Chemical design of (99m)Tc-labeled probes for targeting osteogenic bone region.

Radionuclide bone imaging using polynuclear (99m)Tc complexes of bisphosphonates is the most common clinical practice in nuclear medicine. However, the improvement in the contrast between normal and osteogenic bone regions has been required. Herein we reported a new (99m)Tc-labeled compound considering the increased vascular permeability of osteogenic region. We selected penta-d-Asp as both a targeting motif to hydroxyapatite (HA) and a molecular size modifier, and two penta-d-Asp molecules were conjugated with the two carboxylate residues of ethylene dicysteine (EC) selected as the (99m)Tc chelating moiety to prepare EC-[(d-Asp)5]2. The molecular size, HA binding, and pharmacokinetics of (99m)Tc-EC-[(d-Asp)5]2 in normal mice and model rats bearing osteogenic tumor were compared to those of (99m)Tc-MDP and (99m)Tc-EC with one (d-Asp)5 motif, (99m)Tc-EC-(d-Asp)5. The molecular size of (99m)Tc-EC-[(d-Asp)5]2 was higher than that of (99m)Tc-MDP and (99m)Tc-EC-(d-Asp)5 when determined by permeability of the (99m)Tc-compounds through a membrane filter (10 kDa). The HA binding of (99m)Tc-EC-[(d-Asp)5]2 was higher than and similar to that of (99m)Tc-EC-(d-Asp)5 and (99m)Tc-MDP. (99m)Tc-EC-[(d-Asp)5]2 exhibited significantly lower accumulation in normal bone of mice than did (99m)Tc-MDP. In osteogenic tumor bearing model rats, (99m)Tc-EC-[(d-Asp)5]2 accumulated in the osteogenic and normal bone region similar to and lower than (99m)Tc-MDP, respectively. Although further studies including the chain length of d-Asp are required, these findings indicated that the present chemical design of (99m)Tc-labeled probe would be applicable to develop (99m)Tc-labeled probes for selective imaging of osteogenic bone region as well as develop therapeutic agents using therapeutic radionuclides such as (90)Y, (177)Lu, (186)Re, or (188)Re and cytotoxic agents to osteogenic bone tumor region.

Quantification of the levels of CYP2D6 mRNA in peripheral blood leukocytes by reverse transcriptase polymerase chain reaction HPLC.

We developed a sensitive high performance liquid chromatography (HPLC) method for determining CYP2D6 mRNA in peripheral blood leukocytes (PBL) by using competitive reverse transcriptase polymerase chain reaction (RT-PCR). The method is specific, reproducible, and sensitive enough to quantify the absolute amount of low and high abundant CYP2D6 mRNA. The native CYP2D6 transcript and the internal standard, a CYP2D6 deletion RNA, were amplified with similar efficiency in RT-PCR. The PCR products were separated as the corresponding peaks in optimized HPLC. The coefficients of variation for competitive RT-PCR and HPLC determination were 1.5-6.5% and 0.6-2.4%, respectively, showing high reproducibility and reliability. This approach could also be applicable to the quantification of mRNA expressing on various tissues, including PBL, of which the expression levels were so low that they were hard to determine by existing agarose gel electrophoresis methods.

In-vitro and clinical evaluation of an oral mucosal adhesive film containing indomethacin.

To develop a new mucoadhesive film containing an analgesic combining clinical efficacy and patient comfort, we prepared and evaluated a two-layered film consisting of an adhesive layer containing indomethacin (IM) as the active ingredient and carboxyvinyl polymer (CP) as a bonding agent and a nonadhesive layer containing polyethylene glycol (PEG) to improve film texture. In in vitro and in vivo adhesive tests, the optimal concentration of CP that could be applied to the mucous membrane was 0.2% or 0.3%. Stability testing determined that the optimal storage conditions and expiration period were 4 degrees C without shade and 4 weeks, respectively. The film was clinically evaluated in patients with oral pain. IM at concentrations of 0.5% and 1% provided optimum analgesic effects, and the effects were the greatest in the 1% IM group. The addition of PEG to the nonadhesive layer reduced the number of patients experiencing discomfort at the site where the film was applied. Therefore this film formulation may be useful for local analgesic application due to its low dose requirement, moderate adhesion, and comfortable texture.

Controlled indomethacin release from mucoadhesive film: in vitro and clinical evaluations.

To develop a film formulation allowing controlled release for long-term analgesia, we selected ethyl cellulose (EC) as a novel additive, prepared a film formulation using indomethacin (IM film), and evaluated it in vitro and clinically. In the in vitro experiments, the effects of the EC concentration on the release rate of IM and on the adhesion force to the mucous membrane were investigated. The addition of 10% EC resulted in more sustained slow release compared with no EC, and the adhesion of the film with 10% EC added was similar to that of films containing carboxyvinyl polymer, which we reported previously showed significantly increased adhesion. A two-layered film consisting of an adhesive layer with 2% or 1% IM and 10% EC and a nonadhesive layer with 2% polyethylene glycol as a softening agent, was investigated for clinical use. Film consisting of an adhesive layer with 2% IM and 10% EC exhibited rapid onset of potent analgesia and was expected to prolong the duration of analgesia. These results suggest that IM film with EC added may be useful clinically, since it shows both immediate analgesic effects and prolonged duration of release.

Oral candidiasis deteriorated by local application of a glucocorticoid-containing film in a mouse model.

In order to estimate predisposing activity of oral application of beclomethasone dipropionate (BDP)-containing mucoadhesive films for oral candidiasis, the effects of BDP on growth of Candida albicans were examined in vivo and in vitro. Murine neutrophils inhibited the mycelial growth of C. albicans in vitro, but this anti-Candida activity was clearly suppressed by the presence of 10(-6) M of BDP. In vitro, a BDP-release test showed that the amount of BDP released from BDP-containing films into the fluid phase increased in a time- and concentration-dependent manner and reached about 10-15% of the total amount of BDP in the film within 30 min. When the BDP-containing film was attached to the tongues of mice orally infected with C. albicans, oral infection by C. albicans deteriorated, but not as severely as in mice systemically immunosuppressed with prednisolone. Based on these findings, we also discuss the problems associated with the clinical application of BDP-film as an anti-inflammatory tool.