1. Introduction

2. Molecular biology and

pharmacology of VACC

subtypes

3. Present and potential

therapeutic indications of

VACC ligands

4. New patents related to VACC

ligands in the period

2011 -- 2013

5. Conclusion

6. Expert opinion

Review

Recent patents on calcium channelblockers: emphasis on CNSdiseasesJuan-Alberto Arranz-Tagarro, Cristobal de los Rıos, Antonio G Garcıa† &Juan-Fernando Padın†Servicio de Farmacologıa Clınica, Hospital Universitario de La Princesa, Madrid, Spain

Introduction: Altered homeostasis of cell calciummovement is a central stage in

multiple diseases of CNS. This explains the great therapeutic interest in blockers

for the various subtypes of voltage-activated calcium channels (VACCs)

expressed in neurons. Mitigation of Ca2+ entry excess elicited by those blockers

may restore the altered synaptic transmission, synaptic plasticity and gene

expression to normal parameters, ending the enhanced neuronal vulnerability.

Areas covered: This review summarize 23 patents on ligands for L-, N- or

T-type channels, claimed to have potential therapeutic interest in epilepsy,

pain, migraine and neurodegenerative diseases.

Expert opinion: Collections of compounds are generally screened in cell lines

expressing a given subtype of VACCs. IC50 to block such channels are often,

but not always, provided. In few instances, compounds exhibiting the highest

potency in in vitro experiments are also tested in animal models of pain,

behavior, epilepsy or Alzheimer’s disease. Attempts to develop selectivity for

a given VACC subtype with non-peptidic organic ligands have so far failed.

Due to their wide tissue expression, such selectivity is crucial for minimizing

possible side effects. However, the few data reported by patents does not

allow prediction of selectivity of the new compounds in many cases.

Keywords: calcium channel blockers, epilepsy, neurodegenerative diseases, pain,

voltage-activated calcium channels

Expert Opin. Ther. Patents (2014) 24(9):959-977

1. Introduction

About 130 years ago, Sydney Ringer discovered that calcium was essential for bothheart [1] and skeletal muscle contraction [2]. A few years later, Locke and Overtonfound that calcium was essential for impulse transmission between nerve andmuscle [3,4]. Another crucial experiment demonstrated that the direct injection ofcalcium into muscle fibers caused their rapid and strong contraction, what was adirect proof that calcium acted as a contraction trigger [5]. Other additional relevantdiscovery for our current understanding of the role of calcium as an ubiquitous mes-senger was related to the coupling between calcium entry into the nerve terminalduring action potential (AP) firing and neurotransmitter release at the muscleendplate [6]. In a myriad of subsequent studies, calcium was shown to play a roleas second messenger in practically every cell of the body, from oocyte fertilizationto programmed cell death, hormone secretion, neurotransmitter release, plateletaggregation, smooth muscle contraction, and so on.

Our present detailed understanding on the regulation of cell calcium homeostasisis due to the development of sophisticated techniques to monitor calcium currents(i.e., patch-clamp) or the changes of calcium movements between the extracellularspace, cytosol and organelles, occurring during cell activation (i.e., fluorescenceprobes, aequorins). This helped to identify and characterize the elements of the

10.1517/13543776.2014.940892 © 2014 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674 959All rights reserved: reproduction in whole or in part not permitted

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complex machinery that regulates the transient elevations ofcytosolic calcium concentrations ([Ca2+]c) upon cell stimula-tion, in both physiological and pathological conditions. Theelements of such machinery include various channels; trans-porters, uniporters and antiporters; the endoplasmic andsarcoplasmic reticulums; mitochondria and Ca2+-bindingproteins. The regulation of calcium homeostasis under physi-ological and pathological conditions (e.g., neurodegenerativediseases or stroke) has been analyzed in various recent reviews[7-19].The calcium entry into cells via voltage-activated calcium

channels (VACCs), particularly relevant for this review, wasdiscovered through a pioneering experiment on AP recordingsin crayfish muscle fibers [20]. Since then, a large variety ofVACC subtypes has been identified and characterized invarious cell systems and tissues [21]. Also, many contributionshave been interested in selectively targeting the different chan-nel subtypes, giving rise to the new field of calcium antago-nists or L-type channel blockers, with indications mainly incardiovascular diseases [22]. Here, we review the new patentsfrom the years 2011 to 2013 on new compounds that, bytargeting a specific VACC subtype, may find a therapeuticapplication in the clinic. To place those patents in a physio-pharmacological and clinical context, we first summarize thecurrent understanding of the molecular and pharmacologicalproperties of the discovered VACC subtypes.

2. Molecular biology and pharmacology ofVACC subtypes

VACCs constitute a large family of multi-subunit complexescomposed of up to five distinct proteins, that is, a1, b, a2,d and g [23,24]. The a1 subunits are large proteins with amolecular weight between 212 and 273 kDa. Each subunit

is organized in four homologous repeats (I-IV) of six trans-membrane structures. Each repeat contains a S4 region thatacts as the voltage sensor, a P-loop that forms the selectivefilter and the S5-S6 segments that form the channel pore(Figure 1). The four domains are connected through cyto-plasmic linkers, and both C- and N-termini are cytoplasmic.These regions contain sites of interaction with auxiliarysubunits, various sites for activators and blockers, includingG proteins, as well as several phosphorylation sites [23,24].

The auxiliary intracellular b subunit in all high-VACCsbinds to a conserveda-interaction domain (AID) of thea1 sub-unit, thus modulating the channel gating properties and pro-moting cell surface trafficking (Figure 2). This interaction siteis located at the connector between domains I and II of the a1

subunit [25]. In addition to this regulatory function of the a1

subunit, a role for the b subunit in scaffolding multiple signal-ing pathways around the channel, have also been proposed; thisrole suggests that this subunit belongs to the membrane-associated guanylate kinase family [26]. Moreover, another studyreveals that b3 subunits directly reduce glucose-induced Ca2+

oscillations in pancreatic b cells [27]. Thus, two signaling path-ways are proposed in this b3-subunit-mediated effect: directregulation of the inositol trisphosphate receptor and indirectreduction of phospholipase Cb. Thus, b subunits are nowhypothesized to be independent regulatory proteins.

The a2/d subunit is translated as a single protein, butcleaved into d (a single transmembrane-spanning helix) anda2 (the extracellular domain), which are linked by a disulfidebond. The g subunit, characterized by four transmembranedomains, was formerly found in skeletal muscle and later onin the heart and brain VACCs [28]. Functional studies suggestthat some g subunits of VACCs may behave as regulators ofpostsynaptic membrane AMPA-type glutamate receptors [29].

On the basis of their biophysical and pharmacological prop-erties, several types of VACCs have been identified andclassified. They are primarily defined from the nature of theprincipal pore-forming a1 subunit; 10 different a1 subunitshave been characterized by cDNA cloning and functionalexpression in mammalian cells or Xenopus laevis oocytes [23].These subunits are classified into three structurally and func-tionally different a1 subtypes of VACCs that require strongdepolarizing pulses to open (high-VACCs) and others thatopen at lower voltages (low-VACCs) [30]. The first subfamily(Cav1.1 -- Cav1.4) belongs to the group of high-VACCsand includes channels containing a1 subunits that mediate L-type currents (a1S, a1C, a1D, a1F). The second subfamily(Cav2.1 -- Cav2.3) also belongs to the group of high-VACCsand comprises VACCs containing a1 subunits that mediateP/Q-type (a1A), N-type (a1B) and R-type (a1D) currents.Finally, the third subfamily (Cav3.1 -- Cav3.3) includes low-threshold-VACCs, containing a1 subunits (a1G, a1H, a1I)that mediate T-type currents. In contrast to the Cav3 channels,which express by themselves as typical T-type channels in heter-ologous systems, Cav1 and Cav2 channels act as oligomericcomplexes containing auxiliary subunits.

Article highlights.

. Altered calcium homeostasis is central stage in thepathogenesis of neurodegenerative diseases, stroke, painand epilepsies; thus, there is high interest in the searchof blockers of voltage-activated calcium channels (L-, N-,P/Q-, R-, T-type) that may have therapeutic indications inthose diseases.

. In the period 2011 -- 2013, most of the 23 patents herereviewed deal with N- and T-type channel blockers, withpotential therapeutic interest in pain, epilepsy, migraineand neurodegenerative diseases.

. Compounds blocking L-type channels focus thetreatment of Parkinson’s disease, those blocking N- andT-type channels address the treatment of epilepsy and,particularly, neuropathic pain.

. A major drawback to drug development in this field is thewide distribution of various calcium channel subtypes withsimilar molecular structure in the brain and peripheraltissues, which may give rise to intolerable side effects.

This box summarizes key points contained in the article.

J.-A. Arranz-Tagarro et al.

960 Expert Opin. Ther. Patents (2014) 24(9)

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Table 1 summarizes the current and classical nomenclaturefor the diverse VACC subtypes, the type of current carried byeach channel, their blockers with their pIC50, their activatorsand their location in human tissues. L-type channels are themolecular targets of several organic ligands, including the1,4-dihydropyridine family (DHP), the phenylalkylaminesverapamil and D600, and the benzothiazepine diltiazem.Photoaffinity labeling and mutation analysis have revealedthat these three groups of blockers act at three separate, butallosterically coupled receptor sites (Figure 2) [31]. For instance,verapamil is thought to enter the pore from the cytoplasmicside to block it, where its receptor site is formed by amino-acid residues in the S6 segments of domains III and IV [32].

On the other hand, DHPs can be activators (e.g., BayK8644)or blockers (e.g., nifedipine); therefore, they are thought to actallosterically to shift the channel towards its open or closed state,

rather than occluding the ion-conducting pore. Their receptorsites consist of amino acids located in the S6 segments ofdomains III and IV, and the S5 segment of domain III [33].This DHP receptor site shares some common amino acidswith the binding site for verapamil. Finally, diltiazem binds toa third receptor site. Interestingly, the amino acids that arerequired for its interaction also overlap with those required forverapamil binding [34].

Members of the Cav2 family are relatively insensitive toDHPs but are specifically blocked by peptide toxins fromspiders and marine snails (Figure 2). For instance, N-type chan-nels are selectively blocked by w-conotoxin GVIA [35]. Thereceptor site for this toxin comprises amino-acids residues inthe extracellular loop between segments S5 and S6 of domainIII, consistent with a direct pore-blocking mechanism. P/Q-type channels are blocked by w-agatoxin IVA from the funnel

SS

SS

HOOC

S2S3

S4 S5S6

S1

S1S2

S3 S4S5

S6

S4S5

S6 S1S2

S3

S5S6

S1 S2S3

S4

NH2

NH2 COOH

H2N

HOOC

H2N

COOH

I

II III

IV

α2

γδ

NH2 COOH

β

Extracellular fluid

Cytosol

α1

Figure 1. Subunit arrangement for a typical VACC. VACCs are complex proteins composed of four or five different subunits

encoded by multiple genes. The largest a1 subunit incorporates the conduction pore, the voltage sensor and gating

apparatus. It is organized in four homologous domains (I -- IV) with six transmembrane segments (S1 -- S6) in each. The

S4 serves as the voltage sensor and the P-loop between S5 and S6 in each domain determines ion conductance and selectivity.

The a2 and b subunits are hydrophilic proteins located in the extra- and the intracellular space, respectively, whereas the

highly lipophilic g and d subunits are transmembrane proteins. a2 and d subunits are linked by disulfide bonds.P-loop: Pore loop; VACC: Voltage activated calcium channel.

Recent patents on calcium channel blockers

Expert Opin. Ther. Patents (2014) 24(9) 961

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web spider venom and by w-conotoxin MVIIC from themarine snail Conus geographus [36,37]. Cav2.3 channels areblocked by the synthetic peptide toxin SNX-482 derivedfrom tarantula venom; the presence of domains III and IV arenecessary for toxin-mediated channel blockade [38].The Cav3 family of VACCs is insensitive to the blockers

mentioned above. Although there are no compounds thatspecifically target T-type channels, some clinically used drugsare able to block this channel subtype, in addition to otherpharmacological activities [39]. These Cav3 channel blockersinclude antiepileptics, such as ethosuximide, or antipsy-chotics, such as pimozide. In addition, Ni2+ is somewhatspecific for T-type versus other classes of calcium currents.Interestingly, the scorpion venomous kurtoxin binds to thea1G T-type channel with high affinity inhibiting the channelby modifying voltage-dependent gating [40].

3. Present and potential therapeuticindications of VACC ligands

The development of a defined pharmacology for the variousVACC subtypes has been intensely pursued during the last

two decades. Ligands for L-type channels have given riseto a new chapter of pharmacology with several therapeuticapproaches to treat cardiovascular diseases, such as arrhythmias,hypertension or angina. In contrast, selective non-peptide smallmolecules, which block or regulate N-, P/Q- or R-type chan-nels, are lacking. By contrast, recent contributions have affordedsome selective T-type channels ligands [41,42]. Hence, amajor goal in this field is the search for selective blockers ormodulators of these channels that could eventually be used astherapeutic tools in diseases.

There are several compounds that block the varioussubtypes of VACCs with poor selectivity. Such is the case ofthe piperazine derivatives flunarizine, R56865, lubeluzoleand dotarizine. These compounds have been grouped aswide-spectrum blockers of VACCs [43-45]. Fluspirilene, amember of the diphenylbutylpiperidine class of neurolepticdrugs (which also includes pimozide, clopimozide andpenfluridol) blocks N-type channels in PC12 cells [46]. Pen-fluridol also blocks T-type channels [47]. Thus, inhibition orfacilitation of a given neurotransmitter release by blockers oractivators of VACC subtypes may have interesting functionalconsequences. For instance, synthetic w-conotoxin MVIIA

H2N

COOH

Extracellular fluid

Cytosol

S1 S6S5S2 S3 S4 S1 S6S5S2 S3 S4 S1 S6S5S2 S3 S4

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

S1 S6

P-loop

P-loop

P-loop

P-loop

S5S2 S3 S4

I II III IV

P PP

H2N COOH

β

SNAREs

+ +

+ +

+ +

P

G protein

AID

NH2

α2

δ

HOOC

H2N

HOOCSS

SS

DHPs • BTZs• PAAs

• Gabapentin• Pregabalin

α2/δ

Toxins

α1

Figure 2. Membrane topology of a1 and a2/d subunits of the VACC illustrating proteins and blockers interaction sites. The a1

subunit incorporates most of the known sites of VACCs regulation by second messengers, drugs and toxins, except in the case

of gabapentin and pregabalin that interact with the a2/g subunits.AID: a interaction domain; BTZs: Benzothiazepines; DHPs: Dihydropyridines; PAAs: Phenylalkylamines; SNAREs: Soluble NSF attachment protein receptors VACC:

Voltage activated calcium channel.

J.-A. Arranz-Tagarro et al.

962 Expert Opin. Ther. Patents (2014) 24(9)

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Table 1. Characteristics of the various calcium channel subtypes according to their a1-containing subunit.

Calcium channel subtype Type of

current

Blockers

(affinity; pIC50*)

Activators Tissue location

(human)

Current

nomenclature

Traditional

nomenclature

Cav1.1 a1S L Nifedipine (6.3)NisoldipineNitrendipine (6)(+)-Isradipine (6.7 -- 8.2)(-)-Devapamil (8.2 -- 8.7)Cd2+ (4.3)

BayK8644FPL64176SZ(+)-(S)-202 -- 791

Skeletal muscleBasal gangliaCerebral cortexHippocampusSubstantia nigraSpinal cordLymphocytes

Cav1.2 a1C L Nifedipine (7 -- 8)Nimodipine (6.8)Nisoldipine (7.1)Nitrendipine (6)Kurtoxin (7.2)(+)-Isradipine (7.5)(-) -Devapamil (7 -- 8.4)Cd2+ (5.7 -- 6)Mibefradil (4.9)

BayK8644(-) -(S)-BayK8644FPL64176PCA50941SZ(+)-(S)-202 -- 791

HeartSmooth muscleBrainPituitaryAdrenal medulla

Cav1.3 a1D L NifedipineCalciseptineCalcicludineNisoldipine (6.4 -- 7)Nimodipine (5.7 -- 6.6)

BayK8644(-)-(S)-BayK8644FPL64176PCA50941

BrainPancreasAdrenal medullaCochleaKidneyOvary

Cav1.4 a1F L Nifedipine (6)Isradipine (6.7)

BayK8644FPL64176

RetinaLymphoid tissuesMast cells

Cav2.1 a1A P/Q w-aga-IIIA (9.3)w-aga-IVA (6.8 -- 8.7)w-aga-IVB (8.5)w-ctx-MVIIC (8 -- 9.5)w-ctx-MVIID (9)w-ctx-CVIB (8)w-ctx-CVIC (7.5)w-phonetoxin-IIA (9.2)DW13.3 (8.4)Kurtoxin (7.8)

Brain (hippocampus,entorhinal cortex,subiculum)CerebellumPituitaryCochleaAdrenal medulla

Cav2.2 a1B N w-ctx-GVIA (9.2 -- 10.4)w-ctx-MVIIA (7.7 -- 10.3)w-ctx-MVIIC (6.1 -- 8.5)w-ctx-CVIA (9.3)w-ctx-CVIB (8.1)w-ctx-CVIC (8.1)w-ctx-CVID (10.2)DW13.3 (7.7 -- 8.6)Huwertoxin I (7)w-ctx-SO-3 (6.8)

Glycerotoxin Brain (hippocampus andparahippocampal gyrus)Peripheral nervous systemAdrenal medulla

Peptide toxins from venom of marine snails: w-ctx-GVIA, w-conotoxin GVIA; w-ctx-MVIIA, w-conotoxin MVIIA; w-ctx-MVIIC, w-conotoxin MVIIC; w-ctx-MVIID,

w-conotoxin MVIID; w-ctx-CVIA, w-conotoxin CVIA; w- ctx-CVIB, w-conotoxin CVIB; w- ctx-CVIC, w-conotoxin CVIC; w- ctx-CVID, w-conotoxin CVID; w-ctx-SO-3,w-conotoxin SO-3.

Peptide toxins from different species of venom spiders: DW13.3 (74-amino acid peptide toxin); w-aga-IVA, w-agatoxin IVA; w-aga-IVB, w-agatoxin-IVB; w-aga-IIIA,w-agatoxin-IIIA; ProTx-I, Protoxin-I or b-theraphotoxin-Tp1a; SNX-482 (homologous to the spider peptides grammatoxin S1A and hanatoxin); w-PnTx3-3, w-ctenitoxin-Pn2a; w-PnTx3-6, PhTx-3 toxin fraction.

Drug from chemical synthesis: NNC55-0396, (1S,2S)-2-[2-[[3-(1H-Benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-

2-naphthalenyl cyclopropanecarboxylate dihydrochloride; ML 218, 3,5-Dichloro-N-[[(1a,5a,6-exo,6a)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-

benzamide hydrochloride.

Adapted from [148].

*pIC50 is the negative logarithm of the molar IC50 concentrations.

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Table 1. Characteristics of the various calcium channel subtypes according to their a1-containing subunit

(continued).

Calcium channel subtype Type of

current

Blockers

(affinity; pIC50*)

Activators Tissue location

(human)

Current

nomenclature

Traditional

nomenclature

Cav2.3 a1E R SNX-482 (7 -- 8.2)DW13.3 (7)w-PnTx3-3 (7.9)w-PnTx3-6 (6.9)w-phonetoxin-IIA (7.2)Pb2+ (7)Ni2+ (3.6 -- 4.7)

BrainCochleaRetinaHeartPituitaryAdrenal medulla

Cav3.1 a1G T ProTx-I (7,3)Kurtoxin (7.3 -- 7.8)Mibefradil (6 -- 6.6)NNC55-0396Pimozide (7.5)(-) -(R)-efonidipine (5 -- 7)Ni2+ (3.6 -- 3.8)Anandamide (5.4)

Brain(cerebellum>thalamus>n-eocortex > substantianigra >medulla > striatum)

Peripheral nervous systemAdrenal medulla

Cav3.2 a1H T Kurtoxin (7.3 -- 7.6)Mibefradil (5.9 -- 7.2)NNC55-0396ML 218 (6.5)Ni2+ (4.9 -- 5.2)Anandamide (6.5)Pimozine (7.3)

KidneyLiverHeartBrain(putamen > amygdale,caudate nucleus frontallobe, hippocampus,cerebellum, substantianigra>thalamus>medulla,spinal cord, occipital lobe,temporal lobe)LungSkeletal musclePancreasPlacentaAdrenal glomerulosaAdrenal medulla

Cav3.3 a1I T Mibefradil (5.8)NNC55-0396Pimozide (7.5)Anandamide (6)ML 218 (6.6)Ni2+ (3.7 -- 4.1)

Brain (cerebellum,occipital lobe, frontallobe, amygdale, caudatenucleus,hippocampus > temporallobe,putamen > substantianigra, medulla)

Peptide toxins from venom of marine snails: w-ctx-GVIA, w-conotoxin GVIA; w-ctx-MVIIA, w-conotoxin MVIIA; w-ctx-MVIIC, w-conotoxin MVIIC; w-ctx-MVIID,

w-conotoxin MVIID; w-ctx-CVIA, w-conotoxin CVIA; w- ctx-CVIB, w-conotoxin CVIB; w- ctx-CVIC, w-conotoxin CVIC; w- ctx-CVID, w-conotoxin CVID; w-ctx-SO-3,w-conotoxin SO-3.

Peptide toxins from different species of venom spiders: DW13.3 (74-amino acid peptide toxin); w-aga-IVA, w-agatoxin IVA; w-aga-IVB, w-agatoxin-IVB; w-aga-IIIA,w-agatoxin-IIIA; ProTx-I, Protoxin-I or b-theraphotoxin-Tp1a; SNX-482 (homologous to the spider peptides grammatoxin S1A and hanatoxin); w-PnTx3-3, w-ctenitoxin-Pn2a; w-PnTx3-6, PhTx-3 toxin fraction.

Drug from chemical synthesis: NNC55-0396, (1S,2S)-2-[2-[[3-(1H-Benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-

2-naphthalenyl cyclopropanecarboxylate dihydrochloride; ML 218, 3,5-Dichloro-N-[[(1a,5a,6-exo,6a)-3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hex-6-yl]methyl]-

benzamide hydrochloride.

Adapted from [148].

*pIC50 is the negative logarithm of the molar IC50 concentrations.

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protects hippocampal CA1 pyramidal neurons from damagecaused by transient global forebrain ischemia in the rat [48].

Some medicines that are currently used to treat the epilep-sies are known to target some VACC subtypes. The linkbetween channel blockade and the antiepileptic effect couldbe based in the mitigation of excessive glutamate release partlycontrolled by P/Q-type channels [49], which are believed to beinvolved in idiopathic generalized epilepsies [50]. For instance,gabapentin and pregabalin bind to the a2/d subunit ofVACCs [51-53] and attenuate neurotransmitter release throughinhibition of P/Q-type channels [54,55]. In addition, gabapen-tin reduces excitatory and inhibitory postsynaptic currents inthe spinal cord through P/Q-type channel blockade [56]. Fur-thermore, levetiracetam blocks VACC currents in pyramidalneurons of hippocampal slices [57] and diminishes excitatorypostsynaptic potentials in granule cells through a P/Q-typechannel blockade [58]. In addition, levetiracetam attenuatesthe paroxysmal depolarization shift through N- and P/Q-type channel blockade [59]. Also, the antiepileptics lamotri-gine, ethosuximide, zonisamide and carbamazepine blockhigh-VACCs in cortical neurons [60-62].

Another therapeutic application of VACC ligands is pain.Such is the case of w-conotoxin MVIIA, also known asSNX-111 or ziconotide [63]. Thus, the approval of Prialt�, asynthetic version of w-conotoxin MVIIA, for the treatmentof severe chronic pain associated with cancer, AIDS and neu-ropathies represents a significant advance in analgesia. Itspotent analgesic effects, manifested even in patients resistantto opioids, are due to the inhibition of proprioceptive neuro-transmitters and neuromodulators from the nerve terminals ofprimary afferent neurons in the dorsal horn of the spinalcord [64,65]. Ziconotide has shown potent efficacy in a postsur-gical setting [66] as well as in patients suffering from a varietyof chronic, intractable severe pain syndromes [67].

T-type channel currents were first discovered and charac-terized by Carbone and Lux in dorsal root ganglion (DRG)neurons [68] T-type channels have been deeply studied forpain treatment [63,69-71]. So, T-type knockout mice are hyper-algesic in a model of visceral pain, which is likely related to aT-type channel-dependent antinociceptive mechanismoperating in the thalamus [72].

Some studies have implicated the T-channel isoformCav3.2 in the sensitization of pain responses by enhancingexcitability of nociceptors, in animal models of type 1 andtype 2 peripheral diabetic neuropathies (PDN). In thetype 1 diabetic model, Cav3.2 T-type currents are upregulatedthreefold [73-75]. Furthermore, the Cav3.2 channel is known toregulate presynaptic glutamate release in nociceptive dorsalhorn neurons of the spinal cord [76]. Due to the diabetichyperglycemia, it has been proposed that post-translationalmodification via glycosylation leading to upregulation of theCav3.2 channel in painful PDN, could become a useful target.Hence, by blocking the glycosylation of the Cav3.2 channelwith enzymes, the hyperexcitability of DRG neurons couldbe mitigated, thus preventing or reversing neuropathic pain

symptoms such as hyperalgesia and allodynia in PDN [71].Other state-independent blockers of the Cav3.2 channel,such as TTA-A2 or Z123212, which preferentially interactwith inactivated T-type channels, elicit analgesia in rodentmodels of pain. [77-79].

On the other hand, the antiepileptic ethosuximide blocksT-type currents [80] and reverses tactile allodynia and thermalhyperalgesia in nerve-ligated rats [81]. Other interestingtherapeutic target in analgesia is the auxiliary subunit a2/dof high-VACCs. Gabapentin binds to this subunit with highaffinity [82] and blocks the calcium current, particularly afterconstriction of the sciatic nerve in rats [83]. In clinical trials,gabapentin has shown moderate efficacy in postherpeticneuralgia, diabetic neuropathy, trigeminal neuralgia, lowback pain and cancer pain. More recently, pregabalin, anothera2/d ligand, has shown higher efficacy in animal models andin diseases developing neuropathic pain [84]. Gabapentin hasalso shown efficacy as a prophylactic drug in migraine [85],which agrees with its ability to block spreading depression [86].

L-type channels play multiple roles in the control of neuro-transmitter release. Spontaneous AP firing, low-thresholdexocytosis and compensatory/excess endocytosis are likelycontrolled by the Cav1.3 channel. The delayed inactivationof the Cav1.3 channel could be physiologically relevant forsustaining prolonged Ca2+ influxes that support the endocy-totic process [87,88]. The Cav1.3 channel is also critical forthe control of vital functions, such as heart beating [89], hear-ing [90] and dopamine release [91]. In this context, ongoingresearch focuses on the synthesis of new L-type channelligands with potential therapeutic interest in Parkinson’sdisease (PD), cardiac arrhythmias, chronic stress and otherneuro- and cardiovascular pathologies in which the Cav1.3channel is likely to be involved [92].

Concerning PD, it is worth to note that dopaminergicneurons of the substantia nigra pars compacta (SNc) utilizethe Cav1.3 channel to drive spontaneous pacemaking activity.The continuous Ca2+ influx creates an excessive metabolicload, rendering dopaminergic neurons particularly vulnerableto secondary insults on the mitochondrial function. Theseneurons become more vulnerable with the age, what couldexplain why ageing is a significant risk factor for developingPD. In this context, it is interesting that the L-type channelblocker isradipine restored the Ca2+-independent ‘juvenile’pacemaking activity in those neurons and the finding thatsubcutaneous delivery of isradipine-caused neuroprotection[93]. In fact, hypertensive patients treated with L-type channelblockers had a diminished risk of developing PD [94].

The fact that nimodipine reduced the infarct size in a ratmodel of focal ischemia indicated that the L-type channelblockade could be an effective neuroprotective strategy instroke [95]. Unfortunately, clinical trials have not providedpositive outcomes [96-98]. High-VACCs have also been linkedto Huntington’s disease. Huntingtin directly binds to thea2/d subunit of VACCs and to the pore-forming subunit ofN-type channels, suggesting their involvement in disease

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pathogenesis, together with the promising potential of thesechannel ligands to treat the disease [99]. Finally, amyloid bpeptide (A�) pathology has been associated to a gain-of-func-tion of P/Q-type channels, what could explain that pirace-tam [100] and levetiracetam [101] have shown efficacy inimproving cognitive performance and in slowing down thecognitive decline in patients of Alzheimer’s disease (AD).

4. New patents related to VACC ligands inthe period 2011 -- 2013

Patents that claim various potential therapeutic uses of differ-ent novel molecular entities targeting one or more of theVACC subtypes are summarized in this section. Out of58 patents reviewed dealing with VACC subtypes, but only23 of them included pharmacological tests to confirm theirVACC blocking activity, and these were that we selected fordiscussing in this review.

4.1 Blockers of L-type VACCsThree patents on chemistry and pharmacology of new ligandsfor L-type channels are reviewed. Their molecular structuresare shown in Figure 3.The patent by Delgado-Martın et al. from Consejo Supe-

rior de Investigaciones Cientıficas, Spain [102]. deals with adetailed procedure to extract and purify crambescidine800 and 816 (ratio 1:3), from the sponge Crambe crambe(Figure 3,1). The authors found that the mixture relaxed therat myometrium pre-contracted with high-K+, with an IC50

about 100 µM; veratridine-elicited contractions were notaltered by the mixture, nor those oxytocin or methacholine-induced contractions. Thus, the authors conclude that thecrambescidine mixture is not acting on sodium channels;rather, they attribute its relaxing effect to the blockade ofCa2+ entry through L-type channels that are expressed in therat myometrium. Although the potency of the crambescidinemixture is low, its molecular structure may inspire the synthe-sis and optimization of new more potent ligands for thesubtypes of L-type channels with potential therapeutic indica-tions in cardiovascular diseases.

A second patent focuses on new ligands for L-type channelswith therapeutic potential in PD [103]. The general structure ofthe claimed compounds is a pyrimidine trione core, where Natoms are substituted with alkyl groups, and the heterocycliccarbonyl groups facilitate the generation of either dipole orhydrogen bonds (Figure 3,2). The ligands were tested inHEK 293 cells expressing either human Cav1.3 or Cav1.2 L-type channels. The entry of Ca2+ through those channelswas monitored by using fluorescent imaging plate reader(FLIPR) technology using calcium-4 dye as indicator. Thehighest selectivity for the Cav1.3 channel was achieved withcompounds bearing cyclohexyl or cyclopentyl substituents atR1 and an m-substituted phenethyl moiety at R2 of the pyrim-idine trione core. The authors found that compoundSKP004C08 (Figure 3; 2) had an IC50 of 1.61 µM to blockCav1.3 channels, having > 1000-fold selectivity with respectto the Cav1.2 channel. With the patch-clamp technique,SKP004C08 blocks by 35% Cav1.3 currents at 1 µM, show-ing no effect on Cav1.2 currents. In addition, reportedcompounds presented a good blood--brain barrier (BBB) pen-etration, with blood:brain concentration ratios between10:1 and 1:1, when administered either intraperitoneally ororally. The Cav1.3 channel controls pacemaking in dopami-nergic neurons at the SNc. The fast AP firing of these neuronsgives rise to elevated intracellular Ca2+ trafficking, enhancedoxidative stress and greater neuronal vulnerability. Thus,blocking the Cav1.3 channel is expected to mitigate thoseevents, leading to neuronal protection. This is the basis forthe proposal of using L-type channel blockers to treat PD.To achieve this, the blocker should readily cross the BBBand displays selectivity for the dopaminergic Cav1.3 channelversus the Cav1.2 channel, widely expressed in cardiovasculartissues. For this reason, SKP004C08 could potentiallybecome a useful pharmacological tool to treat PD. Up tonow, however, all known L-type channel blockers exhibitcardiovascular effects.

The third patent deals with the synthesis of dihydropyri-dine-benzodiazepine tetra- and tricyclic derivatives, for thetreatment of both central and vascular diseases (Figure 3,3)disclosed by Verdecia-Reyes et al., from Centro de

X = H, Crambescidine 800X = OH, Crambescidine 816

2 31

SKP004C08

NH

NH

N O N

OO

XH O O

NH2

NH2

OH

14 N N

OO

O

Cl

NH

N

NR1

R2

R3

R4

Ar

Figure 3. Chemical structures of compounds (1) and (2), and general formula (3), from patents on L-type channel ligands,

discussed in Section 4.1. SKP004C08 (2) is the best compound of a pyrimidine-derived family disclosed by Surmeier’s group,

where N atoms are substituted with alkyl groups. General formula (3) exemplifies a juxtaposition of both dihydropyridine and

benzodiazepine moieties, to target VACCs and GABAA receptors, respectively.VACC: Voltage activated calcium channel.

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Investigacion y Desarrollo de Medicamentos [104]. The com-pounds block both GABAA receptors and VACCs. Thispharmacological profile makes Verdecia-Reyes et al. suggesttheir potential therapeutic use for cardiovascular, cerebrovas-cular, neurodegenerative and neurologic diseases. Somein vivo experiments are commented, such as the open-fieldtests to monitor the sedative effects of compounds in AlbinoSwiss mice at the dose of 4 mg/kg, observing a differential neu-rosedative behavior, depending on the structural modificationproposed.

4.2 Blockers of N-type VACCsThe discovery of new ligands for N-type channels (Cav2.2)has attracted much attention and efforts, as they are nowproposed as good targets for pain relief. This action is likelydue to the fact that, at the dorsal horn of the spinal cord,N-type channels modulate the release of key pronociceptiveneurotransmitters, such as glutamate, substance P and calcito-nin-gene-related peptide. Seven patents dealing with newN-type channel blockers are reviewed in this section.

Searle et al. from Abbot Laboratories, synthesizedcyclopenta-fused pyrroles with a general formula showing anamino substitution at C4 on the fully hydrogenated bicyclicring (Figure 4,4) [105]. Near 800 compounds were tested onIMR32 cells that endogenously expressed human N-typechannels, using FLIPR technology to monitor [Ca2+]c eleva-tions elicited by K+. Some hit compounds with IC50 below1 µM were found. Mechanical hyperalgesia elicited by capsa-icin was inhibited 90% by two compounds administeredorally at 30 mg/kg. Furthermore, in the Bennett model ofneuropathic pain-related allodynia, the blockade was 80%.These small molecules may have a better activity than thew-conotoxins peptides for the treatment of the intractablepain, due to their blockade of N-type channels.

Convergence Pharmaceuticals Ltd. has synthesized200 compunds showing a general formula defined by a piper-azinylsulfone, when the aryl group is a differently substitutedbenzene (Figure 4,5). Using the patch-clamp technique in

HEK 293 cells expressing human N-type channels, the bestcompounds caused use-dependent inhibition of calcium cur-rents when administered at 30 µM. However, no in vivodata have been reported [106]. Use-dependent channel block-ade is interesting because, in spite of low potency, thecompounds may efficiently target highly active channelsunder conditions of increased neuronal excitability, whichare contributing to the pathophysiology of chronic pain.

In another series of N-aryltetrazoles, where R2 and R3 canbe part of an aza-heterocycle nucleus (Figure 4,6), Beswicket al., from the same company, found that the use-dependentchannel blockade was achieved at concentrations 10-foldlower than in the previous family [107].

Li et al., from Abbvie, Inc., synthesized 212 octahydropyr-rolo[1,2-a]pyrazine derivatives. Compounds were screened inIMR32 cells expressing human N-type channels, monitoringCa2+ influx through FLIPR technology. For instance, example47 had an IC50 of 0.29 µM (Figure 5,7). Ten compounds wereselected to test in the Bennett model of neurophatic pain.Example 186 (Figure 5,7) exhibited 91% of maximum possi-ble analgesic effect [108]. More recently, the same group hasreported the synthesis of 231 pyrrolo[1,2-a]pyrazine sulfona-mides. In the case of compounds 203 and 223 (Figure 5,8),IC50 to block N-type channels were even lower (0.13 µM).However, the analgesic effect for compound 21 (Figure 5;8)was only 43% of the maximum posible analgesic effect [109].

Khanna et al., from Indiana University Research and Tech-nology Corp., have found an original strategy to block N-typechannels with peptides based on transactivator of transcription(TAT) CB3. The collapsin response mediator protein2 (CRMP-2) upregulates N-type currents. Thus, those peptidesuncouple the CRMP-2-Cav2.2 complex and elicit analgesia inanimal models of pain [110]. A more recent patent of the samegroup reports new TAT-conjugated peptides with the sequenceYGRKKRRQRRRARSRLAELRGVPRGL for compoundST1-104. In the compound ST1-106, arginine-9 is replacedby leucine. These peptides were studied in several pain animalmodels including diabetic neuropathy or retroviral-inducedneuropathy. Interestingly, ST1-106 blocked R-type currentsin addition to the blockade of N-type currents, whereasST1-104 did not target R-type currents [111].

4.3 Blockers of T-type VACCsT-type channels (Cav3) are involved in the regulation of anincreasing number of physiological functions, as well as inthe pathophysiology of diseases like pain, epilepsy, cancer,AD, essential tremor or hypertension. Although severalcompounds have been synthesized and tested in the last twodecades, a selective T-type channel blocker has not yet reacheda clinically useful status. However, much efforts and invest-ment are being devoted to the discovery and development ofnew ligands for T-type channels. The nine patents herereviewed exemplify such effort.

Zalicus Pharmaceuticals Ltd. has synthesized about 600 com-pounds derivates of N-piperidinylacetamide. The compounds

4

N

NR3R2

R1

5

N

N

S

Ar

OO

NO

6

N N

NN

R1

N

R2

R3

Figure 4. General formula (4) to (6), from patents on N-type

channel ligands, discussed in Section 4.2, are grouped.

Piperazinylsulfones disclosed by Convergence Pharmaceuti-

cals present different fusion with pyridine nuclei, and ‘Ar’

defines a differentially substituted benzene, as shown in

the general formula (5). R2 and R3 can be part of an

aza-heterocycle nucleus in the general formula (6).

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present general Markush structures where ‘Ar’ can be eitherphenyl or heterocyclic rings (Figure 6,9). Compound screeningwas performed in HEK 293 cells expressing human T-typechannels, using the patch-clamp technique. Some compoundshad IC50 of 0.1 µM, indicating a high potency to blockT-type channels. Two lead compounds were tested in a neuro-pathic painmodel consisting in L5/L6 spinal nerve ligation, andin two epilepsy models: the electroconvulsive shock thresholdtest and the genetic absence epilepsy rats from Strasbourg(GAERS). Their efficacy led the authors to claim theirtherapeutic use in both pain and epilepsy [112].Gray and Haverstick, from University of Virginia, have

synthesized a series of arylalkylpyrroles analogues to TH-1177, shown in Figure 6,10. This compound inhibitedCav3.2 channel currents with an IC50 of 0.8 µM in Jurkatcancer cells expressing the Cav3.2 channel. Because T-typechannels are associated with prostate cancer, cell proliferationand inflammation [113,114], the authors claim the use ofTH-1177 to treat androgen-sensitive human prostate adeno-carcinoma. In fact, the compound inhibited PC13 humanprostate cancer cell proliferation, with an IC50 of 14 µM [115].Merck Sharp & Dohme Corp. has disclosed the synthesis

of pyrazynilphenylamide derivatives with the general formulashown in Figure 6,11. For some of the compounds, blockadeof T-type currents in HEK 293 cells expressing human T-type channels, using the patch-clamp technique, showedIC50 of < 1 µM, indicating a high potency to block T-typechannels [116]. At this company, Schlegel et al. have patentedthe synthesis and use of heterocycle derivatives having acentral amide group with a general formula, where R1 canbe alkyl or hydrogen. In a few examples, triazole nuclei arereplaced by tetrazole, imidazole or oxazole (Figure 6,12).Some compounds exhibited IC50 lower than 1 µM to block

T-type currents [117]. Another patent from the same companyreports the synthesis of imidazolylmethylpiperidine deriva-tives (Figure 6,13). Again, some of these compounds hadactivities with IC50 below 1 µM to block T-type currents [118].

Tung et al., from Concert Pharmaceuticals, Inc., havepatented a family of deuterated mibefradil derivatives with atetrahydronaphtalene structure. Replacement of hydrogen bydeuterium is aimed to improve the metabolic stability of themibefradil derivatives. Mibefradil is a T-type channel blockerthat inhibits hepatic CYP, more specifically CYP3A4, pre-sumably by some of its oxidized metabolites. This gave riseto fatal drug interactions that caused its withdrawal from themarket. The lead deuterated derivative 500 (Figure 6,14) didnot inhibit CYP3A4 [119]. This effect is due to a slower rateof its enzymatic metabolism by either CYP3A4-catalyzed oxi-dation or ester hydrolysis by esterases. The physicochemicalfundament of this slowing lies in the so-called kinetic isotopeeffect. Basically, the rate of the chemical reaction to oxidize acarbon-deuterium bond is much higher than that one tooxidize a regular carbon-hydrogen bond. Thus, CYP3A4-ad-dressed metabolism over deuterated analogues of mibefradilwill be quite slower than over mibefradil.

Weber et al., from Zenyaku Kogyo Kabushikikaisha, havepatented the use of new heterocyclic compounds, analogues ofST101, that contain a central imidazol-4-one nucleus(Figure 6,15). In HEK 293 cells expressing human T-type chan-nel subtypes, compound ST101 blocked the Cav3.1 channelwith an IC50 of 42.9 nM, while it was unable to blockCav3.2 and Cav3.3 currents with IC50 below 100 µM [120]. Itis important to note that the same compound has been recentlydescribed to show agonist activity of this channel at 0.1 nM[121]. Otherwise, Weber et al. discuss that T-type currents havebeen linked to the activation of A-type potassium currents

CF3

HN

N

NO

N N

OH

SO

ONH

CF3

N

NH

N

NSO

OF3C

O

N N

H

N

NO

N N

SO

ON

H

N

N

O

SO

OF3C

CF3H

7

Example 47 Example 186

8

Example 21 Example 203 Example 223

Figure 5. Chemical structures of (7) to (8), showing the most active compounds from patents of Abbvie corp. on N-type

channels ligands, discussed in Section 4.2, are grouped. on N-type channels ligands, discussed in Section 4.2, are grouped.

This company focuses on pyrrolopyrazine derivatives, mainly showing arylsulfonamide moieties.

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through Kv4 channels [122]. Thus, authors hypothesize that theblockade of T-type channels would increase the firing of APsto indirectly increase neurotransmitter release. Low-thresholdneurotransmitter release may also contribute to an enhancedsynaptic transmission [39]. The parent compound inhibits A�deposition and improves cognition, implying that T-type chan-nel blockade could be beneficial in AD, too [120].

Bourinet et al., from the Universit�e de Nice Sophia Antip-olis, have synthesized a peptide with the following amino-acidsequence: YCQKFLWTCDSERPCCEGLVCRLWCKIN.

The peptide was synthesized either by expression of theencoding DNA into a plasmid or by solid phase chemicalsynthesis. In HEK 293 cells expressing the human Cav3.2channel, the peptide blocked T-type currents with an IC50

in the low micromolar range. In a mouse model of pain eli-cited by thermal stimuli, treatment with the peptide showedmuch longer reaction time to heat, what was not observedin Cav3.2 channel-knockout mice, demonstrating its selectiv-ity of action. Authors also show in vivo studies to testboth antihypertensive and antiproliferative effects, as the

9

NHN

OR

R

NNH

Ar

R

O

10

NO

Cl

O

11

N

N O

HN

N

O CF3

R1 R2

12

O

HN

X

N NNAlkyl

R

R1

13

NN

NH

O

R3

R4

R2

R1

14

N

NHN

O

OO

FD D

DD

15

N

N

O

ST101

16

HN NN

O

O

Figure 6. Chemical structures and general formula (9) to (16), of the patents on T-type Ca2+ channels ligands described in

Section 4.3, are grouped. Acetamides disclosed by Zalicus (9) can present as ‘Ar’ phenyl or heterocyclic rings, while those

patented by Merck are limited to the general formula (11) and (12), where R1 and R2 can be alkyl or hydrogen. In the case of

acetamide lactams disclosed by Abbott, compound (16) showed the best profile in the reduction of the secondary mechanical

hyperalgesia exerted by capsaicin. Structure (10) represents the unique compound disclosed by University of Virginia Patent

Foundation, proposed to block T-type Ca2+ channels in adenocarcinoma cells, thus possessing antiproliferative activity. Merck

has also patented T-type Ca2+ channel ligands with piperidine-imidazole double core, as represented by general formula (13).

Compound (14) showed the best pharmacokinetic profile of all the deuterated analogues of mibefradil disclosed by Concert.

Finally, ST101 (15) is the compound selected by Zeniaku from a one-hundred piridoimidazole-derived family, to carry out

in vivo experiments related to the T-type Ca2+ channel blockade.

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Cav3.2 channel is also involved in cell proliferation. Its anti-proliferative effects suggest this peptide could be useful inthe treatment of cancer [123].Abbott Laboratories has disclosed a series of acetamide

lactams, with similar formula of the selected compound dis-played in Figure 6,16. In IMR32 cells expressing the humanCav3.2 channel, compounds showed IC50 between 10 and15 µM to block [Ca2+]c transients. However, monitoring T-type currents, some compounds exhibited IC50 in the submi-cromolar range. In an animal model of hyperalgesia inducedby capsaicin, compound 16 (Figure 6) reduced it by 74% [124].

4.4 Blockers of L- and T-type VACCsHilpert has reported the synthesis of 25 bridged tetrahydro-naphthalene derivatives (Figure 7,17). In HEK 293 cellsexpressing the human Cav1.2 channel, IC50 to block [Ca2+]ctransients were in the range 1.02 -- 8.7 µM. In HEK 293 cellsexpressing human Cav3.1, Cav3.2 or Cav3.3 channels, IC50

were in the range 0.19 -- 2.9 µM. In the perfused mouse heart,IC50 values to depress contractility ranged between 6 and42 µM. Thus, the compounds are claimed to be useful inthe treatment of cardiovascular diseases [125].

4.5 Blockers of N- and T-type VACCsNon-selective blockers of N- and T-type channels have beenreported in three patents, where the claimed therapeuticindications were inflammatory pain, migraine and epilepsy.Merck Sharp & Dohme Corp. reports a family of N-

substituted oxindoline derivatives with the general structuregiven in Figure 7,18. ‘Ar’ was either a substituted phenyl ringor a pyrimidine heterocycle; X substituents were mostly halogenatoms and the N-anchored heterocycle was 5- and 6-memberedaza-heterocycles. The 59 compounds were tested in HEK293 cells expressing human Cav2.2, Cav3.1 or Cav3.2 channels.Authors used recordings of calcium currents and monitored

[Ca2+]c transients with fluorescent probes. To block N-typecurrents (Cav2.2), preferred compounds exhibited IC50 below1 µM. This was also the case for T-type currents. The bestcompounds were tested in an acute inflammatory pain modelin rats treated with complete Freund’s adjuvant. Based on theirefficacy to inhibit pain in this model, the authors claim theycould be useful in the treatment of acute pain [126].

Pajouhesh et al., from Zalicus Pharmaceuticals Ltd., havepatented 200 bis-aryl and alkylarylsulfones. Most compoundsare exemplified by the Markush structures indicated inFigure 7,19. In cells expressing N- or T-type channel subtypesusing [Ca2+]c transient monitoring, bis-arylsulfones-derivedcompounds inhibited both channel types with IC50 in thesubmicromolar range but with some selectivity for N-typechannels. However, alkylarylsulfones blocked selectively N-type channels with IC50 in the range 0.1 -- 1 µM. The authorsclaim these compounds could be useful in the treatment ofpain and epilepsy [127].

Heer et al., from Convergence Pharmaceuticals Ltd., havereported the synthesis of piperazine derivatives with the gen-eral molecular structure shown in Figure 7,20, where R1 iseither hydrogen or a methyl group and the phenyl group iscommonly mono-substituted with either CF3 or CN groups.The compounds were tested in HEK 293 cells expressinghuman Cav2.2 or Cav3.2 channels, using the patch-clamptechnique. Several compounds blocked both N- and T-typecurrents with IC50 in a range between 3.5 and 30 µM. Onthe basis of these results, the authors claim that these com-pounds may have therapeutic interest in neuropathic pain,inflammatory pain and migraine [128].

5. Conclusion

In these 23 recent patents reviewed, a few thousand newlysynthesized compounds have been screened to assess their

17

OO

CF3

N

N

HN

O 18

NO

Ar

Het

X

19

SO O

NH

OArR

SO O

RHN

O

R

20

R2

SN

NQuinoline

OO

O

R1

Figure 7. Chemical structure of (17) from the patent on L- and T-type channel ligands described in Section 4.4, and chemical

structures (18) to (20) from patents on N- and T-type channels in Section 4.5, are grouped.

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ability to target a given subtype of VACC expressed in celllines. It is highlighted the increasing interest in T-type chan-nels (nine patents), particularly of its isoform Cav3.2 thathas been implicated in the transmission of neuropathic painat the spinal cord. Great efforts are also being made in block-ers of N-type channels (seven patents) and ligands for thea2/d subunit of VACCs, also focused on pain. Intractablepain of peripheral diabetes neuropathy, herpes, cancer, tri-geminal neuralgia or post-surgery is of particular relevance.Thus, pain is the main focus of most patents (16 out of 23).T-type channel blockers mitigating increased neuronal excit-ability in the epilepsy is another area of interest. Finally,some patents on ligands for L-type channels are also discussed,where blockers of the Cav1.3 isoform are being considered forthe treatment of PD. Also, some T-type channel ligands arebeing tested to mitigate Ab burden in AD. Information onselectivity for a given VACC subtype, on pharmacokinetics,particularly the ability of selected ligands to cross the BBB,and to interact with other drugs at the CYP system is requiredbefore positioning compounds as drug-able potential medi-cines indicated in the treatment of those diseases. Because ofthe widespread expression of various of the VACCs in differ-ent brain areas, it is anticipated that unless highly selectivecompounds were found, intolerable side effects may seriouslylimit the clinical use of the compounds here reviewed.

6. Expert opinion

In this review, 23 patents claiming selectivity of newly synthe-sized compounds for L-type channels (three patents), N-typechannels (seven patents) or T-type channels (nine patents)have been selected. Some additional patents have beenreviewed on compounds that target L- and T-type channels(one patent) and others targeting N- and T-type channels(three patents). The most common therapeutic claim isneuropathic pain; occasionally, inflammatory pain has alsobeen claimed. The success of peptidic w-conotoxin MVIIA(ziconotide) in intractable pain has stimulated the search ofnon-peptide compounds that, by targeting N- and/or T-typechannels or both together, could be therapeutically useful totreat severe pain. The second most often claimed indicationis epilepsy, particularly with T-type channel blockers. It isimportant to highlight that Cav3.1 ligands may enhance cog-nition in AD patients by augmenting neurotransmitterrelease. Another interesting claim is that made for ligands ofL-type channels in PD, that could protect dopaminergicneurons from calcium overload, secondary to fast AP firing.We found also some therapeutic claims for these L-type chan-nel ligands in cerebrovascular and neurodegenerative diseases.

Three patents on L-type channel ligands are presented. Inthe patent of Delgado-Martın et al. [102], the original aspect isthe extraction procedure for marine crambescidines. However,the low potency of the crambescidine mixture should beenhanced with new semisynthetic compounds. On theother hand, the patent covering DHP-benzodiazepine hybrid

structures are interesting from a multitarget point of view;although some in vivo experiments are commented, no biolog-ical data are reported [104]. Finally, the patent of new ligandsfor the L-type channel isoform Cav1.3 aims the importantgoal of slowing down PD progression [103]. Channel selectivityis critical here to avoid intolerable side effects. Also, the abilityof readily crossing the BBB is imperative in this type of drug.

The seven patents covering N-type channel ligands focusmainly on the treatment of neuropathic pain. Some patentsare more valuable because they include data obtained in ani-mal models of neuropathic pain. This is the case of the patentsby Searle et al. [105], Khanna et al. [110] and Li et al. [108]. Someselected compounds from these patents exhibited notable effi-cacy when administered orally to murine models of neuro-pathic pain. An interesting new target is that on the N-typechannel regulatory protein CRMP-2. However, the limitationof this strategy is the peptidic nature of the ligands for suchprotein, thus requiring parenteral administration [111].

In some of the nine patents on T-type channel ligandsreviewed, in vivo experiments are given. This is the case for apatent from Zalicus Pharmaceuticals [112] that reports experi-ments on animal models of epilepsy and pain, showing goodefficacy. Blockade of the Cav3.2 channel by compounds synthe-sized by Abbott [124] is also effective in animal models of hyper-algesia induced by capsaicin, indicating the relationshipbetween those channels and neuropathic pain. The patent ofWeber et al. [120] is interesting taking into account the inhibitionof Ab deposition and improved cognition in animal models ofAD. This is interesting because presynaptic T-type channelsare known to enhance transmitter release and thus blockerscould decrease neurotoxicity linked to excess glutamate release.However, other authors have described that the hit compoundof this patent, ST101, is a T-type Ca2+ channel agonist at sub-nanomolar concentrations. A possibility exists that the in vivooutcomes with ST101 would come from an indirect pharmaco-logical effect on several kinase enzymes, as hypothesized byMoriguchi et al. [121], a Ca2+ promoting action or the expressedCa2+ antagonist activity proposed by Weber et al. Otherwise,the physiological relevance of this compound derives from itscognition enhancer activity.

The three patents reporting ligands for both N- and T-typechannels are interesting because these compounds may exhibitsynergy in treating painful conditions in inflammatory pain,neuropathic pain and migraine. This is the case of the patentof Merck Sharp & Dohme Corp. [126]. that shows in vitro andin vivo data. A major limitation of this multitarget strategy isthat the simultaneous blockade of N- and T-type channelsmay give rise to greater effectiveness but also to additive tox-icity. In fact, the great challenge for future research is tofind out more selective ligands not only for the different iso-forms of VACC subtypes L, N, P/Q, R and T, but also forisoforms that may proof to be tissue specific. In fact, marketedCa2+ antagonists present side effects mainly due to their lackof selectivity. One can realize the opposite is also true, as ahuge inhibition of a specific isoform can derive in severe

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adverse effects. We emphasize that, for obtaining selective Ca2+

channel ligands, it is not necessary to reach nanomolar activi-ties, but simply that IC50 data for the inhibition of otherisoforms were 10-fold higher. An emerging discussion iswhether a therapeutically useful Ca2+ antagonist has to be asmuch selective as possible, or possessing a broad-spectrumblocking activity. The only way to resolve this argument is todevelop a real highly selective Ca2+ channel blocker, with theaim to figure out the real contribution of any specific isoformto the pathology object of study, and its therapeutic range.Should a dual, or multitargeted, blocking activity on severalCav isoforms the cause of the therapeutic activity of a certainCa2+ antagonist, this has to be empirically characterized bystudying the contribution of each isoform to blockade. Toachieve this difficult goal, it could be interesting to pay moreattention to structural molecular studies that compare severalof the natural toxins that target with high selectivity some ofthe VACC subtypes. These studies would help to the develop-ment of future peptidomimetic drugs and, in addition, to findthe key pharmacophoric groups in the binding with the biolog-ical target, which in turn would be essential for the develop-ment of small drugs, through an epitope-based chemicalconstruction. Sparse information on structure--activity rela-tionships and limited high-throughput electrophysiologicalmethods have hampered the development of the appropriatelead molecule for a given channel type. The knowledge of thethree-dimensional structure in solution of w-toxins is criticalto study the specificity of their interactions with the varioussubtypes of VACCs as well as to define active sites that canserve as models to design and synthesize non-peptide blockers.The w-conotoxins are small peptides containing 24 -- 29amino-acid residues. A surprising finding is that the amino-acid sequence of w-conotoxin MVIIA (a reversible N-typechannel blocker) is closer to that of w-conotoxin MVIIC (anN- and P/Q-type channel blocker) than to the w-conotoxinGVIA (an irreversible N-type channel blocker). These differ-ences are important in order to define the toxin selectivity forN- or P/Q-type channels. The three-dimensional structuresof w-conotoxin GVIA [129], w-conotoxin MVIIA [130] andw-conotoxinMVIIC [131] have been elucidated. The definitionof the structure of other toxins will facilitate their comparisonsand the definition of structural determinants for specific bind-ing to VACC subtypes. This will allow the identification ofpharmacophores to facilitate the synthesis of non-peptidechannel blockers of therapeutic interest [132].Some recent examples may illustrate the challenge to

develop a highly specific molecule to target one singleVACC type. For instance, compound A-1048400 (AbbotLaboratories) is a small molecule with high potency for theN-, P/Q- and T-type channels, lacking L-type channel activ-ity, with no hemodynamic effects, and having antinocipeptiveactions [133]. Also, Neuromed has described a number of com-pounds that show some selectivity for the N-type channelsversus the P/Q-type channels, and with potential therapeuticindications in pain and stroke [134]. The aliphatic monoamine

dodecylamine has also been suggested to be largely selectivefor P/Q-type channels [135]. A major limitation of thesestudies, however, is that authors do not provide a clear struc-ture--activity relationship for a further lead optimizationfocusing on small P/Q-type channel blockers.

A similar strategy to that followed to develop the N-typechannel blocker ziconotide for severe pain treatment [136] couldbe applied to the w-toxin P/Q-type channel blockers withpotential indications in migraine and AD. Thus, peptide cycli-zation has improved the biophysical properties and the activityof w-agatoxins [137]. However, good bioavailability of peptidetoxins is still a challenge, and these molecules do not penetratethe BBB. Furthermore, even if given intrathecally, like the caseof ziconotide, w-agatoxin IVA may have strong adverse effects,as it irreversibly blocks P/Q-type channels. However, structuralinformation from the binding domain of w-agatoxin IVA andw-agatoxin IVB to the a1 subunit of P/Q channels may inspirethe development of selective and more appropriate peptideanalogues. The strategy of narrowing down the amino-acidsequence of the pharmacophore has met with some success.For instance, the analogue compound 4a of w-conotoxinGVIA, which mimics three of its side chains, potently blocksN-type channels in the micromolar range [138]. Additionally,Pfizer designed a small-molecule mimicking three residues ofw-conotoxin MIIA that blocks N-type channels [139]. Furtherdevelopment resulted in the generation of an orally availablesmall molecule that blocked N-type channels, displayingimproved physicochemical properties [140].

A major limitation to the development of selective modula-tors of VACC subtypes is their wide distribution in the CNS.For instance, the P/Q-type channels are expressed in all brainareas, especially in the cerebellum. This could be a challengefor drug development, as P/Q-type channel blockade in thecerebellum may cause gait and movement disorders. In fact,P/Q-type channel knock-out mice exhibit symptoms of ataxiaand dystonia [141,142]. Addressing a particular splice variantexpressed in the brain region of interest could become anapproach to bypass the effects on the cerebellum of such P/Q-type channel ligands [143,144]. Another interesting approachis the development of state-dependent ligands that preferen-tially bind to the inactivated state of the channel. In this man-ner, the ligand will target channels at overactive synapsesunder pathological conditions, thus sparing normal synapses.This kind of compounds could be indicated in migraine,pain and epilepsy, where the pathophysiology involves pro-longed depolarizations of the membrane over seconds orminutes [145]. Conversely, some compounds bind to theopen state of the channel and thereby delay its deactivation.This leads to a facilitation of Ca2+ influx that can be beneficialin certain types of ataxia. These limitations have precludedthe development of non-toxin, small molecules to targetselectively P/Q-type channels for CNS diseases [146].

The hypothesis on a potential therapeutic application ofVACC agonists deserves attention. In a recent study, Arm-strong and Drupeau [147] have expressed the human gene

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encoding TDP43 (TAR DNA-binding protein) in zebrafishlarvae, with the mutation G348C found in patients of amyo-trophic lateral sclerosis (ALS). These larvae showed impairedswimming and increased motor neuron vulnerability, withreduced synaptic fidelity, reduced quantal transmission andmore orphaned presynaptic and postsynaptic structures atthe neuromuscular junction. Remarkably, all behavioral andcellular features were stabilized by chronic treatment witheither L-type VACC agonists FPL64176 or BayK8644, sug-gesting that these compounds could be a novel therapeutic

approach for ALS. A major limitation of these compoundscould be their arrhythmogenic cardiac effects. Thus, VACCagonists, selective for Cav1.3 channels, could be an interestingnovel approach to treat ALS patients.

Declaration of interest

The authors declare no conflict of interest and have receivedno payment in preparation of this manuscript.

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IUPHAR/BPS Guide to

PHARMACOLOGY. Available from:

http://www.guidetopharmacology.org/

GRAC/FamilyDisplayForward?

familyId=80 [Accessed on 15 June 2014]

AffiliationJuan-Alberto Arranz-Tagarro1,2,3,

Cristobal de los Rıos1,3,

Antonio G Garcıa†1,2,3,4 &

Juan-Fernando Padın1,2,3

†Author for correspondence1Instituto Teofilo Hernando, Universidad

Autonoma de Madrid, Madrid, Spain2Departamento de Farmacologıa y Terap�eutica,

Facultad de Medicina, Universidad Autonoma de

Madrid, Madrid, Spain3Servicio de Farmacologıa Clınica, Instituto de

Investigacion Sanitaria, Hospital Universitario de

La Princesa, Madrid, Spain4Professor,

Hospital Universitario de La Princesa, Spain

Tel: +34 914973120 3121;

Fax: +34 914975380;

E-mail: agg@uam.es

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