WBQ5187, a Multitarget Directed Agent, Ameliorates Cognitive Impairment in a Transgenic Mouse Model of Alzheimer's Disease and Modulates Cerebral ß-Amyloid, Gliosis, cAMP Levels, and Neurodegeneration.

Previously, we designed, synthesized, and evaluated a series of quinolone-benzofuran derivatives as multitargeted anti-Alzheimer's disease (anti-AD) compounds, and we discovered that WBQ5187 possesses superior anti-AD bioactivity. In this work, we investigated the pharmacokinetics of this new molecule, as well as its therapeutic efficacy in restoring cognition and neuropathology, in the APP/PS1 mouse model of AD. Pharmacokinetic analyses demonstrated that WBQ5187 possessed rational oral bioavailability, metabolic stability, and excellent blood-brain barrier (BBB) permeability. Pharmacodynamics studies indicated that a 12-week treatment with the lead compound at doses of 40 mg/kg or higher significantly enhanced the learning and memory performance of the APP/PS1 transgenic mice, and the effect was more potent than that of clioquinol (CQ). Furthermore, WBQ5187 notably reduced cerebral ß-amyloid pathology, gliosis, and neuronal cell loss and increased the levels of cAMP in the hippocampus of these mice. The surrogate measures of emesis indicated that WBQ5187 had no effect at its cognitive effective doses. Overall, our results demonstrated that this compound markedly improves cognitive and spatial memory functions in AD mice and represents a promising pharmaceutical agent with potential for the treatment of AD.

Metal Protein-Attenuating Compound for PET Neuroimaging: Synthesis and Preclinical Evaluation of [11C]PBT2.

Dyshomeostasis or abnormal accumulation of metal ions such as copper, zinc, and iron have been linked to the pathogenesis of multiple neurodegenerative disorders including Alzheimer's disease (AD) and Huntington's disease (HD). 5,7-Dichloro-2-((dimethylamino)methyl)quinolin-8-ol, PBT2, is a second generation metal protein-attenuating compound that has recently advanced in Phase II clinical trials for the treatment of AD and HD based on promising preclinical efficacy data. Herein, we report the first radiosynthesis and preclinical positron emission tomography (PET) neuroimaging evaluation of [11C]PBT2 in rodents and nonhuman primates. Carbon-11 labeled PBT2 was synthesized in 4.8 ± 0.5% (nondecay corrected) radiochemical yield (RCY) at end-of-synthesis, based upon [11C]CH3I (n = 6), with >99% radiochemical purity and 80-90 GBq/µmol molar activity (Am) from the corresponding normethyl precursor. In the nonhuman primate brain, [11C]PBT2 uptake was extensive with peak concentration SUVpeak of 3.2-5.2 within 2.5-4.5 min postinjection in all cortical and subcortical gray matter regions (putamen > caudate > cortex ≫ white matter) followed by rapid washout from normal brain tissues. Furthermore, it is shown that [11C]PBT2 binds specifically in AD human brain tissue in vitro. The results presented here, combined with the clinical data available for PBT2, warrant the evaluation of [11C]PBT2 as an exploratory PET radiotracer in humans.

Development of a copper-clioquinol formulation suitable for intravenous use.

Clioquinol (CQ) is an FDA-approved topical antifungal agent known to kill cancer cells. This facilitated the initiation of clinical trials in patients with refractory hematologic malignancies. These repurposing efforts were not successful; this was likely due to low intracellular levels of the drug owing to poor absorption and rapid metabolism upon oral administration. CQ forms a sparingly soluble copper complex (Cu(CQ)2) that exhibits enhanced anticancer activity in some cell lines. We have utilized a novel method to synthesize Cu(CQ)2 inside liposomes, an approach that maintains the complex suspended in solution and in a format suitable for intravenous administration. The complex was prepared inside 100-nm liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/cholesterol (55:45). The therapeutic activity of the resultant formulation was evaluated in two subcutaneous tumor models (glioblastoma and ovarian cancers) but was not active. We also assessed the ability of the Cu(CQ)2 formulation to increase copper delivery to cancer cells in vitro and its potential to be used in combination with disulfiram (DSF). The results suggested that addition of Cu(CQ)2 enhanced cellular copper levels and the activity of DSF in vitro; however, this combination did not result in a statistically significant reduction in tumor growth in vivo. These studies demonstrate that a Cu(CQ)2 formulation suitable for intravenous use can be prepared, but this formulation used alone or in combination with DSF was not efficacious. The methods described are suitable for development formulations of other analogues of 8-hydroxyquinoline which could prove to be more potent.

A phase I study of the metal ionophore clioquinol in patients with advanced hematologic malignancies.

UNLABELLED: Clioquinol is a small-molecule metal ionophore that inhibits the proteasome through a metal-dependent mechanism. Here, we report a phase I study of clioquinol in patients with refractory hematologic malignancies. Neuropathy and abdominal pain were dose-limiting toxicities. Minimal pharmacodynamic effects were observed, and there were no clinical responses. BACKGROUND: Clioquinol is a small-molecule metal ionophore that inhibits the enzymatic activity of the proteasome and displays preclinical efficacy in hematologic malignancies in vitro and in vivo. Therefore, we conducted a phase I clinical trial of clioquinol in patients with refractory hematologic malignancies to assess its safety and determine its biological activity in this patient population. METHODS: Patients with refractory hematologic malignancies were treated with increasing doses of oral clioquinol twice daily for 15 doses. Plasma and intracellular levels of clioquinol were measured. Enzymatic activity of the proteasome was measured before and after drug administration. RESULTS: Sixteen cycles of clioquinol were administered to 11 patients with 5 patients reenrolled at the next dose level as per the permitted intrapatient dose escalation. Dose-limiting neurotoxicity and abdominal pain were observed at a dose of 1600 mg twice daily. Intracellular drug levels were low. Minimal inhibition of the proteasome was observed. No clinical responses were observed. CONCLUSION: In patients with refractory hematologic malignancies, the maximal tolerated dose of clioquinol was determined. Minimal inhibition of the proteasome was observed at tolerable doses, likely due to low intracellular levels of the drug.

Clioquinol: review of its mechanisms of action and clinical uses in neurodegenerative disorders.

Clioquinol was produced as a topical antiseptic and marketed as an oral intestinal amebicide in 1934, being used to treat a wide range of intestinal diseases. In the early 1970s, it was withdrawn from the market as an oral agent because of its association with subacute myelo-optic neuropathy (SMON), a syndrome that involves sensory and motor disturbances in the lower limbs and visual changes. The first methods for determining plasma and tissue clioquinol (5-chloro-7-iodo-8-quinolinol) levels were set up in the 1970s and involved HPLC separation with UV detection, these were followed by a more sensitive GC method with electron capture detection and a gaschromatographic-massspectrometric (GC-MS) method. Finally, an HPLC method using electrochemical detection has proved to be as highly sensitive and specific as the GC-MS. In rats, mice, rabbits, and hamsters, clioquinol is rapidily absorbed and undergoes first-pass metabolization to glucuronate and sulfate conjugates; the concentrations of the metabolites are higher than those of free clioquinol. Bioavailabilty versus intraperitoneal dosing is about 12%. Dogs and monkeys form fewer conjugates. In man, single-dose concentrations are dose related, and the drug's half-life is 11-14 h. There is no accumulation, and the drug is much less metabolized to conjugates. Clioquinol acts as a zinc and copper chelator. Metal chelation is a potential therapeutic strategy for Alzheimer's disease (AD) because zinc and copper are involved in the deposition and stabilization of amyloid plaques, and chelating agents can dissolve amyloid deposits in vitro and in vivo. In general, the ability of clioquinol to chelate and redistribute metals plays an important role in diseases characterised by Zn, Cu, Fe dyshomeostasis, such as AD and Parkinson's disease, as it reduces oxidation and the amyloid burden. Zinc chelators may also act as anticancer agents. Animal toxicity studies have revealed species-specific differences in neurotoxic responses that are related to the serum levels of clioquinol and metabolites. This is also true in humans, who form fewer conjugates. The results of studies of Alzheimer patients are conflicting and need further confirmation. The potential therapeutic role of the two main effects of MPACs (the regulation of the distribution of metals and antioxidants) has not yet been fully explored.

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.

Pharmacokinetics and distribution of clioquinol in golden hamsters.

Clioquinol (5-chloro-7-iodo-8-quinolinol) is a zinc and copper chelator that can dissolve amyloid deposits and may be beneficial in Alzheimer's disease. Prion diseases are also degenerative CNS disorders characterised by amyloid deposits. The pharmacokinetics and tissue distribution of drugs active against prions may clarify their targets of action. We describe the pharmacokinetics of clioquinol in hamster plasma, spleen and brain after single and repeated oral or intraperitoneal administration (50 mg kg(-1)), as well as after administration with the diet. A single intraperitoneal administration led to peak plasma clioquinol concentrations after 15 min (Tmax), followed by a decay with an apparent half-life of 2.20 +/- 1.1 h. After oral administration, Tmax was reached after 30 min and was followed by a similar process of decay; the AUC(0-last) was 16% that recorded after intraperitoneal administration. The Cmax and AUC values in spleen after a single administration were about 65% (i.p.) and 25% (p.o.) those observed in blood; those in liver were 35% (p.o.) those observed in blood and those in brain were 20% (i.p.) and 10% (p.o.) those observed in plasma. After repeated oral doses, the plasma, brain and spleen concentrations were similar to those observed at the same times after a single dose. One hour after intraperitoneal dosing, clioquinol was also found in the ventricular CSF. Clioquinol was also given with the diet; its morning and afternoon concentrations were similar, and matched those after oral administration. No toxicity was found after chronic administration. Our results indicate that clioquinol, after oral administration with the diet, reaches concentrations in brain and peripheral tissues (particularly spleen) that can be considered effective in preventing prion accumulation, but are at least ten times lower than those likely to cause toxicity.

Determination of 5-chloro-7-iodo-8-quinolinol (clioquinol) in plasma and tissues of hamsters by high-performance liquid chromatography and electrochemical detection.

This paper describes a method of determining clioquinol levels in hamster plasma and tissue by means of HPLC and electrochemical detection. Clioquinol was separated on a Nucleosil C18 300 mm x 3.9 mm i.d. 7 microm column at 1 ml/min using a phosphate/citrate buffer 0.1M (400 ml) with 600 ml of a methanol:acetonitrile (1:1, v/v) mobile phase. The retention times of clioquinol and the IS were, respectively, 11.6 and 8.1 min; the quantitation limit (CV>8%) was 5 ng/ml in plasma and 10 ng/ml in tissues. The intra- and inter-assay accuracies of the method were more than 95%, with coefficients of variation between 3.0 and 7.7%, and plasma and tissue recovery rates of 72-77%. There was a linear response to clioquinol 5-2000 ng/ml in plasma, and 10-1000 ng/g in tissues. The method is highly sensitive and selective, makes it possible to study the pharmacokinetics of plasma clioquinol after oral administration and the distribution of clioquinol in tissues, and could be used to monitor plasma clioquinol levels in humans.

1,1'-Xylyl bis-1,4,8,11-tetraaza cyclotetradecane: a new potential copper chelator agent for neuroprotection in Alzheimer's disease. Its comparative effects with clioquinol on rat brain copper distribution.

Dysfunction of copper metabolism leading to its excess or deficiency results in severe ailments. Recently, neurodegenerative disorders such as Alzheimer's disease have been associated with copper metabolism. Compounds having the ability to reduce copper levels in brain or to affect its distribution could have neuroprotective effects, mainly through a downregulation of the transcription of amyloid peptide precursor (APP). We report here the biological effect of compound 1,1'-xylyl bis-1,4,8,11-tetraaza cyclotetradecane, which specifically affects copper concentration in the brain cortex region. Its copper homeostatic activity is compared with that of clioquinol, a well-known drug, which has been recently reported as an active A beta-peptide clearance drug in vivo for Alzheimer's patients.

Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease.

Neocortical beta-amyloid (Abeta) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with Abeta and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([(125)I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [(125)I]CQ to synthetic Abeta precipitated by Zn(2+) (K(d)=0.45 and 1.40 nm for Abeta(1-42) and Abeta(1-40), respectively), which was fully displaced by free Zn(2+), Cu(2+), the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [(125)I]CQ concentrated in a fraction enriched for both Abeta and Zn, which was modulated by exogenous addition of Zn(2+) or DTPA. APP transgenic (Tg2576) mice injected with [(125)I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [(125)I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [(125)I]CQ. A pilot SPECT study of [(123)I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated Abeta species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology.

PBT-1 Prana Biotechnology.

PBT-1 (clioquinol), is undergoing phase II clinical trials by Prana Biotechnology Ltd for the potential treatment of Alzheimer's disease. By October 2004, the company had planned phase III trials for the first half of 2005.

[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.