of paroxetine.

OBJECTIVE: To develop a pharmacokinetic model able to describe the nonlinear pharmacokinetics of paroxetine (PRX) and to predict the drug-drug interaction between PRX and metoprolol under various dosage regimens. METHODS: A pharmacokinetic model of PRX incorporating mechanism-based inhibition was developed. This model was fitted to the drug concentration profiles obtained after single and repeated administrations of PRX to estimate the pharmacokinetic parameters of PRX and degradation rate constant of cytochrome P450 (CYP) 2D6. It was also fitted to the time profile of S-metoprolol after coadministration of metoprolol and PRX, and the fractional contribution of CYP2D6 to overall clearance of S-metoprolol was estimated. Using the developed model and estimated parameters, an optimal dosage regimen for metoprolol during withdrawal of PRX was simulated. RESULTS: The developed model well described the time profiles of both PRX and metoprolol concentration during concomitant administration. The estimated parameters were consistent with reported values. The nonlinear and accumulation properties of PRX could be explained by mechanism-based inhibition of CYP2D6 by PRX. Upon tapering PRX from 20 mg/ day to 10 mg/day for 14 days then 5 mg/day for 14 days until cessation, the optimal dosage regimen to resume 120 mg/day of metoprolol based on the developed model was as follows: 30 mg/day during concomitant administration, 40 mg/day for the next 14 days, 60 mg/day for the next 14 days, and finally 120 mg/day. CONCLUSIONS: The developed model enabled us to quantitatively estimate drug-drug interactions of PRX and CYP2D6 substrate drugs, and to predict optimal dosage regimens.

Construction of highly reactive probes for abasic site detection by introduction of an aromatic and a guanidine residue into an aminooxy group.

Abasic sites (AP sites) arise from hydrolysis of glycosidic bonds of DNA that is damaged by various external and internal processes; unrepaired AP sites give rise to genetic mutations. We have constructed highly reactive AP-site-detecting probes by introducing a hydrophobic and a hydrophilic residue in an aminooxy group. Synthesized probes containing either a naphthalene or a guanidine residue conjugate effectively with AP sites. In particular, a probe containing both functional groups shows the highest reaction rate, indicating that the hydrophobic and hydrophilic interactions act cooperatively in reaction with AP sites. The guanidine residue also contributes to the solubility of the molecules in aqueous media. The biotinylated probes provide much more sensitive detection of AP sites in genomic DNA than the conventional aldehyde-reactive probe.

Development of novel chemical probes to detect abasic sites in DNA.

We chemically synthesized a series of aminooxy derivatives to develop novel probes for sensitive detection of abasic (AP) sites in DNA. The results of the conjugation reactions showed that the probes could efficiently react to AP sites by introducing an aromatic or a guanidino group in their structures. In particular, the probe having both functional groups showed the most effective reactivity, indicating that hydrophobic and electrostatic interactions cooperatively acted in the reaction of the probe to AP sites. We then synthesized a biotinylated probe and succeeded in more sensitive detection of AP sites in genomic DNA than with the conventional aldehyde reactive probe (ARP).

Efficient synthesis of oligonucleotide conjugates on solid-support using an (aminoethoxycarbonyl)aminohexyl group for 5'-terminal modification.

Solid-support conjugation at the 5'-terminal primary amine of oligonucleotides is a convenient and powerful method for introducing various functional groups. However, conventional aliphatic amines do not necessarily provide conjugates with sufficient yields. To improve the modification efficacy, we used the amino-linker (aminoethoxycarbonyl)aminohexyl group (ssH-linker), for solid-support conjugation. In the ssH-linker terminal modification, reactive free amino group could be easily presented onto a solid-support due to rapid removal of the amino-protecting group, and activated amino acids or cholesterol molecules could be covalently connected more efficiently than to typical 6-aminohexyl-linkers. Based on these results, the ssH-linker can be a useful terminal modification for the solid-support conjugation of functional molecules.

Construction of an aminooxy derivative for RNA and DNA labeling.

We previously reported that the reactivity of a primary amine is increased by connecting the amine to an aromatic residue with an alkyl linker. This strategy was applied to the construction of a reagent that could form a covalent bond with an aldehyde group. We synthesized a new reagent that consisted of aminooxy and aromatic groups and biotin. The new reagent showed a higher reaction rate to apurinic/apyrimidinic (AP) sites in DNA than a conventional aldehyde-reactive probe (ARP). This reagent also efficiently reacted with 2', 3'-dialdehyde groups, which were generated by the oxidation of the 2', 3'-terminal vicinal hydroxy groups of RNA. The reaction efficiency of the reagent closely related to the structure of the target molecules. We describe the synthesis and chemical properties of the new reagent.

Comparison of the chemical properties of a novel amino-linker with various amino modifications.

We previously reported a series of new amino-linkers, consisting of an aminoethyl carbamate structure (Komatsu, 2008). We have now examined the chemical properties of oligonucleotides modified with an ssH-linker, which is the simplest and most cost-effective derivative of the series. Although it was previously shown that monomethoxytrityl protection on a primary amine of the ssH-linker was cleaved under weakly acidic conditions (1% acetic acid), we found that the deprotection also proceeded in aqueous buffer solutions (pH 6.0, 7.0). The MMT group was removed much faster than other commercially available amino-linkers, and this property enabled the ssH-modified oligonucleotides to be conveniently purified with a cartridge column. Furthermore, the ssH-modified oligonucleotides were utilized in on-support labeling reactions. As compared with other amino-linkers, the ssH-linker was superior in terms of its purification and reaction efficiencies.

Novel amino linkers enabling efficient labeling and convenient purification of amino-modified oligonucleotides.

We developed new amino linker reagents for an oligonucleotide (ONT) terminus. These reagents consist of an aminoethyl carbamate main linkage and a side-chain residue, which was a naphthylmethoxymethyl, methoxymethyl, or methyl group or a hydrogen atom. The primary amine was protected with a monomethoxytrityl (MMT) group. The chemical properties of ONTs containing these amino-modifications were investigated. The MMT group of these amino-modifications could be quite rapidly removed from the amine under very mild acidic conditions, which are not strong enough for the deprotection of a conventional aliphatic amine. This significant feature enabled the amino-modified ONTs to be conveniently purified with a reverse phase column. Furthermore, the amino-modifications efficiently reacted to active esters, as compared with other amino-modifications. We also found that the pK(a) values of the amino-modifications were lower than that of the aliphatic amine. All of the experimental results showed that these chemical properties are closely related to their structures. We report here the chemical properties and the availability of the new amino linker reagents.

Enhanced reactivity of amino-modified oligonucleotides by insertion of aromatic residue.

We developed novel amino-modifying reagents, of which an amino group was connected with an aromatic residue by aliphatic linker. It was proved that the insertion of the aromatic residue could increase the reactivity of the amino group on oligonucleotides in comparison with conventional amino-modification.

Generation of an abasic site in an oligonucleotide by using acid-labile 1-deaza-2'-deoxyguanosine and its application to postsynthetic modification.

We developed a new convenient method for generation of an abasic site at the 3'-terminus of an oligonucleotide. This method uses a 1-deaza-2'-deoxyguanosine residue, which easily undergoes depurination under acidic conditions. The abasic site of the oligonucleotide can be further modified with external functional groups. We report herein the chemical stability of 1-deaza-2'-deoxyguanosine in the oligodeoxynucleotide and the application to the postsynthetic modification of an oligonucleotide by utilizing the chemical property of 1-deaza-2'-deoxyguanosine. [Structure: see text]

High throughput purification of amino-modified oligonucleotides and its application to novel detection system of gene expression.

We report here a new amino-modifier reagent, which enable high-throughput purification of amino-modified oligonucleotides. Either monomethoxytrityl (MMT) or trifluoroacetyl (TFA) group has been used as the protecting group for the primary amine when amino-terminal oligonucleotides are prepared. Generally, the removal of MMT requires the stringent acidic treatment after the oligonucleotide synthesis. We chemically synthesized a novel amino-modifier with the MMT protection. It was found that the new amino-modifier released MMT group under mild acidic condition, and then rapid purification of diverse amino-modified oligonucleotides could be achieved with cartridge column of reverse phase. Furthermore, we tried to construct the new detection system to study gene expression using the amino-modified oligonucleotides. The new amino-modifier will be useful for molecular biology by facilitating the construction of oligonucleotide library and improving the chemical reactivity.

Synthesis and application of new amino modification analogues for functional oligonucleotide.

We synthesized new analogues to functionalize oligonucleotides. These analogues included a primary amino group tethering to an aromatic ring, and we introduced them into the 5'-end of each oligonucleotide. The oligonucleotides with the aromatic amino group (OAA) were easily purified from impurities by using their hydrophobic property of the aromatic residue. These OAA probes reacted with activated ester groups more efficiently than the conventional probe, which was modified with 6-aminohexyl group. Furthermore, we applied these OAAs to oligonucleotide array probes. The OAA probes were efficiently immobilized on the array surface, and the hybridization intensity on these probes increased as compared with the conventional probes. These new probes can be a new nucleic acid tool for biological assay systems.

Distribution of tenascin-c and -X in rat TMJ development.

In the neonatal and postnatal development of rat TMJ, tenascin-C and -X were detected in the muscle, bone matrix, connective tissue around the bone, and blood vessel of rats at E18 (18-days old embryo), 0-, and 5-days postnatal. The reaction of tenascin-X was also found in the connective tissue around the mandibular condyle. The mRNA of tenascin-C (600 bp) and -X (588 bp) was also detected in the developmental muscle with the level of tenascin-C mRNA moderately decreased during development. Therefore, tenascin-C and -X may have different effects on the connective tissue during development of TMJ.