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the free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies. The free-living amoebae (FLA) Naegleria spp. and Acanthamoeba spp. are mainly distributed in ponds, rivers, and fresh waters worldwide. Their existing stages are trophozoite and cyst and additional biflagellate form in in case of Naegleria. Trophozoites shows moving, feeding, and proliferation activity. However, cysts are formed in poor environments, such as under abrupt temperature changes, drying, and food depletion, and can survive for long periods 1,2. N. fowleri is a pathogenic agent that causes primary amoebic meningoencephalitis (PAM), which is fatal to humans and laboratory animals 2,3. Acanthamoeba spp. and Balamuthia mandrillaris cause chronic granulomatous amebic encephalitis (GAE) 4-6. Further, A. castellanii and A. polyphaga can infect the eye, resulting in acanthamoeba keratitis (AK) 2,7,8. PAM is mainly associated with activities in amoeba-contaminated water (swimming or water leisure activity), use of Neti-pots in rhinitis treatment, and religious ceremonies in some Asian countries 5,9,10. The amoeba enters through the nasal cavity to invade the mucosal membrane. Subsequently, it moves into the olfactory bulb and meninges via the nasal nerve system, leading to development of meningoencephalitis 11-13. Symptoms of PAM include acute headache, anorexia, nausea, vomiting, high fever (38-40 °C), and limb dysfunction symptoms. It also progresses acutely, with a mortality rate of over 95%. Amphotericin B is mainly used as a therapeutic agent, as a combination treatment by mixing with micronazole, rifampin, and doxycycline; however, only limited therapeutic effects have been demonstrated 1,2,14-16. AK usually occurs after wearing contaminated contact lenses, improper ophthalmic surgery, or corneal injury in various cases. With the popularization of contact lenses and careless lens management, the number of AK patients continues to increase 2,7,17,18. PAM due to N. fowleri occurs annually worldwide. In the United States, 143 cases of PAM occurred from 1962 to...

Leuprolide is a synthetic nonapeptide drug (pyroGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt) that acts as a gonadotropin-releasing hormone agonist. The continuous administration of therapeutic doses of leuprolide inhibits gonadotropin secretion, which is used in androgen-deprivation therapy for the treatment of advanced prostate cancer, central precocious puberty, endometriosis, uterine fibroids, and other sex-hormone-related conditions. To improve the pharmacokinetic properties of peptide drugs, a fatty acid was conjugated with leuprolide for long-term action. In this study, we developed a simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of leuprolide and leuprolide–oleic acid conjugate (LOC) levels. The developed method was validated in terms of linearity, precision, accuracy, recovery, matrix effect, and stability according to the US Food and Drug Administration guidelines, and the parameters were within acceptable limits. Subsequently, the pharmacokinetics of leuprolide and LOCs were evaluated. In vivo rat subcutaneous studies revealed that conjugation with fatty acids significantly altered the pharmacokinetics of leuprolide. After the subcutaneous administration of fatty-acid-conjugated leuprolide, the mean absorption time and half-life were prolonged. To the best of our knowledge, this is the first study showing the effects of fatty acid conjugates on the pharmacokinetics of leuprolide using a newly developed UPLC-MS/MS method for the simultaneous quantification of leuprolide and LOCs.