Baricitinib improves symptoms in patients with moderate-to-severe atopic dermatitis and inadequate response to topical corticosteroids: patient-reported outcomes from two randomized monotherapy phase III trials.

BACKGROUND: Itch, skin pain, and sleep disturbance are burdensome symptoms in atopic dermatitis (AD) that negatively influence a patient's quality of life (QoL). OBJECTIVE: To evaluate the impact of baricitinib on patient-reported outcomes (PROs) in adult patients with moderate-to-severe AD, and explore the association between improvement in key signs and symptoms of AD with improvements in QoL and patient's assessment of disease severity. METHODS: Data were analyzed from two phase III monotherapy trials (BREEZE-AD1/BREEZE-AD2) in which patients were randomized 2:1:1:1 to once-daily placebo, baricitinib 1-mg, 2-mg, or 4-mg for 16 weeks and assessed using PRO measures. RESULTS: At week 16, baricitinib 4-mg and 2-mg significantly reduced itch severity (Itch Numeric Rating Scale (NRS) (BREEZE-AD1: percent change from baseline -36.6% and -29.4% vs. placebo (-12.0%), p≤.001 and p≤.05; BREEZE-AD2: -47.2% and -46.9% vs. placebo (-16.6%), p≤.001). Baricitinib significantly reduced SCORing AD (SCORAD) pruritus (4-mg in BREEZE-AD1 and 2-mg in BREEZE-AD2) and Patient Oriented Eczema Measure (POEM) itch (both doses). Improvements in skin pain severity and sleep disturbance were also observed. Improvements in AD symptoms showed higher correlations with patients' assessment of AD severity and QoL than improvements in skin inflammation. CONCLUSIONS: Baricitinib significantly improved symptoms in patients with moderate-to-severe AD. CLINICALTRIALS.GOV IDENTIFIERS: NCT03334396 (BREEZE-AD1) and NCT03334422 (BREEZE-AD2).

Interactions between the F1 and F0 parts in the Escherichia coli ATP synthase. Associations involving the loop region of C subunits.

The N-ethylmaleimide reactivity of c subunits in Escherichia coli F1F0 ATP synthase (ECF1F0) isolated from five mutants, each with a cysteine at a different position in the polar loop region (positions 39, 40, 42, 43, and 44), has been investigated. The maleimide was found to react with Cys placed at positions 42, 43, and 44 but not at 39 or 40. All copies of the c subunit reacted similarly when the Cys was at position 43 or 44. In contrast, the Cys in the mutant cQ42C reacted as two classes, with 60% reacting relatively rapidly and 40% reacting at a rate 40-fold slower. After removing F1, all copies of the c subunit in this mutant reacted equally fast. Therefore, the slow class in the cQ42C mutant represents c subunits shielded by, and probably involved directly in, the interaction of the F0 with gamma and epsilon subunits of the F1 part. Based on the estimated stoichiometry of c subunits in the ECF1F0 complex, 4 or 5 c subunits are involved in this F1 interaction. N-Ethylmaleimide modification of all of the c subunits reduced ATPase activity by only 30% in ECF1F0 from mutant cQ42C. Modification of the more rapidly reacting class had little effect on ATP hydrolysis-driven proton translocation, and did not alter the DCCD inhibition of ATPase activity. However, as those c subunits involved in the F1 interaction became modified, DCCD inhibition was progressively lost, as was coupling between ATP hydrolysis and proton translocation.

The stalk region of the Escherichia coli ATP synthase. Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer.

The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF1F0. The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping. ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39. Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition. The covalent linkage of the gamma to a c subunit had little effect on ATPase activity. However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+. No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit. Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed. Our results show that there is close interaction of the gamma and epsilon subunits for the full-length of the stalk region in ECF1F0. We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF1F0.

The gamma subunit in the Escherichia coli ATP synthase complex (ECF1F0) extends through the stalk and contacts the c subunits of the F0 part.

A mutant, in which a cysteine has been site-directed into the polar loop region of the c subunit at residue 44, has been studied. Cross-linking of the c subunit to both the gamma and epsilon subunits was observed with cupric 1,10-phenanthrolinate treatment. The linkage between the c and gamma subunits was localized to that part of the gamma subunit between residues 202-286, based on peptide analysis. Reference to the high resolution structure of F1 [Abrahams et al. (1994) Nature 370, 621-628] appears to limit this contact site to the region including residues 202-230. This segment contains 4 tyrosines and 1 tryptophan as possible reactive residues for cross-linking with the c subunit cysteine.

Synthesis of antitumour carbon-bridged imidazopyridazine carbamates.

Two carbon-bridged analogues 11 and 15 of the potent microtubule inhibitor BW1069C85 (1) have been synthesised and evaluated for antitubulin and antitumour activity in vitro. Though the compounds were somewhat less potent than BW1069C85, significant activity against tubulin polymerisation and cell proliferation was demonstrated in the assays.

Schistosoma mansoni: nitrothiazolines and the male tegument.

Within 24 hr of treatment of the mouse host with BW484C, 2-[5-nitro-2-(pivaloylimino)-4-thiazoline-3-yl]diacetamide, pairs of Schistosoma mansoni exhibited "hepatic shift" and began to leave the mesenteric veins. The tegument of the males was altered, both morphologically and physiologically, while that of females was unaffected. This morphological damage to males correlated well with therapeutic efficacy against both sexes in a range of analogues of BW484C. However, parasites removed from mice after treatment but before the hepatic shift and then maintained in vitro were far from moribund as treated males could be maintained for 8 days in vitro, although this was 5 days less than males from untreated mice. Females survived as well as control worms. In contrast, male and female S. mansoni remaining in their host after therapy were invaded by host cells in the liver after 2 days. The morphological effects and reduction of the in vitro survival of males treated in the mouse and removed after 24 hr could be simulated by in vitro exposure for 24 hr to 10(-5) M BW484C. Females were not susceptible to this regime. It was concluded that worm pairs were swept to the liver as a result of drug dependent damage to the tegument of the male and that phagocytic invasion of male and female schistosomes by host cells within the liver was an important factor in the efficacy of BW484C. The biochemical events underlying the effects on the tegument of male worms remain unknown.

Kinetics of 4-aminobutyrate:2-oxoglutarate aminotransferase from Nippostrongylus brasiliensis.

A gamma-aminobutyric acid transferase (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) preparation from Nippostrongylus brasiliensis was found to contain only one peak of enzyme activity with a highly basic pI of 10.5 when analysed by isoelectric focusing and chromatofocusing. This material was used in kinetic studies to demonstrate that the parasite enzyme reaction mechanism conforms to the usual binary, non-sequential ('Bi Bi Ping Pong') type found with aminotransferases. The Km for 4-aminobutyrate was 0.33 mM, the Km for 2-oxoglutarate was 0.57 mM and Ki for glutamate was 0.35 mM. In holoenzyme reconstitution experiments with the cofactor, pyridoxal 5-phosphate, the KD was 1.54 microM. The values are comparable to those reported for other tissues. Only 2-oxoglutarate could function as the keto acid substrate whereas several amino acids besides 4-aminobutyrate (beta-alanine, alpha-L-alanine, L-aspartate and L-arginine) could apparently act as substrate although the possible presence of other amino acid:2-oxoglutarate aminotransferases was not excluded. In preliminary studies on the usefulness of conventional substrate analogues as parasite gamma-aminobutyric acid transferase inhibitors only canaline was effective.

Application of a quick, simple and direct radiometric assay for 4-aminobutyrate:2-oxoglutarate aminotransferase to studies of the parasitic nematode Nippostrongylus brasiliensis.

A preparation of rat brain was used to develop a quick and simple radiometric assay for 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-T), an important enzyme in neural tissue of vertebrates and invertebrates. Application of the methodology to the parasitic nematode Nippostrongylus brasiliensis revealed that a soluble preparation of partially purified GABA-T could be recovered in high yield. This enzyme had a specific activity comparable to that observed in rat brain after similar treatment, was very stable when frozen, depended for activity upon tightly-bound pyridoxal 5-phosphate, had an apparent molecular weight of 72,000 and was strongly inhibited by NaCl. The inhibition was competitive with 4-aminobutyrate (Ki = 20 mM) but uncompetitive with 2-oxoglutarate (Ki = 160 mM).

Inhibition of acetylcholinesterase secretion from Nippostrongylus brasiliensis by benzimidazole anthelmintics.

Treatment of rats infected with Nippostrongylus brasiliensis with a single, oral therapeutic dose of the anthelmintic benzimidazole carbamates oxfendazole or mebendazole resulted, 24 hr later, in a marked reduction (60-90%) in the secretion of a low molecular weight acetylcholinesterase from the parasites when they were incubated in vitro. This effect coincided with the expulsion of parasites from the host as a result of the therapy. When parasites were incubated in vitro with 0.1 mM oxfendazole, mebendazole, flubendazole, parbendazole, cambendazole or thiabendazole a similar effect was observed; with oxfendazole and mebendazole the effect was apparent within 1 hr and lasted for at least 4 hr after removal to fresh, drug-free medium. Whether treated in the host or in vitro the reduction in secretion was balanced by an equivalent rise in acetylcholinesterase activity within the parasites. It is suggested that the inhibition of protein secretion may be a specific manifestation of a general effect of these compounds on microtubule function.

Changes in the acetylcholinesterase activity of the nematode Nippostrongylus brasiliensis following treatment with benzimidazoles in vivo.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) activities of Nippostrongylus brasiliensis were measured following treatment of the hosts with oxfendazole and mebendazole. Drug treatment of N. Brasiliensis caused large, sustained increases in enzyme activity in male and female worms which coincided with the expulsion of the nematodes from the small intestine of the host. It is suggested that the increases in acetylcholinesterase activity may be due to an inhibition by the benzimidazole carbamates of the secretion of enzyme to the exterior of the worms which, in turn, leads to expulsion through failure of the so-called "chemical holdfast".

Colchicine binding in the rat tapeworm, Hymenolepis diminuta.

A fraction with enriched colchicine-binding properties prepared from Hymenolepis diminuta was found to possess many of the properties of tubulin isolated from other sources. Colchicine was bound by a simple saturable process with a dissociation constant of 13.2 microM. The binding capability decayed with a half-life of about 5 h. Binding was unaffected by lumicolchicine, was competitively inhibited by podophyllotoxin (inhibition constant of 4.8 microM) and showed an apparent stimulation by vinblastine sulphate. Sodium chloride also appeared to stimulate the binding process. The ligand/receptor complex had a molecular weight of approx. 112 000 as determined by gel filtration. On the basis of this biochemical and pharmacological evidence it was concluded that the colchicine receptor in the supernatant fraction of the H. diminuta homogenate was almost certainly tubulin. Refinement of the preparation should facilitate further studies on the mode of action of certain types of anthelmintic compound.

Lysosomes and tegument pathology in the chemotherapy of schistosomiasis with 1,7-bis(p-aminophenoxy)heptane (153C51).

One therapeutic oral dose (400 mg/kg) of 153C51 administered to infested mice caused pathological changes in the dorsal region of the tegument of male Schistosoma mansoni during the period 3--24 h after treatment. These changes occurred prior to the 'hepatic shift'. At the ultrastructural level they consisted of a gradual accumulation in the tegument epidermis of numerous membranous inclusions with the characteristics of residual lysosomes and changes in the localization of acid phosphatase, a lysosomal enzyme. It seemed likely that these changes were due to inhibition or exhaustion of enzyme in the epidermis, followed by re-synthesis of enzyme in the cell bodies and its export to the epidermis. The elimination of hydrolytic activity from lysosomes in the epidermis would explain the accumulation of residual lysosomes. Drug-treated parasites retained their disguise of host red blood cell ghost antigens as shown by indirect fluorescent antibody-labelling and, therefore, it seemed unlikely that immunological factors could be important in producing the tegument pathology.