Zezhou Wang

INHIBITION OF MITOCHONDRIAL TRANSLATION AS A THERAPEUTIC STRATEGY FOR HUMAN ACUTE MYELOID LEUKEMIA

To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.

Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

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.

FV-082: A safer orally active broad-spectrum antiepileptic drug candidate

Broad spectrum Anti-Epileptic Drugs (AED) are valued for their potential to treat refractory epilepsy and as general ‘neurostabilizers’ have been employed for a variety of neurological diseases including migrane, biopolar disorder and neuropathic pain. However, the benefits of historical AEDs have been constrained by unintended CNS side effects. Combining proprietary breakthrough chemistry with Fluorinov Pharma and NIH’s in vivo phenotypic screening strategy identified three completely novel candidate drugs, including FV-082 with superior safety and efficacy profiles. FV-082 was screened across available in vitro and rodent seizure models at ASP, and was found to display an excellent spectrum of anti-seizure activity and safety profiles superior to that produced by Depakote, Neurontin and Keppra. FV-082 has an oral ED50 of 20.1 ± 0.7 and 17.4 ± 0.6 mg/kg in the rat MES and mouse 6Hz (ip) assays, respectively. FV-082 prevented seizures induced by sound in the Audiogenic Seizure (AGS)-susceptible Frings mouse model (i.p) with an ED50 value of 13.0 ± 2.0 mg/kg. In addition, FV-082 displayed significant efficacy in the electrically induced corneally kindled mouse, kindled rat amygdala and hippocampal seizures models. Importantly, FV-082 was also found to limit seizure spread and elevate seizure threshold in preclinical animal models, therefore, it has the potential to be effective in refractory, or pharmacoresistant, epilepsy. Also, the anticonvulsant pharmacology suggests applicability for treating multiple forms of epilepsy, such as generalized tonic−clonic, complex partial, and myoclonic seizures, at dose levels that confer a favorable safety margin (therapeutic index ≥ 34). Safety assessment of FV-082 suggests no interaction with the major CYP isoenzymes when tested up to 500 μM and no functional hERG activity when tested up to 20 μM. Further, acute toxicity studies revealed that FV082 exhibited no adverse effects at oral and intravenous doses in rats and mice with a maximally tolerated dose greater than 600 mg/kg in the mice (i.p) and rats (p.o). Also, there was a pronounced safety margin between behavioral efficacy (rat oral MES neurotoxicity and (rat TD50 > 600 mg/kg) for a protective index (rat oral TD50/MES ED50) of >34-fold. Hence, FV-082 displays a considerably broader CNS therapeutic index superior to historical AEDs. Additionally, a convenient and cost effective manufacturing process for FV-082 has been developed allowing for access to multi-kilogram scale synthesis. In-vivo pharmacokinetic studies in rat and dog revealed that FV-082 had excellent oral bioavailability and good oral half-life, and excellent plasma drug levels across multiple models of epilepsy and pain. In addition, drug exposure as measured by Cmax and AUC was shown to be linear with dose. Mechanistically, broad ion channel profiling in combination with a CEREP broad profiling screen (10 μM) across 90 known GPCR, ion channel, transporter and enzyme targets studies suggest that several mechanisms contribute to the observed pharmacological profile of FV-082 but that no single mechanism is likely to be a major contributor. In fact, in whole-cell patch-clamp recordings FV-082 inhibited voltage-gated Na + channel Nav1.7, and the CEREP panel indicated that FV-082 interacted with androgen receptor (AR) and (h) recombinant enzyme monoamine-oxidase-B (MAO-B). The anticonvulsant profile of FV-082 suggests that it may be useful for treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, absence seizures), including refractory (or pharmacoresistant) epilepsy, at dose levels that confer a good safety margin. Furthermore, in models of neuropathic pain (spinal nerve injury [SNI] and formalin inflammatory pain, administration of a single dose showed robust efficacy (SNI: 50 mg/kg ip; and formalin: 71 mg/kg ip). Based on these promising preclinical data, FV-082 warrants clinical investigation.

FV-137 candidate drug: Novel and selective ion channel modulation for epilepsy and pain

Broad spectrum Anti-Epileptic Drugs (AED) are valued for their potential to treat refractory epilepsy and as general ‘neurostabilizers’ have been employed for a variety of neurological diseases including migrane, biopolar disorder and neuropathic pain. However, the benefits of historical AEDs have been constrained by unintended CNS side effects of these non-selective medicines. Combining proprietary breakthrough chemistry with Fluorinov Pharma and NIH’s in vivo phenotypic screening strategy identified three completely novel candidate drugs, including FV-137 with superior safety and efficacy profiles targeting selective ion channels. FV-137 is a novel, orally administered AED, structurally distinct than any known AED, displaying promising preclinical efficacy and safety. FV-137 emerged from a program whose goal was to identify broad-spectrum anti-epileptic drugs (AED) using gold standard benchmarks such as Depakote ® , Neurontin ® and Keppra ® as primary comparators. FV-137 was extensively screened across all available in vitro and rodent seizure models at ASP, and was found to display an excellent spectrum of anti-seizure activity and safety profiles superior to that produced by Depakote, Neurontin and Keppra. FV-137 has an oral ED50 of 22.7 ± 0.6 and 104.9±4.3 mg/kg in the rat MES (po), mouse PTZ (ip) assays, respectively. FV-137 also produced a dose related protection of a psychomotor seizure syndrome induced by a 6-Hz electrical stimulus with an oral ED50 of 48.5 ± 1mg/kg, and prevented seizures induced by sound in the Audiogenic Seizure (AGS)-susceptible Frings mouse model (i.p) with an ED50 value of 28.9 ± 3.0 mg/kg. In addition, FV-137 displayed significant efficacy in the electrically induced corneally kindled mouse, kindled rat amygdala, kindled rat hippocampal and lamotrigineresistant kindled rat seizures models. Additionally, FV-137 also increased seizure threshold in a dose– dependent manner, therefore, it has the potential to be effective in refractory, or pharmacoresistant, epilepsy. In-vivo pharmacokinetic studies in rat revealed that FV-137 had excellent oral bioavailability (F% = 85%), moderate oral half-life (~3h), and predicted plasma drug levels in the range 5-50μM across multiple models of epilepsy. In addition, drug exposure as measured by Cmax and AUC was shown to be linear with dose. Safety assessment of FV-137 suggests no interaction with the major CYP isoenzymes when tested up to 500 μM, except for 1A2 and 2D6 with IC50 values of 19 uM and 156 uM respectively and no functional hERG activity when tested up to 50 μM. Irwin and acute toxicity studies suggest a maximally tolerated oral dose of greater than 500 mg/kg in the mice and rats. Mechanistically, in whole-cell patch-clamp recordings FV-137 inhibited P/Q-type Ca 2+ Channels, Cav2.1/β4/α2δ1 and Cav2.2/β3/α2δ1, and voltage-gated Na + channels, Nav1.6 and Nav1.7 and no inhibition up to 100 μM in other related ion channels. Importantly, no significant affinities were observed in a CEREP broad profiling screen (10 μM) across 90 known GPCR, ion channel, transporter and enzyme targets. The anticonvulsant profile of FV-137 suggests that it may be useful for treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, absence seizures), including refractory (or pharmacoresistant) epilepsy, at dose levels that confer a good safety margin. Furthermore, in models of neuropathic pain (spinal nerve injury [SNI] and spinal nerve ligation models [SNL]) and formalin inflammatory pain, administration of a single dose showed robust efficacy (SNI: 23 mg/kg ip; SNL: 60mg/kg and formalin: 41 mg/kg ip). On the basis of its pharmacology and superior drug-like properties, FV-137 was identified as a promising AED candidate.