Combined Inhibition of C5 and CD14 Attenuates Systemic Inflammation in a Piglet Model of Meconium Aspiration Syndrome.

BACKGROUND: Meconium aspiration syndrome (MAS) is a severe lung condition affecting newborns and it can lead to a systemic inflammatory response. We previously documented complement activation and cytokine release in a piglet MAS model. Additionally, we showed ex vivo that meconium-induced inflammation was dependent on complement and Toll-like receptors. OBJECTIVES: To assess the efficacy of the combined inhibition of complement (C5) and CD14 on systemic inflammation induced in a forceful piglet MAS model. METHODS: Thirty piglets were randomly allocated to a treatment group receiving the C5-inhibitor SOBI002 and anti-CD14 (n = 15) and a nontreated control group (n = 15). MAS was induced by intratracheal meconium instillation, and the piglets were observed for 5 h. Complement, cytokines, and myeloperoxidase (MPO) were measured by ELISA. RESULTS: SOBI002 ablated C5 activity and the formation of the terminal complement complex in vivo. The combined inhibition attenuated the inflammasome cytokines IL-1ß and IL-6 by 60 (p = 0.029) and 44% (p = 0.01), respectively, and also MPO activity in the bronchoalveolar fluid by 42% (p = 0.017). Ex vivo experiments in human blood revealed that the combined regimen attenuated meconium-induced MPO release by 64% (p = 0.008), but there was only a negligible effect with single inhibition, indicating a synergic cross-talk between the key molecules C5 and CD14. CONCLUSION: Combined inhibition of C5 and CD14 attenuates meconium-induced inflammation in vivo and this could become a future therapeutic regimen for MAS.

Stratification of responders towards eculizumab using a structural epitope mapping strategy.

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.

Analysis of mammalian alcohol dehydrogenase 5 (ADH5): characterisation of rat ADH5 with comparisons to the corresponding human variant.

Alcohol dehydrogenase 5 (ADH5) is a member of the mammalian alcohol dehydrogenase family of yet undefined functions. ADH5 was first identified at the DNA level in human and deer mouse. A rat alcohol dehydrogenase structure of similar type has been isolated at the cDNA level using human ADH5 as a screening probe, where the rat cDNA structure displayed several atypical properties. mRNA for rat ADH5 was found in multiple tissues, especially in the kidney. In vitro translation experiments indicated that rat ADH5 is expressed as efficiently as ADH1 and furthermore, rat ADH5 was readily expressed in COS cells fused to Green Fluorescent Protein. However, no soluble ADH5 protein could be heterologously expressed in Escherichia coli cells with expression systems successfully used for other mammalian ADHs, including fused to glutathione-S-transferase. Molecular modelling of the enzyme indicated that the protein does not fold in a productive way, which can be the explanation why no stable and active ADH5 has been isolated. These results indicate that ADH5, while readily expressed at the mRNA level, does not behave similarly to other mammalian ADHs investigated. The results, in vitro and in silico, suggest an unstable ADH5 structure, which can explain for why no active and stable protein can be isolated. Further possibilities are conceivable: the ADH5 protein may have to interact with a stabiliser, or the gene is actually a pseudogene.

Endotoxin reduction in monoclonal antibody preparations using arginine.

A monoclonal antibody preparation was found to be contaminated with endotoxin. Several commercial endotoxin removal steps were attempted but failed to produce a significant reduction due to the fact that the endotoxin was associated with the antibody. Here, several methods for endotoxin removal based on immobilizing monoclonal antibodies to chromatographic media have been evaluated. A crucial step in this process was to dissociate the endotoxin from the protein surface for subsequent removal. This was accomplished by introducing different buffer additives in the mobile phase. In agreement with previous reports, non-ionic detergents efficiently removed endotoxin, but it was also found that 0.5M arginine performed equally well. Since arginine is a non-toxic common amino acid that can be readily removed, it was selected and successfully used in large-scale experiments. With this method, endotoxin could be reduced to <0.2 EU mg(-1) with recovery of the target protein being >95%. Since this procedure is easily integrated into the existing processes of mAb purification, it offers advantages in speed, cost and effort.

Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump.

A parallel chromatographic procedure for the purification of milligram amounts of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quarternary ammonium group with respect to elution and regeneration properties and the 75 cm(2) Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimised. By introducing this step into the protocol, endotoxin levels could be reduced approximately 100-fold to

Preparative parallel protein purification (P4).

In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.

Enzymatic mechanism of low-activity mouse alcohol dehydrogenase 2.

ADH2 is a member of one of the six classes of mammalian alcohol dehydrogenases, which catalyze the reversible oxidation of alcohols using NAD(+) as a cofactor. Within the ADH2 class, the rodent enzymes form a subgroup that exhibits low catalytic activity with all substrates that were examined, as compared to other groups, such as human ADH2. The low activity can be ascribed to the rigid nature of the proline residue at position 47 as the activity can be increased by approximately 100-fold by substituting Pro47 with either His (as found in human ADH2), Ala, or Gln. Mouse ADH2 follows an ordered bi-bi mechanism, and hydride transfer is rate-limiting for oxidation of benzyl alcohols catalyzed by the mutated and wild-type enzymes. Structural studies suggest that the mouse enzyme with His47 has a more closed active site, as compared to the enzyme with Pro47, and hydride transfer can be more efficient. Oxidation of benzyl alcohol catalyzed by all forms of the enzyme is strongly pH dependent, with pK values in the range of 8.1-9.3 for turnover numbers and catalytic efficiency. These pK values probably correspond to the ionization of the zinc-bound water or alcohol. The pK values are not lowered by the Pro47 to His substitution, suggesting that His47 does not act as a catalytic base in the deprotonation of the zinc ligand.

The mammalian alcohol dehydrogenases interact in several metabolic pathways.

Mammalian alcohol dehydrogenases (ADHs), including ADH1-ADH5/6, interact extensively in the oxidation and reduction of alcohols and aldehydes. ADH1 and ADH2 are involved in several metabolic pathways besides the oxidation of ethanol and have also been shown to be involved in drug transformations. The ADH2 enzymes show further complexity among the species, e.g. in enzymatic characteristics where the rodent forms essentially lack ethanol-oxidizing capacity. ADH3 (glutathione-dependent formaldehyde dehydrogenase) has been shown to catalyze the reductive breakdown of S-nitrosoglutathione, indicating involvement in nitric oxide metabolism. Mass spectrometry identified the major enzymatic product as glutathione sulfinamide. This reductive breakdown directly interferes with the formaldehyde scavenging that has been proposed to be the physiological action of ADH3. The human ADH5 and rodent ADH6 seem to be the corresponding enzymes due to their similar behavior. None of these latter ADHs have so far been assigned to any function. They can be expressed as recombinant proteins but no enzymatic activity has been detected.

Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase.

AIMS: Celecoxib is a novel selective cyclooxygenase-2 inhibitor, which is subject to extensive hepatic metabolism. The aims of the present in vitro investigation were 1) to compare the rate of celecoxib hydroxylation by different genetic variants of cytochrome P450 2C9 (CYP2C9), and 2) to identify the enzyme(s) involved in the formation of the major metabolite carboxycelecoxib. METHODS: Hydroxycelecoxib formation was studied in human liver microsomes from 35 genotyped livers, as well as in yeast microsomes with recombinant expression of different P450 variants. Carboxycelecoxib formation was studied in liver microsomes incubated in the absence or presence of liver cytosol. The metabolites were identified and quantified by h.p.l.c. In addition, hydroxycelecoxib oxidation by different variants of recombinant human alcohol dehydrogenase (ADH1-3) was analysed by spectrophotometric monitoring of NADH generation from NAD+. RESULTS: The intrinsic clearance of celecoxib hydroxylation was significantly lower for yeast-expressed CYP2C9.3 (0.14 ml min-1 nmol-1 enzyme) compared with CYP2C9.1 (0.44 ml min-1 nmol-1 enzyme). In human liver microsomes, a significant 2-fold decrease in the rate of hydroxycelecoxib formation was evident in CYP2C9*1/*3 samples compared with CYP2C9*1/*1 samples. There was also a marked reduction (up to 5.3 times) of hydroxycelecoxib formation in a liver sample genotyped as CYP2C9*3/*3. However, the CYP2C9*2 samples did not differ significantly from CYP2C9*1 in any of the systems studied. Inhibition experiments with sulphaphenazole (SPZ) or triacetyloleandomycin indicated that celecoxib hydroxylation in human liver microsomes was mainly dependent on CYP2C9 and not CYP3A4. The further oxidation of hydroxycelecoxib to carboxycelecoxib was completely dependent on liver cytosol and NAD+. Additional experiments showed that ADH1 and ADH2 catalysed this reaction in vitro with apparent K m values of 42 micro m and 10 micro m, respectively, whereas ADH3 showed no activity. CONCLUSIONS: The results confirm that CYP2C9 is the major enzyme for celecoxib hydroxylation in vitro and further indicate that the CYP2C9*3 allelic variant is associated with markedly slower metabolism. Furthermore, it was shown for the first time that carboxycelecoxib formation is dependent on cytosolic alcohol dehydrogenase, presumably ADH1 and/or ADH2.