再利用抗精神疾病藥物Loxapine作為宿主標的性抗菌藥物對抗鼠傷寒沙門氏菌感染之效力

Abstract

抗生素可用於治療大部分細菌感染疾病，然而不當的濫用使得許多抗藥性的產生，而部分具有兼性胞內寄生特性的病原菌，甚至可以透過入侵宿主細胞，來逃避外在抗生素的作用，並持續在細胞內存活(例：Salmonella spp.)，更凸顯出尋找對抗多重抗藥性胞內寄生菌藥物的急迫性。本實驗中，我們以受鼠傷寒沙門氏菌感染的巨噬細胞進行篩選，發現了一個具有抑菌效力的抗精神疾病藥物─Loxapine，而本篇研究即為探討Loxapine 的抑菌能力和與抗生素組合作用的差異。首先，我們以廣大黴素(Gentamicin)與不同濃度Loxapine 共同作用。得知在4 μM下，Loxapine 不僅能夠有效抑制細菌於巨噬細胞內的增生，同時也抑制胞外細菌，使總菌量降低至百分之五十以下，而此藥的半數致死量(IC50)高達108 μM，與抑菌所需濃度相差25 倍之多，確定了抑菌效果並非細胞的死亡；而以此濃度，與其它胺基醣苷類抗生素合併處理，或改以多重抗藥性菌株為對象，也會展現出相同的抑菌效果。其次，為了確認Loxapine 抑菌作用的特異性，我們先以生長曲線實驗確定了Loxapine 並不會對沙門氏菌有直接的抑制作用；而在後續的實驗中，我們也得知Loxapine 單獨作用下會有些許的抑菌效果，對於感染的細胞也有保護的效力。而在Loxapine 與胺基醣苷類類抗生素的增益上，我們以測試巨噬細胞內抗生素的堆積以及活性來觀察，發現在這兩者Loxapine 對並無直接性的影響。最後，我們以感染了鼠傷寒沙門氏菌的BALB/c 老鼠進行實驗，結果卻發現同時給予Loxapine 與胺基糖苷類抗生素的組別，其存活率與單獨給予抗生素的組別，並無顯著性的差異，我們懷疑造成此結果的主因，可能是由於Loxapine 的血清濃度無法達到抑菌所需濃度。從前面的體外實驗中，證實了Loxapine 具有做為以宿主為導向的抗細菌藥物，可作為胺基醣苷類抗生素的佐劑共同使用。

Antibiotics are medicines used to treat bacterial infections. Due to the inappropriate use of antibiotics, many pathogenic bacteria resistant to multiple-antibiotic have emerged worldwide. Moreover, some facultative intracellular bacterial pathogen can evade the bactericidal effect of antibiotics by invading into host cell and survival in cytoplasm (ex. Salmonella spp.). Thus, there is an urgent need to find out an innovative therapeutic approach for diseases caused by intracellular multiple-drug resistant bacteria. Here, we used Salmonella Typhimurium ATCC14028 strain infected RAW 264.7 cell to identify an antipsychotic drug, loxapine, having the ability to be used as a new antibacterial agent. We first showed that 4μM of loxapine can lower total bacteria to less than 50% of that in control group combined with gentamicin, which was 1/27 of its IC50 (108 μM) toward RAW264.7 cells. Loxapine at 4μM displayed the same inhibitory activity in combined with different aminoglycosides and was effective against multidrug-resistant S. Typhimurium strains. Next, the growth curve of S. Typhimurium proved that loxapine doesn’t suppress the bacteria directly, and the viability of host cells also proved that loxapine might have the ability to protect infected cells. To further investigate the complementary between loxapine and aminoglycosides, we tested the intracellular accumulation and uptake of aminoglycosides, but the results just indicate that the increase inhibition was not caused by the loxapine co treatment. Finally, we evaluate the in vivo efficacy of loxapine combined gentamicin in Salmonella-infected BALB/c mice. Surprisingly, loxapine didn't present the same effect in the in vivo test. The survival rate of combination group didn't show any significant difference compared with aminoglycoside-only group. The lack of efficacy might be due to the low serum concentration of loxapine, increasing the difficulty for loxapine to reach the antibacterial concentration in vivo. According to these findings, loxapine possesses a unique host-targeting antibacterial effect against intracellular pathogen and would be a potential antibiotic adjuvant for the treatment of intracellular bacterial-infection.