Early combination therapy of COVID-19 in high-risk patients.

PURPOSE: Prolonged shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been observed in immunocompromised hosts. Early monotherapy with direct-acting antivirals or monoclonal antibodies, as recommended by the international guidelines, does not prevent this with certainty. Dual therapies may therefore have a synergistic effect. METHODS: This retrospective, multicentre study compared treatment strategies for corona virus disease-19 (COVID-19) with combinations of nirmatrelvir/ritonavir, remdesivir, molnupiravir, and/ or mABs during the Omicron surge. Co-primary endpoints were prolonged viral shedding (≥ 106 copies/ml at day 21 after treatment initiation) and days with SARS-CoV-2 viral load ≥ 106 copies/ml. Therapeutic strategies and risk groups were compared using odds ratios and Fisher's tests or Kaplan-Meier analysis and long-rank tests. Multivariable regression analysis was performed. RESULTS: 144 patients were included with a median duration of SARS-CoV-2 viral load ≥ 106 copies/ml of 8.0 days (IQR 6.0-15.3). Underlying haematological malignancies (HM) (p = 0.03) and treatment initiation later than five days after diagnosis (p < 0.01) were significantly associated with longer viral shedding. Prolonged viral shedding was observed in 14.6% (n = 21/144), particularly in patients with underlying HM (OR 3.5; 95% CI 1.2-9.9; p = 0.02). Clinical courses of COVID-19 were mild to moderate with only few adverse effects potentially related to combination treatment. CONCLUSION: Early combination treatment of COVID-19 effectively prevented prolonged viral shedding in 85.6% of cases. Considering the rapid viral clearance rates and low toxicity, individualized dual therapy approaches may be beneficial in high-risk patients.

Discovery of TD-0212, an Orally Active Dual Pharmacology AT1 Antagonist and Neprilysin Inhibitor (ARNI).

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy relative to ACE inhibitors but is associated with a higher risk of life-threatening angioedema due to bradykinin elevations. We hypothesized that dual AT1 (angiotensin II type 1 receptor) blockade and NEP inhibition with a single molecule would produce similar antihypertensive efficacy to omapatrilat without the risk of angioedema since ACE (the rate limiting enzyme in bradykinin metabolism) would remain uninhibited. Merging the structures of losartan (an AT1 antagonist) and thiorphan (a NEP inhibitor) led to the discovery of a novel series of orally active, dual AT1 antagonist/NEP inhibitors (ARNIs) exemplified by compound 35 (TD-0212). In models of renin-dependent and -independent hypertension, 35 produced blood pressure reductions similar to omapatrilat and combinations of AT1 receptor antagonists and NEP inhibitors. Upper airway angioedema risk was assessed in a rat tracheal plasma extravasation (TPE) model. Unlike omapatrilat, 35 did not increase TPE at antihypertensive doses. Compound 35 therefore provides the enhanced activity of dual AT1/NEP inhibition with a potentially lower risk of angioedema relative to dual ACE/NEP inhibition.

Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications.

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO2-fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The Vmax and Km for the oxygenase and carboxylase activities at 25 °C and the specificity factors (Sc/o) at 15, 25, and 35 °C were determined. The Sc/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO2 relative to O2 Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short ßA-ßB loop and a C-terminal extension that forms a ß-hairpin structure (ßE-ßF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, ß-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO2 fixation in arctic diatom Rubiscos.

Policy trends and reforms in the German DRG-based hospital payment system.

A central structural point in all DRG-based hospital payment systems is the conversion of relative weights into actual payments. In this context policy makers need to address (amongst other things) (a) how the price level of DRG-payments from one period to the following period is changed and (b) whether and how hospital payments based on DRGs are to be differentiated beyond patient characteristics, e.g. by organizational, regional or state-level factors. Both policy problems can be and in international comparison often are empirically addressed. In Germany relative weights are derived from a highly sophisticated empirical cost calculation, whereas the annual changes of DRG-based payments (base rates) as well as the differentiation of DRG-based hospital payments beyond patient characteristics are not empirically addressed. Rather a complex set of regulations and quasi-market negotiations are applied. There were over the last decade also timid attempts to foster the use of empirical data to address these points. However, these reforms failed to increase the fairness, transparency and rationality of the mechanism to convert relative weights into actual DRG-based hospital payments.

Discovery of TD-4306, a long-acting ß2-agonist for the treatment of asthma and COPD.

A multivalent approach focused on amine-based secondary binding groups was applied to the discovery of long-acting inhaled ß2-agonists. Addition of amine moieties to the neutral secondary binding group of an existing ß2-agonist series was found to provide improved in vivo efficacy, but also led to the formation of biologically active aldehyde metabolites which were viewed as a risk for the development of these compounds. Structural simplification of the scaffold and blocking the site of metabolism to prevent aldehyde formation afforded a potent series of dibasic ß2-agonists with improved duration of action relative to their monobasic analogs. Additional optimization led to the discovery of 29 (TD-4306), a potent and selective ß2-agonist with potential for once-daily dosing.

Multivalent design of long-acting ß(2)-adrenoceptor agonists incorporating biarylamines.

A series of potent ß2-adrenoceptor agonists incorporating a biarylamine secondary binding group was identified. The previously reported milveterol (5), identified by a multivalent approach and containing a typical ß2-agonist primary binding group linked via a phenethylamine linker to a hydrophilic secondary binding group, served as an initiation point. A more hydrophobic set of secondary binding groups was explored, prepared rapidly from a common intermediate by Buchwald-Hartwig amination. TD-5471 (25), a potent and selective full agonist of the human ß2-adrenoceptor, was identified as the most promising agent. It is potent, with slow onset in an in vitro guinea pig trachea model and shows a dose-dependent and long duration of action in an in vivo guinea pig model of bronchoprotection. TD-5471 is structurally differentiated from milveterol and its long duration of action is consistent with a correlation with hydrophobicity observed in other long-acting ß2-agonist discovery programs.

A multivalent approach to the discovery of long-acting ß(2)-adrenoceptor agonists for the treatment of asthma and COPD.

A multivalent approach was applied to the design of long-acting inhaled ß(2)-adrenoceptor agonists. A series of dimeric arylethanolamines based on the short acting ß(2)-adrenoceptor agonist albuterol were prepared, varying the nature and length of the linker between the basic nitrogens. None of the C(2)-symmetric dimers demonstrated increased potency, however dimer 5j, derived from 4-phenethylamine, was found to have increased binding potency in vitro relative to the parent monomer. Optimization of this structure led to the identification of 22 (milveterol) which demonstrates high potency in vitro and long duration of action in a guinea pig model of bronchoprotection.

Messenger RNA degradation is initiated at the 5' end and follows sequence- and condition-dependent modes in chloroplasts.

Using reporter gene constructs, consisting of the bacterial uidA (GUS) coding region flanked by the 5' and 3' regions of the Chlamydomonas rbcL and psaB genes, respectively, we studied the degradation of mRNAs in the chloroplast of Chlamydomonas reinhardtii in vivo. Extending the 5' terminus of transcripts of the reporter gene by more than 6 nucleotides triggered rapid degradation. Placing a poly(G) tract, known to pause exoribonucleases, in various positions downstream of the 5' terminus blocked rapid degradation of the transcripts. In all these cases the 5' ends of the accumulating GUS transcripts were found to be trimmed to the 5' end of the poly(G) tracts indicating that a 5' → 3' exoribonuclease is involved in the degradation process. Several unstable variants of the GUS transcript could not be rescued from rapid degradation by a poly(G) tract showing that sequence/structure-dependent modes of mRNA breakdown exist in the Chlamydomonas chloroplast. Furthermore, degradation of poly(G)-stabilized transcripts that accumulated in cells maintained in the dark could be augmented by illuminating the cells, implying a photo-activated mode of mRNA degradation that is not blocked by a poly(G) tract. These results suggest sequence- and condition-dependent 5' → 3' mRNA-degrading pathways in the chloroplast of C. reinhardtii.

Concomitant angiotensin AT1 receptor antagonism and neprilysin inhibition produces omapatrilat-like antihypertensive effects without promoting tracheal plasma extravasation in the rat.

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy but cause high incidence of angioedema. We examined whether dual inhibition of angiotensin AT1 receptor (ARB) and NEP (ARB-NEPI, valsartan-candoxatril) provides similar efficacy to omapatrilat without the risk of angioedema. Activity of test compounds at the targets was assayed using fluorescence-based enzyme assays (ACE, NEP, aminopeptidase P) or competition binding assays (AT1). Target engagement in vivo (ACE, AT1, and NEP) was quantified by measuring inhibition of angiotensin-pressor responses and potentiation of atrial natriuretic peptide-induced urinary cyclic guanosine monophosphate (cGMP) output in rats. Tracheal plasma extravasation (TPE) was used as a surrogate to assess propensity of compounds to promote upper airway angioedema. Antihypertensive efficacy in renin-dependent and -independent states was measured in spontaneously hypertensive rats and deoxycorticosterone acetate salt hypertensive rats, respectively. Administration of omapatrilat and coadministration of valsartan and candoxatril blocked angiotensin induced vasopressor responses and potentiated atrial natriuretic peptide-induced increase in urinary cGMP output. In spontaneously hypertensive rats, valsartan, omapatrilat, and valsartan-candoxatril combination all produced reduction in blood pressure to a similar extent, whereas candoxatril was ineffective. In deoxycorticosterone acetate rats, omapatrilat, candoxatril, and valsartan-candoxatril combination but not valsartan produced reduction in blood pressure. Antihypertensive doses of omapatrilat produced robust increases in TPE; by contrast, valsartan, candoxatril, or their combination did not increase TPE. Pretreatment with icatibant, a bradykinin B2 antagonist, abolished omapatrilat-induced TPE but not its antihypertensive effects. On the background of NEP inhibition, suppression of the renin-angiotensin system through ARB and ACE inhibition shows a similar antihypertensive efficacy but exerts differential effects on bradykinin metabolism and TPE indicative of reduced risk of angioedema. Thus, dual AT1 receptor blockade and NEP inhibition is potentially an attractive approach to retain the excellent antihypertensive effects of omapatrilat but with a superior safety profile.

THRX-198321 is a bifunctional muscarinic receptor antagonist and beta2-adrenoceptor agonist (MABA) that binds in a bimodal and multivalent manner.

Biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester (THRX-198321) is a single molecule composed of a muscarinic acetylcholine receptor (mAChR) antagonist moiety, represented by the fragment MA, linked by a C9 polymethylene chain to a ß(2)-adrenoceptor (ß(2)AR) agonist moiety, represented by the fragment 8-hydroxy-5-((R)-1-hydroxy-2-methylamino-ethyl)-1H-quinolin-2-one (BA). THRX-198321 exhibited high affinity for mAChR (M(2) pK(I,App) = 10.57 ± 0.09; M(3) pK(I,App) = 10.07 ± 0.11) and ß(2)AR (pK(I,App) = 9.54 ± 0.15), with potent mAChR antagonist (M(2) pK(I,Fn) = 9.69 ± 0.23; M(3) pK(I,Fn) = 10.05 ± 0.17) and ß(2)AR agonist (pEC(50) = 9.25 ± 0.02) activities. Consistent with multivalent interactions, THRX-198321 binding affinity was >300-fold higher at mAChR and 29-fold higher at ß(2)AR relative to its monovalent fragments biphenyl carbamic acid piperidinyl ester (MA) and BA, respectively. THRX-198321 was a competitive antagonist at mAChR (M(2) pK(B) = 9.98 ± 0.13; M(3) pK(B) = 10.31 ± 0.89), whereas THRX-198321 agonist activity at ß(2)AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on ß(2)AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-(3)H]scopolamine methyl chloride (M(2) pEC(50,diss) = 6.73 ± 0.10; M(3) pEC(50,diss) = 5.02 ± 0.14) and [4,6-propyl-(3)H]dihydroalprenolol (ß(2)AR pEC(50,diss) = 3.82 ± 0.25). The carbostyril-linker fragment (BA-L) binds to the allosteric site of mAChR (M(2) pEC(50,diss) = 5.06 ± 0.03; M(3) pEC(50,diss) = 4.15 ± 0.25), whereas the MA fragment binds to the allosteric site of ß(2)AR (pEC(50,diss) = 3.60 ± 0.18). Collectively, these observations suggest that THRX-198321 exhibits a multivalent bimodal orientation in the orthosteric and allosteric binding pockets of mAChR and ß(2)AR, a phenomenon that may be unique to this class of molecule.

Next generation multifunctional angiotensin receptor blockers.

Angiotensin receptor blockers (ARBs) are well-tolerated drugs that are known to be useful for inhibiting activity of the renin-angiotensin (RAS) system, treating hypertension and reducing the risk for cardiovascular disease. However, inhibition of the RAS does not control all pathophysiological mechanisms of hypertension or cardiovascular risk and many patients continue to suffer from cardiovascular events and metabolic disturbances despite being treated with an ARB, an angiotensin-converting enzyme inhibitor or both, in addition to other standard therapies for cardiovascular disease. Recently, it has become apparent that bifunctional molecules can be designed that do more than just block AT(1) receptors and that can target additional mechanisms of hypertension, cardiovascular disease and diabetes besides just increased activity of the renin-angiotensin system. Specifically, next generation ARBs are becoming available that are intended to not only antagonize AT(1) receptors, but also block endothelin receptors, function as nitric oxide donors, inhibit neprilysin activity and increase natriuretic peptide levels, or stimulate the peroxisome proliferator-activated receptor gamma (PPARgamma). In this review, we: (1) discuss the potential importance of multifunctional ARBs that can reduce cardiovascular and metabolic risk through multiple mechanisms that go beyond just inhibition of the renin-angiotensin system and (2) describe specific examples of next generation ARBs in development that are intended to do more than simply block AT(1) receptors.

Designing selective, high affinity ligands of 5-HT1D receptor by covalent dimerization of 5-HT1F ligands derived from 4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide.

We demonstrate here that covalent dimerization of 5-HT 1 ligands is an effective design strategy to modulate affinity and selectivity of 5-HT 1 ligands. This approach was applied to LY-334370, a selective agonist of 5-HT 1F receptor, to generate structurally well-defined divalent molecules. Radioligand binding assays to three cloned 5-HT 1 receptor subtypes (5-HT 1B, 5-HT 1D, 5-HT 1F) demonstrated that the affinity of a series of homologous dimers varied significantly upon exploration of three structural variables (linker length, attachment position, functionality). In particular, the series of C 3-to-C 3 linked dimers derived from a monomer ( 3) showed high binding affinity to 5-HT 1D (for example, K i approximately 0.3 nM for dimer 8) but did not bind to 5-HT 1F ( K i > 0.01 mM), providing >10000-fold subtype selectivity. Results from a functional assay (rabbit saphenous vein contraction) demonstrate that certain dimers are 5-HT 1 receptor agonists.

Phosphodiesterase type 4 inhibitors cause proinflammatory effects in vivo.

Phosphodiesterase type 4 (PDE(4)) inhibitors are currently being evaluated as potential therapies for inflammatory airway diseases. However, this class of compounds has been shown to cause an arteritis/vasculitis of unknown etiology in rats and cynomolgus monkeys. Studies in rodents have demonstrated the anti-inflammatory effects of PDE(4) inhibitors on lipopolysaccharide (LPS)-induced airway inflammation. The aim of this work was to assess the direct effects of PDE(4) inhibitors on inflammatory cells and cytokine levels in the lung in relation to therapeutic effects. The effects of the PDE(4) inhibitors 3-cyclo-propylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast) and 3-(cyclopentyloxy)-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide (piclamilast) were assessed in vivo, using BALB/c mice, and in vitro, in unstimulated human endothelial and epithelial cell lines. In BALB/c mice, LPS challenge caused an increase in neutrophils in bronchoalveolar lavage (BAL) and lung tissue and BAL tumor necrosis factor-alpha levels, which were inhibited by treatment with either roflumilast or piclamilast (30-100 mg/kg subcutaneously). However, roflumilast and piclamilast alone (100 mg/kg) caused a significant increase in plasma and lung tissue keratinocyte-derived chemokine (KC) levels, and lung tissue neutrophils. In vitro, both piclamilast and roflumilast caused an increase in interleukin (IL)-8 release from human umbilical vein endothelial cells but not BEAS-2B cells, suggesting that one source of the increased KC may be endothelial cells. At doses that antagonized an LPS-induced inflammatory response, the PDE(4) inhibitors possessed proinflammatory activities in the lung that may limit their therapeutic potential. The proinflammatory cytokines KC and IL-8 therefore may provide surrogate biomarkers, both in preclinical animal models and in the clinic, to assess potential proinflammatory effects of this class of compounds.

Ground and excited states proton transfer reactions of 1,8-diaminonaphthalene in perchloric acid solutions.

The proton-transfer reaction of 1,8-diaminonaphthalene (1,8-DAN) in acidic medium was studied by means of fluorescence and picosecond spectroscopic techniques. It has been found that there are three different forms of 1,8-DAN in the ground state, but only two different forms in the excited state. The absorption of the mono-cation form of 1,8-DAN is found to be a mixture of the neutral form and the di-cation form. However, the emission is found to be the same as the neutral form, due to the fast dissociation of the mono-cation form once it is excited. The fluorescence of the mono-cation form of 1,8-DAN shows a small shift under different excitation wavelengths. The di-cation form only fluoresces if no free water cluster is available as a proton acceptor. The reaction in the excited state is shown to be a diabatic quenching reaction. With the help of quantum yields and fluorescence lifetime measurements these results are interpreted in terms of a new photochemical scheme. All dissociation and quenching rate constants, pKa and kq, have been determined.

Specific roles of 5' RNA secondary structures in stabilizing transcripts in chloroplasts.

RNA secondary structures, e.g. stem-loops that are often found at the 5' and 3' ends of mRNAs, are in many cases known to be crucial for transcript stability but their role in prolonging the lifetime of transcripts remains elusive. In this study we show for an essential RNA-stabilizing stem-loop at the 5' end of rbcL gene transcripts in Chlamydomonas that it neither prevents ribonucleases from binding to the RNA nor impedes their movement along the RNA strand. The stem-loop has a formative function in that it mediates folding of a short sequence around its base into a specific RNA conformation, consisting of a helical and single-stranded region, i.e. the real structure required for longevity of rbcL transcripts in chloroplasts. Disturbing this structure renders transcripts completely unstable, even if the sequence of this element is not altered. The requirement of a specific 5' sequence and structure for RNA longevity suggests an interaction of this element with a trans-acting factor that protects transcripts from rapid degradation in chloroplasts.

Report: workshop on mediastinal grey zone lymphoma.

There are several indications that classical Hodgkin lymphoma (cHL) and at least a proportion of cases of Primary Mediastinal B cell Lymphoma (PMBL) are derived from B cells at similar stages of differentiation and share common pathogenic mechanisms. The first indication was the existence of mediastinal grey zone lymphomas as identified in the 4th International Symposium on HL, with clinical, histological and immunohistochemical features intermediate between cHL and PMBL. Second, both tumor types resemble a cell that is developmentally situated in-between the germinal center reaction and a plasma cell. Third, cHL and PMBL were found to have similar gene expression profiles, including the lack of immunoglobulin expression and low levels of B cell receptor signalling molecules, and the secretion of molecules like the chemokine TARC and the prominent expression of IL-13 receptors. Fourth, both entities were found to have common genomic aberrancies, notably in 2p15 and 9p24, the sites of the REL oncogene and the tyrosine kinase gene JAK2, respectively. Further comparison of both lymphoma types may provide further insight in the pathogenic mechanisms and allow the design of diagnostic algorithms to sort out the small number of so-called mediastinal grey zone lymphomas, that appear to be intermediate between PMBL and cHL.

Nature of the main contaminant in the anti malaria drug primaquine diphosphate: a qualitative isomer analysis.

The main contaminant of primaquine (CAS 90-34-6) has been tentatively identified, by using two liquid chromatography (LC) methods and liquid chromatography-mass spectrometry (LC-MS), as the positional isomer quinocide (CAS 525-61-1). The first LC system was equipped with a chiral Chirex (S)-VAL and (R)-NEA column and the second system was equipped with an Adsorbosphere Nucleotide-Nucleoside 7 micro column. Comparison of the main contaminant of primaquine with an authentic quinocide standard by using co-chromatography in both LC systems and LC-MS (mass fragmentation) supported the hypothesis. The toxicity of quinocide batch 17172, primaquine batch 16039, and the drug primaquine diphosphate batch 20107 used in pharmaceutical industry, and the effect of the substances on respiratory and electron transport chain were compared in the eucaryotic unicellular fresh water green alga Chlamydomonas reinhardtii as a model system. These studies suggest that minor amount of other related substances can contribute more to the toxicity of the drug primaquine diphosphate than the positional isomer quinocide.

Changes in the 5'-untranslated region of the rbcL gene accelerate transcript degradation more than 50-fold in the chloroplast of Chlamydomonas reinhardtii.

Using uidA (beta-glucuronidase; GUS) reporter gene constructs, the 5'-untranslated region (UTR) of the Chlamydomonas chloroplast rbcL gene was screened by deletion and mutational analysis for the presence of a promoter element that previous studies implied to reside within the first 63 base pairs of the UTR. Deleting a large segment of the rbcL 5'UTR in a 3'-->5' direction to position +36, changing the remaining 36 base pairs at the 5' end of the UTR, and increasing by five base pairs the distance between the rbcL 5'UTR and the basic promoter element located at position -10 did not abolish transcription from the basic rbcL promoter. It is concluded that the apparent loss of transcriptional activity found in earlier studies after deletion of sequences downstream of the transcription initiation site is due to the synthesis of very unstable transcripts that escape detection by Northern analysis and in vivo transcription assays. Chimeric rbcL:GUS transcripts containing changes in the beginning of the 5'UTR that affect RNA secondary structure are estimated to be at least 50 times less stable than rbcL:GUS transcripts containing the non-modified rbcL 5'UTR sequence.