Cryptotanshinone,an orally bioactive herbal compound from Danshen,attenuates atherosclerosis in apolipoprotein E‐deficient mice: role of lectin‐like oxidized LDL receptor‐1 (LOX‐1)

Background and Purpose

Cryptotanshinone (CTS) is a major bioactive diterpenoid isolated from Danshen, an eminent medicinal herb that is used to treat cardiovascular disorders in Asian medicine. However, it is not known whether CTS can prevent experimental atherosclerosis. The present study was designed to investigate the protective effects of CTS on atherosclerosis and its molecular mechanisms of action.

Experimental Approach

Apolipoprotein E‐deficient (ApoE ^−/−) mice, fed an atherogenic diet, were dosed daily with CTS (15, 45 mg kg⁻¹ day⁻¹) by oral gavage. In vitro studies were carried out in oxidized LDL (oxLDL)‐stimulated HUVECs treated with or without CTS.

Key Results

CTS significantly attenuated atherosclerotic plaque formation and enhanced plaque stability in ApoE ^−/− mice by inhibiting the expression of lectin‐like oxLDL receptor‐1 (LOX‐1) and MMP‐9, as well as inhibiting reactive oxygen species (ROS) generation and NF‐κB activation. CTS treatment significantly decreased the levels of serum pro‐inflammatory mediators without altering the serum lipid profile. In vitro, CTS decreased oxLDL‐induced LOX‐1 mRNA and protein expression and, thereby, inhibited LOX‐1‐mediated adhesion of monocytes to HUVECs, by reducing the expression of adhesion molecules (intracellular adhesion molecule 1 and vascular cellular adhesion molecule 1). Furthermore, CTS inhibited NADPH oxidase subunit 4 (NOX4)‐mediated ROS generation and consequent activation of NF‐κB in HUVECs.

Conclusions and Implications

CTS was shown to have anti‐atherosclerotic activity, which was mediated through inhibition of the LOX‐1‐mediated signalling pathway. This suggests that CTS is a vasculoprotective drug that has potential therapeutic value for the clinical treatment of atherosclerotic cardiovascular diseases.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ApoE^−/−

apolipoprotein E deficient

CD36

cluster of differentiation 36

CTS

cryptotanshinone

HCD

high cholesterol diet

ICAM‐1

intracellular adhesion molecule 1

LOX‐1

lectin‐like oxidized low‐density lipoprotein receptor‐1

NOX4

NADPH oxidase subunit 4

oxLDL

oxidized low‐density lipoprotein

ROS

reactive oxygen species

SMCs

smooth muscle cells

SR‐A

scavenger receptor‐A

VCAM‐1

vascular cellular adhesion molecule 1

Tables of Links

Meningeal blood flow is controlled by H2S‐NO crosstalk activating a HNO‐TRPA1‐CGRP signalling pathway

Background and Purpose

Meningeal blood flow is controlled by CGRP released from trigeminal afferents and NO mainly produced in arterial endothelium. The vasodilator effect of NO may be due to the NO–derived compound, nitroxyl (HNO), generated through reaction with endogenous H₂S. We investigated the involvement of HNO in CGRP release and meningeal blood flow.

Experimental Approach

Blood flow in exposed dura mater of rats was recorded by laser Doppler flowmetry. CGRP release from the dura mater in the hemisected rat head was quantified using an elisa. NO and H₂S were localized histochemically with specific sensors.

Key Results

Topical administration of the NO donor diethylamine‐NONOate increased meningeal blood flow by 30%. Pretreatment with oxamic acid, an inhibitor of H₂S synthesis, reduced this effect. Administration of Na₂S increased blood flow by 20%, an effect abolished by the CGRP receptor antagonist CGRP _8‐37 or the TRPA1 channel antagonist HC030031 and reduced when endogenous NO synthesis was blocked. Na₂S dose‐dependently increased CGRP release two‐ to threefold. Co‐administration of diethylamine‐NONOate facilitated CGRP release, while inhibition of endogenous NO or H₂S synthesis lowered basal CGRP release. NO and H₂S were mainly localized in arterial vessels, HNO additionally in nerve fibre bundles. HNO staining was lost after treatment with L‐NMMA and oxamic acid.

Conclusions and Implications

NO and H₂S cooperatively increased meningeal blood flow by forming HNO, which activated TRPA1 cation channels in trigeminal fibres, inducing CGRP release. This HNO‐TRPA1‐CGRP signalling pathway may be relevant to the pathophysiology of headaches.

Abbreviations

CBS

cystathionine β‐synthase

CSE

cystathionine γ‐lyase

Cy3

cyanine dye 3

DAF

4‐amino‐5‐methylamino‐2′,7′‐difluoresceine diacetate

HNO

nitroxyl

iNOS

inducible NOS

L‐NMMA

L‐NG‐monomethylarginine acetate

MMA

middle meningeal artery

MST

mercaptopyruvate sulfurtransferase

nNOS

neuronal NOS

NONOate

diethylamine‐NONOate, DEANONOate

ODQ

1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one

sGC

soluble GC

SIF

synthetic interstitial fluid

TRPA1

transient receptor potential ankyrin 1 channel

Tables of Links

Pro‐cognitive activity in rats of 3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide,a positive allosteric modulator of the α7 nicotinic acetylcholine receptor

Background and Purpose

α7 nicotinic acetylcholine receptors (α7 nAChRs) may represent useful targets for cognitive improvement. The aim of this study is to compare the pro‐cognitive activity of selective α7‐nAChR ligands, including the partial agonists, DMXBA and A‐582941, as well as the positive allosteric modulator, 3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide (PAM‐2).

Experimental Approach

The attentional set‐shifting task (ASST) and the novel object recognition task (NORT) in rats, were used to evaluate the pro‐cognitive activity of each ligand [i.e., PAM‐2 (0.5, 1.0, and 2.0 mg·kg⁻¹), DMXBA and A‐582941 (0.3 and 1.0 mg·kg⁻¹)], in the absence and presence of methyllycaconitine (MLA), a selective competitive antagonist. To determine potential drug interactions, an inactive dose of PAM‐2 (0.5 mg·kg⁻¹) was co‐injected with inactive doses of either agonist ‐ DMXBA: 0.1 (NORT); 0.3 mg·kg⁻¹ (ASST) or A‐582941: 0.1 mg·kg⁻¹.

Key Results

PAM‐2, DMXBA, and A‐582941 improved cognition in a MLA‐dependent manner, indicating that the observed activities are mediated by α7 nAChRs. Interestingly, the co‐injection of inactive doses of PAM‐2 and DMXBA or A‐582941 also improved cognition, suggesting drug interactions. Moreover, PAM‐2 reversed the scopolamine‐induced NORT deficit. The electrophysiological results also support the view that PAM‐2 potentiates the α7 nAChR currents elicited by a fixed concentration (3 μM) of DMXBA with apparent EC₅₀ = 34 ± 3 μM and E_max = 225 ± 5 %.

Conclusions and Implications

Our results support the view that α7 nAChRs are involved in cognition processes and that PAM‐2 is a novel promising candidate for the treatment of cognitive disorders.

Abbreviations

α7 nAChR

nicotinic acetylcholine receptor with α7 subunit

AD

Alzheimer''s disease

apparent EC₅₀

enhancement potency

ASST

attentional set‐shifting task

CD

compound discrimination

DI

discrimination index

E

exploration time

ED

extra‐dimensional

E_max

ligand efficacy

ID

intra‐dimensional

ITI

inter‐trial interval

MLA

methyllycaconitine

NORT

novel object recognition task

n_H

Hill coefficient

PAM

positive allosteric modulator

PAM‐2

3‐furan‐2‐yl‐N‐p‐tolyl‐acrylamide

Rev

reversal of discrimination

SD

simple discrimination

T1

familiarisation trial

T2

retention trial

     

Intravesical delivery of 5‐aminolevulinic acid from water‐in‐oil nano/submicron‐emulsion systems

The present work reports on the development of water‐in‐oil (w/o) emulsions for the intravesical administration of 5‐aminolevulinic acid (ALA). The physicochemical properties of droplet size, zeta potential, and viscosity of the emulsions are characterized and the ability of the emulsions to release ALA following in vitro application is tested. The delivery systems are administered intravesically for 1 and 3 h in rats to examine the drug accumulation in bladder tissue. The mean size and zeta potential of the emulsions are 50–200 nm and ?3 to ?14 mV, respectively. The loading of ALA into the emulsions resulted in a slower and sustained release. The release extent was found to be inversely related to the droplet size of the emulsions. The emulsions did not increase the drug permeation into tissues during short exposure duration (1 h). When the dwell time was extended to 3 h, the systems showed a 2.7‐fold increase in the ALA concentration in the bladder wall. Images of confocal laser scanning microscopy demonstrated a higher and deeper fluorescence signal, with emulsion administration, as compared to the aqueous control. Intravesical emulsion delivery provides a significant advantage for drugs targeting bladder tissues. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2375–2385, 2010     

Texture Optimization of Water‐in‐Oil Emulsions

The aim of this research is to demonstrate the effect of variations in certain parameters of the oily phase (OP) in water‐in‐oil (W/O) emulsions on rheological and texture properties of finished products. The formulated emulsions were selected according to an optimal experimental procedure. The applied variations were nature of the OP, its volume fraction, the hydrophilic‐lipophilic balance (HLB) value, and the surfactant proportion. Results are presented for the followed tests carried out on the emulsions: texture analysis, rheology, and particle size analysis. The oils used in the study were sweet almond oil, liquid paraffin, maize oil, cyclomethicone, dimethicone, and wheat germ oil. The resulting data demonstrate a notable influence of the volume fraction oil on hardness, viscosity, adhesiveness, and cohesiveness of W/O emulsions. Emulsion hardness and viscosity increased as the OP percentage increased; this effect being even more pronounced for the vegetable oils. In contrast, emulsion adhesiveness and cohesiveness decreased as the volume fraction oil increased. The HLB value of the surfactant mixture of the emulsion also influenced hardness, adhesiveness, and elasticity, increasing or decreasing as HLB value did.     

In Vitro Regioselective Stability of β‐1‐O‐ and 2‐O‐Acyl Glucuronides of Naproxen and Their Covalent Binding to Human Serum Albumin

β‐1‐O‐ (NAG) and 2‐O‐glucuronides (2‐isomer) of (S)‐naproxen (NA) were prepared to determine which positional isomer‐(s) of the acyl glucuronide of NA is responsible for forming covalent adducts with human serum albumin (HSA). Their comparative stability and covalent binding adduct formation with HSA were investigated at pH 7.4 and at 37 °C. NA and its acyl glucuronides were simultaneously determined by HPLC. Three positional isomers were formed successively after incubation of NAG in the buffer only. However, when NAG was incubated with HSA (30 mg/mL), isomers other than the 2‐isomer were formed in little or negligible quantities. In HSA solution, NAG (k_d = 2.08 ± 0.08 h⁻¹) was four times less stable than 2‐isomer (k_d = 0.51 ± 0.02 h⁻¹). NAG was degraded by hydrolysis (k_hyd = 1.01 ± 0.10 h⁻¹) and isomerization (k_iso = 1.07 ± 0.07 h⁻¹) to the same extent; however, hydrolysis was predominant for the 2‐isomer (k_d = 0.51 ± 0.02 h⁻¹). The incubation of both NAG and 2‐isomer with HSA led to the formation of a covalent adduct; however, the adduct formation from the 2‐isomer proceeded more slowly than that from NAG. The present results suggest that the covalent binding of NA to HSA via its acyl glucuronides proceeds through both transacylation (direct nucleophilic displacement) and glycation mechanisms; NAG rapidly forms an adduct that may be unstable, and the protein adduct from the 2‐O‐acyl glucuronide is as important for the covalent binding as those from the 1‐O‐acyl glucuronides.     

Differences in perimenstrual symptoms between cocaine‐abusing and non‐cocaine‐abusing women

Cocaine abuse among women has become a major health problem in the United States, yet there is little information in the literature concerning the effects of this form of substance abuse on a woman's reproductive system. This study of 65 women in residential treatment for cocaine abuse and 65 non‐cocaine‐abusing women was undertaken to determine if there are differences in frequency or severity of perimenstrual symptoms between these two groups of women. Data were collected by questionnaire and included demographics, a substance use history, and the Menstrual Distress Questionnaire developed by Moos. Findings suggested that there are statistically significant differences in frequency and severity of perimenstrual symptoms between the two groups of women.     

Mirabegron relaxes urethral smooth muscle by a dual mechanism involving β3‐adrenoceptor activation and α1‐adrenoceptor blockade

Linked Article

This article is commented on by Michel, M. C., pp. 429‐430 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.13379.

Background and Purpose

Mirabegron is the first β₃‐adrenoceptor agonist approved for treatment of overactive bladder syndrome. This study aimed to investigate the effects of β₃‐adrenoceptor agonist mirabegron in mouse urethra. The possibility that mirabegron also exerts α₁‐adrenoceptor antagonism was also tested in rat smooth muscle preparations presenting α_1A‐ (vas deferens and prostate), α_1D‐ (aorta) and α_1B‐adrenoceptors (spleen).

Experimental Approach

Functional assays were carried out in mouse and rat isolated tissues. Competition assays for the specific binding of [³H]prazosin to membrane preparations of HEK‐293 cells expressing each of the human α₁‐adrenoceptors, as well as β‐adrenoceptor mRNA expression and cyclic AMP measurements in mouse urethra, were performed.

Key Results

Mirabegron produced concentration‐dependent urethral relaxations that were shifted to the right by the selective β₃‐adrenoceptor antagonist L‐748,337 but unaffected by β₁‐ and β₂‐adrenoceptor antagonists (atenolol and ICI‐118,551 respectively). Mirabegron‐induced relaxations were enhanced by the PDE4 inhibitor rolipram, and the agonist stimulated cAMP synthesis. Mirabegron also produced rightward shifts in urethral contractions induced by the α₁‐adrenoceptor agonist phenylephrine. Schild regression analysis revealed that mirabegron behaves as a competitive antagonist of α₁‐adrenoceptors in urethra, vas deferens and prostate (α_1A‐adrenoceptor, pA₂ ≅ 5.6) and aorta (α_1D‐adrenoceptor, pA₂ ≅ 5.4) but not in spleen (α_1B‐adrenoceptor). The affinities estimated for mirabegron in functional assays were consistent with those estimated in radioligand binding with human recombinant α_1A‐ and α_1D‐adrenoceptors (pK _i ≅ 6.0).

Conclusion and Implications

The effects of mirabegron in urethral smooth muscle are the result of β₃‐adrenoceptor agonism together with α_1A and α_1D‐adrenoceptor antagonism.

Abbreviations

CR

concentration ratios

CRC

concentration–response curve

KHS

Krebs–Henseleit solution

LUTS

lower urinary tract symptoms

OAB

Overactive bladder syndrome

     

A comparison of medical symptoms reported by cocaine‐, opiate‐, and alcohol‐dependent patients

Substance abuse is frequently associated with adverse medical consequences. The differences in medical symptoms reported by 101 alcohol‐, 113 cocaine‐, and 107 opiate‐dependent individuals receiving outpatient treatment were studied using a 134‐item questionnaire (MILCOM). Data analysis revealed interesting and unexpected findings, with cocaine patients reporting the fewest total symptoms among the three groups. Moreover, cocaine patients reported significantly fewer CNS and musculo‐skeletal symptoms compared to both alcohol and opiate patients and significantly fewer GI and urinary symptoms than the alcohol but not the opiate patients. In addition, there were sex‐ and race‐related differences in the pattern of symptoms reported. Women reported significantly more CVS, mood, nose/throat, CNS, skin, and GI symptoms than men. Similarly, Caucasians reported significantly more mood, CNS, nose/throat, head/neck, musculoskeletal, and GI symptoms than African‐Americans. The study highlights the influence of drug of choice, gender, and race on medical needs of substance‐abusing persons.     

High‐throughput screening assays for SARS‐CoV‐2 drug development: Current status and future directions

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic, a panel of assays has been developed and applied to screen collections of approved and investigational drugs for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity in a quantitative high-throughput screening (qHTS) format. In this review, we applied data-driven approaches to evaluate the ability of each assay to identify potential anti-SARS-CoV-2 leads. Multitarget assays were found to show advantages in terms of accuracy and efficiency over single-target assays, whereas target-specific assays were more suitable for investigating compound mechanisms of action. Moreover, strict filtering with counter screens might be more detrimental than beneficial in identifying true positives. Thus, developing novel HTS assays acting simultaneously against multiple targets in the SARS-CoV-2 life cycle will benefit anti-COVID-19 drug discovery.     

NVP‐TAE684 reverses multidrug resistance (MDR) in human osteosarcoma by inhibiting P‐glycoprotein (PGP1) function

Background and purpose

Increased expression of P‐glycoprotein (PGP1) is one of the major causes of multidrug resistance (MDR) in cancer, including in osteosarcoma, which eventually leads to the failure of cancer chemotherapy. Thus, there is an urgent need to develop effective therapeutic strategies to override the expression and function of PGP1 to counter MDR in cancer patients.

Experimental Approach

In an effort to search for new chemical entities targeting PGP1‐associated MDR in osteosarcoma, we screened a 500+ compound library of known kinase inhibitors with established kinase selectivity profiles. We aimed to discover potential drug synergistic effects among kinase inhibitors and general chemotherapeutics by combining inhibitors with chemotherapy drugs such as doxorubicin and paclitaxel. The human osteosarcoma MDR cell lines U2OSR2 and KHOSR2 were used for the initial screen and secondary mechanistic studies.

Key Results

After screening 500+ kinase inhibitors, we identified NVP‐TAE684 as the most effective MDR reversing agent. NVP‐TAE684 significantly reversed chemoresistance when used in combination with doxorubicin, paclitaxel, docetaxel, vincristine, ET‐743 or mitoxantrone. NVP‐TAE684 itself is not a PGP1 substrate competitive inhibitor, but it can increase the intracellular accumulation of PGP1 substrates in PGP1‐overexpressing cell lines. NVP‐TAE684 was found to inhibit the function of PGP1 by stimulating PGP1 ATPase activity, a phenomenon reported for other PGP1 inhibitors.

Conclusions and Implications

The application of NVP‐TAE684 to restore sensitivity of osteosarcoma MDR cells to the cytotoxic effects of chemotherapeutics will be useful for further study of PGP1‐mediated MDR in human cancer and may ultimately benefit cancer patients.

Abbreviations

ABC

ATP‐binding cassette

ALK

anaplastic lymphoma kinase

CsA

cyclosporin A

MDR

multidrug resistance

MTT

3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide

PGP1

P‐glycoprotein

Rh123

rhodamine 123

     

Actions of KMUP‐1, a xanthine and piperazine derivative,on voltage‐gated Na+ and Ca2+‐activated K+ currents in GH3 pituitary tumour cells

Background and Purpose

7‐[2‐[4‐(2‐Chlorophenyl)piperazinyl]ethyl]‐1,3‐dimethylxanthine (KMUP‐1) is a xanthine‐based derivative. It has soluble GC activation and K⁺‐channel opening activity. Effects of this compound on ion currents in pituitary GH₃ cells were investigated in this study.

Experimental Approach

The aim of this study was to evaluate effects of KMUP‐1 on the amplitude and gating of voltage‐gated Na⁺ current (I _Na) in pituitary GH₃ cells and in HEKT293T cells expressing SCN5A. Both the amplitude of Ca²⁺‐activated K⁺ current and the activity of large‐conductance Ca²⁺‐activated K⁺ (BK_Ca) channels were also studied.

Key Results

KMUP‐1 depressed the transient and late components of I _Na with different potencies. The IC₅₀ values required for its inhibitory effect on transient and late I _Na were 22.5 and 1.8 μM respectively. KMUP‐1 (3 μM) shifted the steady‐state inactivation of I _Na to a hyperpolarized potential by −10 mV, despite inability to alter the recovery of I _Na from inactivation. In cell‐attached configuration, KMUP‐1 applied to bath increased BK_Ca‐channel activity; however, in inside‐out patches, this compound applied to the intracellular surface had no effect on it. It prolonged the latency in the generation of action currents elicited by triangular voltage ramps. Additionally, KMUP‐1 decreased the peak I _Na with a concomitant increase of current inactivation in HEKT293T cells expressing SCN5A.

Conclusions and Implications

Apart from activating BK_Ca channels, KMUP‐1 preferentially suppresses late I _Na. The effects of KUMP‐1 on ion currents presented here constitute an underlying ionic mechanism of its actions.

Abbreviations

AC

action current

AP

action potential

BK_Ca

channel large‐conductance Ca²⁺‐activated K⁺ channel

I_K(Ca)

Ca²⁺‐activated K⁺ current

I_Na

voltage‐gated Na⁺ current

I–V

current versus voltage

K_ATP

channel ATP‐sensitive K⁺ channel

ODQ

1H‐[1,2,4]oxadiazolo‐[4,3‐a] quinoxalin‐1‐one

TEA

tetraethylammonium chloride

τ_inact(S)

slow component of inactivation time constant for I _Na

YC‐1

3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole

     

Antisense‐mediated reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9): a first‐in‐human randomized,placebo‐controlled trial

AimsLDL‐receptor expression is inhibited by the protease proprotein convertase subtilisin/kexin type 9 (PCSK9), which is considered a pharmacological target to reduce LDL‐C concentrations in hypercholesterolaemic patients. We performed a first‐in‐human trial with SPC5001, a locked nucleic acid antisense inhibitor of PCSK9.MethodsIn this randomized, placebo‐controlled trial, 24 healthy volunteers received three weekly subcutaneous administrations of SPC5001 (0.5, 1.5 or 5 mg kg^–1) or placebo (SPC5001 : placebo ratio 6 : 2). End points were safety/tolerability, pharmacokinetics and efficacy of SPC5001.ResultsSPC5001 plasma exposure (AUC(0,24 h)) increased more than dose‐proportionally. At 5 mg kg^–1, SPC5001 decreased target protein PCSK9 (day 15 to day 35: −49% vs. placebo, P < 0.0001), resulting in a reduction in LDL‐C concentrations (maximal estimated difference at day 28 compared with placebo −0.72 mmol l^–1, 95% confidence interval − 1.24, −0.16 mmol l^–1; P < 0.01). SPC5001 treatment (5 mg kg^–1) also decreased ApoB (P = 0.04) and increased ApoA1 (P = 0.05). SPC5001 administration dose‐dependently induced mild to moderate injection site reactions in 44% of the subjects, and transient increases in serum creatinine of ≥20 μmol l^–1 (15%) over baseline with signs of renal tubular toxicity in four out of six subjects at the highest dose level. One subject developed biopsy‐proven acute tubular necrosis.ConclusionsSPC5001 treatment dose‐dependently inhibited PCSK9 and decreased LDL‐C concentrations, demonstrating human proof‐of‐pharmacology. However, SPC5001 caused mild to moderate injection site reactions and renal tubular toxicity, and clinical development of SPC5001 was terminated. Our findings underline the need for better understanding of the molecular mechanisms behind the side effects of compounds such as SPC5001, and for sensitive and relevant renal toxicity monitoring in future oligonucleotide studies.     

Novel Starch‐Based PVA Thermoplastic Capsules for Hydrophilic Lipid‐Based Formulations

For decades, gelatin has been used in the rotary die process as a shell‐forming material of soft capsules because of its unique physicochemical properties. However, with respect to the encapsulation of comparatively hydrophilic lipid‐based formulations, gelatin has one considerable drawback: Immediately after production, the capsule shell contains a large amount of water (up to 35%). There is the potential for water to migrate from the capsule shell into the formulation, which will lead to a decrease in drug solubility and, in turn, the potential for drug crystallization. The present study introduces a novel capsule material that was obtained from extrusion. The starch‐based polyvinyl alcohol thermoplastic capsules (S‐PVA‐C) mainly comprised a blend of starch and PVA. Gelatin and the novel material were used to encapsulate a hydrophilic lipid‐based system of fenofibrate. Considerable water migration was observed from the soft gelatin shell to the hydrophilic formulation during drying and drug crystallization resulted in soft gelatin capsules. In contrast, S‐PVA‐C displayed no substantial water exchange or drug crystallization upon storage. The thermoplastic capsule material further exhibited more surface roughness and higher resistance to mechanical deformation compared with gelatin. In conclusion, S‐PVA‐C provided a robust drug product following encapsulation of a rather hydrophilic lipid‐based formulation.     

Pharmacodynamics,pharmacokinetics and safety of GSK2190915, a novel oral anti‐inflammatory 5‐lipoxygenase‐activating protein inhibitor

Aim

To assess the pharmacokinetics, pharmacodynamics, safety and tolerability of the 5‐lipoxygenase‐activating protein inhibitor, GSK2190915, after oral dosing in two independent phase I studies, one in Western European and one in Japanese subjects, utilizing different formulations.

Method

Western European subjects received single (50–1000 mg) or multiple (10–450 mg) oral doses of GSK2190915 or placebo in a dose‐escalating manner. Japanese subjects received three of four GSK2190915 doses (10–200 mg) plus placebo once in a four period crossover design. Blood samples were collected for GSK2190915 concentrations and blood and urine were collected to measure leukotriene B₄ and leukotriene E₄, respectively, as pharmacodynamic markers of drug activity.

Results

There was no clear difference in adverse events between placebo and active drug‐treated subjects in either study. Maximum plasma concentrations of GSK2190915 and area under the curve increased in a dose‐related manner and mean half‐life values ranged from 16–34 h. Dose‐dependent inhibition of blood leukotriene B₄ production was observed and near complete inhibition of urinary leukotriene E₄ excretion was shown at all doses except the lowest dose. The EC₅₀ values for inhibition of LTB₄ were 85 nm and 89 nm in the Western European and Japanese studies, respectively.

Conclusion

GSK2190915 is well‐tolerated with pharmacokinetics and pharmacodynamics in Western European and Japanese subjects that support once daily dosing for 24 h inhibition of leukotrienes. Doses of ≥50 mg show near complete inhibition of urinary leukotriene E₄ at 24 h post‐dose, whereas doses of ≥150 mg are required for 24 h inhibition of blood LTB₄.     

Bcl‐2 phosphorylation confers resistance on chronic lymphocytic leukaemia cells to the BH3 mimetics ABT‐737, ABT‐263 and ABT‐199 by impeding direct binding

Background and Purpose

Although the ongoing clinical trials of ABT‐263 and ABT‐199 in chronic lymphocytic leukaemia (CLL) have indicated that BH3 mimetics hold considerable promise, understanding the mechanism of CLL resistance to BH3 mimetics remains a challenge.

Experimental Approach

The LD₅₀ values of ABT‐737, ABT‐263 and ABT‐199 in a number of primary CLL cells from 40 patients, were determined. The levels of Bcl‐2 family proteins, including phosphorylated Bcl‐2 (pBcl‐2) and their interactions were measured by immunoblotting and co‐immunoprecipitation. In vitro binding assays were performed by isothermal titration calorimetry and ELISA. BH3 profiling in isolated mitochondria was analysed.

Key Results

The ratio of (Mcl‐1 + pBcl‐2) to Bcl‐2 expression provided the most significant predictive marker for the cytotoxic potential of ABT‐737, ABT‐263 and ABT‐199 in the panel of CLL samples. Mechanistically, pBcl‐2 inhibited the effects of the ABT compounds on the displacement of Bax and Bim from Bcl‐2, thereby suppressing mitochondrial apoptosis. The ABT compounds exhibited 100–300‐fold lower binding affinity to the glutamic acid, phosphomimetic, mutant of Bcl‐2 (T69E, S70E and S87E; EEE‐Bcl‐2). BH3 peptides exhibited different rank orders of binding affinities to full‐length WT‐Bcl‐2 and full‐length EEE‐Bcl‐2.

Conclusions and Implications

Our study suggested that a structural alteration in the BH3‐binding groove was induced by phosphorylation of Bcl‐2. Our data also provided a framework to overcome resistance of CLL cells to the ABT compounds by combining pBcl‐2 kinase inhibitors with the ABT compounds.

Abbreviations

CLL

chronic lymphocytic leukaemia

ITC

isothermal titration calorimetry

MOMP

mitochondrial outer membrane permeabilization