A3907, a systemic ASBT inhibitor, improves cholestasis in mice by multiorgan activity and shows translational relevance to humans.

BACKGROUND AND AIMS: Cholestasis is characterized by intrahepatic accumulation of bile constituents, including bile acids (BAs), which promote liver damage. The apical sodium-dependent BA transporter (ASBT) plays an important role in BA reabsorption and signaling in ileum, bile ducts, and kidneys. Our aim was to investigate the pharmacokinetics and pharmacological activity of A3907, an oral and systemically available ASBT inhibitor in experimental mouse models of cholestasis. In addition, the tolerability, pharmacokinetics, and pharmacodynamics of A3907 were examined in healthy humans. APPROACH AND RESULTS: A3907 was a potent and selective ASBT inhibitor in vitro. In rodents, orally administered A3907 distributed to the ASBT-expressing organs, that is, ileum, liver, and kidneys, and dose dependently increased fecal BA excretion. A3907 improved biochemical, histological, and molecular markers of liver and bile duct injury in Mdr2-/- mice and also had direct protective effects on rat cholangiocytes exposed to cytotoxic BA concentrations in vitro . In bile duct ligated mice, A3907 increased urinary BA elimination, reduced serum BA levels, and prevented body weight loss, while improving markers of liver injury. A3907 was well tolerated and demonstrated target engagement in healthy volunteers. Plasma exposure of A3907 in humans was within the range of systemic concentrations that achieved therapeutic efficacy in mouse. CONCLUSIONS: The systemic ASBT inhibitor A3907 improved experimental cholestatic disease by targeting ASBT function at the intestinal, liver, and kidney levels, resulting in marked clearance of circulating BAs and liver protection. A3907 is well tolerated in humans, supporting further clinical development for the treatment of cholestatic liver diseases.

Effects of odevixibat on pruritus and bile acids in children with cholestatic liver disease: Phase 2 study.

PURPOSE: Ileal bile acid transporter inhibition is a novel therapeutic concept for cholestatic pruritus and cholestatic liver disease progression. Odevixibat, a potent, selective, reversible ileal bile acid transporter inhibitor, decreases enteric bile acid reuptake with minimal systemic exposure. Oral odevixibat safety, tolerability, and efficacy in pediatric patients with cholestatic liver disease and pruritus were evaluated. PATIENTS AND METHODS: In this phase 2, open-label, multicenter study, children received 10‒200 µg/kg oral odevixibat daily for 4 weeks. Changes in serum bile acid levels (primary efficacy endpoint), pruritus, and sleep disturbance were explored. RESULTS: Twenty patients were enrolled (8 females; 1‒17 years; 4 re-entered at a different dose). Diagnoses included progressive familial intrahepatic cholestasis (n = 13; 3 re-entries), Alagille syndrome (n = 6), biliary atresia (n = 3), and other intrahepatic cholestasis causes (n = 2; 1 re-entry). Mean baseline serum bile acid levels were high (235 µmol/L; range, 26‒564) and were reduced in the majority (-123.1 µmol/L; range, -394 to 14.5, reflecting reductions of up to 98%). Patient-reported diary data documented improved pruritus (3 scales) and sleep. With 100 µg/kg, mean (SEM) decrease was 2.8 (1.1) points for pruritus (visual analogue itch scale 0-10) and 2.9 (0.9) points for sleep disturbance (Patient-Oriented Scoring Atopic Dermatitis scale 0-10). Reduced pruritus correlated significantly with reduced serum bile acids (P ≤ 0.007). Significant correlations were also observed between autotaxin levels and pruritus. All patients completed the study. No serious adverse events were treatment related; most adverse events, including increased transaminases, were transient. CONCLUSIONS: Orally administered odevixibat was well tolerated, reduced serum bile acids, and improved pruritus and sleep disturbance in children with cholestatic diseases.

Systematic Review and Meta-analysis: Partial External Biliary Diversion in Progressive Familial Intrahepatic Cholestasis.

OBJECTIVES: We assessed available data on impact of partial external biliary diversion (PEBD) surgery on clinical outcomes in patients with progressive familial intrahepatic cholestasis (PFIC). METHODS: We performed a systematic literature review (PubMed) and meta-analysis to evaluate relationships between liver biochemistry parameters (serum bile acids, bilirubin, and alanine aminotransferase [ALT]) and early response (pruritus improvement) or long-term outcomes (need for liver transplant) in patients with PFIC who underwent PEBD. RESULTS: Searches identified 175 publications before September 2018; 16 met inclusion criteria. Receiver operating characteristic (ROC) analysis examined ability of liver biochemistry parameters to discriminate patients who demonstrated early and long-term response to PEBD from those who did not. Regarding pruritus improvement in 155 included patients in aggregate, 104 (67%) were responders, 14 (9%) had partial response, and 37 (24%) were nonresponders. In ROC analyses of individual patient data, post-PEBD serum concentration of bile acids, in particular, could discriminate responders from nonresponders for pruritus improvement (area under the curve, 0.99; P < 0.0001; n = 42); to a lesser extent, this was also true for bilirubin (0.87; P = 0.003; n = 31), whereas ALT could not discriminate responders from nonresponders for pruritus improvement (0.74; P = 0.06; n = 28). Reductions from pre-PEBD values in serum bile acid concentration (0.89; P = 0.0003; n = 32) and bilirubin (0.98; P = 0.002; n = 18) but not ALT (0.62; P = 0.46; n = 18) significantly discriminated decreased aggregate need for liver transplant. CONCLUSION: Changes in bile acids seem particularly useful in discriminating early and long-term post-PEBD outcomes and may be potential biomarkers of response to interruption of enterohepatic circulation in patients with PFIC.

Application of a Sulfoxonium Ylide Electrophile to Generate Cathepsin X-Selective Activity-Based Probes.

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.

Changes in vascular permeability in the spinal cord contribute to chemotherapy-induced neuropathic pain.

Chemotherapy-induced neuropathic pain is a dose-limiting side effect of many cancer therapies due to their propensity to accumulate in peripheral nerves, which is facilitated by the permeability of the blood-nerve barrier. Preclinically, the chemotherapy agent vincristine (VCR) activates endothelial cells in the murine peripheral nervous system and in doing so allows the infiltration of monocytes into nerve tissue where they orchestrate the development of VCR-induced nociceptive hypersensitivity. In this study we demonstrate that VCR also activates endothelial cells in the murine central nervous system, increases paracellular permeability and decreases trans endothelial resistance. In in vivo imaging studies in mice, VCR administration results in trafficking of inflammatory monocytes through the endothelium. Indeed, VCR treatment affects the integrity of the blood-spinal cord-barrier as indicated by Evans Blue extravasation, disrupts tight junction coupling and is accompanied by the presence of monocytes in the spinal cord. Such inflammatory monocytes (Iba-1+ CCR2+ Ly6C+ TMEM119- cells) that infiltrate the spinal cord also express the pro-nociceptive cysteine protease Cathepsin S. Systemic treatment with a CNS-penetrant, but not a peripherally-restricted, inhibitor of Cathepsin S prevents the development of VCR-induced hypersensitivity, suggesting that infiltrating monocytes play a functional role in sensitising spinal cord nociceptive neurons. Our findings guide us towards a better understanding of central mechanisms of pain associated with VCR treatment and thus pave the way for the development of innovative antinociceptive strategies.

Cathepsin S acts via protease-activated receptor 2 to activate sensory neurons and induce itch-like behaviour.

Chronic itch is a debilitating condition characterised by excessive scratching and is a symptom frequently reported in skin diseases such as atopic dermatitis. It has been proposed that release of the cysteine protease Cathepsin S (CatS) from skin keratinocytes or immune cells resident in or infiltrating the skin could act as a pruritogen in chronic itch conditions. CatS is known to activate protease-activated receptor 2 (PAR2). We therefore hypothesised that enzymatic activation of neuronally expressed PAR2 by CatS was responsible for activation of sensory neurons and transmission of itch signals. Intradermally-injected human recombinant (hr)-CatS or the PAR2 agonist, SLIGRL-NH2 behaved as pruritogens by causing scratching behaviour in mice. Hr-CatS-induced scratching behaviour was prevented by CatS inhibitors and PAR2 antagonists and reduced by 50% in TRPV1-/- mice compared with wild-type mice, whilst no significant reduction in scratching behaviour was observed in TRPA1-/- mice. Cultured dorsal root ganglion (DRG) cells showed an increase in [Ca2+]i following incubation with hr-CatS, and the percentage of neurons that responded to hr-CatS decreased in the presence of a PAR2 antagonist or in cultures of neurons from TRPV1-/- mice. Taken together, our results indicate CatS acts as a pruritogen via PAR2 activation in TRPV1-expressing sensory neurons.

Pharmacological modulation of colorectal distension evoked potentials in conscious rats.

BACKGROUND: Cerebral evoked potentials (CEP) induced by colorectal distension (CRD) in conscious rats provides a novel method in studies of visceral sensitivity. The aim of this study was to explore the pharmacological effect on CEP of compounds known to reduce the visceromotor response to CRD. METHODS: Epidural electrodes were chronically implanted in eight female Sprague-Dawley rats. Evoked potentials were elicited by colorectal rapid balloon distensions (100 ms, 80 mmHg) and the effect of WIN55 (cannabinoid CB receptor agonist), clonidine (adrenergic α2 receptor agonist), MPEP (mGluR5 receptor antagonist), pregabalin (ligand of α2δ subunits in voltage-gated calcium channels) and baclofen (GABA-B receptor agonist) on amplitudes and latency of CEP were determined. RESULTS: WIN55 (0.1 µmol kg-1), clonidine (0.05 µmol kg-1), MPEP (10 µmol kg-1) and pregabalin (200 µmol kg-1) caused a significant, p < 0.05, reduction of the N2 to P2 peak-to-peak amplitude by 23 ±â€¯8%, 25 ±â€¯8%, 39 ±â€¯5%, and 47 ±â€¯6% respectively. Baclofen (9 µmol kg-1) induced a prolongation of the N2 peak latency of 18 ±â€¯4% but had no significant effect on the amplitudes. CONCLUSION: The obtained results suggest that MPEP, WIN55, clonidine, and pregabalin reduce visceral nociceptive input to the brain, whereas the lack of effect of baclofen on CRD evoked CEP amplitudes suggest that the effect on VMR is not due to a direct analgesic effect. Brain responses to colorectal distension provide a useful tool to evaluate pharmacological effects in rats and may serve as a valuable preclinical model for understanding pharmacological mechanisms related to visceral sensitivity.

Nonclinical and clinical pharmacological characterization of the potent and selective cathepsin K inhibitor MIV-711.

BACKGROUND: Cathepsin K is an attractive therapeutic target for diseases in which bone resorption is excessive such as osteoporosis and osteoarthritis (OA). The current paper characterized the pharmacological profile of the potent and selective cathepsin K inhibitor, MIV-711, in vitro and in cynomolgus monkeys, and assessed translation to human based on a single dose clinical study in man. METHODS: The potency and selectivity of MIV-711 were assessed in vitro using recombinant enzyme assays and differentiated human osteoclasts. MIV-711 was administered to healthy cynomolgus monkeys (3-30 µmol/kg, p.o.). Plasma levels of MIV-711 and the bone resorption biomarker CTX-I were measured after single dose experiments, and urine levels of CTX-I, NTX-I and CTX-II biomarkers were measured after repeat dose experiments. The safety, pharmacokinetics and pharmacodynamics (serum CTX-I) of MIV-711 were assessed in human healthy subjects after single ascending doses from 20 to 600 mg. RESULTS: MIV-711 was a potent inhibitor of human cathepsin K (Ki: 0.98 nmol/L) with > 1300-fold selectivity towards other human cathepsins. MIV-711 inhibited human osteoclast-mediated bone resorption with an IC50 value of 43 nmol/L. Single oral doses of MIV-711 to monkeys reduced plasma levels of CTX-I in a dose-dependent fashion by up to 57% at trough. The effect on CTX-I was linearly correlated to the plasma exposure of MIV-711, while the efficacy duration outlasted plasma exposure. Repeat oral dosing with MIV-711 also reduced urinary levels of the bone resorption biomarkers CTX-I (by 93%) and NTX-I (by 71%) and the cartilage degradation biomarker CTX-II (by 71%). MIV-711 was safe and well-tolerated when given as single ascending doses to healthy subjects. MIV-711 reduced serum CTX-I levels in a dose-dependent manner by up to 79% at trough. The relationship between MIV-711 exposure and effects on these biomarkers in humans was virtually identical when compared to the corresponding monkey data. CONCLUSIONS: MIV-711 is a potent and selective cathepsin K inhibitor with dose-dependent effects on biomarkers of bone and cartilage degradation in monkey and human. Taken together, MIV-711 shows promise for the treatment of bone and cartilage related disorders in humans, such as OA. Trial Registration EudraCT number 2011-003024-12, registered on June 22nd 2011.

The selective cathepsin K inhibitor MIV-711 attenuates joint pathology in experimental animal models of osteoarthritis.

BACKGROUND: MIV-711 is a highly potent and selective cathepsin K inhibitor. The current article summarizes the therapeutic effects of MIV-711 on joint pathology in rabbits subjected to anterior cruciate ligament transection (ACLT), and the prophylactic effects on joint pathology in dogs subjected to partial medial meniscectomy, two surgical models of osteoarthritis (OA). METHODS: Starting 1 week after surgery, rabbits were dosed daily via oral gavage with either MIV-711 or vehicle (n = 7/group) for 7 weeks. The four treatment groups were: (1) sham + vehicle; (2) ACLT + vehicle; (3) ACLT + MIV-711, 30 µmol/kg and (4) ACLT + MIV-711, 100 µmol/kg. Subchondral bone and articular cartilage structures were assessed by µCT, histomorphometry, and scoring. Dogs subjected to partial medial meniscectomy received either MIV-711 (30 µmol/kg) or vehicle (n = 15/group) via oral gavage once daily, starting 1 day before meniscectomy, for 28 days. Cartilage degradation was assessed at the macroscopic and microscopic levels. The exposures of MIV-711 were assessed in both studies and biomarkers reflecting bone resorption (HP-1 in rabbits, CTX-I in dogs) and cartilage degradation (CTX-II) were measured. RESULTS: In ACLT rabbits, MIV-711 decreased HP-1 levels by up to 72% (p < 0.001) and CTX-II levels by up to 74% (p < 0.001) compared to ACLT vehicle controls. ACLT surgery significantly reduced the total thickness of the subchondral bone plate and reduced trabecular bone volume in the femur and tibia. These effects were reversed by MIV-711. ACLT resulted in cartilage thickening, which was attenuated by MIV-711. MIV-711 did not affect osteophyte formation or Mankin scores. In dogs, MIV-711 reduced CTX-I and CTX-II levels by 86% (p < 0.001) and 80% (p < 0.001), respectively. Synovial CTX-II levels were reduced by 55-57% (p < 0.001) compared to baseline. MIV-711-treated animals had 25-37% lower macroscopic scores in the femur condyles and 13-33% lower macroscopic scores in the tibial plateaus. CONCLUSIONS: MIV-711 prevents subchondral bone loss and partially attenuates cartilage pathology in two animal models of OA. These beneficial effects of MIV-711 on joint pathology are observed in conjunction with decreases in bone and cartilage biomarkers that have been shown to be clinically attainable in human. The data support the further development of MIV-711 for the treatment of OA.

Analgesic effects of the cathepsin K inhibitor L-006235 in the monosodium iodoacetate model of osteoarthritis pain.

INTRODUCTION: The mounting evidence that osteoclasts play an important role in osteoarthritis (OA) pain lead us to investigate the effects of L-006235, a potent and selective inhibitor of cathepsin K, on pain behaviour and joint pathology in a model of OA pain. METHODS: Effects of preventative (30 and 100 mg/kg) and therapeutic (100 mg/kg) oral dosing with L-006235 on weight-bearing asymmetry, hind paw withdrawal thresholds, cartilage and bone pathology, synovial inflammation, and drug exposure were studied in the monosodium iodoacetate rat model of OA pain. RESULTS: Preventative L-006235 inhibited weight-bearing asymmetry from day 14, with this measure nearly abolished by the higher dose. In the same treatment setting, L-006235 prevented lowering of hind paw withdrawal thresholds from day 7. Exposure to L-006235 in plasma was higher for the 100 mg/kg dose, compared with 30 mg/kg. Therapeutic dosing with L-006235 from day 14 significantly inhibited weight-bearing asymmetry, compared with monosodium iodoacetate vehicle rats. Regression analysis revealed a significant interaction coefficient of the effects of L-006235 on weight-bearing asymmetry and synovitis score, but not for cartilage damage nor osteophyte scores. CONCLUSION: Our novel finding that cathepsin K inhibition is analgesic in a clinically relevant model of OA pain provides new evidence for the therapeutic potential of this target.

Selective Cathepsin S Inhibition with MIV-247 Attenuates Mechanical Allodynia and Enhances the Antiallodynic Effects of Gabapentin and Pregabalin in a Mouse Model of Neuropathic Pain.

Cathepsin S inhibitors attenuate mechanical allodynia in preclinical neuropathic pain models. The current study evaluated the effects when combining the selective cathepsin S inhibitor MIV-247 with gabapentin or pregabalin in a mouse model of neuropathic pain. Mice were rendered neuropathic by partial sciatic nerve ligation. MIV-247, gabapentin, or pregabalin were administered alone or in combination via oral gavage. Mechanical allodynia was assessed using von Frey hairs. Neurobehavioral side effects were evaluated by assessing beam walking. MIV-247, gabapentin, and pregabalin concentrations in various tissues were measured. Oral administration of MIV-247 (100-200 µmol/kg) dose-dependently attenuated mechanical allodynia by up to approximately 50% reversal when given as a single dose or when given twice daily for 5 days. No behavioral deficits were observed at any dose of MIV-247 tested. Gabapentin (58-350 µmol/kg) and pregabalin (63-377 µmol/kg) also inhibited mechanical allodynia with virtually complete reversal at the highest doses tested. The minimum effective dose of MIV-247 (100 µmol/kg) in combination with the minimum effective dose of pregabalin (75 µmol/kg) or gabapentin (146 µmol/kg) resulted in enhanced antiallodynic efficacy without augmenting side effects. A subeffective dose of MIV-247 (50 µmol/kg) in combination with a subeffective dose of pregabalin (38 µmol/kg) or gabapentin (73 µmol/kg) also resulted in substantial efficacy. Plasma levels of MIV-247, gabapentin, and pregabalin were similar when given in combination as to when given alone. Cathepsin S inhibition with MIV-247 exerts significant antiallodynic efficacy alone, and also enhances the effect of gabapentin and pregabalin without increasing side effects or inducing pharmacokinetic interactions.

Cathepsin S causes inflammatory pain via biased agonism of PAR2 and TRPV4.

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR2) at R(36)↓S(37) and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR2 at E(56)↓T(57), which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR2 coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca(2+), activate ERK1/2, recruit ß-arrestins, or induce PAR2 endocytosis. Cat-S caused PAR2-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR2 in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR2 that causes PAR2- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain.

Design and synthesis of potent hydroxyethylamine (HEA) BACE-1 inhibitors carrying prime side 4,5,6,7-tetrahydrobenzazole and 4,5,6,7-tetrahydropyridinoazole templates.

A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC(50) values of 44 nM (enzyme assay) and 65 nM (cell-based assay).

Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S.

Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.

Aliskiren displays long-lasting interactions with human renin.

Aliskiren is a selective renin inhibitor recently approved for use in hypertension. Efficacy duration appears longer than what would be expected based on its circulating half-life. The aim was therefore to characterize the kinetics of the interaction between aliskiren and renin. The interaction was evaluated in three assays and compared with two other renin inhibitors including remikiren. First, the inhibition of recombinant human renin was assessed by monitoring the cleavage of fluorescent substrate. Second, human plasma renin activity (PRA) was monitored by measuring generated angiotensin I over 1 h in the presence or absence of inhibitor. Finally, the affinity, association and dissociation rate constants were determined by using a surface plasmon resonance (SPR) biosensor assay. Aliskiren and remikiren were found to be equipotent inhibitors of recombinant renin activity (K(i) ≤ 0.04 nM) while compound 1 displayed a K (i) value of 1 nM. PRA was efficiently inhibited by both aliskiren and remikiren with IC50 values of 0.2-0.3 nM. Remikiren and aliskiren also displayed long-lasting interactions with immobilized renin having k (off) values of 0.18 and 0.11 × 10⁻³ s⁻¹ respectively. These dissociation rate constants corresponded to residence times of 1.5 and 2.5 h, respectively, while compound 1 had a residence time lasting only 3 min. It is therefore concluded that the long-lasting interaction between aliskiren and human renin may contribute to the 24 h anti-hypertensive effect seen in clinical trials and possibly also to target-mediated drug disposition.

BACE-1 inhibition prevents the γ-secretase inhibitor evoked Aß rise in human neuroblastoma SH-SY5Y cells.

BACKGROUND: Accumulation of amyloid ß-peptide (Aß) in the plaques is one of the major pathological features in Alzheimer's disease (AD). Sequential cleavage of amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE-1) and γ-secretase results in the formation of Aß peptides. Preventing Aß formation is believed to attenuate AD progression and BACE-1 and γ-secretase are thus attractive targets for AD drug development. METHODS: Combining BACE-1 and γ-secretase inhibition on Aß secretion from human neuroblastoma SH-SY5Y cells was evaluated in this study. Secreted Aß40 and Aß42 levels were measured from SH-SY5Y cells stably transfected with APPwt or APPswe genes. A selective BACE inhibitor and the γ-secretase inhibitor LY450139 (semagacestat) were used to inhibit respective secretase. RESULTS: LY450139 increased Aß40 and Aß42 secretion from SH-SY5Y APPwt cells at low concentrations (by 60% at 3 nM) followed by subsequent inhibition at higher concentrations (IC(50) 90 nM). Washout studies showed that the Aß increase evoked by 3 nM LY450139 was not due to enhanced cleavage following substrate accumulation but rather to activation of Aß formation. By contrast, LY450139 inhibited Aß formation from SH-SY5Y APPswe in a monophasic manner (IC(50) 18 nM). The BACE inhibitor per se inhibited Aß secretion from both SH-SY5Y APPwt and SH-SY5Y APPswe cells with IC(50)s ranging between 7 - 18 nM and also prevented the increased Aß secretion evoked by 3 nM LY450139. Combining the BACE inhibitor with higher inhibitory concentrations of LY450139 failed to demonstrate any clear additive or synergistic effects. CONCLUSION: BACE-1 inhibition attenuates the Aß increase evoked by LY450139 while not providing any obvious synergistic effects on LY450139-mediated inhibition.

A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane.

A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (ß-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.

Oral baclofen reduces visceral pain-related pseudo-affective responses to colorectal distension in rats: relation between plasma exposure and efficacy.

OBJECTIVE: We previously showed that activation of GABA(B) receptors by intravenous baclofen reduces pseudo-affective responses to colorectal distension in rats. Here we evaluate the potential clinical significance of these observations. MATERIAL AND METHODS: Clinically relevant colorectal distension protocols were used to assess the effects of oral baclofen on visceromotor and autonomic cardiovascular responses in conscious rats. Plasma levels of baclofen were monitored to provide clinical relevance to the doses used. Conscious female Sprague-Dawley rats were subjected to repeated noxious colorectal distension (12 × 80 mmHg), ascending-phasic colorectal distension (10-80 mmHg, 10 mmHg increments) or ramp colorectal distension (10 min ramp at 8 mmHg/min). Visceromotor and cardiovascular responses (mean arterial blood pressure and heart rate) were monitored. Pain-related response thresholds were assessed using ascending-phasic and ramp colorectal distension. RESULTS: Baclofen (1-10 µmol/kg, p.o.) reduced the visceromotor response to colorectal distension, reaching a 40% maximal inhibition (p < 0.05). The highest dose (10 µmol/kg, p.o.) also inhibited pain-related cardiovascular responses in telemetrized rats (50-55% reduction in colorectal distension-evoked hypertensive and tachycardic responses; p < 0.05). Similar thresholds for pain-related visceromotor responses were determined during ramp or ascending-phasic colorectal distension (34.1 ± 1.9 and 31.7 ± 3.2 mmHg, respectively). Baclofen (10 µmol/kg, p.o.) increased thresholds to 71.1 ± 3.7 and 77.5 ± 1.8 mmHg during ramp and ascending-phasic colorectal distension, respectively (p < 0.001). Plasma levels of baclofen were 3.3 ± 0.2 µmol/l at 90 min post-dosing, corresponding to the end of the colorectal distension procedure. CONCLUSIONS: Oral baclofen, at plasma levels similar to those reported safe and within a therapeutic range in humans, produced significant visceral anti-nociceptive effects in rats.

Effect of the protonation state of the titratable residues on the inhibitor affinity to BACE-1.

BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimer's disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.