Evaluation of activity and toxicity of combining clioquinol with ciclopirox and terbinafine in alternative models of dermatophytosis.

Dermatophytosis is a superficial fungal infection that affects humans and is very common in small animals. The treatment using the most commonly used antifungals is failing, and new therapeutic alternatives are required to combat the resistance of these fungal infections. Previous studies by the group have shown that clioquinol is an important therapeutic alternative in the treatment of dermatophytosis. The object was to conduct studies of antidermatophytic activity and the irritant potential from the double and triple combinations of clioquinol, terbinafine and ciclopirox in ex vivo and in vivo alternative models. To evaluate the irritant potential of antifungal combinations, the alternative HET-CAM method (chicken egg test chorioallantoic membrane) was used. Ex vivo models were used to assess the effectiveness of antifungal combinations, using pig hooves and veterinary fur. Any possible tissue damage was to assess through in histopathology of swine ears. HET-CAM results showed that all combinations can be classified as non-irritating, corroborated by the results of the histopathological evaluation of the pig's ear skin. Only the double combinations managed to remove 100% of the colony-forming units (CFU) formed on the pig's hooves. The clioquinol + terbinafine combination and the triple combination were more effective than clioquinol + ciclopirox in eradicating the preformed biofilm in fur of veterinary origin. These results show the potential of formulations of clioquinol in combination with antifungals for use in humans and in the veterinary field to combat dermatophytosis, as an important alternative therapy, for use in the near future.

[Fifity years after the identification of the cause of SMON].

SMON (subacute myelo-optico-neuropathy) is toxic neurological disease which had a profound impact on the population in Japan in 1960's. The clinical characteristics of SMON includes an ascending sensory disturbance, spasticity, and visual impairment typically following abdominal symptoms. Infection was first suspected as an underlying cause of this epidemic. The disorder was ultimately attributed to the overuse of clioquinol, based on the analysis of green urine from affected patients and confirmed by the epidemiological surveys and experimental animal studies. The factors that contributed to the prevalence of SMON which remains the worst example of drug-associated toxicity in Japan to date include the conversion of clioquinol from a purely topical agent to an orally-administered drug, dogma associated with drug safety, relatively limited regulation of drug use, an increase in the number of prescriptions due to the availability of universal insurance, as well as the complexity of the associated abdominal symptoms. Periodical examination of the patients diagnosed with SMON continues to this day. As such, it is important to have a better understanding of clioquinol-induced neurotoxicity together with the mechanisms underlying drug susceptibility; we should not permit the memory of this severe and prominent drug-associated toxicity fade from view.

Clioquinol inhibits dopamine-ß-hydroxylase secretion and noradrenaline synthesis by affecting the redox status of ATOX1 and copper transport in human neuroblastoma SH-SY5Y cells.

Clioquinol (5-chloro-7-indo-8-quinolinol), a chelator and ionophore of copper/zinc, was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. Although pathological studies demonstrated axonopathy of the spinal cord and optic nerves, the underlying mechanisms of clioquinol toxicity have not been elucidated in detail. In the present study, a reporter assay revealed that clioquinol (20-50 µM) activated metal response element-dependent transcription in human neuroblastoma SH-SY5Y cells. Clioquinol significantly increased the cellular level of zinc within 1 h, suggesting zinc influx due to its ionophore effects. On the other hand, clioquinol (20-50 µM) significantly increased the cellular level of copper within 24 h. Clioquinol (50 µM) induced the oxidation of the copper chaperone antioxidant 1 (ATOX1), suggesting its inactivation and inhibition of copper transport. The secretion of dopamine-ß-hydroxylase (DBH) and lysyl oxidase, both of which are copper-dependent enzymes, was altered by clioquinol (20-50 µM). Noradrenaline levels were reduced by clioquinol (20-50 µM). Disruption of the ATOX1 gene suppressed the secretion of DBH. This study suggested that the disturbance of cellular copper transport by the inactivation of ATOX1 is one of the mechanisms involved in clioquinol-induced neurotoxicity in SMON.

Clioquinol kills astrocyte-derived KT-5 cells by the impairment of the autophagy-lysosome pathway.

Clioquinol has been implicated as a causative agent for subacute myelo-optico-neuropathy (SMON) in humans, although the mechanism remains to be elucidated. In this study, we utilized astrocyte-derived cell line, KT-5 cells to explore its potential cytotoxicity on glial cells. KT-5 cells were exposed in vitro to a maximum of 50 µM clioquinol for up to 24 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte trazolium bromide (MTT) assay of the cells revealed that clioquinol induced significant cell damage and death. We also found that clioquinol caused accumulation of microtubule-associated protein light chain-3 (LC3)-II and sequestosome-1 (p62) in a dose- and time-dependent manner, suggesting the abnormality of autophagy-lysosome pathway. Consistent with these findings, an exposure of 20 µM clioquinol induced the accumulation of cellular autophagic vacuoles. Moreover, an exposure of 20 µM clioquinol provoked a statistically significant reduction of intracellular lysosomal acid hydrolases activities but no change in lysosomal pH. It also resulted in a significant decline of intracellular ATP levels, enhanced cellular levels of reactive oxygen species, and eventually cell death. This cell death at least did not appear to occur via apoptosis. 10 µM Chloroquine, lysosomal inhibitor, blocked the autophagic degradation and augmented clioquinol-cytotoxicity, whereas rapamycin, an inducer of autophagy, rescued clioquinol-induced cytotoxicity. Thus, our present results strongly suggest clioquinol acts as a potentially cytotoxic agent to glial cells. For future clinical application of clioquinol on the treatment of neurological and cancer disorders, we should take account of this type of cell death mechanism.

Synergistic association of clioquinol with antifungal drugs against biofilm forms of clinical Fusarium isolates.

BACKGROUND: The influence of biofilm on the complexity of fungal diseases has been reported in recent years, especially in non-invasive mycoses such as keratitis and onychomycosis. The difficulty in treating cases of fusariosis in the human medical clinic exemplifies this situation, because when Fusarium spp. are present in the form of biofilm, the permeation of antifungal agents is compromised. OBJECTIVES: This study proposes an association of clioquinol, an inhibitor of fungal cells with antifungal drugs prescribed to combat fusariosis in humans. METHODS: Susceptibility was assessed by microdilution in broth. Formation of biofilm by staining with violet crystal. Inhibition and removal of biofilm using the MTT colorimetric reagent. Time-kill combination, hypoallergenicity test, cytotoxicity test and toxicity prediction by computer analysis were also performed. RESULTS: Clioquinol associated with voriconazole and ciclopirox inhibited biofilm formation. Possibly, clioquinol acts in the germination and elongation of hyphae, while voriconazole prevents cell adhesion and ciclopirox the formation of the extracellular polymeric matrix. The CLIO-VRC association reduced the biofilm formation by more than 90%, while the CLIO-CPX association prevented over 95%. None of the association was irritating, and over 90% of the leucocytes remained viable. Computational analysis does not reveal toxicity relevant to CLIO, whereas VRC and CPX showed some risks for systemic use, but suitable for topical formulations. CONCLUSIONS: The combination of CLIO-VRC or CLIO-CPX proved to be a promising association strategy in the medical clinic, both in combating fungal keratitis and onychomycosis, since they prevent the initial process of establishing an infection, the formation of biofilm.

Preparation and characterization of gellan gum/glucosamine/clioquinol film as oral cancer treatment patch.

To administer cancer drugs with improved convenience to patients and to enhance the bioavailability of cancer drugs for oral cancer therapy, this study prepared gellan gum/glucosamine/clioquinol (GG/GS/CQ) film as the oral cancer treatment patch. GG/GS/CQ film fabricated through the EDC-mediated coupling reactions (GG/GS/CQ/EDC film). The film of the physicochemical properties and drug release kinetics were studied. The effectiveness of GG/GS/CQ/EDC film as oral cancer treatment patch were evaluated with the animal model. The results confirmed that CQ can be incorporated via EDC-mediated covalent conjugation to gellan gum/glucosamine. Mechanical testing revealed that the maximum tensile strength and elongation percentage at break were 1.91kgf/mm2 and 5.01% for GG/GS/CQ/EDC film. After a drug release experiment lasting 45days, 86.8% of CQ was released from GG/GS/CQ/EDC film. The Huguchi model fit the GG/GS/CQ/EDC drug release data with high correlation coefficients (R2=0.9994, respectively). The effect of the CQ dose on oral cancer cells (OC-2) was tested, and the IC50 of CQ alone and CQ with 10µM CuCl2 were 9.59 and 2.22µM, respectively. The animal testing indicated that GG/GS/CQ/EDC film was decreased epidermal growth factor receptor (EGFR) expression and suppress tumor progression. These findings provide insights into a possible use for GG/GS/CQ/EDC film for oral ca in clinical practice. The GG/GS/CQ/EDC film is suitable as the dressing for use in the treatment of early-stage cancer or as wound care after surgery in late-stage of oral cancer treatment.

Dihydropyrimidine-Thiones and Clioquinol Synergize To Target ß-Amyloid Cellular Pathologies through a Metal-Dependent Mechanism.

The lack of therapies for neurodegenerative diseases arises from our incomplete understanding of their underlying cellular toxicities and the limited number of predictive model systems. It is critical that we develop approaches to identify novel targets and lead compounds. Here, a phenotypic screen of yeast proteinopathy models identified dihydropyrimidine-thiones (DHPM-thiones) that selectively rescued the toxicity caused by ß-amyloid (Aß), the peptide implicated in Alzheimer's disease. Rescue of Aß toxicity by DHPM-thiones occurred through a metal-dependent mechanism of action. The bioactivity was distinct, however, from that of the 8-hydroxyquinoline clioquinol (CQ). These structurally dissimilar compounds strongly synergized at concentrations otherwise not competent to reduce toxicity. Cotreatment ameliorated Aß toxicity by reducing Aß levels and restoring functional vesicle trafficking. Notably, these low doses significantly reduced deleterious off-target effects caused by CQ on mitochondria at higher concentrations. Both single and combinatorial treatments also reduced death of neurons expressing Aß in a nematode, indicating that DHPM-thiones target a conserved protective mechanism. Furthermore, this conserved activity suggests that expression of the Aß peptide causes similar cellular pathologies from yeast to neurons. Our identification of a new cytoprotective scaffold that requires metal-binding underscores the critical role of metal phenomenology in mediating Aß toxicity. Additionally, our findings demonstrate the valuable potential of synergistic compounds to enhance on-target activities, while mitigating deleterious off-target effects. The identification and prosecution of synergistic compounds could prove useful for developing AD therapeutics where combination therapies may be required to antagonize diverse pathologies.

Novel 8-Hydroxyquinoline Derivatives as Multitarget Compounds for the Treatment of Alzheimer's Disease.

We discovered a small series of hit compounds that show multitargeting activities against key targets in Alzheimer's disease (AD). The compounds were designed by combining the structural features of the anti-AD drug donepezil with clioquinol, which is able to chelate redox-active metals, thus decreasing metal-driven oxidative phenomena and ß-amyloid (Aß)-mediated neurotoxicity. The majority of the new hybrid compounds selectively target human butyrylcholinesterase at micromolar concentrations and effectively inhibit Aß self-aggregation. In addition, compounds 5-chloro-7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (1 b), 7-((4-(2-methoxybenzyl)piperazin-1-yl)methyl)-8-hydroxyquinoline (2 b), and 7-(((1-benzylpiperidin-4-yl)amino)methyl)-5-chloro-8-hydroxyquinoline (3 a) are able to chelate copper(II) and zinc(II) and exert antioxidant activity in vitro. Importantly, in the case of 2 b, the multitarget profile is accompanied by high predicted blood-brain barrier permeability, low cytotoxicity in T67 cells, and acceptable toxicity in HUVEC primary cells.

Histone deacetylase inhibitor attenuates neurotoxicity of clioquinol in PC12 cells.

Clioquinol is considered to be a causative agent of subacute myelo-optico neuropathy (SMON), although the pathogenesis of SMON is yet to be elucidated. We have previously shown that clioquinol inhibits nerve growth factor (NGF)-induced Trk autophosphorylation in PC12 cells transformed with human Trk cDNA. To explore the further mechanism of neuronal damage by clioquinol, we evaluated the acetylation status of histones in PC12 cells. Clioquinol reduced the level of histone acetylation, and the histone deacetylase (HDAC) inhibitor Trichostatin A upregulated acetylated histones and prevented the neuronal cell damage caused by clioquinol. In addition, treatment with HDAC inhibitor decreased neurite retraction and restored the inhibition of NGF-induced Trk autophosphorylation by clioquinol. Thus, clioquinol induced neuronal cell death via deacetylation of histones, and HDAC inhibitor alleviates the neurotoxicity of clioquinol. Clioquinol is now used as a potential medicine for malignancies and neurodegenerative diseases. Therefore, HDAC inhibitors can be used as a candidate medicine for the prevention of its side effects on neuronal cells.

[SMON: toxicity of clioquinol and the status quo].

Subacute myelo-optico-neuropathy (SMON) is a disease characterized by subacute onset of sensory and motor disorders in the lower half of the body and visual impairment preceded by abdominal symptoms. A large number of SMON were observed throughout Japan, and the total number of cases reached nearly 10,000 by 1970. Despite clinical features mimicking infection or multiple sclerosis, SMON was confirmed as being caused by ingestion of clioquinol, an intestinal antibacterial drug, based on extensive epidemiological studies. After the governmental ban on the use of clioquinol in September 1970, there was a dramatic disappearance of new case of SMON. In the 1970s, patients with SMON initiated legal actions against the Government and pharmaceutical companies, and the court ruled that the settlements would be made as health management allowances and lasting medical check-ups. The physical condition of patients with SMON remains severe owing to SMON as well as gerontological complications. The pathological findings in patients with SMON included symmetrical demyelination in the lateral and posterior funiculi of the spinal cord and severe demyelination of the optic nerve in patients with blindness. Although clioquinol may show activity against Alzheimer's disease or malignancy, its toxic effects cause severe irreversible neurological sequelae. Thus, caution must be exercised in the clinical use of clioquinol.

Superoxide dismutase as a target of clioquinol-induced neurotoxicity.

Subacute myelo-optico-neuropathy (SMON) is a progressive neurological disorder affecting the spinal cord, peripheral nerves and optic nerves. Although it has been assumed that SMON was caused by intoxication of clioquinol, the mechanism underlying clioquinol-induced neurotoxicity is not fully understood. This study aimed to clarify the relevance of oxidative stress to clioquinol-induced neurotoxicity and the cause of the enhanced oxidative stress. Clioquinol induced cell death in human-derived neuroblastoma cell line, SH-SY5Y, in a dose-dependent manner. This process was accompanied by activation of caspase-3 and enhanced production of reactive oxygen species (ROS). We examined whether clioquinol inhibited the activity of superoxide dismutase-1 (SOD1), based on its metal chelating properties. Clioquinol inhibited activities of purified SOD1 in a dose-dependent manner. Cytosolic SOD activities were also inhibited in SH-SY5Y cells treated with clioquinol. Finally, addition of exogenous SOD1 to the culture significantly reduced enhanced ROS production and cell death induced by clioquinol in SH-SY5Y cells. These findings suggested that enhanced oxidative stress caused by inhibition of SOD1 undelay clioquinol-induced neurotoxicity and was relevant to the pathogenesis of SMON.

Clioquinol induces DNA double-strand breaks, activation of ATM, and subsequent activation of p53 signaling.

Clioquinol, a Cu²âº/Zn²âº/Fe²âº chelator/ionophor, was used extensively in the mid 1900s as an amebicide for treating indigestion and diarrhea. It was eventually withdrawn from the market because of a link to subacute myelo-optic neuropathy (SMON) in Japan. The pathogenesis of SMON, however, is not fully understood. To clarify the molecular mechanisms of clioquinol-induced neurotoxicity, a global analysis using DNA chips was carried out on human neuroblastoma cells. The global analysis and quantitative PCR demonstrated that mRNA levels of p21(Cip1), an inhibitor of cyclins D and E, and of GADD45α, a growth arrest and DNA damage-inducible protein, were significantly increased by clioquinol treatment in SH-SY5Y and IMR-32 neuroblastoma cells. Activation of p53 by clioquinol was suggested, since clioquinol induced phosphorylation of p53 at Ser15 to enhance its stabilization. The phosphorylation of p53 was inhibited by KU-55933, an inhibitor of ataxia-telangiectasia mutated kinase (ATM), but not by NU7026, an inhibitor of DNA-dependent protein kinase (DNA-PK). Clioquinol in fact induced phosphorylation of ATM and histone H2AX, a marker of DNA double-strand breaks (DSBs). These results suggest that clioquinol-induced neurotoxicity is mediated by DSBs and subsequent activation of ATM/p53 signaling.

Zinc at cytotoxic concentrations affects posttranscriptional events of gene expression in cancer cells.

Zinc at cytotoxic concentrations has been shown to regulate gene transcription in cancer cells, though zinc's involvement in posttranscriptional regulation is less characterized. In this study, we investigated the involvement of cytotoxic zinc in the posttranscriptional steps of gene expression. Clioquinol, a well-established zinc ionophore, was used to raise intracellular zinc to reported cytotoxic levels. The MCF-7 human cancer cell line was applied as a cell model system. Several parameters were used as indictors of posttranscriptional regulation, including p-body formation, microRNA profiling, expression level of proteins known to regulate mRNA degradation, microRNA processing, and protein translation. p-body formation was observed in MCF-7 cells using several molecules known as p-body components. Clioquinol plus zinc enhanced p-body assembly in MCF-7 cells. This enhancement was zinc-specific and could be blocked by a high affinity zinc chelator. The enhancement does not seem to be due to a stress response, as paclitaxel, a commonly used chemotherapeutic, did not cause enhanced p-body formation at a highly cytotoxic concentration. microRNA profiling indicated that clioquinol plus zinc globally down-regulates microRNA expression in this model system, which is associated with the reduced expression of Dicer, an enzyme key to microRNA maturation, and Ago2, a protein essential for microRNA stability. This study demonstrates that ionophoric zinc can induce cytotoxicity in cancer cells by globally regulating posttranscriptional events.

Impairment of recognition memory and hippocampal long-term potentiation after acute exposure to clioquinol.

Clioquinol (CQ) was associated with cases of transient global amnesia and with the neurodegenerative syndrome subacute myelo-optico-neuropathy (SMON) in humans. However, CQ forms lipophilic chelates with cations and has the potential as a scientific and clinical tool used for selective modulation of histochemically reactive zinc pools. The relationship among transient lack of synaptic zinc release, hippocampal long-term potentiation (LTP) induction and cognitive memory is poorly understood. To evaluate the role of synaptic zinc release, in the present study, hippocampal LTP induction and cognitive behavior were examined in young rats after i.p. injection of CQ (30 mg/kg). Intracellular zinc detected by Timm's stain and extracellular (synaptic cleft) zinc detected by ZnAF-2 were significantly decreased in the hippocampus 6 h after CQ injection. The molecular layer of the dentate gyrus, in which perforant path-granule cell synapses exist, was most responsive to CQ injection. Dentate gyrus LTP was induced similarly to the control 2 h after CQ injection, while significantly attenuated 6-24 h after CQ injection. In the training trial of the object recognition memory 2 h after CQ injection, there was no significant difference in learning behavior between the control and CQ-treated rats. In the test trial, CQ-treated rats showed normal recognition memory 1 h after the training, whereas recognition memory deficit 24 h after the training unlike the control rats. These results indicate that acute exposure to CQ impairs long-term (24 h) memory in the hippocampus of young rats. The CQ-mediated attenuation of dentate gyrus LTP, which may be associated with the transient lack of zinc release from zincergic neurons, seems to be involved in the impairment of the long-term memory.

The toxicology of Clioquinol.

5-Chloro-7-iodo-quinolin-8-ol (Clioquinol) is a halogenated 8-hydroxyquinoline that was used in 1950-1970s as an oral anti-parasitic agent for the treatment and prevention of intestinal amebiasis. However in the 1970s oral Clioquinol was withdrawn from the market due to reports of neurotoxicity in Japanese patients. Recently, reports have demonstrated that Clioquinol has activities beyond its use as an antimicrobial. For example, Clioquinol inhibits the function of the proteasome and displays preclinical efficacy in the treatment of malignancy. In addition, due to its ability to bind copper and dissolve beta-amyloid plaques in the brain, Clioquinol has been investigated for the treatment of Alzheimer's disease. As such, efforts are underway to repurpose Clioquinol. In light of the reemergence of oral Clioquinol, we review the toxicology of this compound in animals and humans with an emphasis on its neurotoxicity. Such information will aid in the design of clinical trials of oral Clioquinol for new indications such as cancer therapy.

The oxidative neurotoxicity of clioquinol.

Clioquinol is a metal chelator that may attenuate beta-amyloid deposition and mitigate the progression of Alzheimer's disease. Its prior use as a systemic antibiotic was associated with a neurodegenerative syndrome, subacute myelo-optico-neuropathy (SMON), although a mechanistic link has not been precisely defined. While testing clioquinol in murine cortical cultures, it was observed to have a pro-oxidant effect. Exposure to 1-3 microM for 24 h increased malondialdehyde, and resulted in death of approximately 40% of neurons; a higher concentration (30 microM) was paradoxically less toxic. Both malondialdehyde production and cell death were attenuated by concomitant treatment with the antioxidants ascorbic acid and Trolox C, or with the lipid-soluble metal chelator 1,10-phenanthroline. In contrast, injury was increased in cultures prepared from mice lacking heme oxygenase-2, which protects against non-heme mediated oxidative injury to neurons. Addition of vitamin B12 to the culture medium was not cytoprotective. These results suggest that therapeutically relevant concentrations of clioquinol are toxic to cultured neurons by an oxidative mechanism that is unrelated to vitamin B12 deficiency. In vivo evaluation of the pro-oxidant effect of clioquinol seems warranted prior to further clinical trials.

[Electrophysiological and histopathological study on visual toxicity of clioquinol].

OBJECTIVE: To evaluate the effect of clioquinol on the optic nerve and retina of rhesus monkeys by ophthalmoscopy, electrophysiology and histopathology. METHODS: Clioquinol was given orally to 5 monkeys, gradually increasingly from 100 mg/kg/day up to 14 months(total dosage 227 g/kg). Ophthalmoscopy, erectroretinogram(ERG), visual evoked potential(VEP) and histopathological examination of enucleated eyeballs were done periodically up to 10 years. RESULTS: The margin of the optic disc was not clear at the early stage, but the colour became atrophic at the late stage. VEP maximum amplitude decreased quickly at the early stage and the amplitude of ERG a and b waves and oscillatory potential decreased gradually. 37 months after the discontinuation of administration of VEP, ERG amplitude increased gradually. Swelling of axons and disorganization of the myelin sheath were noticed 2.5 months after beginning treatment. Swelling of the peripapillary nerve fiber layer was seen 5.5 months after beginning treatment. Karyorrhexis was seen in the inner layer of the retina after 12.5 months. Axonal swelling disappeared and the myelin sheath became reorganized 9 months after the discontinuation of treatment. CONCLUSIONS: Clioquinol produced an early decrease of electrophysiological function, but recovery of function was seen after discontinuation of treatment. The degeneration of axons and myelin sheath continued during treatment, and interruption of the degeneration was seen after discontinuation of treatment.

Subacute myelo-optico-neuropathy: clioquinol intoxication in humans and animals.

It remains a tragic event that some 10,000 individuals in Japan developed a unique neurologic disease, subacute myelo-optico-neuropathy (SMON). Many of the affected patients still suffer serious sequelae, such as dysesthesia and muscle weakness in the lower extremities, and loss or deficits in visual acuity. Neuropathologic studies on SMON patients and experimental reproduction of the disease in animals which had been administered clioquinol helped resolve the etiology of this disease. Common pathologic features seen in SMON patients and in dogs and cats chronically intoxicated with clioquinol were distal dominant axonopathy, mainly in the spinal long tracts and optic tracts. Particular abdominal symptoms present in patients after clioquinol ingestion could also be reproduced experimentally in dogs. SMON research in Japan may be worth reviewing for determining the etiology and preventing similar neurotoxic diseases in the future.

[SMON and pharmacokinetics of chinoform with special reference to animal species difference].

The experimental reproduction of SMON using several kinds of animals given a prolonged administration of chinoform has been carried out by many investigators because of the importance to solve the problem of etiology in the SMON. In these experiments, it is demonstrated that a marked species difference was observed in the relationship between the doses given to animals and the frequency of the onset of neurologic symptoms. Although dogs were accepted as the most suitable animal model for SMON, pathological changes in the peripheral nerve of the dog were not observed. The blood level or tissue distribution of chinoform after oral, intravenous or intraperitoneal administration of the drug differed in the animal species. Thus, it is considered that the species difference in the onset of neurologic symptoms is principally caused by the difference in pharmacokinetics of chinoform in each animal. Moreover, for the onset of neurologic symptoms in animals, perhaps it is necessary to maintain the level of unconjugated chinoform in the nerve tissues around several to over ten micrograms/ml for three or four weeks as well as that in SMON patients while the neurologic symptoms or pathological changes do not appear in some kinds of animals at these levels. In a study on the cellular toxicity of chinoform, many other problems remain to be solved although degeneration or uncoupling on oxidative phosphorylation in mitochondria of the axons by chinoform and lipid peroxidation of the membrane by chinoform-ferric chelate have been already shown.