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Natural products (NPs) as source of new antibiotics
There is a renewed interest in NPs as a source of chemical diversity and lead generation in drug discovery. The availability of new analytical chemistry and molecular biology methods and the gradual transition away from 11 the mono-substance therapy towards multi-targeted therapy have resumed research in NPs[47]. The Plant Kingdom for example, represents an enormous reservoir of the most structurally diverse compounds, offering rich mines of biologically valuable molecules. In this respect, plant secondary metabolites (PSMs) are proven to be active against a vast number of microbial species, making these molecules an interesting starting point in drug development[48].
Why NPs as new antibacterials?
There are several reasons why NPs are of main interest in the discovery of drugs as new antibacterials:
• The abundant scaffold diversity of NPs is coupled with a “purposeful design”. Most microbes and plants make by-products with a purpose, usually to afford advantages for survival in environments threatening their growth. It is reasoned that these defense systems produced would have an inherent antimicrobial activity, giving an evolutionary advantage to the producer organism. In the search for novel antibiotics, it would be difficult to imagine a more specific source of naturally occurring antimicrobials than nature itself[49].
• NPs structures have a high chemical diversity, biochemical specificity and other molecular properties that make them favorable as lead structures for drug discovery. This serves to differentiate them from libraries of synthetic and combinatorial compounds[50].
• Suitability of NPs for modulating chemical reactions and protein interactions: NPs can be viewed as a population of privileged structures selected by evolutionary pressures to interact with a wide variety of proteins and other biological targets for specific purposes[50].
• NPs identified as antibacterial leads typically possess bacterial permeability, i.e. access to the target. Hence, avoiding the need for engineering in bacterial membrane and cell wall permeability, a situation often encountered with synthetic leads[49].
Therefore, it seems that the main task in NPs research is to identify and isolate appropriate PSMs from a rich pool provided by the Plant Kingdom. The fact that one of the reasons plants biosynthesize these compounds is the defense against microorganisms justifies the efforts aimed towards finding PSMs with antimicrobial activity[48].
Which are the shortcomings of NPs?
Despite the above-mentioned advantages, NPs have some limitations that could explain the fact that these mixtures are currently not commercialized as antibacterials:
• NPs library and high-throughput screening (HTS): The heterogeneity of NPs library samples adds
two additional levels of complexity to the screening process[50]:
o Once a response for the sample is detected by HTS, one or more rounds of chemical purification and biological assays might be necessary for identifying and isolating the active components in the mixture.
o The complexity of crude or semi-pure NPs libraries challenges the robustness of HTS technology.
• Usefulness of in vitro data: Active concentrations in in vitro conditions frequently cannot be reached in vivo and so in vitro results cannot correlate with in vivo activities of the tested compounds[48].
• Therapeutic window: most of the time therapeutic windows of active PSMs are not specified as they are for other drugs, leaving a risk of appearance of side effects. Toxicity studies, for example towards host cells, have to be carried out. Side effects of NP drugs with synthetic drugs in co-medication must also been studied[48].
• Variable composition of NP drugs: relative concentrations of individual components in a sample are not know precisely, and are highly susceptible to external geographical, climatic and ecological factors that influence plant metabolism[48,50].
In such way, biological activity is just one of the necessary prerequisites for NP substances to be applied as pharmaceuticals. Despite of the shortcomings, PSMs are still considered a good starting point in the search for/design/development of new antimicrobial agents[48]. Biological activities of NPs aiming a therapeutic application must also be verified in controlled clinical trials, before they can be submitted as conventional drugs[47].
Intelligent mixtures
Plant species often respond to stress by increasing the biosynthesis of different classes of molecules, rather than just an individual PSM[48]. In light of the cost-benefit aspect, it is beneficial for a plant to produce mixtures of small molecules made of simple and ubiquitous building blocks that easily diffuse to reach a maximal number of potential targets. Networks of synergic molecules could greatly enhance the chance for fitness, closer to a trial and error-type of selection[51].
Mixtures of bioactive compounds are widely claimed to be superior over monosubstances, and a synergistic therapeutic effect might be mainly responsible for this[48]. Thus, a PSM does not need to completely inhibit a target but partially inhibit different targets within a network. Other authors state that multi-target effects predominate over synergistic mechanisms[51]. Of importance is the fact that production of diverse compounds is an evolutionary advantage developed by plants to overcome emerging resistance. It is much harder for microorganisms to shield themselves simultaneously against all these different PSMs. In this way, it is highly likely that the mixture of these compounds is in fact responsible of the onset of their activity[48].
Therefore, in the search for new potent antimicrobials, research should move towards the investigation of combination of substances to achieve efficacy[48]. However, we need to scientifically address the therapeutic potential of NPs. The reengineering of botanical drugs would make the area of medicinal plant research more reproducible, and may even lead to the de novo engineering of more intelligent mixtures than the ones provided by plants. By doing so, our understanding of pharmacological efficacy would be based on measurable parameters, and also avoid the problem of variable concentrations[51].
Probably the best way of corroborating the pharmacological efficacy of NP drugs would be to reengineer the mixtures. By taking apart and reassembling all bioactive constituents, one would be able to find out which ones contribute to the final pharmacological effect either directly (interacting with particular targets) or indirectly (modulating solubility bioavailability)[51].
Mechanisms of synergy in NPs: Based on studies, the following mechanisms can be observed[47]:
• Synergistic multi-target effects: The single constituents of a natural blend affect not only one single, but also several targets; cooperating therefore in an agonistic, synergistic way.
• Pharmacokinetic or physicochemical effects: Certain components that do not possess specific pharmacological effects themselves may increase the solubility and/or the resorption rate of other constituents; enhancing thereby its bioavailability and resulting in higher effectiveness of the mixture rather than an isolated constituent.
• Interactions with resistance mechanisms of bacteria: some PSMs are able to partly or completely suppress bacterial resistance mechanisms working synergistically with antibiotics.
• The elimination or neutralization of adverse effects by compounds in the mixture: even if this is not a real synergistic effect, it generates better effectiveness in therapy. It occurs when a constituent of the mixture neutralizes or destroys another possessing toxicity.
Essential oils (EOs) as antibacterial agents
EOs are PSMs composed of complex mixtures of several components at quite different concentrations. They are characterized by 2 or 3 major components at fairly high concentrations compared to the others. Generally, these major components determine the biological properties of the EOs. The EOs components include 2 groups of distinct biological origin: terpenes and terpenoids; and phenylpropane derivatives[52].
EOs can be present in all plant organs, but are generally stored by the plant in secretory cells, cavities, canals, glandular trichomes or epidermic cells[53]. They do not appear to participate directly in plant growth and development. Instead, EOs play an important role in the protection of plants against microbial infection[48].
EOs possess antibacterial properties and have been screened worldwide as potential sources of novel antimicrobial compounds as alternatives to treat infectious diseases and promote food preservation, and for the fight against resistant bacteria[54].
Mechanisms of action of EOs: The activity of an EO can affect both the external envelope of the cell and the cytoplasm. The antimicrobial activity of EOs, similar to all natural extracts, is dependent on their chemical composition and on the concentration of the components. Generally, the antimicrobial activity is not attributable to an unique mechanism, but is instead a cascade of reactions involving the entire
bacterial cell[53].
• Targeting the membranes:
The lipophilicity typical of EOs is responsible for their interaction with bacterial membranes[55]. The hydrophobicity of EOs enable them to partition with lipids of bacterial cell membrane, disturbing the phospholipid bilayers, leading to an increased permeability[53,54,56,57].
This might cause degradation of the cell wall, damage of the cytoplasmic membrane, damage of membrane proteins involved in active transport, inactivation of enzymatic mechanisms like reduction of the intracellular adenosine triphosphate (ATP) pool via decreased ATP synthesis, leakage of cell contents, reduction of the proton motive force and membrane potential, disruption of the electron transport system and coagulation of cell contents[53,55,58].
Furthermore, some EOs can cause a change on the fatty acids profile of the bacterial cell membrane. This is induced by an increase in the percentage of unsaturated fatty acids (UFAs)
responsible for the fluidity of the membrane, causing membrane structural alterations[59]. Indeed, Kwon et al.[60] showed that EO components caused elongation and filamentation in B. cereus because septa formation was incomplete. Other effects on bacterial cell morphology, like swelling of bacterial surfaces, have also been reported[61].
• Other targets
o Components of EOs can also act on cell proteins embedded in the cytoplasmic membrane. ATPases are located in the cytoplasmic membrane and bordered by lipid molecules. Hence two mechanisms have been suggested whereby EOs components could act: lipophilic hydrocarbons could accumulate in the lipid bilayer distorting the lipid-protein interaction; or a
direct interaction of the lipophilic compounds with hydrophobic parts of the protein[55,62]. Components of EOs can directly bind to proteins affecting cell division[63] and to enzymes[64]. EOs may also affect the enzymes that are involved in fatty acid synthesis[65].
o EOs can also inhibit bacterial toxin production[66].
o Intracellular processes such as DNA/RNA synthesis can also be affected[67] as well as protein expression[68].
o Effect on ATP: EOs disrupt the cell membrane alter the intracellular and external ATP balance
such that ATP is lost through the disturbed membrane[69,70]. Other events may contribute to the intracellular ATP decrease like disrupted balance of K+ and H+ by EOs[71]and decrease ATP
synthesis as mentioned before[53].
o Effects on the metabolome: Picone et al.[72] found that glucose tends to accumulate when microbial cells are treated with EOs components and that cells are unable to metabolize the
glucose, leading to a loss of viability. An important change in the production of bacterial metabolites has also been shown[73].
o Communication between bacterial cells involves the production and detection of diffusible signal molecules and it is known as Quorum Sensing (QS). The discovery that many pathogenic bacteria employ QS to regulate their virulence makes this system interesting as target for antimicrobial therapy[74]. EOs components can affect QS in bacteria[75,76].
Considering the large number of different groups of chemical compounds present in EOs, it is most likely that their antibacterial activity is not attributable to one specific mechanism but that there are several targets in the cell. Not all of the mechanisms are separate targets; some are affected as a consequence of another mechanism being targeted. Hence, studies have concluded that whole EOs have a greater antibacterial activity than the major components alone, suggesting that the minor components are critical to the activity and many have a synergistic effect or potentiating influence[58].
Furthermore, EOs in combination with other antimicrobial can improve antimicrobial effectiveness. EOs could have important implications for the development and implementation of therapeutic antimicrobial strategies[54].
Moreover, the presence of multiple compounds and a possible multiplicity of action in EOs is favorable since drugs that interact with multiple targets are highly desirable because they have a low likelyhood for or can delay development of bacterial resistance[77,78]. It is likely more difficult for bacteria to develop resistance to the multi-component EOs than to common antibiotics that are often composed of only a single molecular entity[54].
EOs vs. MDR bacteria
In the fight against MDR strains, multidrug therapy has become of paramount importance[48] and EOs are good sources for combination therapy[79]. In this context, EOs can overcome several mechanisms of bacterial resistance:
• Receptor or active site modification: Nicolson et al.[80], have shown that the phenolic diterpene totarol potentiated the activity of methicillin against MRSA by significantly reducing the expression of penicillin-binding protein 2a (PBP 2a). This protein, encoded by MecA has reduced affinity for β-lactam antibiotics (BLA).
• Enzymatic degradation and modification of the drug: ESBL are enzymes that confer resistance to BLA like third generation cephalosporins. Cinnamaldehyde and eugenol hydrogen bonded with catalytic and other crucial amino acid residues of ESBL enzymes of pathogenic bacteria[81], which may restore BLA activity.
• Decreased outer membrane (OM) permeability: The OM of Gram-negative bacteria functions as a permeability barrier for many agents. EOs like thymol and carvacrol are membrane permeabilizers[57] that could enhance penetration of antibiotics.
• Active efflux: Efflux pumps are one of the defense mechanisms employed by bacteria to reduce the accumulation of antibiotics inside the cell. EOs were able to block efflux pumps in Gram-negative bacteria[82].
Table of contents :
Abbreviations
Research context
Objectives
Presentation of the work
CHAPTER I
1. The threat of resistant bacteria
1.1. Principles of antimicrobial resistance
1.2. Sources of resistant bacteria
1.3. Multiple-drug resistant (MDR) bacteria
1.4. There is no ESKAPE?
1.5. Costs of Bacterial Resistance
1.6. Pharmaceutical industry and antibiotic discovery
1.6.1. The need of new antimicrobials
1.6.2. Why have the pharmaceutical companies abandoned antibiotic R&D?
1.6.3. What can be done to encourage antibiotic R&D?
2. Natural products (NPs) as source of new antibiotics
2.1. Why NPs as new antibacterials?
2.2. Which are the shortcomings of NPs?
2.3. Intelligent mixtures
2.4. Essential oils (EOs) as antibacterial agents
2.5. EOs vs. MDR bacteria
3. Inflammatory Bowel Diseases (IBD) and its link to bacteria
3.1. Etiology of IBD
3.1.1. Genetic factors
3.1.1.1. NOD2/CARD15
3.1.1.2. ATG16L1 and IRGM
3.1.1.3. SLC22A4/OCTN1 and SLC22A5/OCTN2
3.1.1.4. DLG5
3.1.1.5. HLA
3.1.1.6. MDR-1
3.1.2. Immune factors
3.1.2.1. Innate immunity
3.1.2.2. Adaptive immunity
3.1.2.3. Non-immune cells
3.1.3. Environmental factors
3.1.4. Bacterial factors
3.1.4.1. Evidence of the microbial influences in chronic mediated intestinal inflam
3.1.4.2. Role of microbes in the pathogenesis of IBD
3.2. Treatment of IBD
3.2.1. Anti-inflammatory and immunomodulatory drugs
3.2.1.1. 5-Aminosalicylates (5-ASA)
3.2.1.2. Corticosteroids
3.2.1.3. Thiopurines
3.2.1.4. Methotrexate
3.2.1.5. Calcineurin inhibitors
3.2.1.6. Anti-tumor necrosis factor alpha (anti-TNFα)
3.2.1.7. Anti-adhesion molecules
3.2.1.8. Agents in the pipeline
3.2.2. Microbial modulatory drugs
3.2.2.1. Antibiotics
3.2.2.2. Probiotics
3.2.2.3. Prebiotics
3.2.2.4. Enteral nutrition
3.2.2.5. Fecal microbiota transplantation (FMT)
4. Colonic-targeted delivery of drugs
4.1. pH-dependent
4.2. Time-dependent
4.3. Microbial enzyme-dependent
4.3.1. Prodrugs
4.3.2. Azo-polymeric coatings
4.3.3. Polysaccharide delivery systems
4.4. Newer technologies
4.4.1. Pressure-dependent
4.4.2. Osmotic-dependent
4.4.3. CODES™
CHAPTER II
1. Introduction
2. Materials and methods
2.1. Antibacterial agents and growth media
2.2. Microorganisms, storage and growth conditions
2.3. Determination of the Minimal Inhibitory Concentration (MIC)
2.4. Time of logarithmic reduction
2.5. Post-Antibiotic Effect (PAE)
2.6. Bacterial growth at sub-inhibitory (subMIC) concentrations
2.7. Interaction with antibiotics
3. Results and discussion
3.1. MIC and spectrum of action of CIN-102
3.2. Logarithmic reduction time of CIN-102
3.3. PAE of CIN-102
3.4. Bacterial growth a CIN-102 subMICs
3.5. Interaction of CIN-102 with antibiotics
4. Conclusion
CHAPTER III
1. Introduction
2. Materials and methods
2.1. Materials
2.2. Preparation of coated pellets
2.2.1. Preparation of drug-laded pellet starter cores
2.2.2. Pellet coating
2.3. Preparation of mini-tablets
2.4. In vitro drug release
2.5. Equilibrium solubility measurements
2.6. mDSC analysis
2.7. Mouse study
3. Results and discussion
3.1. CIN-102-loaded coated pellets
3.2. CIN-102 mini-tablets
3.3. In vivo evaluation
4. Conclusion
CHAPTER IV
1. Introduction
2. Materials and methods
2.1. Antibacterial agents and growth media
2.2. Microorganisms, storage and growth conditions
2.3. Determination of the Minimal Inhibitory Concentration (MIC)
3. Results
4. Discussion
5. Conclusion
GENERAL CONCLUSION
PERSPECTIVES
ANNEXES
RÉSUMÉ EN FRANÇAIS
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