Refining the therapeutic range of posaconazole and isavuconazole for efficient therapeutic drug monitoring using a bioassay approach

Therapeutic drug monitoring (TDM) of antifungal triazole was recommended, except for isavuconazole (ISA) whose target trough concentrations (C_min) need to be specified. Concerning posaconazole (POS), tablet formulation results in higher exposure but no upper C_min threshold has been yet proposed. We aimed to investigate the pharmacokinetic–pharmacodynamic relationship of POS and ISA, using a bioassay approach as surrogate marker of antifungal activity, in order to refine the therapeutic C_min of both antifungals. A bioassay using a cellulose disk diffusion method was performed to determine the growth inhibition zone (GIZ) of POS and ISA on Aspergillus fumigatus and Candida parapsilosis (ISA only). GIZs of plasma from patients undergoing TDM for POS (n = 136) or ISA (n = 40) were determined. GIZs of plasma patients and antifungal C_min were highly correlated for ISA (A. fumigatus: ρ = 0.942, P < 0.0001; C. parapsilosis: ρ = 0.949, P < 0.0001) and POS (ρ = 0.922, P < 0.0001), and these relationships were represented with a Michaelis–Menten model. Based on this modeling, the recommended thresholds of 0.7, 1, and 1.25 mg/L for the POS C_min corresponded to 50.1, 55.2, and 59.1% of the maximal GIZ, respectively. We propose an upper threshold of 4.8 mg/L for the POS C_min and a lower threshold of 2.0 mg/L for the C_min of ISA, as they respectively corresponded to concentrations leading to 90% and 50% of the maximal GIZ on A. fumigatus. The determination of antifungal activity using this bioassay allowed refining target C_min of POS and ISA, especially the upper threshold of POS (4.8 mg/L) and the lower threshold of ISA (2.0 mg/L).     

第5批人绝经尿促性腺素国家标准品的制备与标定

目的:制备和标定第5批人绝经尿促性腺素(HMG)国家标准品。方法:以WHO第5批国际标准品10/286作为标准,选择6个实验室,采用《中华人民共和国药典》2015年版四部1216卵泡刺激素(FSH)测定法和1217黄体生成素(LH)测定法对第5批HMG国家标准品待标品的生物效价进行标定;按照2015年版药典四部通则1431生物统计法中的量反应平行线测定法,采用BS 2000软件计算生物效价,并进行合并计算。结果:第5批HMG国家标准品待标品经协作标定,确定其生物效价FSH为220 IU·安瓿^-1,LH为203 IU·安瓿^-1。结论:本批待标品可作为第5批HMG国家标准品供HMG及相关制剂的生物效价测定用。     

Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of Nifurtimox, an antitrypanosomiasis drug

The chronic toxicity and carcinogenicity of Nifurtimox (NFX), a 5-nitrofuran derivative used in the treatment of American trypanosomiasis, were studied in male and female Wistar rats in an accelerated cancer bioassay (ACB). The ACB is a mechanistic initiation/promotion chronic toxicity and carcinogenicity bioassay designed to assess potential carcinogenic activity of a test substance in critical organs and tissues of rodents in which human carcinogens are active. The organs studied were liver, lungs, urinary bladder (UB), mammary gland (MG), bone marrow, spleen, kidneys, colon, stomach and any grossly observed lesions.

NFX is a genotoxin which has been reported previously to exert a variable degree of carcinogenic activity in rat liver, kidney, UB and MG. The present study was undertaken to assess whether NFX has initiating activity in these four named target sites. In the initiation phase, groups of 20 Wistar rats were given NFX daily in the diet at 0.2% for the first 12 weeks of the study to assess initiating activity, followed by promoters (PROs) for four organs for an additional 24 weeks. NFX was compared to the following known initiators (INs) for each of these four tissues: diethylnitrosamine (DEN) for liver and kidney, N-butyl-N(4-hydroxybutyl)nitrosamine (BBN) for UB and 7,12-dimethylbenz(a)anthracene (DMBA) for MG. PROs included phenobarbital (PB) for liver and kidney, nitrilotriacetic acid (NTA) for UB, and diethylstilbestrol (DES) for MG. NFX was also administered continuously without PROs for 40 weeks. At the end of dosing (40 weeks) and at the end of recovery (52 weeks), animals were sacrificed and subjected to complete gross and histopathological examinations, along with evaluations of body weight gain over time and terminal body weights.

Mortality was highest with DEN+PB (group 6) (40%), followed by BBN+NTA (group 7) (15%) and NFX+DES (group 5) and DMBA+DES (group 8) (10% each). The same groups also showed significant reductions in body weight gain over time and terminal body weights at sacrifice. In these groups, the expected preneoplastic, neoplastic and metastatic neoplastic lesions were produced, demonstrating the sensitivity of the model.

In groups given NFX+PROs (groups 3–5), either no neoplasms occurred (group 4) or only single neoplasms (groups 3 and 5). In contrast, the PROs all elicited tumors in groups given INs (groups 6–8). Also, NFX given alone for 40 weeks did not produce any chronic toxicity, preneoplastic or neoplastic lesions. Thus, in this study, NFX did not demonstrate chronic toxicity or carcinogenicity. Moreover, in four target sites, i.e., liver, kidney, UB and MG, it exhibited no neoplastic initiating activity manifested by PROs for these four target sites.     

Brine shrimp bioassay: importance of correct taxonomic identification of Artemia (Anostraca) species

Despite the common use of the brine shrimp bioassay in toxicology, there is confusion in the literature regarding citation of the correct taxonomic identity of the Artemia species used. The genus Artemia, once thought to be represented by a single species Artemia salina, is now known to be composed of several bisexual species as well as parthenogenetic populations. Artemia franciscana is the best studied of the Artemia species and is considered to represent the vast majority of studies in which Artemia is used as an experimental test organism. We found that in studies referring to the use of A. salina, the zoogeography of the cyst harvest site indicated that the species used was actually A. franciscana. Those performing bioassays with Artemia need to exercise diligence in assigning correct species identification, as the identity of the test organism is an important parameter in assuring the validity of the results of the assay.     

Rat urinary bladder-derived relaxant factor: studies on its nature and release by coaxial bioassay system

The present study was designed to characterize the urinary bladder-derived relaxant factor that was demonstrated by acetylcholine-induced relaxation response in a coaxial bioassay system consisting of rat bladder as the donor organ and rat anococcygeus muscle as the assay tissue. The concentration-dependent relaxation to acetylcholine (10 nM-1 mM) was inhibited by atropine but was not altered by the antagonists of calcitonin gene-related peptide (CGRP 8-37), vasoactive intestinal peptide (VIP 6-28), tachykinin NK1 (L-732138), tachykinin NK2 (MEN-10376), tachykinin NK3 (SB-218795), purinergic P2 (PPADS) and adenosine (CGS 15943) receptors as well as alpha-chymotrypsin. Adenylate cyclase inhibitor SQ-22536 and protein kinase A inhibitor KT-5720 significantly inhibited the acetylcholine response while guanylate cyclase inhibitor ODQ, and protein kinase C inhibitor H-7 did not have any effect. The P2X agonist alpha,beta-methylene ATP (10 nM-0.1 mM) also produced concentration-dependent relaxation response that was inhibited by PPADS, SQ-22536 and KT-5720 in the coaxial bioassay system. In bladder strips, acetylcholine and alpha,beta-methylene ATP elicited concentration-dependent contractions that were not altered in the presence of SQ-22536 and KT-5720. In conclusion, the urinary bladder-derived relaxant factor that was recognized by the coaxial bioassay system is neither a peptide of the bladder neurons nor a purinergic mediator but adenylate cyclase and protein kinase A are involved in its release and/or relaxant effect. Furthermore, activation of purinergic P2X receptors besides the muscarinic receptors leads to the release of this factor.     

Prediction and assessment of mixture toxicity of compounds in antifouling paints using the sea-urchin embryo-larval bioassay

The ecotoxicological assessment of alternative "booster" biocides is urgently needed in order to develop environmentally acceptable antifouling paints. However, research has focused mainly on single compounds, and there is still a lack of data on their mixture toxicity. The present study investigated the single and mixture toxicity of three of the most widely used antifouling biocides: zinc pyrithione, chlorothalonil and Sea-Nine, using the sea-urchin (Paracentrotus lividus) embryo-larval bioassay. Also, the predictive ability of the concentration addition (CA) and independent action (IA) concepts for antifouling mixtures was evaluated. Both concepts failed to accurately predict the toxicity of the antifouling mixtures, with the exception of the zinc pyrithione and Sea-Nine mixture, which was accurately predicted by the IA concept, suggesting a dissimilar mode of action of those substances. In general, CA predicted consistently higher toxicity than IA; however, CA overestimated the toxicity of the studied mixtures by a factor of only 1.6, representing a reasonable worst-case approach to be used in the predictive hazard assessment of antifouling mixtures. Finally, the present study demonstrates that the risk of antifouling mixtures for the early developmental stages of sea urchin is higher than the risk of each single substance, and therefore, the inclusion of mixture considerations in the development of water quality criteria for antifouling compounds is strongly recommended.     

Toxic marine puffer fish in Thailand seas and tetrodotoxin they contained

A total of 155 puffers caught from two of Thailand's seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.     

Human ScFv that block sodium ion channel activity of tetrodotoxin

Tetrodotoxin (TTX) is a heterocyclic guanidinium alkaloid (C₁₁H₁₇N₃O₈) with molecular mass of ∼320 Da. The TTX and toxic analogs block sodium ion activity of mammalian nerve cells resulting in failure to conduct nerve impulse which manifested clinically in host as variable degrees of organ paralysis. Human intoxication occurs after consuming food containing the toxins. Current treatment of the poisoning is supportive and symptomatic. There has been no specific drug or antidote for the TTX mediated malady. In this study, phage clones displaying human single chain antibody fragments (HuScFv) were selected from a human ScFv phage display library. HuScFv derived from phagemid transformed Escherichia coli clones (clones s16 and s35) bound to the TTX as tested by indirect ELISA and band shift assay. Homology modeling and molecular docking revealed that VL domain of the s16-HuScFv interacted with the hydroxyl groups of C6, C9, C10 and C11 of the TTX by means of Tyr 223, Ser226 and Tyr228, while the Asp53 and Asp55 of the VH domain of s35-HuScFv interacted with the positions 1 and 2 of the guanidinium group and the hydroxyl groups at C9 and C10 of the TTX. The s16- and s35-HuScFv neutralized the TTX bioactivity in nerve cell based- and mouse bio-assays. Moreover, the HuScFv could rescue the intoxicated mice from the TTX mediated lethality. Thus, the HuScFv derived from the transformed E. coli clones have high potential as a safe, effective and specific therapeutic remedy for TTX intoxication in humans and warrant further trials.