A Study to Evaluate Relative Bioavailability, Food Effect, and Pharmacodynamics of Tropifexor, a Farnesoid X Receptor Agonist, in Healthy Participants.

This open-label, randomized, 3-treatment, 3-period, 6-sequence, crossover study in healthy subjects compared the pharmacokinetic and pharmacodynamic properties of a lipid-based (soft gelatin capsule) prototype final market image (pFMI) formulation of tropifexor (90-µg) to its clinical service form (CSF) and assessed the food effect for the pFMI formulation. In the fasted state, drug exposure was higher for the pFMI. The geometric mean ratios for pFMI versus CSF of peak concentration and area under the concentration-time curve were 2.0 and 1.5, respectively. No food effect was apparent for the pFMI formulation, and the geometric mean ratios for pFMI fed versus pFMI fasted of peak concentration and area under concentration-time curve were 1.0 and 1.0 respectively. Despite having lower systemic exposure, the CSF formulation provided a higher pharmacological response for the gut biomarker fibroblast growth factor 19. Under fasted conditions, fibroblast growth factor 19 maximum change from baseline serum concentration after drug administration and area under the change from baseline serum concentration-time curve from time 0 to 24 hours were 36% for CSF and 12% for FMI. For a second biomarker, serum 7-alpha hydroxy-4-cholest-3-one, the pharmacological activity was comparable between CSF (fasted) and pFMI (both fasted and fed states). The pFMI offers advantages over the CSF in terms of insensitivity to food effect, lower intersubject variability, and overcoming solubility limitations.

Synergistic Strategies to Accelerate the Development of Function-Promoting Therapies: Lessons From Operation Warp Speed and Oncology Drug Development.

BACKGROUND: Functional limitations and physical disabilities associated with aging and chronic disease are major concerns for human societies and expeditious development of function-promoting therapies is a public health priority. METHODS: Expert panel discussion. RESULTS: The remarkable success of Operation Warp Speed for the rapid development of COVID-19 vaccines, COVID-19 therapeutics, and of oncology drug development programs over the past decade have taught us that complex public health problems such as the development of function-promoting therapies will require collaboration among many stakeholders, including academic investigators, the National Institutes of Health, professional societies, patients and patient advocacy organizations, the pharmaceutical and biotechnology industry, and the U.S. Food and Drug Administration. CONCLUSIONS: There was agreement that the success of well designed, adequately powered clinical trials will require careful definitions of indication/s, study population, and patient-important endpoints that can be reliably measured using validated instruments, commensurate resource allocation, and versatile organizational structures such as those used in Operation Warp Speed.

Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation.

BACKGROUND: Elastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases. METHODS: Elastin fragments were identified by mass-spectrometry and one sequence, generated by MMP-9 and -12 (ELN-441), was selected for monoclonal antibody generation and used in the development of an ELISA. Soluble and insoluble elastin from lung was cleaved in vitro and the time-dependent release of fragments was assessed in the ELN-441 assay. The release of ELN-441 in human serum from patients with chronic obstructive pulmonary disease (COPD) (n = 10) and idiopathic pulmonary fibrosis (IPF) (n = 29) were compared to healthy matched controls (n = 11). RESULTS: The sequence ELN-441 was exclusively generated by MMP-9 and -12 and was time-dependently released from soluble lung elastin. ELN-441 levels were 287% higher in patients diagnosed with COPD (p < 0.001) and 124% higher in IPF patients (p < 0.0001) compared with controls. ELN-441 had better diagnostic value in COPD patients (AUC 97%, p = 0.001) than in IPF patients (AUC 90%, p = 0.0001). The odds ratios for differentiating controls from COPD or IPF were 24 [2.06-280] for COPD and 50 [2.64-934] for IPF. CONCLUSIONS: MMP-9 and -12 time-dependently released the ELN-441 epitope from elastin. This fragment was elevated in serum from patients with the lung diseases IPF and COPD, however these data needs to be validated in larger clinical settings.

Farnesoid X receptor agonist tropifexor attenuates cholestasis in a randomised trial in patients with primary biliary cholangitis.

Background & Aims: The safety, tolerability, and efficacy of the non-bile acid farnesoid X receptor agonist tropifexor were evaluated in a phase II, double-blind, placebo-controlled study as potential second-line therapy for patients with primary biliary cholangitis (PBC) with an inadequate ursodeoxycholic acid response. Methods: Patients were randomised (2:1) to receive tropifexor (30, 60, 90, or 150 µg) or matched placebo orally once daily for 28 days, with follow-up on Days 56 and 84. Primary endpoints were safety and tolerability of tropifexor and reduction in levels of γ-glutamyl transferase (GGT) and other liver biomarkers. Other objectives included patient-reported outcome measures using the PBC-40 quality-of-life (QoL) and visual analogue scale scores and tropifexor pharmacokinetics. Results: Of 61 enrolled patients, 11, 9, 12, and 8 received 30-, 60-, 90-, and 150-µg tropifexor, respectively, and 21 received placebo; 3 patients discontinued treatment because of adverse events (AEs) in the 150-µg tropifexor group. Pruritus was the most frequent AE in the study (52.5% [tropifexor] vs. 28.6% [placebo]), with most events of mild to moderate severity. Decreases seen in LDL-, HDL-, and total-cholesterol levels at 60-, 90-, and 150 µg doses stabilised after treatment discontinuation. By Day 28, tropifexor caused 26-72% reduction in GGT from baseline at 30- to 150-µg doses (p <0.001 at 60-, 90-, and 150-µg tropifexor vs. placebo). Day 28 QoL scores were comparable between the placebo and tropifexor groups. A dose-dependent increase in plasma tropifexor concentration was observed, with 5- to 5.55-fold increases in AUC0-8h and Cmax between 30- and 150-µg doses. Conclusions: Tropifexor showed improvement in cholestatic markers relative to placebo, predictable pharmacokinetics, and an acceptable safety-tolerability profile, thereby supporting its potential further clinical development for PBC. Lay summary: The bile acid ursodeoxycholic acid (UDCA) is the standard-of-care therapy for primary biliary cholangitis (PBC), but approximately 40% of patients have an inadequate response to this therapy. Tropifexor is a highly potent non-bile acid agonist of the farnesoid X receptor that is under clinical development for various chronic liver diseases. In the current study, in patients with an inadequate response to UDCA, tropifexor was found to be safe and well tolerated, with improved levels of markers of bile duct injury at very low (microgram) doses. Itch of mild to moderate severity was observed in all groups including placebo but was more frequent at the highest tropifexor dose. Clinical Trials Registration: This study is registered at ClinicalTrials.gov (NCT02516605).

Ligation of erythrocyte CR1 induces its clustering in complex with scaffolding protein FAP-1.

The primary identified function of complement receptor 1 (CR1/CD35) on primate erythrocytes is to bind complement-tagged inflammatory particles including microbes and immune complexes. When erythrocytes circulate through liver and spleen, sinusoidal phagocytes remove CR1-adherent particles and erythrocytes return to the circulation. This process of immune adherence clearance is important for host defense and prevention of autoimmunity. CR1 was previously described as clustered in the human erythrocyte membrane, which was thought to be necessary for binding complement-opsonized particles. In contrast, we demonstrate that on erythrocytes CR1 is not clustered, but dispersed, and able to bind complement-tagged particles. When fresh erythrocytes are solubilized by nonionic detergent, CR1 partitions to the cytoskeleton fraction. Using a PDZ-peptide array, CR1's cytoplasmic tail, which contains 2 PDZ-motifs, binds PDZ domains 2, 3, and 5 of Fas-associated phosphatase 1 (FAP-1), a scaffolding protein. We show that FAP-1, not previously recognized as an erythroid protein, is expressed on circulating erythrocytes. CR1 and FAP-1 coimmunoprecipitate, which confirms their molecular association. Disperse CR1 on erythrocytes may be advantageous for capturing immune-complexes, while ligation-induced CR1 clustering may prevent ingestion of the erythrocyte during the immune-complex transfer to the macrophages by keeping the opsonic stimulus localized thus preventing phagocyosis.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the Novel Non-Bile Acid FXR Agonist Tropifexor (LJN452) in Healthy Volunteers.

Tropifexor (LJN452) is a potent, orally available, non-bile acid farnesoid X receptor agonist under clinical development for chronic liver diseases. Here, we present results from a first-in-human study of tropifexor following single- and multiple-ascending doses (SAD/MAD) and food effect substudy in healthy volunteers. The SAD study included 6 fasted cohorts receiving 10- to 3000-µg tropifexor or placebo and 1 cohort receiving 300-µg tropifexor with a high-fat meal. The MAD study included 4 lean cohorts receiving 10 to 100 µg and 1 obese cohort receiving 30-µg once-daily doses or placebo for 14 days. Pharmacodynamic assessment of fibroblast growth factor 19 and fasting plasma lipids was performed after dosing. Overall, 95 volunteers received at least 1 tropifexor or placebo dose. Tropifexor was well tolerated up to 3000 µg and 100 µg in the SAD and MAD studies, respectively; however, 2 subjects discontinued the MAD study due to asymptomatic elevation of liver transaminases. At single doses, tropifexor showed a moderate rate of absorption (median time to maximum concentration, 4 hours), dose-proportional increases in exposure, and elimination half-life of 13.5 to 21.9 hours. When taken with food, tropifexor exposure increased by ∼60%. With multiple dosing, steady state was reached on day 4 with <2-fold accumulation. Single and multiple doses showed dose-dependent increases in fibroblast growth factor 19. No changes in serum lipids were observed in tropifexor- vs placebo-treated obese subjects. In conclusion, tropifexor was well tolerated, had a pharmacokinetic profile suitable for once-daily dosing and showed dose-dependent target engagement without altering plasma lipids in healthy volunteers.

Tropifexor-Mediated Abrogation of Steatohepatitis and Fibrosis Is Associated With the Antioxidative Gene Expression Profile in Rodents.

Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid-derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin-resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion: Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.

Characterization of two novel LPS-binding sites in leukocyte integrin betaA domain.

Lipopolysaccharide (LPS), a bacterial endotoxin, triggers deleterious systemic inflammatory responses when released into blood circulation, causing organ dysfunction and death. In response to LPS stimulation, CD14 and toll-like receptor (TLR)-4 elicit inflammatory signaling cascades. Although leukocyte integrins (CD11b/CD18 and CD11c/CD18) were reported to bind LPS and induce NF-kappaB translocation, the evidence on such epitope location remains elusive. The present study aims to delineate the LPS-binding sites on the integrin CD18 antigen and to design peptide(s) as potential prophylactic and/or therapeutic agents to modulate LPS effects in activated Jurkat cells. Epitope mapping analysis using a series of CD18 truncated variants revealed two putative LPS-binding sites within the betaA region (216-248 and 266-318 a.a.), which were further confirmed by point mutation studies. Inhibition assay demonstrated that the CD18-betaA(266-318) peptide could block LPS binding in a dose-dependent manner. Our data also indicated that treatment with the CD18-peptide modulated TNF-alpha mRNA transcription via the NF-kappaB signaling pathway in LPS-activated Jurkat cells. In conclusion, we have identified two novel LPS-binding sites located at the CD18 betaA domain of leukocyte integrin, and the integrin peptide betaA(266-318) is shown to inhibit LPS binding and subsequent inflammatory events, having therapeutic implications to cure gram-negative endotoxemia.

TORC1 inhibition enhances immune function and reduces infections in the elderly.

Inhibition of the mechanistic target of rapamycin (mTOR) protein kinase extends life span and ameliorates aging-related pathologies including declining immune function in model organisms. The objective of this phase 2a randomized, placebo-controlled clinical trial was to determine whether low-dose mTOR inhibitor therapy enhanced immune function and decreased infection rates in 264 elderly subjects given the study drugs for 6 weeks. A low-dose combination of a catalytic (BEZ235) plus an allosteric (RAD001) mTOR inhibitor that selectively inhibits target of rapamycin complex 1 (TORC1) downstream of mTOR was safe and was associated with a significant (P = 0.001) decrease in the rate of infections reported by elderly subjects for a year after study drug initiation. In addition, we observed an up-regulation of antiviral gene expression and an improvement in the response to influenza vaccination in this treatment group. Thus, selective TORC1 inhibition has the potential to improve immune function and reduce infections in the elderly.

Short-term Low-Dose mTORC1 Inhibition in Aged Rats Counter-Regulates Age-Related Gene Changes and Blocks Age-Related Kidney Pathology.

Rapalogs, inhibitors of mTORC1 (mammalian target of rapamycin complex 1), increase life span and delay age-related phenotypes in many species. However, the molecular mechanisms have not been fully elucidated. We determined gene expression changes comparing 6- and 24-month-old rats in the kidney, liver, and skeletal muscle, and asked which of these changes were counter-regulated by a clinically-translatable (short-term and low-concentration) treatment, with a rapalog (RAD001). Surprisingly, RAD001 had a more pronounced effect on the kidney under this regimen in comparison to the liver or skeletal muscle. Histologic evaluation of kidneys revealed that the severity of chronic progressive nephropathy lesions was lower in kidneys from 24-month-old rats treated with RAD001 compared with vehicle. In addition to other gene expression changes, c-Myc, which has been shown to regulate aging, was induced by aging in the kidney and counter-regulated by RAD001. RAD001 caused a decrease in c-Myc protein, which could be rescued by a proteasome inhibitor. These findings point to settings for use of mTORC1 inhibitors to treat age-related disorders, and highlight c-Myc regulation as one of the potential mechanisms by which mTORC1 inhibition is perturbing age-related phenotypes.

A role for lipid rafts in C1q-triggered O2- generation by human neutrophils.

Calreticulin, a candidate C1q receptor, was shown recently to be present on the surface of human neutrophils in association with glycosylphosphatidylinositol (GPI) anchored proteins, particularly CD59. In this study, we show that antibodies to CD59, as well as to every other GPI-anchored protein tested, inhibited the C1q-triggered release of O(2)(-) from PMN. Methyl beta cyclodextrin (M beta CD) treatment of the cells to disrupt lipid rafts also prevented C1q-triggered O(2)(-) production. beta(2) integrin-dependent co-stimulation is required for O(2)(-) production from PMN, however M beta CD had no effect on LFA-1 or Mac-1-mediated adhesion, soluble iC3b binding to PMN, or spreading and migration, all of which suggested that PMN integrin function remained intact. Flow cytometric analysis of PMN treated with M beta CD showed upregulation of PMN granule-associated integrins and a corresponding increase in integrin activation-reporter epitopes, in contrast to the decreased expression of GPI-anchored antigens. These data support a model where lipid rafts and their associated GPI-anchored proteins are critical for C1q-triggered O(2)(-) production, consistent with a model where calreticulin serves as the C1q receptor for O(2)(-) production from PMN.

A comparison of type I collagen, fibronectin, and vitronectin in supporting adhesion of mechanically strained osteoblasts.

We used an adhesion assay for cells cultured under high dynamic strain to measure human osteoblast-like HOS cell adherence to immobilized type I collagen, fibronectin, and vitronectin. These conditions were designed to model the increased forces present at unstable fractures or loose joint prostheses. At a constant, low protein-coating density (1000 molecules/microm2) and 20% cyclic strain for 24 h, type I collagen, fibronectin, and vitronectin supported 24.6 +/- 2%, 16.7 +/- 3%, and 1.1 +/- 1% adherence, respectively, which paralleled the relative number of integrin-binding sites in each protein. Thus, when the number of available binding sites was limited, strain resistance was proportional to the number of integrin-ligand interactions. In contrast, at high protein-coating densities (> or = 2,500 molecules/microm2), vitronectin supported greater adherence (45.7 +/- 2%) when compared with type I collagen (37 +/- 2%) or fibronectin (34.8 +/- 2%) and directed constitutive expression of osteopontin (OPN), which suggested that there exist discrete signals on vitronectin receptor occupancy that promoted cell adherence and survival under strain. Integrin-mediated binding was necessary for resistance to strain, as evidenced by the low levels of strain resistance observed when cells were adherent in a nonintegrin-dependent manner. These findings support the utilization of at least two distinct mechanisms (i.e., tensegrity and integrin-mediated signal transduction) by HOS cells to remain adherent and viable on exposure to mechanical forces.

Expression and function of C1q receptors and C1q binding proteins at the cell surface.

C1q is the recognition unit of the first component of complement that binds not only IgG and IgM containing immune complexes, but also recognizes foreign structures such as the lipid A of endotoxin, and molecules expressed at the surface of apoptotic cells. In this review, the plasma membrane receptors and binding proteins for C1q are discussed and new data are presented on calreticulin expression on human peripheral blood cells. Although much is known about C1q receptors and binding molecules there are still many questions regarding their role in vivo.

Sialylation of ICAM-2 on platelets impairs adhesion of leukocytes via LFA-1 and DC-SIGN.

Intercellular adhesion molecule (ICAM)-2 is highly expressed on platelets and endothelium and is a counter-receptor for the leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1) and for the dendritic cell-specific, ICAM-grabbing non-integrin (DC-SIGN) protein. In this study, we investigated structural and functional differences between ICAM-2 from platelets and that from endothelial cells. The isoelectric point (pI) of ICAM-2 from HUVEC was pH 3.5-4.3, whereas that of platelet ICAM-2 was more acidic at pH 3.0-3.7. This charge difference was abolished by treatment with N-glycanase or neuraminidase, thus it was due to cell-specific N-linked glycosylation. Purified, immobilized platelet ICAM-2 supported 50% less adhesion of LFA-1-bearing T cells than did purified HUVEC ICAM-2 and no adhesion was observed of monocyte-derived immature dendritic cells via DC-SIGN to platelet ICAM-2. Treatment of platelet ICAM-2 with neuraminidase abolished these functional differences. These findings demonstrated that physiologic sialylation of platelet ICAM-2 renders it less able than endothelial ICAM-2 to support adherence of leukocytes.

mTOR inhibition improves immune function in the elderly.

Inhibition of the mammalian target of rapamycin (mTOR) pathway extends life span in all species studied to date, and in mice delays the onset of age-related diseases and comorbidities. However, it is unknown if mTOR inhibition affects aging or its consequences in humans. To begin to assess the effects of mTOR inhibition on human aging-related conditions, we evaluated whether the mTOR inhibitor RAD001 ameliorated immunosenescence (the decline in immune function during aging) in elderly volunteers, as assessed by their response to influenza vaccination. RAD001 enhanced the response to the influenza vaccine by about 20% at doses that were relatively well tolerated. RAD001 also reduced the percentage of CD4 and CD8 T lymphocytes expressing the programmed death-1 (PD-1) receptor, which inhibits T cell signaling and is more highly expressed with age. These results raise the possibility that mTOR inhibition may have beneficial effects on immunosenescence in the elderly.

AXT914 a novel, orally-active parathyroid hormone-releasing drug in two early studies of healthy volunteers and postmenopausal women.

Antagonism of the calcium-sensing receptor in the parathyroid gland leads to parathyroid hormone (PTH) release. Calcilytics are a new class of molecules designed to exploit this mechanism. In order to mimic the known bone-anabolic pharmacokinetic (PK) profile of s.c. administered PTH, such molecules must trigger sharp, transient and robust release of PTH. The results of two early clinical studies with the orally-active calcilytic AXT914, a quinazolin-2ne derivative are reported. These were GCP-compliant, single and multiple dose studies of PK/PD and tolerability in healthy volunteers and postmenopausal women. The first study, examined single ascending doses (4 to 120 mg) and limited multiple doses (60 or 120 mgq.d. for 12 days) of AXT914. The second study was a randomized, double-blind, active- and placebo-controlled, 4-week repeat-dose parallel group study of healthy postmenopausal women (45 and 60 mg AXT914, placebo, 20 µg Forteo/teriparatide/PTH(1-34) fragment). AXT914 was well tolerated at all doses and reproducibly induced the desired PTH-release profiles. Yet, 4 weeks of 45 or 60 mg AXT914 did not result in the expected changes in circulating bone biomarkers seen with teriparatide. However total serum calcium levels increased above baseline in the 45 and 60 mg AXT914 treatment groups (8.0% and 10.7%, respectively), compared to that in the teriparatide and placebo groups (1.3% and 1.0%, respectively). Thus the trial was terminated after a planned interim analysis due to lack of effect on bone formation biomarkers and dose-limiting effects on serum calcium. In conclusion, AXT914 was well tolerated but the observed transient and reproducible PTH-release after repeat oral administration of AXT914 which showed an exposure profile close to that of s c. PTH, did not translate into a bone anabolic response and was associated with a persistent dose-related increase in serum calcium concentrations.

Serological investigation of the collagen degradation profile of patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis.

In both chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), abnormally high collagen remodeling occurs within the lung tissue. Matrix metalloproteinase (MMP)-degraded type I, III, IV, V and VI collagen and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-degraded type III collagen were assessed in serum of patients diagnosed with mild COPD (n = 10) or IPF (n = 30), and healthy controls (n = 15). The collagen degradation markers C1M, C3M, C5M and C6M were significantly elevated in serum of both mild COPD and IPF patients, versus controls. C3A and C4M were only elevated in patients with mild COPD, compared with controls. The most reliable indicators of mild COPD versus controls were: C1M (area under the receiver-operating characteristics (AUROC = 0.94, P < 0.0001), C3M (AUROC = 0.95, P < 0.0001), and C5M (AUROC = 0.95, P < 0.0001). The most reliable markers for the diagnosis of IPF were achieved by C1M (AUROC = 0.90, P < 0.0001) and C3M (AUROC = 0.93, P < 0.0001). Collagen degradation was highly up-regulated in patients with IPF and mild COPD, indicating that degradation fragments of collagens are potential markers of pulmonary diseases. Interestingly, C4M and C3A were only elevated in patients with mild COPD, indicating that these markers could be used to distinguish between the two pathologies.

ATF936, a novel oral calcilytic, increases bone mineral density in rats and transiently releases parathyroid hormone in humans.

Parathyroid hormone (PTH), when injected daily as either the intact hormone PTH(1-84) or the active fragment PTH(1-34) (teriparatide), is an efficacious bone anabolic treatment option for osteoporosis patients. Injections lead to rapid and transient spikes in hormone exposure levels, a profile which is a prerequisite to effectively form bone. Oral antagonists of the calcium-sensing receptor (calcilytics) stimulate PTH secretion and represent thus an alternative approach to elevate hormone levels transiently. We report here on ATF936, a novel calcilytic, which triggered rapid, transient spikes in endogenous PTH levels when given orally in single doses of 10 and 30mg/kg to growing rats, and of 1mg/kg to dogs. Eight weeks daily oral application of 30mg/kg of ATF936 to aged female rats induced in the proximal tibia metaphysis increases in bone mineral density, cancellous bone volume and cortical and trabecular thickness as evaluated by computed tomography. In healthy humans, single oral doses of ATF936 produced peak PTH levels in plasma after a median time of 1h and levels returned to normal at 24-h post-dose. The average maximum PTH concentration increase from baseline was 1.9, 3.6, and 6.0-fold at doses of 40, 70, and 140mg. ATF936 was well tolerated. The sharp, transient increase in PTH levels produced by the oral calcilytic ATF936 was comparable to the PTH profile observed after subcutaneous administration of teriparatide. In conclusion, ATF936 might hold potential as an oral bone-forming osteoporosis therapy.

Aggregatibacter actinomycetemcomitans leukotoxin requires beta-sheets 1 and 2 of the human CD11a beta-propeller for cytotoxicity.

Aggregatibacter actinomycetemcomitans leukotoxin (Ltx) is a repeats-in-toxin (RTX) cytolysin that kills human leukocyte function-associated antigen-1 (LFA-1; alpha(L)/beta(2))-bearing cells. In order to determine whether the alpha(L) portion of the heterodimer is involved in Ltx recognition, we transfected human, mouse and bovine alpha(L) cDNAs into J-beta(2).7, an alpha(L)-deficient cell line, and looked for restoration of Ltx susceptibility. Cells expressing either bovine or human alpha(L) in conjunction with human beta(2) were efficiently killed by Ltx, an indication that bovine alpha(L) could substitute for its human counterpart in critical regions used by Ltx for attachment to LFA-1. On the other hand, cells expressing murine alpha(L) and human beta(2) were not susceptible to the lethal effects of Ltx indicating that the toxin recognition sites are not present in the corresponding mouse sequence. To further identify the region(s) of alpha(L) recognized by Ltx, we constructed and evaluated a panel of chimeric human/murine alpha(L) genes in J-beta(2).7 cells. Analysis of the alpha(L) mutant panel showed that the presence of human N-terminal 128 amino acids on a mouse CD11a background, a region that includes beta-sheets 1 and 2 of the beta-propeller of the human alpha(L) chain, was sufficient for Ltx cytolysis.

Rituximab for steroid-refractory chronic graft-versus-host disease.

B cells may be implicated in the pathophysiology of chronic graft-versus-host disease (GVHD), as evidenced by antibody production against sex-mismatched, Y chromosome-encoded minor HLA antigens in association with chronic GVHD. We therefore designed a phase 1/2 study of anti-B-cell therapy with rituximab in steroid-refractory chronic GVHD. Twenty-one patients were treated with 38 cycles of rituximab. Rituximab was tolerated well, and toxicity was limited to infectious events. The clinical response rate was 70%, including 2 patients with complete responses. Responses were limited to patients with cutaneous and musculoskeletal manifestations of chronic GVHD and were durable through 1 year after therapy. The median dose of prednisone among treated subjects fell from 40 mg/day to 10 mg/day, 1 year after rituximab therapy (P < .001). A chronic GVHD symptom score improved in the majority of treated patients. Antibody titers against Y chromosome-encoded minor HLA antigens fell and remained low, whereas titers against infectious antigens (EBV, tetanus) remained stable or rose during the treatment period. We conclude that specific anti-B-cell therapy with rituximab may be beneficial for patients with steroidrefractory chronic GVHD. This trial was registered at www.clinicaltrials.gov as #NCT00136396.