Differential effects of ELX/TEZ/IVA on organ-specific CFTR function in two patients with the rare CFTR splice mutations c.273+1G>A and c.165-2A>G

Introduction: Evidence for the efficiency of highly-effective triple-CFTR-modulatory therapy with elexacaftor/tezacaftor/ivacaftor (ETI), either demonstrated in clinical trials or by in vitro testing, is lacking for about 10% of people with cystic fibrosis (pwCF) with rare mutations. Comprehensive assessment of CFTR function can provide critical information on the impact of ETI on CFTR function gains for such rare mutations, lending argument of the prescription of ETI. The mutation c.165-2A>G is a rare acceptor splice mutation that has not yet been functionally characterized. We here describe the functional changes induced by ETI in two brothers who are compound heterozygous for the splice mutations c.273+1G>C and c.165-2A>G.Methods: We assessed the effects of ETI on CFTR function by quantitative pilocarpine iontophoresis (QPIT), nasal potential difference measurements (nPD), intestinal current measurements (ICM), β-adrenergic sweat secretion tests (SST) and multiple breath washout (MBW) prior to and 4 months after the initiation of ETI.Results: Functional CFTR analysis prior to ETI showed no CFTR function in the respiratory and intestinal epithelia and in the sweat gland reabsorptive duct in either brother. In contrast, β-adrenergic stimulated, CFTR-mediated sweat secretion was detectable in the CF range. Under ETI, both brothers continued to exhibit high sweat chloride concentration in QPIT, evidence of low residual CFTR function in the respiratory epithelia, but normalized β-adrenergically stimulated production of primary sweat.Discussion: Our results are the first to demonstrate that the c.165-2A>G/c.273+1G>C mutation genotype permits mutant CFTR protein expression. We showed organ-specific differences in the expression of CFTR and consecutive responses to ETI of the c.165-2A>G/c.273+1G>C CFTR mutants that are probably accomplished by non-canonical CFTR mRNA isoforms. This showcase tells us that the individual response of rare CFTR mutations to highly-effective CFTR modulation cannot be predicted from assays in standard cell cultures, but requires the personalized multi-organ assessment by CFTR biomarkers

Acetato(1,10-phenanthroline-5,6-dione)silver(I) trihydrate

In the structure of the title compound, [Ag(C2H3O2)(C12H6N2O2)]·3H2O, the AgI atom is coordinated by both 1,10-phenanthroline-5,6-dione N atoms and one O atom from the acetate anion. The three water mol­ecules are involved in extensive hydrogen bonding to each other and to the acetate O and 1,10-phenanthroline-5,6-dione O atoms. In addition, there are weak C—H⋯O inter­actions

Novel methods to incorporate photosensitizers into nanocarriers for cancer treatment by photodynamic therapy

Objective A hydrophobic photosensitizer, 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a (HPPH), was loaded into nontoxic biodegradable amine functionalized polyacrylamide (AFPAA) nanoparticles using three different methods (encapsulation, conjugation, and post‐loading), forming a stable aqueous dispersion. Each formulation was characterized for physicochemical properties as well as for photodynamic performance so as to determine the most effective nanocarrier formulation containing HPPH for photodynamic therapy (PDT). Materials and Methods HPPH or HPPH‐linked acrylamide was added into monomer mixture and polymerized in a microemulsion for encapsulation and conjugation, respectively. For post‐loading, HPPH was added to an aqueous suspension of pre‐formed nanoparticles. Those nanoparticles were tested for optical characteristics, dye loading, dye leaching, particle size, singlet oxygen production, dark toxicity, in vitro photodynamic cell killing, whole body fluorescence imaging and in vivo PDT. Results HPPH was successfully encapsulated, conjugated or post‐loaded into the AFPAA nanoparticles. The resultant nanoparticles were spherical with a mean diameter of 29 ± 3 nm. The HPPH remained intact after entrapment and the HPPH leaching out of nanoparticles was negligible for all three formulations. The highest singlet oxygen production was achieved by the post‐loaded formulation, which caused the highest phototoxicity in in vitro assays. No dark toxicity was observed. Post‐loaded HPPH AFPAA nanoparticles were localized to tumors in a mouse colon carcinoma model, enabling fluorescence imaging, and producing a similar photodynamic tumor response to that of free HPPH in equivalent dose. Conclusions Post‐loading is the promising method for loading nanoparticles with hydrophobic photosensitizers to achieve effective in vitro and in vivo PDT. Lasers Surg. Med. 43:686–695, 2011. © 2011 Wiley‐Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86992/1/21113_ftp.pd

Changes in cystic fibrosis transmembrane conductance regulator protein expression prior to and during elexacaftor-tezacaftor-ivacaftor therapy

Background: Defects in expression, maturation or function of the epithelial membrane glycoprotein CFTR are causative for the progressive disease cystic fibrosis. Recently, molecular therapeutics that improve CFTR maturation and functional defects have been approved. We aimed to verify whether we could detect an improvement of CFTR protein expression and maturation by triple therapy with elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA).Methods: Rectal suction biopsies of 21 p.Phe508del homozygous or compound heterozygous CF patients obtained pre- and during treatment with ELX/TEZ/IVA were analyzed by CFTR Western blot that was optimized to distinguish CFTR glycoisoforms.Findings: CFTR western immunoblot analysis revealed that—compared to baseline—the levels of CFTR protein increased by at least twofold in eight out of 12 patients upon treatment with ELX/TEZ/IVA compared to baseline (p < 0.02). However, polydispersity of the mutant CFTR protein was lower than that of the fully glycosylated wild type CFTR Golgi isoform, indicating an incompletely glycosylated p.Phe508el CFTR protein isoform C* in patients with CF which persists after ELX/TEZ/IVA treatment.Interpretation: Treatment with ELX/TEZ/IVA increased protein expression by facilitating the posttranslational processing of mutant CFTR but apparently did not succeed in generating the polydisperse spectrum of N-linked oligosaccharides that is characteristic for the wild type CFTR band C glycoisoform. Our results caution that the lower amounts or immature glycosylation of the C* glycoisoform observed in patients’ biomaterial might not translate to fully restored function of mutant CFTR necessary for long-term provision of clinical benefit

Synthesis of metabolites from the ascomycetes : I. The synthesis and absolute configuration of leptosphaerin : II. Approaches to the synthesis of byssochlamic acid

Part I. The total synthesis of (+)-leptosphaerin (2), a metabolite of the marine Ascomycete Leptosphaeria Oraemaris (Linder) was achieved, unambiguously establishing both the relative and absolute stereochemistry of this natural product. This synthesis also resolved the long-standing question of which of the isomeric structures 1 or 2 correctly represents the structure of leptosphaerin. The target material was prepared in 12 steps, beginning from mannitol and ethyl pyruvate. In the key step a protected form of R-(+)-glyceraldehyde (3) underwent a stereospecific condensation with a 3- lithioacrylate derivative (48) to give a product (55) which was elaborated into leptosphaerin. The stereochemical outcome of this condensation was determined by means of a single crystal X-ray analysis. Part II. Two synthetic approaches to byssochlamic acid (3), a metabolite of Byssochlamys fulva, were studied. The first of these was based on a route in which a suitably functionalized 1,5-hexadiene (23) was intended to provide the substrate for an intramolecular [2+2] photocycloaddition. The impracticability of obtaining a suitable photolysis substrate caused revision of this strategy, for which the macrolide 88 became the focal intermediate. The bis phenol 89 was prepared by a sequence of reactions, the key step of which was the samarium diiodide mediated alkylation of 2',5'-dimethoxybutyrophenone (79) with 2,5-dimethoxybenzyl bromide (77). Reduction of the resulting tertiary alcohol (80) was followed by the selective protection of 80 as bisphenol 89, which underwent esterification with ethylmalonyl dichloride to give macrolide 88