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The abuse of drugs has become a serious social problem worldwide. Amphetamine-type stimulants such as methamphetamine are recreationally abused and can cause toxic effects in the body. Unfortunately, death from drug poisoning can occur due to careless intake. In postmortem examinations, the distribution of drugs in an entire organ gives valuable information for evaluating their toxicity. We developed methods to measure the distribution of drugs in organs using LC/MS and matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). The complementary use of the two methods provides more detailed information on the distribution and concentration of drugs in organs because the accurate quantification in LC/MS and small spatial resolution in MALDI-IMS are combined. On the other hand, it is important to elucidate the drug intake history of suspects and victims in drug-facilitated crimes (DFCs). Hair and nail samples are often used to confirm chronic drug intake because ingested drugs can stably remain in these specimens over several months. However, it is impossible to determine the day of drug ingestion in conventional segmental analysis of bulk samples. Therefore, we developed methods to cut hair strands at 0.4-mm intervals and nails at 0.2-mm intervals, which correspond to their respective growth rates over 1-2 d, to analyze the drugs in each segment efficiently using LC/MS. The microsegmental hair analysis method is applied to estimate the day of drug ingestion in DFC investigations. These methods could be applied to measure the distribution of compounds in various solid samples.

Affinity of Luliconazole (LLCZ), an antifungal drug used for topical treatment of onychomycosis in Japan, to nail keratin was demonstrated. Efinaconazole (EFCZ) was used as a reference drug. Drugs at fixed concentrations were added to 4 ml of buffer solution containing 40 mg of nail keratin powder prepared from healthy volunteers or from tinea unguium patients. The mixtures were shaken at 37℃, and adsorption and desorption rates of the drug in nail keratin were measured. Theoretical analysis using the Freundlich adsorption isotherm was applied to eliminate effects of testing conditions on the results. Results showed that compared with EFCZ, LLCZ exhibited high adsorption rates and low desorption rates in nail keratins. These results were verified by Freundlich analysis, in which adsorption coefficient (K ads F ) and desorption coefficient (K ads F ) of LLCZ were 5-7 times and about 2 times higher than EFCZ, respectively. In addition, antifungal activity against Trichophyton rubrum of the desorbed LLCZ samples was determined using disk diffusion assay. In conclusion, LLCZ is considered to possess high affinity to nail keratin. LLCZ, therefore, can be retained in the nail as a reservoir and continuously desorbed at the infection site to exhibit antifungal activity against pathogenic fungi. The pharmacokinetics of LLCZ in the nail is believed to differ from that of EFCZ. As adsorption and desorption rates of the two drugs in nail keratin tended to be different between healthy volunteers and patients, further detailed study is needed.

To clarify the character of luliconazole nail solution we have developed, we investigated luliconazole distribution and antifungal activity in nail plate. An in vitro permeation study which measured luliconazole concentration of sliced nail in the transverse direction after treatment of luliconazole nail solution was conducted to investigate for concentration dependency and the influences of nail thickness and treatment duration. When 0.2, 1, 3, 5, and 7.5% luliconazole nail solutions were used, luliconazole was detected in the all the layers of nail and there was a concentration gradient from the dorsal side to deep nail layers. The luliconazole concentration was almost same after 14-day treatment with 5% luliconazole nail solution when using nails of different thicknesses. And we confirmed that concentration of luliconazole into the nail was increased depending on the treatment duration. In zone of inhibition test after 14-day treatment, 5% luliconazole nail solution showed statistically high formation rate of zones of inhibition compared to 8% ciclopirox nail lacquer. Above all, these data suggested that 5% luliconazole nail solution has the potential to show high therapeutic effect for onychomycosis.

Two topical therapeutic agents were approved in Japan from 2015 to 2016, adding new options for onychomycosis therapy in the clinical field. In order to confirm the differences of formulation properties and nail pharmacokinetics between 5% luliconazole solution and 10% efinaconazole solution, drug concentration and antifungal activity in the nail were measured after topical treatment using human nail plates. In the in vitro permeation study, concentration of each drug was measured in the transversely sliced nail after single treatment with the two topical therapeutic agents. The results showed that concentration of luliconazole is higher than that of efinaconazole at all nail layers, differing by 1.7-8.4 times at each measurement point. Next, we examined antifungal activities of each drug in sliced nail after 14-day topical treatment. Mean rates of formation of inhibition zones for 5% luliconazole solution and 10% efinaconazole solution were 71.0% and 12.6%, respectively, and were statistically different. These results show that the two topical therapeutic agents have different properties, and suggest that 5% luliconazole solution has good nail permeation and retention characteristics. Moreover, luliconazole was found to retain enough antifungal activity in the nail plate against Trichophyton spp. after treatment with the topical agent.

Luliconazole (LLCZ), an imidazole derivative with a broad spectrum of potent antifungal activity especially for T. rubrum and T. mentagrophytes, is under development as a new drug for treatment of tinea unguium.　It is well known that curative effect of an antifungal agent in dermatophytosis is affected by the pharmacokinetics of an agent at the infection loci as well as its antifungal activity, but there is no report about the affinity of LLCZ to nail keratin. We studied LLCZ affinity to keratin powder prepared from healthy human nail and porcine hoof. The LLCZ adsorbed to keratin preparations was washed with phosphate buffer, and its concentration in the buffer supernatant was measured by HPLC. Antifungal titer of the supernatant was also biologically confirmed by disk diffusion assay. Adsorption rate of LLCZ was 80% or more, and LLCZ was gradually liberated into washing buffer. Cumulative liberation rate in 10 times repeated washing against initially adsorbed drug amount was 47.4% for keratin from human nail and was either 52.5% or 50.8% (depending on the LLCZ concentration) for keratin from porcine hoof. The supernatant showed antifungal potential to T. rubrum. These results indicate that LLCZ applied to the nail surface is fully adsorbed to nail keratin and gradually liberated from it. The nail keratin could function as drug reservoir to supply biologically active LLCZ to the nail tissue region of infection loci. The LLCZ delivered to the loci would exert its antifungal potential on tinea unguium. This study also suggests the versatility of porcine hoof powder as an alternative to human nail keratin preparation for non-clinical study.

We have recently developed target-specific derivatization methods for determining the metabolites in human nails. The nails can be used to obtain physiologic information and may serve as noninvasive biosamples for the diagnosis of chronic disease. Certain endogenous biogenic D,L-amino acids, polyamines, and advanced glycation end-products (AGEs) have been detected in human nails. By identifying biological trace components in human samples, we tested the possibility of using these new methods to diagnose chronic disease, i.e., diabetes and lung cancer. When comparing the index from diabetic patients to that from healthy volunteers, there was no significant difference in the content of L-amino acids, methylglyoxal, and glyoxal in the nails. However, a statistically significant correlation was observed between the D/L-amino acid concentration ratios (Ala, Val, Ile, Leu; p<0.01), and 3-deoxyglucosone concentrations (p<0.001). Additionally, in the lung cancer patient group, as compared with healthy volunteers, the concentrations of spermine showed a statistically significant (p<0.05) correlation. This review describes our current target-specific derivatization UPLC-ESI-MS methods for the determination of metabolites and related compounds, e.g., D,L-amino acids, polyamines, and AGEs, in human nails. Advanced applications of human nails as new, noninvasively obtained biosamples for the diagnosis of chronic disease are also described.

The growth of nails, and their matrix components, are influenced by several physiological, pathological and environmental factors. Because of the slow rate of nail growth, the elemental composition of the nail is also expected to be affected by transient factors controlling serum components. The mineral components of nail clippings may therefore reflect the long-term patterns of mineral metabolism, such as the rise of creatinine concentration seen in nails in patients with renal failure with hypercreatinemia. Fingernail and toenail Ca concentrations decreased with age in both men and women, whereas Mg concentrations tended to increase. Postmenopausal women had lower finger nail Ca concentrations than premenopausal women. LBMD showed a significant positive correlation with finger nail Ca content. The measurement of finger nail Ca content may be useful as a predictor of osteoporosis.

The study protocol was approved by the Ethical Committee of Sakai Municipal Hospital. A total of 40 outpatients with tinea unguium of the toenails, fingernails or both took part after giving voluntary written informed consent. Inclusion criteria were suggestive clinical appearance, a positive KOH preparation and an opaqueness of more than 50% of the nail length. The patients received 125 mg terbinafine once a day. The medication was taken after the evening meal and treatment was continued for 12 weeks. Medication stopped in twenty patients who responded well and a follow-up study was continued for 64 weeks. In the other 20 patients, medication was continued for 24 weeks and the follow-up study continued for 64 weeks. At 4-week intervals, the patients were evaluated for their clinical and mycological statuses and adverse reactions. Clipping of distal nail samples, including any attached subungual tissue, was done using nail clippers at 4-week intervals after cessation of therapy and the level of terbinafine was measured in the laboratory. No adverse reactions were detected. Tinea unguium of fingernails (1) and the third toes (2) were cured easily in the short term in the 12 week therapy group. One case in the 24 week therapy group was excluded because systemic steroid therapy was started for bullous pemphigoid at 32 weeks. The results of treatment of big toe onychomycosis were compared between the 12 week (17) and the 24 week (19). In the former group, 9 (52.9%) showed complete cure and 8 (47.1%) showed relapse or worse after cessation of therapy was. In 24 week therapy group, complete cure was achieved in 14 (73.7%) and relapse or worse in 5 (26.3%). The cure rate between the 2 groups was statistically not significant. Terbinafine was detected in the target nails up to 24 weeks after cessation of medication in both groups. Onychomycosis or tinea pedis reappeared in a few cases 12 to 16 weeks after medication ceased in the 12 week group. Topical antimycological therapy is necessary after cessation of oral terbinafine.