Discovery of GLPG2737, a Potent Type 2 Corrector of CFTR for the Treatment of Cystic Fibrosis in Combination with a Potentiator and a Type 1 Co-corrector.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.

Discovery and Optimization of Orally Bioavailable Phthalazone and Cinnolone Carboxylic Acid Derivatives as S1P2 Antagonists against Fibrotic Diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease. Current treatments only slow down disease progression, making new therapeutic strategies compelling. Increasing evidence suggests that S1P2 antagonists could be effective agents against fibrotic diseases. Our compound collection was mined for molecules possessing substructure features associated with S1P2 activity. The weakly potent indole hit 6 evolved into a potent phthalazone series, bearing a carboxylic acid, with the aid of a homology model. Suboptimal pharmacokinetics of a benzimidazole subseries were improved by modifications targeting potential interactions with transporters, based on concepts deriving from the extended clearance classification system (ECCS). Scaffold hopping, as a part of a chemical enablement strategy, permitted the rapid exploration of the position adjacent to the carboxylic acid. Compound 38, with good pharmacokinetics and in vitro potency, was efficacious at 10 mg/kg BID in three different in vivo mouse models of fibrotic diseases in a therapeutic setting.

TEG001 Insert Integrity from Vector Producer Cells until Medicinal Product.

Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αßT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo.

Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP).

Fibroblast activation protein (FAP) is a proline-selective serine protease. It is hardly expressed in healthy adult tissue but upregulated in tissue remodeling sites associated with several diseases including epithelial cancer types, atherosclerosis, arthritis and fibrosis. Ongoing research aims at clinical implementation of FAP as a biomarker for these diseases. Several immunochemical methods that quantify FAP expression have been reported. An alternative/complementary approach focuses on quantification of FAP's enzymatic activity. Developing an activity-based assay for FAP has nonetheless proven challenging because of selectivity issues with respect to prolyl oligopeptidase (PREP). Here, we present substrate-type FAP probes that are structurally derived from a FAP-inhibitor (UAMC1110) that we published earlier. Both cleavage efficiency and FAP-selectivity of the best compounds in the series equal or surpass the most advanced peptide-based FAP substrates reported to date. Finally, proof-of-concept is provided that 4-aminonaphthol containing probes can spatially localize FAP activity in biological samples.

GMP-Grade Manufacturing of T Cells Engineered to Express a Defined γδTCR.

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αßTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αßTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

Scaling-up of a HepaRG progenitor cell based bioartificial liver: optimization for clinical application and transport.

A new generation of bioartificial livers, based on differentiated proliferative hepatocyte sources, has been developed. Several practicable and regulatory demands have to be addressed before these can be clinically evaluated. We identified three main hurdles: (1) expansion and preservation of the biocomponent, (2) development of scaled-up culture conditions and (3) transport of the device to the bedside. In this study we address these three issues for the HepaRG-progenitor cell line-loaded AMC-Bioartificial Liver. (1) HepaRG cells were expanded in large quantities and then cryopreserved or loaded directly into bioreactors. After 3 weeks of culture, key hepatic functions (ammonia/lactate elimination, apolipoprotein A1 synthesis and cytochrome P450 3A4 activity) did not differ significantly between the two groups. (2) Bioartificial livers were scaled up from 9 ml to 540 ml priming volume, with preservation of normalized hepatic functionality. Quantification of amino acid consumption revealed rapid depletion of several amino acids. (3) Whole-device cryopreservation and cooled preservation induced significant loss of hepatic functionality, whereas simulated transport from culture-facility to the bedside in a clinical-grade transport unit with controlled temperature maintenance, medium perfusion and gas supply did not affect functionality. In addition, we assessed tumorigenicity of HepaRG cells in immune-incompetent mice and found no tumor formation of HepaRG cells (n = 12), while HeLa cells induced formation of carcinomas in eight out of 12 mice in 140 days.

Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-cyanopyrrolidine inhibitors of fibroblast activation protein (FAP).

Fibroblast activation protein (FAP) is a serine protease related to dipeptidyl peptidase IV (DPPIV). It has been convincingly linked to multiple disease states involving remodeling of the extracellular matrix. FAP inhibition is investigated as a therapeutic option for several of these diseases, with most attention so far devoted to oncology applications. We previously discovered the N-4-quinolinoyl-Gly-(2S)-cyanoPro scaffold as a possible entry to highly potent and selective FAP inhibitors. In the present study, we explore in detail the structure-activity relationship around this core scaffold. We report extensively optimized compounds that display low nanomolar inhibitory potency and high selectivity against the related dipeptidyl peptidases (DPPs) DPPIV, DPP9, DPPII, and prolyl oligopeptidase (PREP). The log D values, plasma stabilities, and microsomal stabilities of selected compounds were found to be highly satisfactory. Pharmacokinetic evaluation in mice of selected inhibitors demonstrated high oral bioavailability, plasma half-life, and the potential to selectively and completely inhibit FAP in vivo.

Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold.

Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp(2) hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>10(3)) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory.

Two years after in vivo implantation of poly(DL-lactide-epsilon-caprolactone) nerve guides: has the material finally resorbed?

Long-term nerve guide studies on nerve regeneration in vivo are scarce. Previously, we showed that small fragments of biomaterial could still be found on the edge of the epineurium of the regenerated nerve after implantation of poly(DL-lactide-epsilon-caprolactone) [poly(DLLA-epsilon-CL), Neurolac(R)] nerve guides. As these nerve guides are assumed to fully resorb, we studied the 2-year degradation and possible long-term foreign body reaction against the nerve guides after implantation in the sciatic nerve of the rat. In addition, the distribution of both collagen type III and IV, and nerve regeneration through the nerve guides were studied and compared with the non-operated control side. The results demonstrate that nerve regeneration took place through the poly(DLLA-epsilon-CL) nerve guides. After 2 years of implantation, biomaterial could not be found macroscopically. Biomaterial fragments in company of multi-nucleated giant cells and macrophages were found along the regenerated nerve tissue. Collagen III and IV were both found around the epineurium and perineurium in the regenerated nerve, the organization of these layers resembled that of the contra-lateral non-operated nerve. Although sufficient nerve regeneration was obtained after long-term implantation in the rat sciatic nerve, biomaterial fragments and foreign body reactions against these fragments, even after 24 months of implantation, could still be found. The poly(DLLA-epsilon-CL), Neurolac nerve guides do resorb, however, not complete up to 2 years of implantation. It is not known whether the remaining biomaterial fragments and foreign body reactions may cause granulomas or other complications after longer implantation periods.

Circadian rhythms of C-FOS expression in the suprachiasmatic nuclei of the common vole (Microtus arvalis).

The suprachiasmatic nuclei of the hypothalamus (SCN) are the master circadian clock in mammals. Transcriptional activity in this master clock has a marker in the immediate-early gene c-Fos. Within the SCN, distinct differences in c-Fos in the ventrolateral and the dorsomedial SCN have been reported for rodent species such as rats, mice, and hamsters. We studied C-FOS expression in the common vole (Microtus arvalis) SCN under LD 12:12 h and under constant dim light conditions. In the vole dorsomedial SCN, rhythmic C-FOS expression was seen in LD with a clear peak in the middle of the light period. Under constant dim light, we report constitutive, non-rhythmic expression of C-FOS in the dorsomedial SCN. This pattern is consistent with the circadian organization of behavioral activity, which is weak in voles and may be lost under constant dim-light conditions. In the ventrolateral SCN, we observed a rise in C-FOS expression under LD conditions prior to lights-on, followed by peak expression at lights-on. Another peak was seen at lights-off. In an additional experiment, we subjected animals to LD 16:8 to test the hypothesis that the dawn and dusk peaks in ventrolateral C-FOS expression change phase along with the photoperiod. The peak in C-FOS expression did not shift with the time of lights on, but remained at the same external time 6. The results are consistent with the interpretation that in the vole, c-Fos expression reports transcriptional activity associated more likely with an internal, gating process than with an external effect of light.

Vasopressin immunoreactivity, but not vasoactive intestinal polypeptide, correlates with expression of circadian rhythmicity in the suprachiasmatic nucleus of voles.

In common voles (Microtus arvalis), natural variation in locomotor behavior can be exploited to study the mechanism of pacemaker control over circadian timing of behavior. Here we studied daily patterns in numbers of neuropeptide immunoreactive suprachiasmatic nucleus neurons in rhythmic, weakly rhythmic, and non-rhythmic voles. Circadian rhythmic voles showed circadian variation in numbers of vasoactive intestinal polypeptide and vasopressin immunoreactive suprachiasmatic nucleus neurons with a peak at zeitgeber time 0. In contrast, voles with weak or no circadian rhythmicity exhibited similar fluctuations for vasoactive intestinal polypeptide, but a continuous, non-rhythmic high profile for vasopressin. Vole suprachiasmatic nucleus neurons do not produce somatostatin or substance P. We conclude that the vasopressin system in the common vole suprachiasmatic nucleus acts as a principal correlate with expression of circadian behavior, in contrast to vasoactive intestinal polypeptide, somatostatin, and substance P. We also conclude that high levels of vasopressin immunoreactivity in the non-rhythmic vole suprachiasmatic nucleus is in line with previously demonstrated hampered release, probably resulting in vasopressin accumulation in the suprachiasmatic nucleus. Vasopressin could be a candidate in mediating output of the vole circadian clock, leading to circadian expression of locomotor behavior.