Armando Lagrutta

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K+ channel β-subunits, β2 and β3, which are encoded by distinct genes that map to 3q26.2–27. A single β2 family member and four alternatively spliced variants of β3 were investigated. These subunits have predicted molecular masses of 27.1–31.6 kDa, share ∼30–44% amino acid identity with β1, and exhibit distinct but overlapping expression patterns. Coexpression of the β2 or β3a–c subunits with a BK α-subunit altered the functional properties of the current expressed by the α-subunit alone. The β2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the β3a–c subunits resulted in only partial inactivation of the current, and the β3b subunit conferred an apparent inward rectification. Furthermore, unlike the β1 and β2 subunits, none of the β3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these β-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics.

Biological therapeutic agents (biologicals), such as monoclonal antibodies (mAbs), are increasingly important in the treatment of human disease, and many types of biologicals are in clinical development. During preclinical drug development, cardiovascular safety pharmacology studies are performed to assess cardiac safety in accord with the ICH S7A and S7B regulations that guide these studies. The question arises, however, whether or not it is appropriate to apply these guidelines, which were devised primarily to standardize small molecule drug testing, to the cardiovascular evaluation of biologicals. We examined the scientific literature and formed a consensus of scientific opinion to determine if there is a rational basis for conducting an in vitro hERG assay as part of routine preclinical cardiovascular safety testing for biologicals. We conclude that mAb therapeutics have very low potential to interact with the extracellular or intracellular (pore) domains on hERG channel and, therefore, are highly unlikely to inhibit hERG channel activity based on their targeted, specific binding properties. Furthermore, mAb are large molecules (>140,000 Da) that cannot cross plasma membranes and therefore would be unable to access and block the promiscuous inner pore of the hERG channel, in contrast with typical small molecule drugs. Consequently, we recommend that it is not appropriate to conduct an in vitro hERG assay as part of a preclinical strategy for assessing the heart rate corrected QT interval (QTc) prolongation risk of mAbs and other types of biologicals. It is more appropriate to assess QTc risk by integrating cardiovascular endpoints into repeat-dose general toxicology studies performed in an appropriate non-rodent species. These recommendations should help shape future regulatory strategy and discussions for the cardiovascular safety pharmacology testing of mAbs as well as other biologicals and provide guidance for the preclinical cardiovascular evaluation of such agents.

Novel, Potent Inhibitors of Human Kv1.5 K+ Channels and Ultrarapidly Activating Delayed Rectifier Potassium Current

We have identified a series of diphenyl phosphine oxide (DPO) compounds that are potent frequency-dependent inhibitors of cloned human Kv1.5 (hKv1.5) channels. DPO inhibited hKv1.5 expressed in Chinese hamster ovary cells in a concentration-dependent manner preferentially during channel activation and slowed the deactivating tail current, consistent with a predominant open-channel blocking mechanism. Varying kinetics of DPO interaction with Kv1.5 channels resulted in differing potencies and frequency dependencies of inhibition that were comparable for both expressed hKv1.5 current and native ultrarapidly activating delayed rectifier potassium current (IKur) in human atrial myocytes. Selectivity of DPO versus other cardiac K+ channels was demonstrated in human atrial myocytes (IKur versus transient outward potassium current) and guinea pig ventricular myocytes [IKur versus rapidly activating delayed rectifier potassium current (IKr), slowly activating delayed rectifier potassium current (IKs) and inward rectifier potassium current (IK1), and one compound (DPO-1) was shown to be 15-fold more selective for Kv1.5 versus Kv3.1 channels expressed in Xenopus oocytes. DPO-1 also prolonged action potentials of isolated human atrial but not ventricular myocytes, in contrast to the effect of a selective IKr blocker. The selectivity and kinetics of inhibition hKv1.5 and IKur by DPO and the resulting selective prolongation of atrial repolarization could provide an effective profile for treatment of supraventricular arrhythmias.

The hERG Channel and Risk of Drug-Acquired Cardiac Arrhythmia: An Overview

This review summarizes current knowledge of the cardiac rapidly activating delayed rectifier potassium current (I(Kr)), and its connection to drug-acquired QT prolongation and the associated risk of ventricular arrhythmia and fibrillation. The molecular characterization of hERG as the structural correlate of I(Kr) and the link between inherited long QT and the KCNH2 gene (hERG), have facilitated mechanistic studies of drug-acquired QT prolongation. The development of high throughput assays to evaluate drug effects on hERG has provided an avenue to determine structure activity relations (SAR) within chemical series. More than 10 years of collective data and structural considerations support the notion that hERG is an unusually promiscuous target among potassium channels, but that defining SAR within a chemical series is a viable strategy to reduce or eliminate hERG activity. Despite a critical need to minimize drug effects on hERG, one should always keep in mind that hERG is not the only structural correlate of QT prolongation, and that QT prolongation is a sub-optimal biomarker for ventricular arrhythmia and fibrillation.

Nonclinical safety assessment of the histone deacetylase inhibitor vorinostat.

Vorinostat (SAHA, Zolinza), a histone deacetylase inhibitor, is assessed in nonclinical studies to support its approval for cutaneous T-cell lymphoma. Vorinostat is weakly mutagenic in the Ames assay; is clastogenic in rodent (ie, CHO) cells but not in normal human lymphocytes; and is weakly positive in an in vivo mouse micronucleus assay. No effects are observed on potassium ion currents in the hERG assay up to 300 μM (safety margin ~300-fold the ~1 μM serum concentration associated with the 400 mg/d maximum recommended human dose. No rat respiratory or central nervous system effects are found at 150 mg/kg (>2-fold maximum recommended human dose). No cardiovascular effects, including effects on QTc interval, are observed after a single oral dose (150 mg/kg) in dogs. Vorinostat is orally dosed daily in rats (controls, 20, 50, or 150 mg/kg/d) and dogs (controls, 60, 80, or 100/125/160 mg/kg/d) for 26 weeks with a 4-week recovery. Rat vorinostat-related adverse findings are decreased food consumption, weight loss, and hematologic changes; a no observed adverse effects level is not established. In dogs, adverse effects are primarily gastrointestinal; the no observed adverse effects level is 60 mg/kg/d (~6-fold maximum recommended human dose). Toxicities are reversible and can be monitored in the clinic.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad spectrum antibacterial agents.

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC50 > 18 μM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase (IC50 = 1.02 μM) and topo IV (IC50 = 10.4 μM). AM8191 showed parenteral and oral efficacy (ED50) at less than 2.5 mg/kg doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker.

Tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of left-hand-side moiety (Part-2).

Novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. A series of novel oxabicyclooctane-linked NBTIs with new tricyclic-1,5-naphthyridinone left hand side moieties have been described. Compounds with a (R)-hydroxy-1,5-naphthyridinone moiety (7) showed potent antibacterial activity (e.g., Staphylococcus aureus MIC 0.25 μg/mL), acceptable Gram-positive and Gram-negative spectrum with rapidly bactericidal activity. The compound 7 showed intravenous and oral efficacy (ED50) at 3.2 and 27 mg/kg doses, respectively, in a murine model of bacteremia. Most importantly they showed significant attenuation of functional hERG activity (IC50 >170 μM). In general, lower logD attenuated hERG activity but also reduced Gram-negative activity. The co-crystal structure of a hydroxy-tricyclic NBTI bound to a DNA-gyrase complex exhibited a binding mode that show enantiomeric preference for R isomer and explains the activity and SAR. The discovery, synthesis, SAR and X-ray crystal structure of the left-hand-side tricyclic 1,5-naphthyridinone based oxabicyclooctane linked NBTIs are described.

Use of FDSS/μCell imaging platform for preclinical cardiac electrophysiology safety screening of compounds in human induced pluripotent stem cell-derived cardiomyocytes

FDSS/μCell is a high-speed acquisition imaging platform (Hamamatsu Ltd., Hamamatsu, Japan) that allows for simultaneous high-throughput reading under controlled conditions. We evaluated the Ca(2+) transients or optical membrane potential changes of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (iCells) in the presence or absence of 44 pharmacological agents known to interfere with cardiac ion channels (e.g., hERG, IKs, NaV1.5, CaV1.2). We tested two Ca(2+)-sensitive fluorescence dyes (Codex ACTOne® and EarlyTox®) and a membrane potential dye (FLIPR® membrane potential dye). We were able to quantify and report drug-induced early-after depolarizations (EAD)-like waveforms, cardiomyocyte ectopic beats and changes in beating rate from a subgroup of pharmacological agents acting acutely (within a 1-hour period). Cardiovascular drugs, such as dofetilide and d,l-sotalol, exhibited EAD-like signals at 3nM and 10μM, respectively. CNS drugs, such as haloperidol and sertindole, exhibited EAD-like signals and ectopic beats at 30nM and 1μM, respectively. Other drugs, such as astemizole, solifenacin, and moxifloxacin, exhibited similar arrhythmias at 30nM, 3μM and 300μM, respectively. Our data suggest that the membrane potential and intracellular Ca(2+) signal are tightly coupled, supporting the idea that the EAD-like signals reported are the accurate representation of an EAD signal of the cardiac action potential. Finally, the EAD-like Ca(2+) signal was well correlated to clinically-relevant concentrations where Torsade de Pointes (TdPs) arrhythmias were noted in healthy volunteers treated orally with some of the compounds we tested, as reported in PharmaPendium®.

Hydroxy tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of RHS moiety (Part-3).

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. (R)-Hydroxy-1,5-naphthyridinone left-hand side (LHS) oxabicyclooctane linked pyridoxazinone right-hand side (RHS) containing NBTIs showed a potent Gram-positive antibacterial profile. SAR around the RHS moiety, including substitutions around pyridooxazinone, pyridodioxane, and phenyl propenoids has been described. A fluoro substituted pyridoxazinone showed an MIC against Staphylococcus aureus of 0.5 μg/mL with reduced functional hERG activity (IC50 333 μM) and good in vivo efficacy [ED90 12 mg/kg, intravenous (iv) and 15 mg/kg, oral (p.o.)]. A pyridodioxane-containing NBTI showed a S. aureus MIC of 0.5 μg/mL, significantly improved hERG IC50 764 μM and strong efficacy of 11 mg/kg (iv) and 5 mg/kg (p.o.). A phenyl propenoid series of compounds showed potent antibacterial activity, but also showed potent hERG binding activity. Many of the compounds in the hydroxy-tricyclic series showed strong activity against Acinetobacter baumannii, but reduced activity against Escherichia coli and Pseudomonas aeruginosa. Bicyclic heterocycles appeared to be the best RHS moiety for the hydroxy-tricyclic oxabicyclooctane linked NBTIs.

Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-5)

Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property.