Potential of ileal bile acid transporter inhibition as a therapeutic target in Alagille syndrome and progressive familial intrahepatic cholestasis.

Alagille syndrome (ALGS) and progressive familial intrahepatic cholestasis (PFIC) are rare, inherited cholestatic liver disorders that manifest in infants and children and are associated with impaired bile flow (ie cholestasis), pruritus and potentially fatal liver disease. There are no effective or approved pharmacologic treatments for these diseases (standard medical treatments are supportive only), and new, noninvasive options would be valuable. Typically, bile acids undergo biliary secretion and intestinal reabsorption (ie enterohepatic circulation). However, in these diseases, disrupted secretion of bile acids leads to their accumulation in the liver, which is thought to underlie pruritus and liver-damaging inflammation. One approach to reducing pathologic bile acid accumulation in the body is surgical biliary diversion, which interrupts the enterohepatic circulation (eg by diverting bile acids to an external stoma). These procedures can normalize serum bile acids, reduce pruritus and liver injury and improve quality of life. A novel, nonsurgical approach to interrupting the enterohepatic circulation is inhibition of the ileal bile acid transporter (IBAT), a key molecule in the enterohepatic circulation that reabsorbs bile acids from the intestine. IBAT inhibition has been shown to reduce serum bile acids and pruritus in trials of paediatric cholestatic liver diseases. This review explores the rationale of inhibition of the IBAT as a therapeutic target, describes IBAT inhibitors in development and summarizes the current data on interrupting the enterohepatic circulation as treatment for cholestatic liver diseases including ALGS and PFIC.

Treatment of Clostridium difficile Infection with a Small-Molecule Inhibitor of Toxin UDP-Glucose Hydrolysis Activity.

Clostridium difficile infection (CDI) is the leading cause of hospital-acquired infectious diarrhea, with significant morbidity, mortality, and associated health care costs. The major risk factor for CDI is antimicrobial therapy, which disrupts the normal gut microbiota and allows C. difficile to flourish. Treatment of CDI with antimicrobials is generally effective in the short term, but recurrent infections are frequent and problematic, indicating that improved treatment options are necessary. Symptoms of disease are largely due to two homologous toxins, TcdA and TcdB, which are glucosyltransferases that inhibit host Rho GTPases. As the normal gut microbiota is an important component of resistance to CDI, our goal was to develop an effective nonantimicrobial therapy. Here, we report a highly potent small-molecule inhibitor (VB-82252) of TcdA and TcdB. This compound inhibits the UDP-glucose hydrolysis activity of TcdB and protects cells from intoxication after challenge with either toxin. Oral dosing of the inhibitor prevented inflammation in a murine intrarectal toxin challenge model. In a murine model of recurrent CDI, the inhibitor reduced weight loss and gut inflammation during acute disease and did not cause the recurrent disease that was observed with vancomycin treatment. Lastly, the inhibitor demonstrated efficacy similar to that of vancomycin in a hamster disease model. Overall, these results demonstrate that small-molecule inhibition of C. difficile toxin UDP-glucose hydrolysis activity is a promising nonantimicrobial approach to the treatment of CDI.

Identification and initial optimization of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

The discovery, synthesis and preliminary structure-activity relationship (SAR) of a novel class of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB) is described. A high throughput screening (HTS) campaign resulted in the identification of moderately active screening hits 1-5 the most potent of which was compound 1 (IC50 = 0.77 µM). In silico docking of an early analog offered suggestions for structural modification which resulted in the design and synthesis of highly potent analogs 13j(IC50 = 1 nM) and 13 l(IC50 = 7 nM) which were chosen as leads for further optimization.

Synthesis and SAR studies of novel benzodiazepinedione-based inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

Synthesis and structure-activity relationships (SAR) of a novel series of benzodiazepinedione-based inhibitors of Clostridium difficile toxin B (TcdB) are described. Compounds demonstrating low nanomolar affinity for TcdB, and which possess improved stability in mouse plasma vs. earlier compounds from this series, have been identified. Optimized compound 11d demonstrates a good pharmacokinetic (PK) profile in mouse and hamster and is efficacious in a hamster survival model of Clostridium difficile infection.

NASPGHAN Clinical Report on Postoperative Recurrence in Pediatric Crohn Disease.

Pediatric Crohn disease is characterized by clinical and endoscopic relapses. The inflammatory process is considered to be progressive and may lead to strictures, fistulas, and penetrating disease that may require surgery. In addition, medically refractory disease may be treated by surgical resection of inflamed bowel in an effort to reverse growth failure. The need for surgery in childhood suggests severe disease and these patients have an increased risk for recurrent disease and potentially more surgery. Data show that up to 55% of patients had clinical recurrence in the first 2 years after initial surgery. The current clinical report on postoperative recurrence in pediatric Crohn disease reviews the risk factors for early surgery and postoperative recurrence, operative risk factors for recurrence, and prevention and monitoring strategies for postoperative recurrence. We also propose an algorithm for postoperative management in pediatric Crohn disease.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium.

Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Synthesis and SAR of potent and selective tetrahydropyrazinoisoquinolinone 5-HT(2C) receptor agonists.

The 5-HT2C receptor has been implicated as a critical regulator of appetite. Small molecule activation of the 5-HT2C receptor has been shown to affect food intake and regulate body weight gain in rodent models and more recently in human clinical trials. Therefore, 5-HT2C is a well validated target for anti-obesity therapy. The synthesis and structure-activity relationships of a series of novel tetrahydropyrazinoisoquinolinone 5-HT2C receptor agonists are presented. Several members of this series were identified as potent 5-HT2C receptor agonists with high functional selectivity against the 5-HT2A and 5-HT2B receptors and reduced food intake in an acute rat feeding model upon oral dosing.

Genome-wide association study of N370S homozygous Gaucher disease reveals the candidacy of CLN8 gene as a genetic modifier contributing to extreme phenotypic variation.

Mutations in GBA1 gene result in defective acid ß-glucosidase and the complex phenotype of Gaucher disease (GD) related to the accumulation of glucosylceramide-laden macrophages. The phenotype is highly variable even among patients harboring identical GBA1 mutations. We hypothesize that modifier gene(s) underlie phenotypic diversity in GD and performed a GWAS study in Ashkenazi Jewish patients with type 1 GD (GD1), homozygous for N370S mutation. Patients were assigned to mild, moderate, or severe disease categories using composite disease severity scoring systems. Whole-genome genotyping for >500,000 SNPs was performed to search for association signals using OQLS algorithm in 139 eligible patients. Several SNPs in linkage disequilibrium within the CLN8 gene locus were associated with the GD1 severity: SNP rs11986414 was associated with GD1 severity at P value 1.26 × 10(-6) . Compared to mild disease, risk allele A at rs11986414 conferred an odds ratio of 3.72 for moderate/severe disease. Loss of function mutations in CLN8 causes neuronal ceroid-lipofuscinosis, but our results indicate that its increased expression may protect against severe GD1. In cultured skin fibroblasts, the relative expression of CLN8 was higher in mild GD compared to severely affected patients, in whom CLN8 risk alleles were overrepresented. In an in vitro cell model of GD, CLN8 expression was increased, which was further enhanced in the presence of bioactive substrate, glucosylsphingosine. Taken together, CLN8 is a candidate modifier gene for GD1 that may function as a protective sphingolipid sensor and/or in glycosphingolipid trafficking. Future studies should explore the role of CLN8 in pathophysiology of GD.

Evaluation of high density lipoprotein as a circulating biomarker of Gaucher disease activity.

Circulating biomarkers are important surrogates for monitoring disease activity in type I Gaucher disease (GD1). We and others have reported low high-density lipoprotein (HDL) in GD1. We assessed HDL cholesterol as a biomarker of GD1, with respect to its correlation with indicators of disease severity and its response to imiglucerase enzyme replacement therapy (ERT). In 278 consecutively evaluated GD1 patients, we correlated HDL cholesterol, chitotriosidase, and angiotensin-converting enzyme (ACE) with indicators of disease severity. Additionally, we measured the response of these biomarkers to ERT. HDL cholesterol was negatively correlated with spleen volume, liver volume, and GD severity score index; the magnitude of this association of disease severity with HDL cholesterol was similar to that for ACE and for chitotriosidase. Within individual patients monitored over many years, there was a strikingly strong correlation of HDL with liver and spleen volumes; there was a similarly strong correlation of chitotriosidase and ACE with disease severity in individual patients monitored serially over many years (chitotriosidase r = 0.96 to 0.98, ACE r = 0.88 to 0.94, and HDL r = -0.84 to -0.94, p < 0.001). ERT for 3 years resulted in a striking increase of HDL while serum levels of chitotriosidase and ACE decreased. Our results reveal markedly low HDL cholesterol in untreated GD1, a correlation with indicators of disease severity in GD1, and a rise towards normal after ERT. These findings suggest HDL cholesterol merits inclusion within the "biomarker basket" for monitoring of patients with GD1.

Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors.

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide-valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC(50) of 7 nM and EC(2×PT) of 1.7 µM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide-valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.

Differences in efficacy of monepantel, derquantel and abamectin against multi-resistant nematodes of sheep.

Drug resistance has become a global phenomenon in gastrointestinal nematodes of sheep, particularly resistance to macrocyclic lactones. New anthelmintics are urgently needed for both the control of infections with multi-resistant nematodes in areas where classical anthelmintics are no longer effective, and the prevention of the spread of resistance in areas where the problem is not as severe. Recently, two new active ingredients became commercially available for the treatment of nematode infections in sheep, monepantel (Zolvix®) and derquantel, the latter used only in a formulated combination with the macrocyclic lactone, abamectin (Startect®). In order to assess the potential of the new actives for the control and prevention of spread of anthelmintic resistance, two characterized multi-resistant field isolates from Australia were used in a GLP (good laboratory practice) conducted efficacy study in sheep. Eight infected sheep in each group were treated orally according to the product labels with 2.5 mg/kg body weight monepantel, 0.2 mg/kg abamectin, or with the combination of 2.0 mg/kg derquantel and 0.2 mg/kg abamectin. The results demonstrate that monepantel was fully effective against multi-resistant species, Trichostrongylus colubriformis and Haemonchus contortus (99.9%). In contrast, the combination of derquantel and abamectin was effective against T. colubriformis (99.9%), but was not effective against larval stages of the barber's pole worm H. contortus (18.3%).

Focal splenic lesions in type I Gaucher disease are associated with poor platelet and splenic response to macrophage-targeted enzyme replacement therapy.

Focal splenic lesions (FSL) occur in Gaucher disease type I (GD1), but their clinical significance is not known. Previous studies estimated the prevalence of FSL at 4% (pediatric) to 33% (adult) of GD1 patients and reported an association with splenomegaly. We tested the hypothesis that the presence of FSL is associated with suboptimal response to macrophage-directed enzyme replacement therapy (ERT). Additionally we investigated whether FSL were associated with other phenotypic features of GD1. The splenic parenchyma was assessed by MRI performed for routine evaluation of GD1 in 239 consecutive GD1 patients with intact spleens. The prevalence of FSL was 18.4% (44/239). Following a mean of 3.5 years of ERT, platelet response was inferior among patients with FSL (80,700 ± 9,600 to 90,100 ± 7,200/mm(3) , P = 0.2) compared to patients without FSL in whom there was a robust platelet response: 108,600 ± 5,670 to 150,200 ± 6,710/mm(3), P < 0.001. Compared to patients without FSL, patients harboring FSL had worse thrombocytopenia (platelet count: 83,700 ± 8,800 vs. 112,100 ± 4,200/mm(3), P = 0.004), greater frequency of pre-ERT splenomegaly, and greater post-ERT splenomegaly (8.5 ± 0.77 vs. 4.8 ± 0.25× normal, P < 0.001). Additionally, the prevalence of osteonecrosis was higher among patients with FSL compared to patients without FSL (38 vs. 20.7%, P = 0.026). FSL appear to be a determinant of response to ERT, suggesting studies comparing relative efficacy of newly emerging therapies for GD1 should adjust for this factor. Moreover, occurrences of FSL coincide with more severe manifestations of GD1 such as avascular osteonecrosis.

Hyperferritinemia and iron overload in type 1 Gaucher disease.

Hyperferritinemia occurs in Gaucher disease but its clinical spectrum or its association with systemic iron overload and HFE mutations are not known. In 114 patients with Type 1 Gaucher disease, we determined serum ferritin, transferrin saturation and HFE genotype. The results were correlated with the extent of hepatosplenomegaly, overall Gaucher disease severity score index, and response to enzyme replacement therapy. In a subset of patients with radiological and/or laboratory evidence of systemic iron overload, liver biopsy was performed. There was a mean 3.7-fold elevation of serum ferritin over the upper limit of normal (ULN). Prior splenectomy was associated with most severe hyperferritinemia compared to patients with intact spleen (6.53 x ULN vs. 2.69 x ULN, P = 0.003). HFE genotyping revealed two patients homozygous for H63D mutation and 30% of patients heterozygote carriers of H63D mutation; no patients harbored C282Y mutation; there was no correlation of ferritin with HFE genotype. Ferritin level correlated with liver volume (Pearson correlation coefficient = 0.254, P = 0.035) and it was negatively correlated with hemoglobin (r = -0.315, P = 0.004); there was no relationship with other indicators of Gaucher disease activity. Enzyme replacement therapy (ERT) resulted in amelioration of hyperferritinemia: 707 +/- 898 ng/ml vs. 301 +/- 310 ng/ml (P = 0.001), transferrin saturation remained normal. Three patients were suspected of clinical iron overload, confirmed on liver biopsy. Iron accumulation was variably noted in hepatocytes and Kupffer cells. There is a high prevalence of hyperferritinemia in Type 1 Gaucher disease that is associated with indicators of disease severity, reversed by ERT and is not related to HFE mutations.

Expanding spectrum of the association between Type 1 Gaucher disease and cancers: a series of patients with up to 3 sequential cancers of multiple types--correlation with genotype and phenotype.

In Gaucher disease (GD), inherited deficiency of lysosomal glucocerebrosidase due to mutations in GBA1 gene results in accumulation of glucosylceramide in tissue macrophages, systemic macrophage activation, and a complex multisystemic phenotype. We and others have reported an increased risk of multiple myeloma and other malignancies in non-neuronopathic Type 1 GD (GD1). Here, we describe a subset of GD1 patients with multiple malignancies. In our cohort of 403 patients with GD1, nine patients (2.2%) developed two or three different types of cancers either consecutively or simultaneously. Patients were characterized by age at diagnosis of GD1, GBA1 genotype, disease severity, age at cancer diagnosis, enzyme replacement therapy (ERT) status, and splenectomy status. Of the nine patients, six developed two types of malignancies and three had three cancers each. Overall, the hematologic malignancies comprised lymphoma/leukemia (4) and multiple myeloma (4). Nonhematologic malignancies included colon (2), lung (2), thyroid (2), and prostate cancer (1). Of the seven patients who received ERT, the first cancer was diagnosed before initiation of ERT in all but one. Asplenic patients were more likely to have single or multiple cancers compared with patients with intact spleens (P < 0.0072 and P < 0.0203, respectively). Our data strengthen the association of GD1 and cancer and suggest that patients may be at risk of developing multiple malignancies. We found an association between splenectomy and multiple cancers in GD1. It will be of interest to determine whether timely ERT and declining rates of splenectomy will translate into declining rates of multiple and single cancers.

Ileal bile acid transporter inhibition as an anticholestatic therapeutic target in biliary atresia and other cholestatic disorders.

Biliary atresia is a rare cholestatic liver disease that presents in infants and rapidly advances to death in the absence of intervention. As a result of blockage or destruction of the biliary tract, bile acids accumulate and drive inflammation, fibrosis, and disease progression. The standard of care, Kasai portoenterostomy (KPE), is typically performed shortly after diagnosis (currently at ~ 2 months of age) and aims to restore bile flow and relieve cholestasis. Nevertheless, most patients continue to experience liver injury from accumulation of bile acids after KPE, since there are no known effective therapeutics that may enhance survival after KPE. Improving cholestasis via interruption of the enterohepatic circulation of bile acids may directly attenuate hepatic bile acid retention and reduce the risk of early organ failure. Directly addressing intrahepatic accretion of bile acids to avoid inherent bile acid toxicities provides an attractive and plausible therapeutic target for biliary atresia. This review explores the novel therapeutic concept of inhibiting the sole ileal bile acid transporter (IBAT), also known as ASBT (apical sodium-bile acid transporter, encoded by SLC10A2), as a means to reduce hepatic bile acid concentration after KPE. By reducing return of bile acids to the cholestatic liver, IBAT inhibitors may potentially lessen or delay liver damage associated with the hepatotoxicity and cholangiopathy of bile acid accumulation. The clinical programs of 2 IBAT inhibitors in development for the treatment of pediatric cholestatic liver diseases, maralixibat and odevixibat, are highlighted.

Aroylguanidine-based factor Xa inhibitors: the discovery of BMS-344577.

We report the design and synthesis of a novel class of N,N'-disubstituted aroylguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The structure-activity relationships (SAR) investigation led to the discovery of the nicotinoyl guanidine 22 as a potent FXa inhibitor (FXa IC(50)=4 nM, EC(2xPT)=7 microM). However, the potent CYP3A4 inhibition activity (IC(50)=0.3 microM) of 22 precluded its further development. Detailed analysis of the X-ray crystal structure of compound 22 bound to FXa indicated that the substituent at the 6-position of the nicotinoyl group of 22 would be solvent-exposed, suggesting that efforts to attenuate the unwanted CYP activity could focus at this position without affecting FXa potency significantly. Further SAR studies on the 6-substituted nicotinoyl guanidines resulted in the discovery of 6-(dimethylcarbamoyl) nicotinoyl guanidine 36 (BMS-344577, IC(50)=9 nM, EC(2xPT)=2.5 microM), which was found to be a selective, orally efficacious FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models.

Cyanoguanidine-based lactam derivatives as a novel class of orally bioavailable factor Xa inhibitors.

The N,N'-disubstituted cyanoguanidine is an excellent bioisostere of the thiourea and ketene aminal functional groups. We report the design and synthesis of a novel class of cyanoguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The SAR studies led to the discovery of compound 4 (BMS-269223, K(i)=6.5nM, EC(2xPT)=32muM) as a selective, orally bioavailable FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models. The X-ray crystal structure of 4 bound in FXa is presented and key ligand-protein interactions are discussed.

Efficacy of the amino-acetonitrile derivative, monepantel, against experimental and natural adult stage gastro-intestinal nematode infections in sheep.

Multiple drug resistance by nematodes, against anthelmintics has become an important economic problem in sheep farming worldwide. Here we describe the efficacy of monepantel, a developmental molecule from the recently discovered anthelmintic class, the amino-acetonitrile derivatives (AADs). Efficacy was tested against adult stage gastro-intestinal nematodes (GINs) in experimentally and naturally infected sheep at a dose of 2.5mg/kg body weight when administered as an oral solution. Some of the isolates used in experimental infection studies were known to be resistant to the benzimidazoles or levamisole anthelmintics; strains resistant to the macrocyclic lactones were not available for these tests. Worm count-based efficacies of >98% were determined in these studies. As an exception, Oesophagostomum venulosum was only reduced by 88% in one study, albeit with a low worm burden in the untreated controls (geometric mean 15.4 worms). Similar efficacies for monepantel were also confirmed in naturally infected sheep. While the efficacy against most species was >99%, the least susceptible species was identified as Nematodirus spathiger, and although efficacy was 92.4% in one study it was generally >99%. Several animals were infected with Trichuris ovis, which was not eliminated after the treatment. Monepantel demonstrated high activity against a broad range of the important GINs of sheep, which makes this molecule an interesting candidate for use in this species, particularly in regions with problems of anthelmintic resistance. Monepantel was well tolerated by the treated sheep, with no treatment related adverse events documented.

The Guinea pig as a preclinical model for demonstrating the efficacy and safety of statins.

Statins, because of their excellent efficacy and manageable safety profile, represent a key component in the current armamentarium for the treatment of hypercholesterolemia. Nonetheless, myopathy remains a safety concern for this important drug class. Cerivastatin was withdrawn from the market for myotoxicity safety concerns. BMS-423526 [{(3R,5S)-7-[4-(4-fluorophenyl)-6,7-dihydro-2-(1-methylethyl)-5H-benzo[6,7]cyclohepta[1,2-b]pyridin-3-yl]-3,5-dihydroxy-heptenoic acid} sodium salt], similar to cerivastatin in potency and lipophilicity, was terminated in early clinical development due to an unacceptable myotoxicity profile. In this report, we describe the guinea pig as a model of statin-induced cholesterol lowering and myotoxicity and show that this model can distinguish statins with unacceptable myotoxicity profiles from statins with acceptable safety profiles. In our guinea pig model, both cerivastatin and BMS-423526 induced myotoxicity at doses near the ED(50) for total cholesterol (TC) lowering in plasma. In contrast, wide differences between myotoxic and TC-lowering doses were established for the currently marketed, more hydrophilic statins, pravastatin, rosuvastatin, and atorvastatin. This in vivo model compared favorably to an in vitro model, which used statin inhibition of cholesterol synthesis in rat hepatocytes and L6 myoblasts as surrogates of potential efficacy and toxicity, respectively. Our conclusion is that the guinea pig is a useful preclinical in vivo model for demonstrating whether a statin is likely to have an acceptable therapeutic safety margin.