Bioremediation

From Wikipedia, the free encyclopedia

Bioremediation is a process used to treat contaminated media, including water, soil and subsurface material, by altering environmental conditions to stimulate growth of microorganisms and degrade the target pollutants. In many cases, bioremediation is less expensive and more sustainable than other remediation alternatives.^[1] Biological treatment is a similar approach used to treat wastes including wastewater, industrial waste and solid waste.

Most bioremediation processes involves oxidation-reduction reactions where either an electron acceptor (commonly oxygen) is added to stimulate oxidation of a reduced pollutant (e.g. hydrocarbons) or an electron donor (commonly an organic substrate) is added to reduce oxidized pollutants (nitrate, perchlorate, oxidized metals, chlorinated solvents, explosives and propellants).^[2] In both these approaches, additional nutrients, vitamins, minerals, and pH buffers may be added to optimize conditions for the microorganisms. In some cases, specialized microbial cultures are added (bioaugmentation) to further enhance biodegradation. Some examples of bioremediation related technologies are phytoremediation, mycoremediation, bioventing, bioleaching, landfarming, bioreactor, composting, bioaugmentation, rhizofiltration, and biostimulation.

Chemistry

Most bioremediation processes involve oxidation-reduction (Redox) reactions where a chemical species donates an electron (electron donor) to a different species that accepts the electron (electron acceptor). During this process, the electron donor is said to be oxidized while the electron acceptor is reduced. Common electron acceptors in bioremediation processes include oxygen, nitrate, manganese (III and IV), iron (III), sulfate, carbon dioxide and some pollutants (chlorinated solvents, explosives, oxidized metals, and radionuclides). Electron donors include sugars, fats, alcohols, natural organic material, fuel hydrocarbons and a variety of reduced organic pollutants. The redox potential for common biotransformation reactions is shown in the table.

Process	Reaction	Redox potential (Eh in mV)
aerobic	O2 + 4e− + 4H+ → 2H2O	600 ~ 400
anaerobic
denitrification	2NO3− + 10e− + 12H+ → N2 + 6H2O	500 ~ 200
manganese IV reduction	MnO2 + 2e− + 4H+ → Mn2+ + 2H2O	400 ~ 200
iron III reduction	Fe(OH)3 + e− + 3H+ → Fe2+ + 3H2O	300 ~ 100
sulfate reduction	SO42− + 8e− +10 H+ → H2S + 4H2O	0 ~ −150
fermentation	2CH2O → CO2 + CH4	−150 ~ −220

Aerobic

Aerobic bioremediation is the most common form of oxidative bioremediation process where oxygen is provided as the electron acceptor for oxidation of petroleum, polyaromatic hydrocarbons (PAHs), phenols, and other reduced pollutants. Oxygen is generally the preferred electron acceptor because of the higher energy yield and because oxygen is required for some enzyme systems to initiate the degradation process. Numerous laboratory and field studies have shown that microorganisms can degrade a wide variety of hydrocarbons, including components of gasoline, kerosene, diesel, and jet fuel. Under ideal conditions, the biodegradation rates of the low- to moderate-weight aliphatic, alicyclic, and aromatic compounds can be very high. As the molecular weight of the compound increases, so does the resistance to biodegradation.^{[citation needed]}

Common approaches for providing oxygen above the water table include landfarming, composting and bioventing. During landfarming, contaminated soils, sediments, or sludges are incorporated into the soil surface and periodically turned over (tilled) using conventional agricultural equipment to aerate the mixture. Composting accelerates pollutant biodegradation by mixing the waste to be treated with a bulking agent, forming into piles, and periodically mixed to increase oxygen transfer.  Bioventing is a process that increases the oxygen or air flow into the unsaturated zone of the soil which increases the rate of natural in situ degradation of the targeted hydrocarbon contaminant^[3].

Approaches for oxygen addition below the water table include recirculating aerated water through the treatment zone, addition of pure oxygen or peroxides, and air sparging. Recirculation systems typically consist of a combination of injection wells or galleries and one or more recovery wells where the extracted groundwater is treated, oxygenated, amended with nutrients and reinjected. However, the amount of oxygen that can be provided by this method is limited by the low solubility of oxygen in water (8 to 10 mg/L for water in equilibrium with air at typical temperatures). Greater amounts of oxygen can be provided by contacting the water with pure oxygen or addition of hydrogen peroxide (H₂O₂) to the water. In some cases, slurries of solid calcium or magnesium peroxide are injected under pressure through soil borings. These solid peroxides react with water releasing H₂O₂ which then decomposes releasing oxygen. Air sparging involves the injection of air under pressure below the water table. The air injection pressure must be great enough to overcome the hydrostatic pressure of the water and resistance to air flow through the soil.^{[citation needed]}

Anaerobic

Typical contaminated groundwater systems contain high amounts of organic and inorganic contaminants, and high levels of anaerobic microorganisms.^[4] The anaerobic microorganisms that dominate these environments are widely beneficial due to the difficulty in using aerobic microorganisms in the presence of highly reducing sediment material. Anaerobic bioremediation involves the addition of an electron donor to: 1) deplete background electron acceptors including oxygen, nitrate, oxidized iron and manganese and sulfate; and 2) stimulate the biological and/or chemical reduction of the oxidized pollutants. This approach can be employed to treat a broad range of contaminants including chlorinated ethenes. (PCE, TCE, DCE, VC), chlorinated ethanes (TCA, DCA), chloromethanes (CT, CF), chlorinated cyclic hydrocarbons, various energetics (e.g., perchlorate,^[5] RDX, TNT), and nitrate. Hexavalent chromium (Cr[VI]) and uranium (U[VI]) can be reduced to less mobile and/or less toxic forms (e.g., Cr[III], U[IV]). Similarly, reduction of sulfate to sulfide (sulfidogenesis) can be used to precipitate certain metals (e.g., zinc, cadmium). The choice of substrate and the method of injection depend on the contaminant type and distribution in the aquifer, hydrogeology, and remediation objectives. Substrate can be added using conventional well installations, by direct-push technology, or by excavation and backfill such as permeable reactive barriers (PRB) or biowalls. Slow-release products composed of edible oils or solid substrates tend to stay in place for an extended treatment period. Soluble substrates or soluble fermentation products of slow-release substrates can potentially migrate via advection and diffusion, providing broader but shorter-lived treatment zones. The added organic substrates are first fermented to hydrogen (H₂) and volatile fatty acids (VFAs). The VFAs, including acetate, lactate, propionate and butyrate, provide carbon and energy for bacterial metabolism.^{[citation needed]}

Cadmium

Toxic heavy metals pose an array of dangers for species in over 10% of the world's farmlands and can be traced back to human activities 90% of the time.^[6] One heavy metal that requires much attention is cadmium. Cadmium is an extremely dangerous heavy metal partially due to its long half-life of over twenty years.^[7] These dangers can be felt by plants and animals alike resulting in diseases and ailments such as lung cancer, homeostatic disruption, growth inhibition, and damage to the reproductive, cardiovascular, renal, and hepatic systems.^[7] Although the vast majority of microorganisms are sensitive to heavy metal toxicity, in the recent decades, studies have found seventy different species of bacterium which are resistant to the toxic effects of cadmium.^[6] Studies have been done in order to test the effectiveness of biosorption and bioaccumulation of cadmium when put to use in the field in order to immobilize it from biological influence. Biosorption has been found to be a favorable technique when trying to immobilize cadmium and similar heavy metals, because it is specific to the metal that one is aiming to secure.^[8] These studies have found that there are two specific biological pathways for resistance to toxic metals in microorganisms including metal-binding proteins and efflux systems.^[9] The efflux system of gram-positive bacterium for cadmium is often a single protein known as the CadA protein which binds to the cadmium atom/molecule and removes it from the cell.^[9] In gram-negative bacterium, the mechanisms for ridding toxic metals like cadmium is often a network of multiple proteins which transport the toxic metals out of the cell.^[9]

Uranium

Hexavalent uranium contamination is a large global problem in areas such as, near uranium mines, and near weapon crafting facilities. The typical, very costly, method for removing uranium from ground water is to pump the water out, treat it above ground, then pump it back to its original location. Some ways that have been attempted to remediate the contaminates are biosorption and bioaccumulation^[10], a third option has been shown to have a more stable outcome, which is reduction of uranium (VI) to uranium (IV) which is immobile in solution and settles to the bottom^[11]. A wide range of microorganisms have shown to be able to utilize uranium for this reaction including Pseudomonas sp., Pantoea sp., Enterobacter sp., several Geobacter species, and Thermus scotoductus^[11]. The difficulties with this method are determining what methods and conditions are optimal for this type of bioremediation, including pH, type of substrate (electron donor), and salt concentrations.

Lead

Lead is commonly found in wastewaters as it is released from manufacturing facilities that utilize the metal or that recycle it. Lead as an environmental contaminant can cause many adverse health effects in animals and humans, and higher cases in young children in developing countries . Multiple species of bacteria have been shown to utilize different methods for removing lead from soils or ground water including biosorption, biofilm-mediated, and cell surface hydrophobicity^[11]. The removal of lead from water and soils has many difficulties such as an increased energy consumption, introduction of additional chemicals, and pollution from byproducts. The use of Microorganisms for bioremediation is an environmentally friendly process that can reach the goals of removing the contaminant from the environment. Pseudomonas aeruginosa in a previous study was shown to be able to adsorb 61.2% of lead from a water source in optimal conditions indicating that it could be useful for bioremediation of lead^[11]. This species of Pseudomonas was also shown to be able to remove soil bound lead showing more versatility than only that of water containing lead^[11].

Chromium

Chromium is the second largest heavy metal in contaminated environmental systems like soil and water. It can be introduced into the environment from human activities and through natural processes. Chromium can exist in nature in two forms, Trivalent Chromium (Cr III) and Hexavalent Chromium (Cr VI) with Cr (VI) being the most toxic as it can denature proteins and has mutagenic effects^[12]. Many strategies have been used to remove Chromium from environmental systems. Many methods involve high prices, high energy requirements, and generation of dangerous byproducts^[12]. The eco-friendly way to remediate the system from the Chromium is a method known as bioadsorption as the heavy metal has an affinity for cell wall components. Two species of bacterial organisms with the highest adsorption are Psuedomonas and Aeromonas cavisiae^[12]. The bioremedation of chromium from contaminated systems can take two paths for the most effective route. At concentrations of 50 mg/L of Cr (VI) or lower, biological reduction of Cr (VI) to Cr (III) predominates, at concentrations above 50 mg, bioadsorption dominates^[12]. The adsorption method showed a reduction of 70% for concentrations of 5-500 mg/L with a maximum of 94% at 100 mg/L. The biological reduction pathway had a maximum reduction at 60 mg/L at initial concentrations of 250 mg/L^[12].

Additives

In the event of biostimulation, adding nutrients that are limited to make the environment more suitable for bioremediation, nutrients such as nitrogen, phosphorus, oxygen, and carbon may be added to the system to improve effectiveness of the treatment.^[13]

Many biological processes are sensitive to pH and function most efficiently in near neutral conditions. Low pH can interfere with pH homeostasis or increase the solubility of toxic metals. Microorganisms can expend cellular energy to maintain homeostasis or cytoplasmic conditions may change in response to external changes in pH. Some anaerobes have adapted to low pH conditions through alterations in carbon and electron flow, cellular morphology, membrane structure, and protein synthesis.^[14]

Limitations

Only biodegradable contaminants can be transformed using bioremediation processes.^[15] Some compounds, such as highly chlorinated compounds, heavy metals, and radionuclides are not readily biodegradable.^[16][17][18] Also, microbes sometimes do not fully biodegrade a pollutant and may end up producing a more toxic compound.^[18] For example, under anaerobic conditions, the reductive dehalogenation of TCE may produce vinyl chloride, which is a known carcinogen.^[16] Therefore, more research is required to see if the products from biodegradation are less persistent and less toxic than the original contaminant.^[18] Thus, the metabolic and chemical pathways of the microorganisms of interest must be known.^[16] In addition, knowing these pathways will help develop new technologies that can deal with sites that have uneven distributions of a mixture of contaminants.^[15]

Also, for biodegradation to occur, there must be a microbial population with the metabolic capacity to degrade the pollutant, an environment with the right growing conditions for the microbes, and the right amount of nutrients and contaminants.^[15][17] The biological processes used by these microbes are highly specific, therefore, many environmental factors must be taken into account and regulated as well.^[15][16] Thus, bioremediation processes must be specifically made in accordance to the conditions at the contaminated site.^[16] Also, because many factors are interdependent, small-scale tests must be performed before carrying out the procedure at the contaminated site.^[17] However, it is difficult to extrapolate the results from the small-scale test studies into big field operations.^[15] Lastly, the process of bioremediation is longer and can be more expensive than other conventional options such as land filling and incineration.^[15][16]

Genetic Engineering Applications

The use of genetic engineering to create organisms specifically designed for bioremediation has great potential.^[19] Two category of genes can be inserted in the organism: degradative genes which encode proteins required for the degradation of pollutants, and reporter genes that are able to monitor pollutant levels.^[20] An example of a degradation gene is biphenyl dioxygenase, which has been transformed in E.Coli to degrade PCB (polychlorinated biphenyl).^[20] An example of a reporter gene is lux, which can act as a biosensor for detecting the Hg2+ concentration in E.Coli.^[20] Numerous members of Pseudomonas have also been modified with the lux gene, but for the detection of the polyaromatic hydrocarbon naphthalene. A field test for the release of the modified organism has been successful on a moderately large scale.^[21]

There are concerns surrounding release and containment of genetically modified organisms into the environment due to the potential of horizontal gene transfer.^[22] Genetically modified organisms are classified and controlled under the Toxic Substances Control Act of 1976 under United States Environmental Protection Agency.^[23] Measures have been created to address these concerns. Organisms can be modified such that they can only survive and grow under specific sets of environmental conditions. Outside of environmental conditions they were designed for, they lose their biodegradation ability or undergo self destruction.^[22] To survive, a signal (the pollutant) is required. In the absence of the signal, a suicide gene is expressed which will lead to cell apoptosis.^[22] In addition, the tracking of modified organisms can be made easier with the insertion of bioluminescence genes for visual identification.^[24]

Drug metabolism

From Wikipedia, the free encyclopedia

Cytochrome P450 oxidases are important enzymes in xenobiotic metabolism.

Drug metabolism is the metabolic breakdown of drugs by living organisms, usually through specialized enzymatic systems. More generally, xenobiotic metabolism (from the Greek xenos "stranger" and biotic "related to living beings") is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism's normal biochemistry, such as any drug or poison. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds (although in some cases the intermediates in xenobiotic metabolism can themselves cause toxic effects). The study of drug metabolism is called pharmacokinetics.

The metabolism of pharmaceutical drugs is an important aspect of pharmacology and medicine. For example, the rate of metabolism determines the duration and intensity of a drug's pharmacologic action. Drug metabolism also affects multidrug resistance in infectious diseases and in chemotherapy for cancer, and the actions of some drugs as substrates or inhibitors of enzymes involved in xenobiotic metabolism are a common reason for hazardous drug interactions. These pathways are also important in environmental science, with the xenobiotic metabolism of microorganisms determining whether a pollutant will be broken down during bioremediation, or persist in the environment. The enzymes of xenobiotic metabolism, particularly the glutathione S-transferases are also important in agriculture, since they may produce resistance to pesticides and herbicides.

Drug metabolism is divided into three phases. In phase I, enzymes such as cytochrome P450 oxidases introduce reactive or polar groups into xenobiotics. These modified compounds are then conjugated to polar compounds in phase II reactions. These reactions are catalysed by transferase enzymes such as glutathione S-transferases. Finally, in phase III, the conjugated xenobiotics may be further processed, before being recognised by efflux transporters and pumped out of cells. Drug metabolism often converts lipophilic compounds into hydrophilic products that are more readily excreted.

Permeability barriers and detoxification

The exact compounds an organism is exposed to will be largely unpredictable, and may differ widely over time; these are major characteristics of xenobiotic toxic stress.^[1] The major challenge faced by xenobiotic detoxification systems is that they must be able to remove the almost-limitless number of xenobiotic compounds from the complex mixture of chemicals involved in normal metabolism. The solution that has evolved to address this problem is an elegant combination of physical barriers and low-specificity enzymatic systems.

All organisms use cell membranes as hydrophobic permeability barriers to control access to their internal environment. Polar compounds cannot diffuse across these cell membranes, and the uptake of useful molecules is mediated through transport proteins that specifically select substrates from the extracellular mixture. This selective uptake means that most hydrophilic molecules cannot enter cells, since they are not recognised by any specific transporters.^[2] In contrast, the diffusion of hydrophobic compounds across these barriers cannot be controlled, and organisms, therefore, cannot exclude lipid-soluble xenobiotics using membrane barriers.

However, the existence of a permeability barrier means that organisms were able to evolve detoxification systems that exploit the hydrophobicity common to membrane-permeable xenobiotics. These systems therefore solve the specificity problem by possessing such broad substrate specificities that they metabolise almost any non-polar compound.^[1] Useful metabolites are excluded since they are polar, and in general contain one or more charged groups.

The detoxification of the reactive by-products of normal metabolism cannot be achieved by the systems outlined above, because these species are derived from normal cellular constituents and usually share their polar characteristics. However, since these compounds are few in number, specific enzymes can recognize and remove them. Examples of these specific detoxification systems are the glyoxalase system, which removes the reactive aldehyde methylglyoxal,^[3] and the various antioxidant systems that eliminate reactive oxygen species.^[4]

Phases of detoxification

Phases I and II of the metabolism of a lipophilic xenobiotic.

The metabolism of xenobiotics is often divided into three phases:- modification, conjugation, and excretion. These reactions act in concert to detoxify xenobiotics and remove them from cells.

Phase I – modification

In phase I, a variety of enzymes act to introduce reactive and polar groups into their substrates. One of the most common modifications is hydroxylation catalysed by the cytochrome P-450-dependent mixed-function oxidase system. These enzyme complexes act to incorporate an atom of oxygen into nonactivated hydrocarbons, which can result in either the introduction of hydroxyl groups or N-, O- and S-dealkylation of substrates.^[5] The reaction mechanism of the P-450 oxidases proceeds through the reduction of cytochrome-bound oxygen and the generation of a highly-reactive oxyferryl species, according to the following scheme:^[6]

O₂ + NADPH + H⁺ + RH → NADP⁺ + H₂O + ROH

Phase I reactions (also termed nonsynthetic reactions) may occur by oxidation, reduction, hydrolysis, cyclization, decyclization, and addition of oxygen or removal of hydrogen, carried out by mixed function oxidases, often in the liver. These oxidative reactions typically involve a cytochrome P450 monooxygenase (often abbreviated CYP), NADPH and oxygen. The classes of pharmaceutical drugs that utilize this method for their metabolism include phenothiazines, paracetamol, and steroids. If the metabolites of phase I reactions are sufficiently polar, they may be readily excreted at this point. However, many phase I products are not eliminated rapidly and undergo a subsequent reaction in which an endogenous substrate combines with the newly incorporated functional group to form a highly polar conjugate.

A common Phase I oxidation involves conversion of a C-H bond to a C-OH. This reaction sometimes converts a pharmacologically inactive compound (a prodrug) to a pharmacologically active one. By the same token, Phase I can turn a nontoxic molecule into a poisonous one (toxification). Simple hydrolysis in the stomach is normally an innocuous reaction, however there are exceptions. For example, phase I metabolism converts acetonitrile to HOCH₂CN, which rapidly dissociates into formaldehyde and hydrogen cyanide.^[7]

Phase I metabolism of drug candidates can be simulated in the laboratory using non-enzyme catalysts.^[8] This example of a biomimetic reaction tends to give products that often contains the Phase I metabolites. As an example, the major metabolite of the pharmaceutical trimebutine, desmethyltrimebutine (nor-trimebutine), can be efficiently produced by in vitro oxidation of the commercially available drug. Hydroxylation of an N-methyl group leads to expulsion of a molecule of formaldehyde, while oxidation of the O-methyl groups takes place to a lesser extent.

Oxidation

• Cytochrome P450 monooxygenase system
• Flavin-containing monooxygenase system
• Alcohol dehydrogenase and aldehyde dehydrogenase
• Monoamine oxidase
• Co-oxidation by peroxidases

Reduction

• NADPH-cytochrome P450 reductase

Cytochrome P450 reductase, also known as NADPH:ferrihemoprotein oxidoreductase, NADPH:hemoprotein oxidoreductase, NADPH:P450 oxidoreductase, P450 reductase, POR, CPR, CYPOR, is a membrane-bound enzyme required for electron transfer to cytochrome P450 in the microsome of the eukaryotic cell from a FAD- and FMN-containing enzyme NADPH:cytochrome P450 reductase The general scheme of electron flow in the POR/P450 system is: NADPH → FAD → FMN → P450 → O₂

• Reduced (ferrous) cytochrome P450

During reduction reactions, a chemical can enter futile cycling, in which it gains a free-radical electron, then promptly loses it to oxygen (to form a superoxide anion).

Hydrolysis

• Esterases and amidase
• Epoxide hydrolase

Phase II – conjugation

In subsequent phase II reactions, these activated xenobiotic metabolites are conjugated with charged species such as glutathione (GSH), sulfate, glycine, or glucuronic acid. Sites on drugs where conjugation reactions occur include carboxyl (-COOH), hydroxyl (-OH), amino (NH₂), and sulfhydryl (-SH) groups. Products of conjugation reactions have increased molecular weight and tend to be less active than their substrates, unlike Phase I reactions which often produce active metabolites. The addition of large anionic groups (such as GSH) detoxifies reactive electrophiles and produces more polar metabolites that cannot diffuse across membranes, and may, therefore, be actively transported.

These reactions are catalysed by a large group of broad-specificity transferases, which in combination can metabolise almost any hydrophobic compound that contains nucleophilic or electrophilic groups.^[1] One of the most important classes of this group is that of the glutathione S-transferases (GSTs).

Mechanism	Involved enzyme	Co-factor	Location
methylation	methyltransferase	S-adenosyl-L-methionine	liver, kidney, lung, CNS
sulphation	sulfotransferases	3'-phosphoadenosine-5'-phosphosulfate	liver, kidney, intestine
acetylation	N-acetyltransferases bile acid-CoA:amino acid N-acyltransferases	acetyl coenzyme A	liver, lung, spleen, gastric mucosa, RBCs, lymphocytes
glucuronidation	UDP-glucuronosyltransferases	UDP-glucuronic acid	liver, kidney, intestine, lung, skin, prostate, brain
glutathione conjugation	glutathione S-transferases	glutathione	liver, kidney
glycine conjugation	Two step process: XM-ligase (forms a xenobiotic acyl-CoA) Glycine N-acyltransferase (forms the glycine conjugate)	glycine	liver, kidney

Phase III – further modification and excretion

After phase II reactions, the xenobiotic conjugates may be further metabolised. A common example is the processing of glutathione conjugates to acetylcysteine (mercapturic acid) conjugates.^[11] Here, the γ-glutamate and glycine residues in the glutathione molecule are removed by Gamma-glutamyl transpeptidase and dipeptidases. In the final step, the cystine residue in the conjugate is acetylated.

Conjugates and their metabolites can be excreted from cells in phase III of their metabolism, with the anionic groups acting as affinity tags for a variety of membrane transporters of the multidrug resistance protein (MRP) family.^[12] These proteins are members of the family of ATP-binding cassette transporters and can catalyse the ATP-dependent transport of a huge variety of hydrophobic anions,^[13] and thus act to remove phase II products to the extracellular medium, where they may be further metabolised or excreted.^[14]

Endogenous toxins

The detoxification of endogenous reactive metabolites such as peroxides and reactive aldehydes often cannot be achieved by the system described above. This is the result of these species' being derived from normal cellular constituents and usually sharing their polar characteristics. However, since these compounds are few in number, it is possible for enzymatic systems to utilize specific molecular recognition to recognize and remove them. The similarity of these molecules to useful metabolites therefore means that different detoxification enzymes are usually required for the metabolism of each group of endogenous toxins. Examples of these specific detoxification systems are the glyoxalase system, which acts to dispose of the reactive aldehyde methylglyoxal, and the various antioxidant systems that remove reactive oxygen species.

Sites

Quantitatively, the smooth endoplasmic reticulum of the liver cell is the principal organ of drug metabolism, although every biological tissue has some ability to metabolize drugs. Factors responsible for the liver's contribution to drug metabolism include that it is a large organ, that it is the first organ perfused by chemicals absorbed in the gut, and that there are very high concentrations of most drug-metabolizing enzyme systems relative to other organs. If a drug is taken into the GI tract, where it enters hepatic circulation through the portal vein, it becomes well-metabolized and is said to show the first pass effect.

Other sites of drug metabolism include epithelial cells of the gastrointestinal tract, lungs, kidneys, and the skin. These sites are usually responsible for localized toxicity reactions.

Factors that affect drug metabolism

The duration and intensity of pharmacological action of most lipophilic drugs are determined by the rate they are metabolized to inactive products. The Cytochrome P450 monooxygenase system is the most important pathway in this regard. In general, anything that increases the rate of metabolism (e.g., enzyme induction) of a pharmacologically active metabolite will decrease the duration and intensity of the drug action. The opposite is also true (e.g., enzyme inhibition). However, in cases where an enzyme is responsible for metabolizing a pro-drug into a drug, enzyme induction can speed up this conversion and increase drug levels, potentially causing toxicity.

Various physiological and pathological factors can also affect drug metabolism. Physiological factors that can influence drug metabolism include age, individual variation (e.g., pharmacogenetics), enterohepatic circulation, nutrition, intestinal flora, or sex differences.

In general, drugs are metabolized more slowly in fetal, neonatal and elderly humans and animals than in adults.

Genetic variation (polymorphism) accounts for some of the variability in the effect of drugs. With N-acetyltransferases (involved in Phase II reactions), individual variation creates a group of people who acetylate slowly (slow acetylators) and those who acetylate quickly, split roughly 50:50 in the population of Canada. This variation may have dramatic consequences, as the slow acetylators are more prone to dose-dependent toxicity.

Cytochrome P450 monooxygenase system enzymes can also vary across individuals, with deficiencies occurring in 1 – 30% of people, depending on their ethnic background.

Dose, frequency, route of administration, tissue distribution and protein binding of the drug affect its metabolism.

Pathological factors can also influence drug metabolism, including liver, kidney, or heart diseases.

In silico modelling and simulation methods allow drug metabolism to be predicted in virtual patient populations prior to performing clinical studies in human subjects.^[15] This can be used to identify individuals most at risk from adverse reaction.

History

Studies on how people transform the substances that they ingest began in the mid-nineteenth century, with chemists discovering that organic chemicals such as benzaldehyde could be oxidized and conjugated to amino acids in the human body.^[16] During the remainder of the nineteenth century, several other basic detoxification reactions were discovered, such as methylation, acetylation, and sulfonation.

In the early twentieth century, work moved on to the investigation of the enzymes and pathways that were responsible for the production of these metabolites. This field became defined as a separate area of study with the publication by Richard Williams of the book Detoxication mechanisms in 1947.^[17] This modern biochemical research resulted in the identification of glutathione S-transferases in 1961,^[18] followed by the discovery of cytochrome P450s in 1962,^[19] and the realization of their central role in xenobiotic metabolism in 1963.^[20][21]

Drug discovery

From Wikipedia, the free encyclopedia

In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which new candidate medications are discovered. Historically, drugs were discovered through identifying the active ingredient from traditional remedies or by serendipitous discovery. Later chemical libraries of synthetic small molecules, natural products or extracts were screened in intact cells or whole organisms to identify substances that have a desirable therapeutic effect in a process known as classical pharmacology. Since sequencing of the human genome which allowed rapid cloning and synthesis of large quantities of purified proteins, it has become common practice to use high throughput screening of large compounds libraries against isolated biological targets which are hypothesized to be disease modifying in a process known as reverse pharmacology. Hits from these screens are then tested in cells and then in animals for efficacy.

Modern drug discovery involves the identification of screening hits, medicinal chemistry and optimization of those hits to increase the affinity, selectivity (to reduce the potential of side effects), efficacy/potency, metabolic stability (to increase the half-life), and oral bioavailability. Once a compound that fulfills all of these requirements has been identified, it will begin the process of drug development prior to clinical trials. One or more of these steps may, but not necessarily, involve computer-aided drug design. Modern drug discovery is thus usually a capital-intensive process that involves large investments by pharmaceutical industry corporations as well as national governments (who provide grants and loan guarantees). Despite advances in technology and understanding of biological systems, drug discovery is still a lengthy, "expensive, difficult, and inefficient process" with low rate of new therapeutic discovery.^[1] In 2010, the research and development cost of each new molecular entity was about US$1.8 billion.^[2] Drug discovery is done by pharmaceutical companies, with research assistance from universities. The "final product" of drug discovery is a patent on the potential drug. The drug requires very expensive Phase I, II and III clinical trials, and most of them fail. Small companies have a critical role, often then selling the rights to larger companies that have the resources to run the clinical trials.

Discovering drugs that may be a commercial success, or a public health success, involves a complex interaction between investors, industry, academia, patent laws, regulatory exclusivity, marketing and the need to balance secrecy with communication.^[3] Meanwhile, for disorders whose rarity means that no large commercial success or public health effect can be expected, the orphan drug funding process ensures that people who experience those disorders can have some hope of pharmacotherapeutic advances.

History

The idea that the effect of a drug in the human body is mediated by specific interactions of the drug molecule with biological macromolecules, (proteins or nucleic acids in most cases) led scientists to the conclusion that individual chemicals are required for the biological activity of the drug. This made for the beginning of the modern era in pharmacology, as pure chemicals, instead of crude extracts of medicinal plants, became the standard drugs. Examples of drug compounds isolated from crude preparations are morphine, the active agent in opium, and digoxin, a heart stimulant originating from Digitalis lanata. Organic chemistry also led to the synthesis of many of the natural products isolated from biological sources.

Historically, substances, whether crude extracts or purified chemicals, were screened for biological activity without knowledge of the biological target. Only after an active substance was identified was an effort made to identify the target. This approach is known as classical pharmacology, forward pharmacology,^[4] or phenotypic drug discovery.^[5]

Later, small molecules were synthesized to specifically target a known physiological/pathological pathway, avoiding the mass screening of banks of stored compounds. This led to great success, such as the work of Gertrude Elion and George H. Hitchings on purine metabolism,^[6][7] the work of James Black^[8] on beta blockers and cimetidine, and the discovery of statins by Akira Endo.^[9] Another champion of the approach of developing chemical analogues of known active substances was Sir David Jack at Allen and Hanbury's, later Glaxo, who pioneered the first inhaled selective beta2-adrenergic agonist for asthma, the first inhaled steroid for asthma, ranitidine as a successor to cimetidine, and supported the development of the triptans.^[10]

Gertrude Elion, working mostly with a group of fewer than 50 people on purine analogues, contributed to the discovery of the first anti-viral; the first immunosuppressant (azathioprine) that allowed human organ transplantation; the first drug to induce remission of childhood leukaemia; pivotal anti-cancer treatments; an anti-malarial; an anti-bacterial; and a treatment for gout.

Cloning of human proteins made possible the screening of large libraries of compounds against specific targets thought to be linked to specific diseases. This approach is known as reverse pharmacology and is the most frequently used approach today.^[11]

Targets

The definition of "target" itself is something argued within the pharmaceutical industry. Generally, the "target" is the naturally existing cellular or molecular structure involved in the pathology of interest that the drug-in-development is meant to act on. However, the distinction between a "new" and "established" target can be made without a full understanding of just what a "target" is. This distinction is typically made by pharmaceutical companies engaged in discovery and development of therapeutics. In an estimate from 2011, 435 human genome products were identified as therapeutic drug targets of FDA-approved drugs.^[12]

"Established targets" are those for which there is a good scientific understanding, supported by a lengthy publication history, of both how the target functions in normal physiology and how it is involved in human pathology. This does not imply that the mechanism of action of drugs that are thought to act through a particular established target is fully understood.^{[citation needed]} Rather, "established" relates directly to the amount of background information available on a target, in particular functional information.^{[citation needed]} The more such information is available, the less investment is (generally) required to develop a therapeutic directed against the target.^{[citation needed]} The process of gathering such functional information is called "target validation" in pharmaceutical industry parlance.^{[citation needed]} Established targets also include those that the pharmaceutical industry has had experience mounting drug discovery campaigns against in the past; such a history provides information on the chemical feasibility of developing a small molecular therapeutic against the target and can provide licensing opportunities and freedom-to-operate indicators with respect to small-molecule therapeutic candidates.^{[citation needed]}

In general, "new targets" are all those targets that are not "established targets" but which have been or are the subject of drug discovery campaigns. These typically include newly discovered proteins, or proteins whose function has now become clear as a result of basic scientific research.^{[citation needed]}

The majority of targets currently selected for drug discovery efforts are proteins. Two classes predominate: G-protein-coupled receptors (or GPCRs) and protein kinases.^{[citation needed]}

Screening and design

The process of finding a new drug against a chosen target for a particular disease usually involves high-throughput screening (HTS), wherein large libraries of chemicals are tested for their ability to modify the target. For example, if the target is a novel GPCR, compounds will be screened for their ability to inhibit or stimulate that receptor (see antagonist and agonist): if the target is a protein kinase, the chemicals will be tested for their ability to inhibit that kinase.^{[citation needed]}

Another important function of HTS is to show how selective the compounds are for the chosen target, as one wants to find a molecule which will interfere with only the chosen target, but not other, related targets.^{[citation needed]} To this end, other screening runs will be made to see whether the "hits" against the chosen target will interfere with other related targets – this is the process of cross-screening.^{[citation needed]} Cross-screening is important, because the more unrelated targets a compound hits, the more likely that off-target toxicity will occur with that compound once it reaches the clinic.^{[citation needed]}

It is very unlikely that a perfect drug candidate will emerge from these early screening runs. One of the first steps is to screen for compounds that are unlikely to be developed into drugs; for example compounds that are hits in almost every assay, classified by medicinal chemists as "pan-assay interference compounds", are removed at this stage, if they were not already removed from the chemical library.^[13][14][15] It is often observed that several compounds are found to have some degree of activity, and if these compounds share common chemical features, one or more pharmacophores can then be developed. At this point, medicinal chemists will attempt to use structure-activity relationships (SAR) to improve certain features of the lead compound:

• increase activity against the chosen target
• reduce activity against unrelated targets
• improve the druglikeness or ADME properties of the molecule.

This process will require several iterative screening runs, during which, it is hoped, the properties of the new molecular entities will improve, and allow the favoured compounds to go forward to in vitro and in vivo testing for activity in the disease model of choice.

Amongst the physico-chemical properties associated with drug absorption include ionization (pKa), and solubility; permeability can be determined by PAMPA and Caco-2. PAMPA is attractive as an early screen due to the low consumption of drug and the low cost compared to tests such as Caco-2, gastrointestinal tract (GIT) and Blood–brain barrier (BBB) with which there is a high correlation.

A range of parameters can be used to assess the quality of a compound, or a series of compounds, as proposed in the Lipinski's Rule of Five. Such parameters include calculated properties such as cLogP to estimate lipophilicity, molecular weight, polar surface area and measured properties, such as potency, in-vitro measurement of enzymatic clearance etc. Some descriptors such as ligand efficiency^[16] (LE) and lipophilic efficiency^[17][18] (LiPE) combine such parameters to assess druglikeness.

While HTS is a commonly used method for novel drug discovery, it is not the only method. It is often possible to start from a molecule which already has some of the desired properties. Such a molecule might be extracted from a natural product or even be a drug on the market which could be improved upon (so-called "me too" drugs). Other methods, such as virtual high throughput screening, where screening is done using computer-generated models and attempting to "dock" virtual libraries to a target, are also often used.^{[citation needed]}

Another important method for drug discovery is de novo drug design, in which a prediction is made of the sorts of chemicals that might (e.g.) fit into an active site of the target enzyme. For example, virtual screening and computer-aided drug design are often used to identify new chemical moieties that may interact with a target protein.^[19][20] Molecular modelling^[21] and molecular dynamics simulations can be used as a guide to improve the potency and properties of new drug leads.^[22][23]. Increasingly accurate computational platforms for modelling interactions are being developed^[24].

There is also a paradigm shift in the drug discovery community to shift away from HTS, which is expensive and may only cover limited chemical space, to the screening of smaller libraries (maximum a few thousand compounds). These include fragment-based lead discovery (FBDD)^{[25][26][27][28]} and protein-directed dynamic combinatorial chemistry.^{[29][30][31][32][33]} The ligands in these approaches are usually much smaller, and they bind to the target protein with weaker binding affinity than those hits that are identified from HTS. Further modified through organic synthesis into lead compounds are often required. Such modifications are often guided by protein X-ray crystallography of the protein-fragment complex.^[34][35][36] The advantages of these approaches are that they allow more efficient screening and the compound library, although small, typically covers a large chemical space when compared to HTS.

Once a lead compound series has been established with sufficient target potency and selectivity and favourable drug-like properties, one or two compounds will then be proposed for drug development. The best of these is generally called the lead compound, while the other will be designated as the "backup".^{[citation needed]}

Nature as source

Traditionally many drugs and other chemicals with biological activity have been discovered by studying allelopathy – chemicals that organisms create that affect the activity of other organisms in the fight for survival.^[37]

Despite the rise of combinatorial chemistry as an integral part of lead discovery process, natural products still play a major role as starting material for drug discovery.^[38] A 2007 report^[39] found that of the 974 small molecule new chemical entities developed between 1981 and 2006, 63% were natural derived or semisynthetic derivatives of natural products. For certain therapy areas, such as antimicrobials, antineoplastics, antihypertensive and anti-inflammatory drugs, the numbers were higher.^{[citation needed]} In many cases, these products have been used traditionally for many years.^{[citation needed]}

Natural products may be useful as a source of novel chemical structures for modern techniques of development of antibacterial therapies.^[40]

Despite the implied potential, only a fraction of Earth’s living species has been tested for bioactivity.^{[citation needed]}

Plant-derived

Many secondary metabolites produced by plants have potential therapeutic medicinal properties. These secondary metabolites contain bind to and modify the function of proteins (receptors, enzymes, etc.). Consequently, plant derived natural products have often been used as the starting point for drug discovery.^{[41][42][43][44]}

History

Until the Renaissance, the vast majority of drugs in Western medicine were plant-derived extracts.^[45] This has resulted in a pool of information about the potential of plant species as important sources of starting materials for drug discovery.^[46] Botanical knowledge about different metabolites and hormones that are produced in different anatomical parts of the plant (e.g. roots, leaves, and flowers) are crucial for correctly identifying bioactive and pharmacological plant properties.^[46][47] Identifying new drugs and getting them approved for market has proved to be a stringent process due to regulations set by national drug regulatory agencies.^[48]

Jasmonates

Chemical structure of methyl jasmonate (JA).

Jasmonates are important in responses to injury and intracellular signals. They induce apoptosis ^[49][50] and protein cascade via proteinase inhibitor,^[49] have defense functions,^[51][52] and regulate plant responses to different biotic and abiotic stresses.^[52][53] Jasmonates also have the ability to directly act on mitochondrial membranes by inducing membrane depolarization via release of metabolites.^[54]

Jasmonate derivatives (JAD) are also important in wound response and tissue regeneration in plant cells. They have also been identified to have anti-aging effects on human epidermal layer.^[55] It is suspected that interact with proteoglycans (PG) and glycosaminoglycan (GAG) polysaccharides, which are essential extracellular matrix (ECM) components to help remodel the ECM.^[56] The discovery of JADs on skin repair has introduced newfound interest in the effects of these plant hormones in therapeutic medicinal application. ^[55]

Salicylates

Chemical structure of acetylsalicylic acid, more commonly known as Aspirin.

Salicylic acid (SA), a phytohormone, was initially derived from willow bark and has since been identified in many species. It is an important player in plant immunity, although its role is still not fully understood by scientists.^[57] They are involved in disease and immunity responses in plant and animal tissues. They have salicylic acid binding proteins (SABPs) that have shown to affect multiple animal tissues.^[57] The first discovered medicinal properties of the isolated compound was involved in pain and fever management. They also play an active role in the suppression of cell proliferation.^[49] They have the ability to induce death in lymphoblastic leukemia and other human cancer cells.^[49] One of the most common drugs derived from salicylates is aspirin, also known as acetylsalicylic acid, with anti-inflammatory and anti-pyretic properties.^[57][58]

Microbial metabolites

Microbes compete for living space and nutrients. To survive in these conditions, many microbes have developed abilities to prevent competing species from proliferating. Microbes are the main source of antimicrobial drugs. Streptomyces isolates have been such a valuable source of antibiotics, that they have been called medicinal molds. The classic example of an antibiotic discovered as a defense mechanism against another microbe is penicillin in bacterial cultures contaminated by Penicillium fungi in 1928.^{[citation needed]}

Marine invertebrates

Marine environments are potential sources for new bioactive agents.^[59] Arabinose nucleosides discovered from marine invertebrates in 1950s, demonstrated for the first time that sugar moieties other than ribose and deoxyribose can yield bioactive nucleoside structures. It took until 2004 when the first marine-derived drug was approved.^{[citation needed][dubious – discuss]} For example, the cone snail toxin ziconotide, also known as Prialt treats severe neuropathic pain. Several other marine-derived agents are now in clinical trials for indications such as cancer, anti-inflammatory use and pain. One class of these agents are bryostatin-like compounds, under investigation as anti-cancer therapy.^{[citation needed]}

Chemical diversity

As above mentioned, combinatorial chemistry was a key technology enabling the efficient generation of large screening libraries for the needs of high-throughput screening. However, now, after two decades of combinatorial chemistry, it has been pointed out that despite the increased efficiency in chemical synthesis, no increase in lead or drug candidates has been reached.^[39] This has led to analysis of chemical characteristics of combinatorial chemistry products, compared to existing drugs or natural products. The chemoinformatics concept chemical diversity, depicted as distribution of compounds in the chemical space based on their physicochemical characteristics, is often used to describe the difference between the combinatorial chemistry libraries and natural products. The synthetic, combinatorial library compounds seem to cover only a limited and quite uniform chemical space, whereas existing drugs and particularly natural products, exhibit much greater chemical diversity, distributing more evenly to the chemical space.^[38] The most prominent differences between natural products and compounds in combinatorial chemistry libraries is the number of chiral centers (much higher in natural compounds), structure rigidity (higher in natural compounds) and number of aromatic moieties (higher in combinatorial chemistry libraries). Other chemical differences between these two groups include the nature of heteroatoms (O and N enriched in natural products, and S and halogen atoms more often present in synthetic compounds), as well as level of non-aromatic unsaturation (higher in natural products). As both structure rigidity and chirality are well-established factors in medicinal chemistry known to enhance compounds specificity and efficacy as a drug, it has been suggested that natural products compare favourably to today's combinatorial chemistry libraries as potential lead molecules.

Screening

Two main approaches exist for the finding of new bioactive chemical entities from natural sources.
The first is sometimes referred to as random collection and screening of material, but the collection is far from random. Biological (often botanical) knowledge is often used to identify families that show promise. This approach is effective because only a small part of earth’s biodiversity has ever been tested for pharmaceutical activity. Also, organisms living in a species-rich environment need to evolve defensive and competitive mechanisms to survive. Those mechanisms might be exploited in the development of beneficial drugs.

A collection of plant, animal and microbial samples from rich ecosystems can potentially give rise to novel biological activities worth exploiting in the drug development process. One example of a successful use of this strategy is the screening for antitumour agents by the National Cancer Institute, started in the 1960s. Paclitaxel was identified from Pacific yew tree Taxus brevifolia. Paclitaxel showed anti-tumour activity by a previously undescribed mechanism (stabilization of microtubules) and is now approved for clinical use for the treatment of lung, breast and ovarian cancer, as well as for Kaposi's sarcoma. Early in the 21st century, Cabazitaxel (made by Sanofi, a French firm), another relative of taxol has been shown effective against prostate cancer, also because it works by preventing the formation of microtubules, which pull the chromosomes apart in dividing cells (such as cancer cells). Other examples are: 1. Camptotheca (Camptothecin · Topotecan · Irinotecan · Rubitecan · Belotecan); 2. Podophyllum (Etoposide · Teniposide); 3a. Anthracyclines (Aclarubicin · Daunorubicin · Doxorubicin · Epirubicin · Idarubicin · Amrubicin · Pirarubicin · Valrubicin · Zorubicin); 3b. Anthracenediones (Mitoxantrone · Pixantrone).

The second main approach involves ethnobotany, the study of the general use of plants in society, and ethnopharmacology, an area inside ethnobotany, which is focused specifically on medicinal uses.

Artemisinin, an antimalarial agent from sweet wormtree Artemisia annua, used in Chinese medicine since 200BC is one drug used as part of combination therapy for multiresistant Plasmodium falciparum.

Structural elucidation

The elucidation of the chemical structure is critical to avoid the re-discovery of a chemical agent that is already known for its structure and chemical activity. Mass spectrometry is a method in which individual compounds are identified based on their mass/charge ratio, after ionization. Chemical compounds exist in nature as mixtures, so the combination of liquid chromatography and mass spectrometry (LC-MS) is often used to separate the individual chemicals. Databases of mass spectras for known compounds are available, and can be used to assign a structure to an unknown mass spectrum. Nuclear magnetic resonance spectroscopy is the primary technique for determining chemical structures of natural products. NMR yields information about individual hydrogen and carbon atoms in the structure, allowing detailed reconstruction of the molecule’s architecture.