Application of hydrogeological and biological research for the lysimeter experiment performance under simulated municipal landfill condition

The size and chemical composition of leachates migrating into the aquifer are dependent on the parameters of the waste and the storage conditions. Lysimeter studies allow us to determine the size and chemical composition of leachates as well as the leachate water balance. Lysimeter studies were conducted on a 230-L municipal waste sample for 6 months. During the tests, the specific electrolyte conductivity, pH, Eh, and temperature, as well as the chemical composition, microbiological analysis, and profiling of physiological population level using EcoPlate™ microarrays were measured in collected leachate samples. During the entire experiment, the amounts of inflow and outflow from lysimeters were measured. To assess the existence of significant differences in the chemical component concentrations in leachates, use of Principal Component Analysis was taken into account. The maximum EC value from leachate from the lysimeter was 33 mS/cm. High concentrations of ammonium ion (up to approx. 1400 mg dm−3), chlorides (up to approx. 6800 mg dm−3), and iron (up to approx. 31 mg dm−3) were observed in the effluents. The number of enterococci in May reached 53,000 cells/100 ml. By contrast, the number of these microorganisms was about 15,000 and 16,000 CFU/100 ml in January and April, respectively. Community-level physiological profiling indicates that the activity and functional diversity of microorganisms were higher in the leachate samples obtained in winter compared to effluents collected from lysimeters in spring

Metagenomic Functional Profiling Reveals Differences in Bacterial Composition and Function During Bioaugmentation of Aged Petroleum-Contaminated Soil

Our objective was to study the bacterial community changes that determine enhanced removal of petroleum hydrocarbons from soils subjected to bioaugmentation with the hydrocarbon-degrading strains Rhodococcus erythropolis CD 130, CD 167, and their combination. To achieve this, a high-throughput sequencing of the 16S rRNA gene was performed. The changes in the bacterial community composition were most apparent the day after bacterial inoculation. These changes represented an increase in the percentage abundance of Rhodococcus and Pseudomonas genera. Surprisingly, members of the Rhodococcus genus were not present after day 91. At the end of the experiment, the bacterial communities from the CD 130, CD 167, and control soils had a similar structure. Nevertheless, the composition of the bacteria in the CD 130 C CD 167 soil was still distinct from the control. Metagenomic predictions from the 16S rRNA gene sequences showed that the introduction of bacteria had a significant influence on the predicted pathways (metabolism of xenobiotics, lipids, terpenoids, polyketides, and amino acids) on day one. On day 182, differences in the abundance of functional pathways were also detected in the CD 130 and CD 130 C CD 167 soils. Additionally, we observed that on day one, in all bioaugmented soils, the alkH gene was mainly contributed by the Rhodococcus and Mycobacterium genera, whereas in non-treated soil, this gene was contributed only by the Mycobacterium genus. Interestingly, from day 91, the Mycobacterium genus was the main contributor for the tested genes in all studied soils. Our results showed that hydrocarbon depletion from the analyzed soils resulted from the activity of the autochthonous bacteria. However, these changes in the composition and function of the indigenous bacterial community occurred under the influence of the introduced bacteria

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia

: Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD+) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD+ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet

Biological characterization and targeted delivery of NAMPT inhibitors in hematological malignancies

Cancer cells must reprogram their metabolism to sustain their continuous growth and proliferation. They rely on fast nicotinamide adenine dinucleotide (NAD+) turnover for their energy supply, which is crucial for their survival. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate limiting enzyme involved in the production of NAD+. The inhibition of NAMPT induces NAD+ depletion and is a therapeutic strategy to selectively eliminate malignant cells highly dependent on NAD+. Until now, several NAMPT inhibitors have been developed and, despite their high efficacy in preclinical studies, their clinical activity was limited. Therefore, there is still a need to synthesize novel more potent NAMPT inhibitors and/or to specifically target them to cancer cells. In this work, new NAMPT inhibitors were characterized for their biological activities in vitro and in vivo on haematological malignancies. Moreover, few selected NAMPT inhibitors were conjugated to an anti-CD138 antibody, targeting inhibitors to multiple myeloma cells. All tested compounds exhibited low nanomolar cytotoxicity against various haematological cancer cell lines. They depleted NAD+ after 24 hours of drug treatment, followed by ATP depletion and mitochondrial membrane depolarization, what finally led to various types of cell death (apoptosis and necrosis). Treatments with NAMPT inhibitors caused time-dependent reactive oxygen species (ROS) production that was rescued by catalase (CAT). Unfortunately, the most potent compounds in vitro (i.e., FEI199) did not translate their efficacy in vivo. Indeed, among all compounds (JJ08, FEI191, FEI199) administered into mouse xenograft model of human Burkitt lymphoma only JJ08 completely eradicated tumour growth. Moreover, the pharmacokinetics (PK) profile of JJ08 was like that of APO866. Some of the synthesized analogues were further modified to be attached to an anti-CD138 antibody to create antibody-drug-conjugates (ADCs). CD138 is an antigen selectively overexpressed on MM cells and its responsible for its malignant growth. The cytotoxicity of the NAMPT inhibitors ADCs (NAMPTi_ADCs) was assessed on target-positive and negative cells. Additionally, a positive control ADC was synthesized with amanitin as a payload (ADC-ama) instead of the inhibitor. The native NAMPT inhibitors used as payloads had IC50 on target-positive (RPMI8226) cells between 2.3-118 nM, and the anti-CD138 antibody itself was not toxic to the cells. The ADC-ama resulted in very potent activity against RPMI8226 cells (IC50 0.1 nM). However, the NAMPTi_ADCs did not exhibit cytotoxicity on RPMI8226 cells up to 500 nM, except for SF316 and SF319, sharing the same payload. The compounds induced 60-70% cell death at 500 nM concentrations, which was considered unspecific. Taken together, these findings demonstrate the mode of action and the very potent cytotoxicity of different NAMPT inhibitors in vitro, and the high efficacy of JJ08 in vivo. Despite the successful conjugation of the selected compounds to the anti-CD138 antibody, they were not effective in vitro. Therefore, further optimization of the NAMPTi_ADCs is needed. -- Les cellules cancéreuses reprogramment leur métabolisme pour maintenir leur prolifération et leur croissance continuelles. Elles se reposent sur un renouvellement rapide du nicotinamide adénine dinucléotide (NAD+) pour leur apport en énergie, ce qui est crucial pour les cellules cancéreuses. La nicotinamide phosphorybosyltransferase (NAMPT) est l’enzyme limitante impliquée dans la production de NAD+. L’inhibition de l’enzyme NAMPT et donc la déplétion du NAD+ est une stratégie thérapeutique pour éliminer de manière sélective les cellules malignes qui sont fortement dépendantes du NAD+. Jusqu’à maintenant, beaucoup d’inhibiteurs de NAMPT ont été développés et malgré leur forte efficacité dans les études précliniques, leur activité clinique était limitée. Il reste donc un besoin de synthétiser de nouveaux inhibiteurs du NAMPT plus performants et/ou spécialement ciblés contre les cellules cancéreuses. Dans ce travail, de nouveaux inhibiteurs du NAMPT ont été caractérisés pour leur activité biologique in vitro et in vivo sur des cellules hématologiques malignes. De plus, certains inhibiteurs de NAMPT sélectionnés ont été conjugués à un anticorps anti-CD138 ciblant les myélomes multiples (MM). Tous les composés ont montré à basses concentrations molaires une cytotoxicité contre diverses lignées cellulaires cancéreuses hématologiques. Le NAD+ était déplété après 24 heures de traitement, suivi par une déplétion de l’ATP et une dépolarisation de la membrane mitochondriale, qui ont finalement amené à différents types de morts cellulaires (apoptose et nécrose). Le traitement avec les inhibiteurs de NAMPT a causé la production d’espèces réactives de l’oxygène (ERO) dépendant du temps, production qui a pu être prévenue par la catalase (CAT). Finalement, le composé le plus efficace in vitro était le 35a (un IC50 de 18pM sur des cellules ML2), malgré ça, l’efficacité in vitro des inhibiteurs les plus efficaces ne s’est pas retranscrite dans les expériences in vivo. Tous les composés ont été administrés dans des modèles de souris xénogreffes soit de lymphomes Burkitt (JJ08, FEI191, FEI199) ou de leucémie myéloïdes aigue (35a, 47), Seul JJ08 a complétement éradiqué la croissance de la tumeur. De plus, le profil pharmacocinétique de JJ08 était similaire à celui d’APO866. Certains des analogues synthétisés ont été modifiés pour permettre leur attachement à l’anticorps anti- CD138 dans le but de créer des conjugués anticorps-médicaments. Le CD138 est un antigène surexprimé chez les myélomes multiples qui est responsable de leur croissance maligne. La cytotoxicité des inhibiteurs de NAMPT conjugués anticorps-médicaments a été testée sur des cellules cibles positives et négatives. De plus, un contrôle positif du conjugué anticorps-médicament a été synthétisé avec l’amanitine comme payload. Les inhibiteurs de NAMPT utilisés comme paylaods avaient un IC50 sur les cellules cibles positives (RPMI8226) entre 2.3 et 118 nM. L’anticorps anti-CD138 n’était pas toxique lui-même pour les cellules. Le conjugué anticorps-médicament amanitine a montré des résultats extrêmement efficaces sur les cellules RPMI8226 (IC50 0.1nM). Malgré cela, les conjugués anticorps-médicaments inhibiteurs de NAMPT n’ont pas montré de cytotoxicité sur les RPMI8226 jusqu’à 500nM, à l’exception de SF316 et SF 319 qui partagent le même payload. Ces composés ont induit une mort cellulaire autour de 60 à 70% avec une concentration de 500nM qui a été considérée comme non spécifique. En conclusion, ces observations démontrent le mode d’action et la forte cytotoxicité de différents inhibiteurs de NAMPT in vitro, ainsi que la grande efficacité de JJ08 in vivo. Bien que la conjugaison des composés choisis à l’anticorps anti-CD138 ai été un succès, ils n’ont pas montré d’efficacité in vitro. Par conséquent, une meilleure optimisation des inhibiteurs de NAMPT conjugués anticorps-médicaments est nécessaire

Method of purification of post-production condensates from polyester polyol production

Nowadays, the topics of closed-loop and eco-design are raised very often, especially in the chemical industry. To combine development with these trends, Purinova Sp. z o.o. has focused on pursuing the closed-loop use of post-production condensate from polyester polyols production. To this end, purification and distillation processes have been adapted, both at the laboratory and production scale, to receive treated condensate with decreased Chemical Oxygen Demand (COD) index. The method involves connected purification of production condensate by returning condensate to the top of the distillation column during polycondensation and two stages distillation system afterwards. The method allows for decreasing COD index and contents of diethylene glycol and 1,4-dioxane. The resulting technology has consequently allowed the use of tailored distillation in the purification of post-production condensates in the production of polyester polyols. Furthermore, the quality of the condensate obtained allowed it to be used in the closed loop of the production plant

Innovative method to prepare polyester polyols intended for the production of footwear soles

Opracowano nową technologię otrzymywania polioli poliestrowych z zastosowaniem surowca stanowiącego nowo wyodrębniony strumień uboczny procesu utleniania cykloheksanu. Innowacja polegała na wprowadzeniu etapu rozdziału strumienia produktów odpadowych, pozwalającego na uzyskanie frakcji o składzie gwarantującym optymalne właściwości zsyntetyzowanego poliolu przeznaczonego do systemów poliuretanowych używanych do produkcji spodów obuwniczych. Otrzymana frakcja prepoliestrowa charakteryzowała się dużą zawartością związków alifatycznych dwufunkcyjnych i niewielkim udziałem małocząsteczkowych monokwasów, przyczyniających się do pogorszenia jakości wyrobu końcowego. Powstająca w wyniku rozdziału frakcja stanowi surowiec alternatywny dla powszechnie stosowanego kwasu adypinowego. Oceniono właściwości spodów obuwniczych wytworzonych z mikroporowatych poliuretanów na bazie uzyskanych poliestrów. Stwierdzono, że otrzymane poliestry wykazują właściwości zbliżone (lub lepsze) do cech poliestrów wytwarzanych dotychczas przez Spółkę Purinova.A new technology for the production of polyester polyols using a newly separated side stream from the cyclohexane oxidation process as a raw material was developed. The innovation consisted in the introduction of the stage of separation of the waste product stream, which allowed to obtain a fraction with composition suitable to ensure optimal properties of the synthesized polyol intended for polyurethane systems used in the production of footwear soles. The obtained prepolyester fraction was characterized by a high content of bifunctional aliphatic compounds, with a minor share of low molecular weight monoacids, contributing to the deterioration of final product quality. The fraction resulting from the separation is an alternative raw material to the commonly used adipic acid. The parameters of footwear soles manufactured from microporous polyurethanes based on the obtained polyesters were evaluated. It was found that polyesters produced from new raw material are characterized by similar or higher physical and mechanical properties in comparison to the existing products of Purinova Company

Nicotinaldehyde, a Novel Precursor of NAD Biosynthesis, Abrogates the Anti-Cancer Activity of an NAD-Lowering Agent in Leukemia

Targeting NAD depletion in cancer cells has emerged as an attractive therapeutic strategy for cancer treatment, based on the higher reliance of malignant vs. healthy cells on NAD to sustain their aberrant proliferation and altered metabolism. NAD depletion is exquisitely observed when NAMPT, a key enzyme for the biosynthesis of NAD, is inhibited. Growing evidence suggests that alternative NAD sources present in a tumor environment can bypass NAMPT and render its inhibition ineffective. Here, we report the identification of nicotinaldehyde as a novel precursor that can be used for NAD biosynthesis by human leukemia cells. Nicotinaldehyde supplementation replenishes the intracellular NAD level in leukemia cells treated with NAMPT inhibitor APO866 and prevents APO866-induced oxidative stress, mitochondrial dysfunction and ATP depletion. We show here that NAD biosynthesis from nicotinaldehyde depends on NAPRT and occurs via the Preiss–Handler pathway. The availability of nicotinaldehyde in a tumor environment fully blunts the antitumor activity of APO866 in vitro and in vivo. This is the first study to report the role of nicotinaldehyde in the NAD-targeted anti-cancer treatment, highlighting the importance of the tumor metabolic environment in modulating the efficacy of NAD-lowering cancer therapy

Synthesis and Structure-activity Relationship of New Nicotinamide Phosphoribosyltransferase Inhibitors with Antitumor Activity on Solid and Haematological Cancer

Cancer cells are highly dependent on Nicotinamide phosphoribosyltransferase (NAMPT) activity for proliferation, therefore NAMPT represents an interesting target for the development of anti-cancer drugs. Several compounds, such as FK866 and CHS828, were identified as potent NAMPT inhibitors with strong anti-cancer activity, although none of them reached the late stages of clinical trials. We present herein the preparation of three libraries of new inhibitors containing (pyridin-3-yl)triazole, (pyridin-3-yl)thiourea and (pyridin-3/4-yl)cyanoguanidine as cap/connecting unit and a furyl group at the tail position of the compound. Antiproliferative activity in vitro was evaluated on a panel of solid and haematological cancer cell lines and most of the synthesized compounds showed nanomolar or sub-nanomolar cytotoxic activity in MiaPaCa-2 (pancreatic cancer), ML2 (acute myeloid leukemia), JRKT (acute lymphobalistic leukemia), NMLW (Burkitt lymphoma), RPMI8226 (multiple myeloma) and NB4 (acute myeloid leukemia), with lower IC50 values than those reported for FK866. Notably, compounds 35a, 39a and 47 showed cytotoxic activity against ML2 with IC50 = 18, 46 and 49 pM, and IC50 towards MiaPaCa-2 of 0.005, 0.455 and 2.81 nM, respectively. Moreover, their role on the NAD+ synthetic pathway was demonstrated by the NAMPT inhibition assay. Finally, the intracellular NAD+ depletion was confirmed in vitro to induced ROS accumulation that cause a time-dependent mitochondrial membrane depolarization, leading to ATP loss and cell death.European Union No671881Ministerio de Ciencia e Innovación PID2020-116460RB-100Associazione Italiana per la Ricerca sul Cancro IG#22098Italian Ministry of Health PE-2016-02362694, PE-2016-0236307

Anticancer Activities of Novel Nicotinamide Phosphoribosyltransferase Inhibitors in Hematological Malignancies

Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD+ production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD+ and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD+ precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies