Multiple-ascending doses of ACT-1014-6470, an oral complement factor 5a receptor 1 (C5a1 receptor) antagonist: Tolerability, pharmacokinetics and target engagement.

AIMS: Targeting the complement factor 5a receptor 1 (C5a1 receptor) offers potential to treat various autoimmune diseases. The C5a1 receptor antagonist ACT-1014-6470 was well tolerated in a single-ascending dose study in healthy subjects. This double-blind, randomized, placebo-controlled study aimed to investigate the safety, tolerability, pharmacokinetics (PK) and target engagement of multiple-ascending doses of ACT-1014-6470. METHODS: Per dose level, 10 healthy male and female subjects of nonchildbearing potential (1:1 sex ratio) were enrolled to assess 30, 60 and 120 mg ACT-1014-6470 administered twice daily for 4.5 days under fed conditions. Adverse events, clinical laboratory data, vital signs, electrocardiogram and PK blood samples were collected up to 120 h post last dose and ex vivo stimulated matrix metalloproteinase 9 was quantified as target engagement biomarker. At the 60-mg dose level, PK samples were collected until 8 weeks post last dose. RESULTS: The total adverse event number was 57 and no treatment-related safety pattern was apparent. At steady state, ACT-1014-6470 reached maximum plasma concentrations after 2-3 h and the half-life estimated up to Day 10 was 115-146 h across dose levels. Exposure parameters increased dose-proportionally, steady state was attained between Day 3-5, and ACT-1014-6470 accumulated 2-fold. At the 60-mg dose level, ACT-1014-6470 was quantifiable until 8 weeks after the last dose. Matrix metalloproteinase 9 release was suppressed to endogenous background concentrations up to the last sampling time point, confirming sustained target engagement of ACT-1014-6470. CONCLUSION: The compound was generally safe and well tolerated at all dose levels, warranting further clinical investigations.

Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients.

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients.

ACT-1004-1239, a first-in-class CXCR7 antagonist with both immunomodulatory and promyelinating effects for the treatment of inflammatory demyelinating diseases.

Current strategies for the treatment of demyelinating diseases such as multiple sclerosis (MS) are based on anti-inflammatory or immunomodulatory drugs. Those drugs have the potential to reduce the frequency of new lesions but do not directly promote remyelination in the damaged central nervous system (CNS). Targeting CXCR7 (ACKR3) has been postulated as a potential therapeutic approach in demyelinating diseases, leading to both immunomodulation by reducing leukocyte infiltrates and promyelination by enhancing myelin repair. ACT-1004-1239 is a potent, selective, insurmountable, and orally available first-in-class CXCR7 receptor antagonist. The effect of ACT-1004-1239 was evaluated in the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and the cuprizone-induced demyelination mouse models. In addition, ACT-1004-1239 was assessed in a rat oligodendrocyte precursor cell (OPC) differentiation assay in vitro. In the MOG-induced EAE model, ACT-1004-1239 treatment (10-100 mg/kg, twice daily, orally) showed a significant dose-dependent reduction in disease clinical scores, resulting in increased survival. At the highest dose tested (100 mg/kg, twice daily), ACT-1004-1239 delayed disease onset and significantly reduced immune cell infiltrates into the CNS and plasma neurofilament light chain concentration. Treatment with ACT-1004-1239 dose-dependently increased plasma CXCL12 concentration, which correlated with a reduction of the cumulative disease score. Furthermore, in the cuprizone model, ACT-1004-1239 treatment significantly increased the number of mature myelinating oligodendrocytes and enhanced myelination in vivo. In vitro, ACT-1004-1239 promoted the maturation of OPCs into myelinating oligodendrocytes. These results provide evidence that ACT-1004-1239 both reduces neuroinflammation and enhances myelin repair substantiating the rationale to explore its therapeutic potential in a clinical setting.

Immunoaffinity Targeted Mass Spectrometry Analysis of Human Plasma Samples Reveals an Imbalance of Active and Inactive CXCL10 in Primary Sjögren's Syndrome Disease Patients.

One of the most important advantages of mass spectrometry is the ability to quantify proteins and their modifications in parallel to obtain a holistic picture of the protein of interest. Here, we present a hybrid immunoaffinity targeted mass spectrometry (MS) method that combines efficient pan-antibody enrichment of a specific protein from plasma with the selectivity of high-resolution targeted MS analysis to quantitate specific proteoforms of interest. We used this approach to quantify plasma levels of the chemokine CXCL10 that has been associated with many immunological disorders such as systemic lupus erythematosus and primary Sjögren's Syndrome (pSS). The hybrid approach enabled sensitive, specific, and simultaneous quantification of total, full-length (active) CXCL101-77 and DPP4-truncated (inactive) CXCL103-77 in human plasma down to the low pg/mL level, reaching ELISA sensitivities. Samples from 30 control subjects and 34 pSS patients (n = 64) were analyzed. The ratio of CXCL101-77 to truncated CXCL103-77 was significantly increased in patients with pSS and provided the highest correlation with pSS disease activity. Therefore, this CXCL10 proteoform ratio represents an interesting exploratory disease activity biomarker to further investigate. As this strategy can be readily adapted to other plasma proteins and proteoforms of interest, we are convinced that it will lead to a more detailed understanding of proteoforms in physiology and pathology yielding more relevant biomarkers and drug targets.

Impact of high-altitude therapy on type-2 immune responses in asthma patients.

BACKGROUND: Asthma patients present with distinct immunological profiles, with a predominance of type 2 endotype. The aim of this study was to investigate the impact of high-altitude treatment on the clinical and immunological response in asthma. METHODS: Twenty-six hospitalized asthma patients (nine eosinophilic allergic; EA, nine noneosinophilic allergic; NEA and eight noneosinophilic nonallergic; NN) and nine healthy controls in high altitude for 21 days were enrolled in the study. We assessed eosinophils, T cells, Tregs, and innate lymphoid cells (ILC) from peripheral blood using flow cytometry. RESULTS: The number of eosinophils (both resting and activated) and chemoattractant receptor homolog expressed on Th2 cells (CRTH2)-expressing CD4+ and CD8+ T cells decreased significantly in EA patients after altitude treatment. The frequency of CRTH2+ Tregs as decreased significantly in all the asthma phenotypes as well as the frequency of ILC2 was significantly reduced in EA after altitude treatment. After 21 days of altitude therapy, CRTH2-expressing ILC2, CD4+ and CD8+ T cells and Treg cells showed attenuated responses to exogenous PGD2. Furthermore, PGD2 signaling via CRTH2 was found to diminish the suppressive function of CRTH2+ Tregs which partially normalized during high-altitude treatment. Improved asthma control was particularly evident in allergic asthma patients and correlated with decreased frequencies of CRTH2+ Treg cells in EA patients. Serum IL-5 and IL-13 decreased during climate treatment in asthma patients with high baseline levels. CONCLUSIONS: Asthma treatment in high altitude reduced the type 2 immune response, corrected the increased CRTH2 expression and its dysregulated functions.

Pharmacokinetics, pharmacodynamics, tolerability and prediction of clinically effective dose of ACT-774312: A novel CRTH2 antagonist.

ACT-774312 is an antagonist of the chemoattractant receptor-homologous molecule expressed on T helper (Th) 2 cells (CRTH2), in development for the treatment of nasal polyposis or other allergic and type-2 inflammatory diseases. Placebo, single doses of 1-1000 mg, or multiple doses of 30-500 mg either once or twice daily for 4 days of ACT-774312 were administered orally to healthy subjects. The single- and multiple-dose pharmacokinetics (PK) of ACT-774312 were dose proportional and characterized by a time to attainment of maximum plasma concentrations between 1 and 3 hours and a terminal elimination half-life of about 12 hours In the presence of food, tmax was delayed by 1 hour and exposure to ACT-774312 slightly decreased. Full blockade (>90% of the maximum effect, Emax ) of CRTH2 as measured in a whole blood internalization assay was observed after 50 mg ACT-774312 twice daily and lasted for at least 9 hours The relationship between ACT-774312 concentration and CRTH2 blockade was described by a Emax model. The estimated twice-daily dose of ACT-774312 at which full blockade of CRTH2 is achieved at trough in at least 80% of subjects was estimated at 109 mg. Administration of ACT-774312 was safe and well tolerated at all doses. For none of the recorded adverse events, a relationship to dose was discerned, and there were no clinically relevant findings on the measured ECG, clinical laboratory and vital signs variables. The observed PK, pharmacodynamics and safety profile warrant further development of ACT-774312.

A novel CRTH2 antagonist: Single- and multiple-dose tolerability, pharmacokinetics, and pharmacodynamics of ACT-453859 in healthy subjects.

The chemoattractant receptor-homologous molecule expressed on T-helper 2 cells (CRTH2) is a G-protein-coupled receptor for prostaglandin D2 , a key mediator in inflammatory disorders. In this randomized, double-blind, placebo-controlled study we investigated the single- and multiple-dose tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) up to a dose of 800 mg once a day of ACT-453859, a potent and selective CRTH2 antagonist. ACT-453859 was moderately rapidly absorbed and followed a biphasic elimination pattern, with an elimination half-life between 11 and 20 hours. Steady-state conditions were reached after 1 day, and ACT-453859 did not accumulate. Urinary excretion of unchanged ACT-453859 did not exceed 1.4% of the administered dose. Administration of ACT-453859 resulted in a dose-dependent blockadeof CRTH2 on the surface of eosinophils. The maximum PD effect of ACT-453859 was reached about 2.0 hours after dosing, which corresponded to the highest concentration at which PD were assessed. At steady state, 100 and 800 mg ACT-453859 once a day resulted in blockade of CRTH2 over 24 hours. In this entry-into-humans study, ACT-453859 showed good tolerability at all doses and a PK and PD profile compatible with once-daily dosing.

Identification of cathepsin L as a potential sex-specific biomarker for renal damage.

The renin-angiotensin system is a well-known regulator of blood pressure and plays an important role in the pathogenesis of cardiovascular disease and renal damage. Genetic factors, including single nucleotide polymorphisms and sex, are increasingly recognized as potential risk factors for the development of cardiovascular disease. Double transgenic rats (dTGRs), harboring human renin and angiotensinogen genes, were used in this study to investigate potential sex differences influencing renal function and renal gene expression. dTGR males and females had comparable increases in blood pressure, whereas body weight, albuminuria/proteinuria, and urine flow rate were higher in males. At 8 weeks of age, renal plasma flow and glomerular filtration rate were proportionally lower in males, and renal vascular resistance tended to be higher. Males developed more severe tubulointerstitial and vascular lesions. By the end of week 8, 40%of the males but none of the females had died. Genome expression studies were performed with RNA from kidneys of 7-week-old male and female dTGRs and control rats to further investigate the sex-related differences on a molecular level. Forty-five genes showed sex-dependent expression patterns in dTGRs that were significantly different compared to controls. Cathepsin L, one of the genes differentially expressed between the sexes, was also shown to be strongly associated with the degree of renal injury. In dTGRs, urinary cathepsin L at week 7 was higher in males (nanograms per 24 hours: male, 512±163; female, 132±70). These results reveal a potential new biomarker for the personalized diagnosis and management of chronic kidney disease.