Appunti Drugs.pdf

BIOTECHNOLOGICAL DRUGS INTRODUCTION We study biotechnological drugs because there is an unmet need in medicine: combining biotechnologies with medicine allowed to apply typical techniques of the biotechnological field to the medical one, in order to find new treat...

BIOTECHNOLOGICAL DRUGS INTRODUCTION We study biotechnological drugs because there is an unmet need in medicine: combining biotechnologies with medicine allowed to apply typical techniques of the biotechnological field to the medical one, in order to find new treatments and so in order to correct the defects that characterize a lot of pathologies (for example, the cloning techniques used in biotechnologies are used to produce proteins that can be used to treat some pathologies). The main problem with these drugs is the delivering into the body, because biotechnological drugs are usually macromolecules (mainly proteins, but also nucleic acids), hence they have a huge molecular weight. So this field of applications is in constant development. The field of biotechnology applied in medicine is usually called red biotechnology and it involves the production of new drugs based on the knowledge of the mechanisms that induce the development of a specific pathology, because thanks to this knowledge we can also find pharmacological targets. The main aim is in fact to correct the defects typically part of that disease, most of the times in order to reconstitute the function of specific molecules (especially proteins) that don’t work anymore in that pathology. In fact, for this reason, the field of red biotechnology is involved also in designing organisms to manufacture antibiotics and vaccines and in engineering genetic defects through genomic manipulation (gene therapy). So one of the main goals of red biotechnology is drug production, which is the process in which pharmaceutical products are produced through application of biotechnological techniques. These drugs are produced for diagnosis (in this case they are just tools, not treatments), cure treatments and prevention of diseases. It’s estimated that by 2030, almost 80% of pharmaceutical will be represented by biotechnological drugs (right now they represent the 20% of marketed drugs and 50% of drugs under development): this is because they are highly effective and they have great advantages. However, biotechnological drugs cannot fully replace chemical drugs because chemical drugs are easier to produce and they can be used against certain types of pathologies that it’s better not to treat with biotechnological drugs (for example, when we have a headache it is better to use chemical drugs like anti-inflammatories, instead of using biotechnological drugs that are harder to produce and that are usually also hard to deliver inside the body). Biotechnological drugs are for this reason really useful mainly for really important and massive diseases, while mild diseases should be treated with small drugs. Right now we have more than 400 new biotechnological drugs and biotechnological vaccines for more than 200 diseases are currently being tested. The disease against which a lot of biotechnological drugs are being developed is cancer, followed by infectious diseases, autoimmune disorders and cardiovascular diseases. Moreover, recent statistics have pointed out that the most researched category of biotechnological drugs are monoclonal antibodies. The most sold drug in the world right now is a biotechnological drug. The main application of biotechnological drugs, that firstly induced the production of these type of molecules, consists in the administration of biotech drugs in pathological situations of either lack or insufficiency of natural proteins. Traditional methods involved the purification from blood, urine, human tissues or animal organs; however these products didn’t have a good efficacy since, if we extract from blood for example, we could have the possible presence of pathogenic agents, or if we purify animal proteins, the products that we obtain could be highly immunogenic for our system. Also, all of these techniques required a lot of money and they eventually allowed us to obtain only a small amount of product that could be used for medical purposes. So there was the need to find a way to synthesize human molecules, and thanks to biotechnology this was possible. With the in vitro production of these proteins the safety of the drugs was improved, moreover we could be able to obtain a modified protein for particular purposes (for example we can engineer the gene to obtain a modified protein with modified characteristics) and in top of that we could produce proteins whose extraction was impossible, so the ones we weren’t able to use for medical purposes until that point (for example erythropoietin, interferon, interleukin and growth factors). The first proteins that were produced in vitro were insulin and human growth hormone. How are biotechnology drugs different from traditional pharmaceuticals? The small molecule synthesis is a very simple and easy process, in two or three reaction is possible to obtain a drug like aspirin. On the other hand, it is very difficult to obtain a protein: we start with cloning, then you must grow the protein in a host cell and then you must purify the protein (very important step). Moreover, the production of small molecules allows you to obtain always the same product by following the same steps of synthesis, meanwhile biologic drugs are highly sensitive to manufacturing conditions. However, we can say that it is quicker to make a biologic drug, in fact a small molecule needs to be discovered and optimized, which takes an average of 5.5 years from target to a drug ready for a Phase 1 clinical trial. Moreover, recent studies suggest that due to the higher target specificity and lower off-target effects, biologics might have a higher rate of success than small molecules. In fact, the likelihood of success from phase I to approval can be as much as twice the rate in biologics compared to small molecules. In fact, generally, only one drug out of 1000 chemical products reaches the final phase of drug discovery, while with biological drugs it is easier because if you know the structure of the protein you know if it works or not. Finally, we could say that the specificity of biologics also means they can have a better side effect profile. It is hard to make a specific small molecule, which means many small molecules hit many targets in the body, leading to unwanted “off- target” side effects. How do we produce a biotech drug? The basic step that is present in almost every preparation of a biotech drug is the cloning of a gene (to make an example, insulin is made by two chains, so two clones have to be produced, one for the A chain and one for the B chain). This mean that the mandatory knowledge at the basis of the development of a biotech drug is in the field of genomics, which is the study of gene functions and of its mutations (whether under normal or pathological conditions). However, also proteomics must be taken in consideration when we develop a biotech drug, allowing us to understand the function and the structure of the proteins of our interest under either normal or pathological conditions. These two fields are important for the first step of drug development, which is the identification of a biological target. Then we can either target a gene or a gene product of transcription (gene therapy or antisense nucleotides, using RNAs), or we can target the gene product of translation, so a protein (using protein as drugs or using proteins as targets of antibodies). In any case though, if you have to produce a protein as a drug for example, you first need to know how it works and then you need to know its structure and the sequence of the gene that encodes for it, in order to produce it, so genomics and proteomics are essential. The manufacturing processes for the production of chemical or biotech drugs are really different: biotech products are produced from purification (we produce a molecule from a living organism and then we have to purify it from the molecules of the organism, so it is possible to find, in biotech products, a complex mix of heterogeneous proteins and impurities), every step has to be finely controlled, so many in-process tests are required, moreover the starting material is variable, the process is product-specific, the production usually lasts longer (some months are generally required, especially for the growth of the organisms that produce our drugs), the final product size is small (in grams or kg) and, even if it is difficult to quantify, it usually varies, for this reason a batch number is always needed (because the protein produced in, for example, March will be different from the protein produced in May, and not only from a quantitative point of view but also from a qualitative one, because the manufacturing process can change and because we usually use biological products also in the manufacturing process so they can influence the production of the drugs). On the other hand, small molecule drugs are produced by formulation, the starting material is always defined, a few in-process tests are required, the process is product type specific (so generalizations are possible, and the batch is not needed). Biotech drugs are highly complex: they are usually really big molecules, especially if they are antibodies, which are one of the biggest molecules present in our body. If we compare it to aspirin we can see that the differences for what concerns the size and the structure are noticeable. So molecular weight is one great difference between small molecules and biotech drugs, and this determines the fact that these molecules are really hard to formulate. Moreover, because of their molecular weights they are unstable, for this reason they have to be kept at very low temperatures (which is a parameter that needs to be taken into consideration for the storage, since if you change also one degree the protein can lose its structure). They can change their tertiary structure depending on the medium in which they are in solution (based on the pH and the salts of the medium), because their structures are held together by weak, non-covalent forces. So it is really important to preserve the integrity and the specific folding of the protein, because otherwise it will not work as it is required. In fact, another important aspect that may be difficult to handle during the production of protein as drugs is the achievement of the post-translational modifications, which are very important in the final, functioning configuration of a protein. For example, some protein cannot work without a glycosylation. So in general biologic drugs are really sensitive to minor changes, on the other hand, small molecules are generally not affected by the manufacturing process and so they are highly reproducible. So there are a lot of aspects that need to be considered when using a protein as a drug: we need to know its mechanism of action, we need to know the tissue distribution, the efficacy, the safety (the majority of biotech drugs do not exert any side effects as we said, but there could still be some safety concerns because they still exert a function that should not overcome the therapeutical aim of the protein) and the immunogenicity level (which is a problem that involves only biotech drugs, since chemical drugs are not immunogenic, even if they could be allergenic). Another difference between small molecules and biological drugs is that the first ones can be found in normal pharmacies, because the patients can generally self-administer these medicines (the preferred route of administration is usually the oral one in this case), while the second ones usually are not sold in pharmacies because their administration is typically intravenous or subcutaneous, so they can only be dispended only from doctors in hospitals. Which are the expression systems that we usually use to produce our biotech drugs? We could use prokaryotes and eukaryotes: among the eukaryotes we could find the yeast (especially Saccharomyces Cerevisiae), insect cells, mammal cells, transgenic animals and transgenic plants; on the other hand, the prokaryote that we usually use the most is E. coli. Which are the advantages of the cloning in bacteria? First of all, they were the first cells used to express a gene, because they are simple organism and they replicate in a few minutes (for example E. col duplicates in a few minutes), they can grow on cheap media and on large scale (production of high quantities of protein in a small amount of time), they cannot be infected by viruses that infect also human and there are no risk of allergies for humans for the proteins that we produce in them. However, there is one important disadvantage that involves the production process: bacteria are not able to glycosylate proteins, so the drugs that need to be glycosylated cannot be produced in these cells (for example, gonadotropins cannot be produced in prokaryotes). In fact, bacteria lack of appropriate systems of post-translational modifications and this has very important consequences on biological activity and immunogenicity of the protein. Moreover, many produced proteins cannot fold in their native forms in bacteria. For what concerns eukaryotic cells, they have a lot of advantages if compared to prokaryotes, however their big disadvantage is that they take a long time to duplicate (24 hours at least), which is a lot of time if compared to the 20 minutes of bacteria. Moreover, the amount of protein produced is usually lower than the quantity produced in prokaryotes (which can also be a direct consequence of the long time of reproduction). However, eukaryotes are able to guarantee the correct folding of proteins (for example by carrying out the correct formation of disulphide bonds), they can perform the proteolytic cleavage from pre-proteins, they can perform glycosylation and they can also allow the modification of amino acids (phosphorylation, acetylation, myristylation, …). Hence, we can assess that the expression system varies based on the needs, so on the type of protein that we have to produce: if the protein must be glycosylated to make it work or if it cannot achieve its correct folding in bacteria, we must use eukaryotes, otherwise we tend to use bacteria because they are easier to manipulate and they produce a lot of product in a fast way. The current biotech drugs used nowadays are: insulin, somatotropin (growth hormone), blood proteins, interferons, cytokines (in general), hematopoietic growth factors, monoclonal antibodies and vaccines. The first biotechnological drug out of these was insulin, approved by the FDA in 1982. Before we were able to reproduce insulin in the lab, it was extracted by the pancreas of pigs and for one patient 70 pigs were needed per year in order to produce the right amount of insulin. This production method was really expensive, it caused multiple problems (high immunogenicity even if our insulin differs from the pig insulin for only one ammino acid) and it wasn’t convenient. Hence, the production of human insulin in bacteria was really a great revolution. The only disadvantage is that the whole insulin molecule cannot be produced in the same bacteria because E. coli is not able to link together the two chains that constitute this protein, so we produce the two chains separately and then we make them bind in vitro. The second protein that was produced as a biotech drug is the growth hormone, before its engineering it was extracted by corpses, because the growth hormone of other species cannot be used in humans. It required at least 80 pituitary glands from 80 corpses to produce the amount of growth hormone needed for 1 patient every year (usually used to treat dwarfism). The method of production from corpses was, other than really expensive, also possibly harmful for the patient, because there was the risk of infections from prions, causing the Creutzfeldt-Jacob disease (also known as “mad-cow” disease, caused by the transfer of neurological material from the corpse to the patient). The production of growth hormone in vitro was, also in this case, a great innovation which had no disadvantage this time. We could say that every biotechnological drug has 5 characteristic features: 1. Gene expression system: in order to produce a protein you have to identify, isolate and clone the gene that encodes for it, but then you also need to choose a vector that will be engineered with the gene and the regulatory elements for the control of the gene expression (like promoters, signal peptides). However, nowadays these vectors are not so difficult to produce because we know how they work and what could be the best organization for the expression of our protein of interest. Then, we need to choose the actual host cell, that is going to produce our cell of interest, and this selected organism where we decide to insert the vector is going to produce our protein. 2. Production system compatible with the host organism: every host cell can be cultured in a specific machinery. The aim of these production systems is to optimize the culture conditions for the genetically modified organism so that it can produce the desired amount of the product protein. We also have to take care of the selection of the culture medium, of the culture conditions and of the tools that we are going to use (for example fermenters). 3. Purification system: the purification is a really important step that allows us to get rid of all the other proteins contained in the host cells (which are the proteins that the cell regularly produces for itself, we have to eliminate them in order to obtain only our protein of interest). The aim in this step is to obtain a purity of about 100% without any protein alteration. Purification is also needed to make sure that we don’t have any contaminants: if we have some, we have to understand where the contamination comes from, how much the contaminants are dangerous, how can they be removed and then how can we be sure that we removed all of them. The impurities that we find in the final product can derive from the production process, so from the host cells (for example mammalian cells can be infected by viruses that could also infect men), from the cell cultures (antibiotics used to grow mammalian cells, solvents, …) or from the last step of the whole process (the cells are usually grown in big tanks that are isolated but can come in contact with external material); they can derive from the produced protein (the protein could be produced in shorter forms, modified from or aggregates that could be dangerous once injected); finally, they could  derive from microbiological contaminants (derived from viruses, as we said before). The risks that we may encounter with one of these impurities are of toxicity (heavy metals, antibiotics, organic solvents), altered pharmacological activity (aggregates or degradation products), immunogenicity, oncogenicity (only if the viruses that infected mammalian cells in culture are oncogenic ones) and infectious diseases (mycoplasma, yeast, viruses). 4. Nature of the active product: once the protein has been produced and purified it must be analysed to see if it will work. So we need to check for the sequence of the protein, if it folded in the correct tertiary folding, if it underwent the post-translational modifications operated by the host cells. 5. Pharmaceutical formulation: once the protein its ready and we are sure it works, we have to preserve its biological activity by preserving the correct folding of the active protein in solution. In order to make sure that this is guaranteed we can use methods that allow a chemical and physical stability (addition of stabilizers and excipients, like albumin, glycerol, …) and then we have to store the protein at low temperatures (-20° to make sure that it still works after months, but if we lyophilize the protein we can also keep it at room temperature and this can save money and it can facilitate the transport of the drug too). At the end of the process you have to further control the protein’s: identity, purity, activity, stability, general safety, sterility and pyrogenicity (bacterial cells have LPS on their cell wall, LPS is really dangerous so it needs to be removed from the final product). The whole process usually takes 8-9 months. An important concept to understand is immunogenicity, which is the main problem related to the use of this kind of drugs. But why? First of all, the use of the antibody in a patient could trigger the production of antibodies against the drug, if this happens then the drug is not going to work so it cannot be used anymore in that patient. However, not every protein drug triggers the production of antibodies, in fact it depends on many factors, for example, it depends on how frequent the administration of this kind of drug is (if the drug is administered once a month maybe the immunogenicity can develop after a year, but if the drug is administered with a lower frequency, the possibility to develop antibodies is lower). Sometimes we can also have reaction like anaphylaxis, or we could have the production of antibodies that can cross react with endogenous proteins (because if we use, as a drug, a protein that has a similar structure to one that we have inside our body we could develop antibodies that do not just react against the drug but they also activate against self-antigens). This last event could also be influenced by the genetics of the patient, because people with autoimmune diseases may be more prone to the development of antibodies against self-antigens (lack of regulation of immunity). So which are the factors influencing immunogenicity? Sequence variations from the original protein (this is actually something that is really useful to prevent the generation of antibodies against self-antigens, but at the same time the protein could be recognized as non-self and so we could still have the blocking of its action through the generation of antibodies), alterations in the glycosylation, but also contaminants and impurities present in the final product could evocate a response from the immune system, as well as a different formulation, addition of particular molecules in the final protein (like PEG), route of application (an intradermal administration usually triggers the production of antibodies in a higher way than when we administer the drug intravenously, because with this route of administration the drug is released slowly into the site of injection, activating also the local production of antibodies), the quantity (the dose), the length of exposure to the drug (period of treatment, we already explained it), patient characteristics and unknown factors too. To see if a protein is immunogenic or not we can test it in vitro with assay technologies, and this is a really useful way to see if what we have the risk of production of antibodies against it, and so we need to change one of the factors that we mentioned above, or if it is safe and we can proceed with the administration. PRODUCTION OF BIOTECHNOLOGICAL DRUGS Let’s first say that when we talk about biotechnological drugs we can also call them biologicals, but when we say biologicals we also refer to whole blood ad its components and organs and tissues used for transplants, other than to antibodies (for passive immunization and monoclonal ones), proteins extracted from animals and derived from DNA technology. So when we want to refer to only these last categories, we say biotechnological drugs. So we said that biotechnological drugs are produced through DNA recombinant technologies. Why does this DNA recombinant technology have advantages over the simple extraction of the same proteins from biological fluids? First of all it overcomes the problem of source availability, it overcomes problems of product safety (for example for the transmission of HIV or B/C hepatitis from blood, Creutzfeldt-Jacob disease from human pituitaries), it provides an alternative to direct extraction from inappropriate/dangerous source material (for example, FSH and hCG were purified from urine of post- menopausal women, which is not so easy to do, or anti-coagulant proteins which were extracted from Malaysian pit viper), it allows to produce a higher quantity of final protein compared to the one that we can extract from biological fluids (and this also makes the whole process cheaper), and finally it facilitates the generation of engineered therapeutic proteins displaying some clinical advantage over the native protein product (for instance we can change the pharmacokinetics of the protein, for example prolonging the half- life to facilitate the administration regimen, simply by changing specific groups from the original protein sequence with a site-directed mutagenesis, or by adding particular molecules to the protein structure, like PEG). The modifications that we could perform are synthesized in the table: The production of small molecules usually begins with a classical screening, which starts from a source of molecules (from microbe, plants, libraries or molecular design) from which we are possibly going to obtain our active molecule, on this source we perform one or more in vitro assays, we select the active compounds, then we perform one or more in vivo assays, to select the in vivo active molecule, and then, once we identified the molecule that could possibly work against that pathology, we have to perform pre- clinical studies (pharmacological studies, PK and PD, toxicological,...). However, we don’t know if at the end of this process we are going to have an active molecule that is actually effective and safe for humans, so that can reach the market, moreover it takes a lot of time and a lot of money to perform all of these steps, so the process is difficult, time consuming and really expensive. Instead, biotech drug discovery is really easy: we select the human molecule (macromolecule) with a known biological activity that could act against our pathology of interest, then we produce either a recombinant protein by genetic engineering in bacteria or eukaryotes, producing enough amount for pre-clinical or clinical studies, or antibodies, through the isolation and production of a monoclonal antibody that is specific and selective towards a distinct molecular or cellular target. So it is really simple to produce a protein, you just need to know the sequence; antibodies are actually a little bit more difficult to produce but overall we know that once we produce the right, selective and specific antibody it will work for sure. So the process is not difficult nor long and in the majority of the cases we end up with an active and working macromolecule without spending a lot of money. So it takes 2 to 10 years to obtain a biotechnological drug and put it in the market (so for FDA and EMA to approve it). So for biotech drugs the knowledge of the molecular mechanism that are at the basis of the disease allows to identify the possible pharmacological target and the possible macromolecule that could work as a drug. At the moment more than 70% of all the marketed medicines are represented by biopharmaceuticals and more than 1000 biotech drugs are under pre-clinical and clinical studies. If we compare a biotech drug to a chemical drug, the biotech one as a 4-fold higher possibility to enter the market compared to a small molecule (so for one chemical molecule that enters the market, 4 biotech drugs do it too). The major field in which biotechnological drugs have been developed and approved so far is the cancer field, especially monoclonal antibodies. Which kind of molecules can we use as biotechnological drugs? We can have human proteins (normally they are used as replacement of proteins that are not produced or don’t work properly in some pathologic conditions, like insulin in diabetes), modified proteins, humanized and human antibodies, nucleic acids and vaccines. As we can see from the list of different types of biotechnological drugs, they are typically large molecules, so we can assess that for biologics size does matter: the bigger is the molecule the more difficult it is to administer it, because molecules need to pass across the tissues, an action which is really simple for smaller molecule (especially if not charged, but also if it is charged we have transported) but it could be very hard for large molecules. In fact, chemical molecules can be administered through oral route, through which they can reach easily the targeted tissue, while for biotech drugs this route of administration is not convenient at all because it is more difficult for them to be absorbed and pass tissues. Which are the steps for the production of biotech drugs? We first have a pre-clinical phase (we have to prove their efficacy and their safety), which is composed of some phases: we first have the target identification (through genomics and proteomics), then we test the administration route, the pharmacokinetics and pharmacodynamic, the bioequivalence and bioavailability and then we have toxicologic studies. We could say that the different steps are very similar to the ones that we have to perform on small molecules to test them, however it is easier for biotech drugs because the identification of the molecule is easier and faster. Usually, 10% of the potential biotech drugs overcomes the pre-clinical phase. Between 3000 and 10 000 of new protein-based drug targets can be found in the human genome sequence, and hundreds of pathogen proteins as potential drug targets are present in the sequence of human pathogens. So thanks to genomics, the identification of previously undiscovered proteins will have potential therapeutic application. PHARMACOKINETICS 1. PROTEINS: For what concerns the pharmacokinetics (how the body modifies the drug once it is administered) of peptides and proteins, it is really different from the one of a chemical drug: the chemical drug is recognized as an exogenous molecule so the body is going to eliminate it (as with any other stranger molecule). In order to eliminate a molecule, it has to become hydrophilic, because it is usually expelled through urine. With biotech drugs it is different because the body doesn’t recognize it as an exogenous molecule and so it doesn’t expel it as it will do with chemical molecules. For example, when we eat an egg, the proteins contained in the eggs are usually not expelled but digested and absorbed, and in the same way when we administer a biotech drug (not from the oral route, as we already saw) it will end up in the same manner, so the protein will be digested by our body and the different parts that constitute it will be absorbed. So let’s start from the administration: the main route is the parenteral one, we cannot administer the biotech drugs through the oral route. So biotech drugs are injected intravenously, subcutaneously and intramuscularly. They cannot be delivered in a non-parenteral way because they have a high molecular mass, they can be inactivated by enzymes and they can aggregate and so their absorption would become impossible. For example, the oral administration is not convenient because we could have inactivation of the biotech drug due to stomach acids (all biopharmaceuticals are acid labile and inactivated at low pH), digestive proteases (pepsin, trypsin and chymotrypsin usually degrade proteins during digestion), the passage through the intestinal mucosa could be difficult because of their large size and their hydrophilic nature (so even if we protect the drug with a capsule from enzymes and the environment, the protein won’t be absorbed still), and then we have the first-pass metabolism (there is a local circulation between the intestine and the liver, once a molecule is absorbed by the intestinal mucosa it passes from the intestine to the liver and then from the liver to the intestine before being absorbed, and once it reaches the liver it will be metabolized; so, even if a protein can pass the intestinal mucosa, then it will be directed to the liver to be metabolized and then it will be absorbed, so it will still be modified before entering the systemic circulation). However, some attempts to avoid these complications have been made, in order to make this kind of drugs more accessible, improve their bioavailability and allow their oral administration. The strategies that the researchers have come up with are: encapsulation within an enteric coat, inclusion of protease inhibitors (like aprotin), permeation enhancers that facilitate the absorption through the gastrointestinal lining, production of mucoadhesive delivery systems to interact with the intestinal mucosa. This last strategy has not yet been established, but it is generally divided into two steps, which can be supported by the nature of the dosage form and how it is directed. The principle is simple: if the formulation is made of polymers which are in deep contact with the drug and that can adhere to the mucins that constitute the mucous layer of the intestinal mucosa, then we can induce the attachment of the so formulated drug on the intestinal mucosa and this attachment will last for enough time to help the protein pass across the mucosa (the time is one of the variables that influences the difficulty of the protein to pass the intestinal mucosa, because the faster the digestion goes the lower is the probability to absorb it). In the first step, the mucoadhesive systems and biological substrate start to come into intimate contact (wetting), which is called the contact stage. The second step is the consolidation, being characterized by penetration of the formulation into the tissue or into the surface of the mucus membrane with several physicochemical interactions to consolidate and support the adhesive joint, and then extend the adhesion. So, in this type of strategy, the aim is not to make the protein reach the blood stream, but it is of making it act locally, for example in intestinal diseases, because in any way the passage through the mucosa is kind of hard. A mucoadhesive polymer is a natural or a synthetic polymer capable of producing an adhesive interaction with a biological membrane or with the mucus lining on the GI mucosal membrane; it is known to have the following molecular characteristics: it has molecular flexibility; it contains hydrophilic functional groups; it poses a specific molecular weight, chain length and conformance. Another strategy is the production of proteins into leaves, for example of lettuce, in particular in their chloroplasts (CTB, chloroplast transformation technology). Foreign genes are first expressed in lettuce chloroplasts by bombardment of leaves with chloroplast vectors using the gene gun. After confirmation of stable integration of foreign genes into all the chloroplast genomes in each plant cell and expression of the correct size protein and functionality, genetically modified lines are transferred to the greenhouse to increase biomass. Harvested leaves are lyophilized, powdered and stored in moisture free environment. Machines are commercially available for processing lyophilized leaf materials into desired particle size and packaging into capsules. Evaluation process includes microbial count in lyophilized materials, integrity of therapeutic proteins after prolonged storage (folding with disulfide bonds, pentameric or multimeric structures) and functionality in conferring immunity with vaccine antigens (protective immunoglobulins IgG1, IgA, cytokines, pathogen/toxin challenge) or developing tolerance with autoantigens (suppression of allergy, formation of IgE, inhibitory antibodies, destruction of pancreatic islets, etc.) or conferring desired functions (regulating blood glucose with insulin, exendin-4, etc.). It is an advantage because it reduces manufacturing costs, it allows a long-term stability and storage, a large-scale production, the elimination of the cold chain, of expensive fermentation, of purification steps, sterile injections and microbes and it presents comparable safety potency and efficacy. Another interesting delivery route could be the buccal delivery: we have a special formulation of the drug, in which the protein is embedded in a muco-acid film (used for a lot of chemical drugs), so that the protein will be directly absorbed in the blood stream from the oral mucosa. An attempt to produce a biotech drug with this formulation was made on insulin: the recombinant human insulin is bound to glycan-coated gold nanoparticles through non-covalent binding and embedded in a polymeric mucoadhesive film for delivery of insulin via the buccal mucosa. However, the trial updates for this formulation have interrupted so the trial was probably halted. Future developments in buccal mucoadhesive drug delivery system for biologics could be directed to vaccines, peptides or proteins. Pulmonary delivery could be favourable because the absorption appears to be inversely related to molecular mass (MM); moreover we have a large absorptive surface (the alveoli have more than 100m 2 of surface of absorption), and from the alveoli the protein could go directly to the bloodstream (because of the this diffusional layer and of the high vascularization), without any alteration in the protein structure (in fact in the lungs we have proteolytic inhibitors and there is a high tolerance to foreign substances). Moreover, through this route we can avoid the first-pass metabolism and we have reliable, metered nebulizer- based delivery systems that have been already created. In this route of delivery, it is easier for the protein to pass the epithelial layer either through transcytosis or through paracellular transport (passage between two cells), because the drug has to cross a monolayer of insoluble lipids (lung surfactants), epithelial cells, the interstitium (fibrosus material) and the vascular endothelium. For this passage, the formulation of the drug can also involve the formation of a liposome nanoparticle, which presents a lipidic bilayer containing the drug (which is water soluble), tagged with specific targeting compounds. So, the strategies that have been conceived for this route of administration are: - Intratracheal instillation: provides information about protein stability, systemic absorption and toxicity (it is still experimental). - Aerosol inhalation: it involves two-phases colloidal systems (very fine liquid droplets dispersed in a gaseous medium); aerosol particle size is one of the most important in determining drug deposition and distribution in the lung. The devices used for this type of delivery are the nebulizers (however they are  non-portable and time-consuming) and the metered dose inhalers (MDI, they are portable and easy to use; they are the ones used for asthma or allergies). - Dry powder inhalers (DPI): they are generally used, but they require a rapid rate of inhalation to provide the necessary energy for aerosolization (difficult for paediatric patients, in general it is easier to inhale liquid droplets than powders). In conclusion, the pulmonary delivery of proteins and peptides is a promising alternative to parental administration, but we still have some obstacles that need to be overcome. For example, only a small fraction of the drug is able to reach the alveoli in this route (the other 90% remains stuck in the mouth or in the trachea; producing biotechnological drugs is very expensive, so if you use only 10% of it is a total waste of money and time), and aerosol particles generally tend to aggregate. However, some years ago one type of insulin delivered through this route of administration was invented, but it eventually failed. The proteins that could be used with this type of application are actually drugs of topic treatments (for asthma or cystic fibrosis for example, so they act locally on the lung). For what concerns nasal delivery route, we can say that it is an easily accessible type of administration, nasal cavities are serviced by a high density of blood vessels, nasal microvilli provide a large potential absorption surface area and the drug is not going to undergo first-pass metabolism (It is going to be absorbed directly in the blood stream). However, in this case we have the problem of the clearance of a portion of the administered drug, moreover the majority of the molecules are not able to pass the mucosa (low uptake rates for larger peptides, so molecules bigger than 10 kDa; for this reason the detergent-like uptake enhancers could be administered) and at nasal level we have extracellular proteases/peptidases. So another delivery route could be the skin: the transdermal delivery system is really useful for topical applications, however it could also be used for the systemic absorption of the drug. In this way we can bypass metabolic and chemical degradation (that is typical of the gastrointestinal route) and we do not have first-pass metabolism. The two strategies that have been conceived for this route are the iontophoresis (which uses low level electric currents, you apply the drug on the outside of the skin and then with a specific device you send the current which allows the transfer of the protein, creating, with a counterion, a flow that sends the drug towards the other side of the device, so that the difference of concentration makes the protein go deeply in the epidermis and reaching blood vessels) and sonophoration (which uses low frequency ultrasounds to create sort of open spaces among the cells facilitating the entrance of the protein from the external side of the skin to the internal, until it reaches the blood stream). One type of transdermal delivery is performed through the use of TPM, which is a sort of patch applied on the skin. It was used for a certain type of insulin formulated for this use; however the trial wasn’t successful (but it wasn’t abandoned, they are still working on it). The patch is based on a Tocopheryl Phosphate Mixture (TPM) and it can transport both small and large molecules, moreover Tocopheryl Phosphate is found as an endogenous molecule in humans (so it’s not going to be neutralized), it is a powerful penetration enhancer that does not disrupt or irritate the dermis; it allows a sustained release of compounds from just one application and rapidly penetrates the dermis (less than 1 hour). The benefits are that this technology is applicable to a wide range of drugs, it is natural and safe, it increases patient comfort and compliance, maintains skin integrity, it allows flexible dosage regimens and therapeutic levels can be maintained for longer. So, we can say that there could be a lot of different routes of distributions for biotech drugs, however they are all still in development (also the oral one). What happens when the drug has been administered? We have the phase of distribution: the bloodstream is going to distribute it to the tissues after it absorbed the drug (if we inject it intravenously, the drug is already in the blood stream, but if we inject it subcutaneously or intramuscularly it has to be absorbed by the local vessels first). At the level of the tissues we can find proteases, so for biotech drugs the result of distribution is digestion; however, not every molecule that has been administered is going to be directly digested, so a quantitative of protein could bind other proteins and avoid degradation, while a good amount of protein will still reach the target site and do its work before getting degraded. So, whole-body distribution studies are essential for classical small molecule drugs in order to exclude any tissue accumulation of potentially toxic metabolites, but since protein drugs are degraded and recycled in the endogenous amino acid pool, biodistribution (the distribution of biotech drugs) studies for peptides and proteins are performed primarily to assess targeting to specific tissues and to identify the major elimination organs. Factors that influence the distribution of therapeutic peptides and proteins are: an active tissue uptake and binding to intra- and extravascular proteins (small Vd, volume distribution, despite specific binding to receptors in the tissue), binding to endogenous protein structures (for example transport proteins), specific binding to cytokine-binding proteins (antibodies or soluble cytokine receptors, it prolongs the cytokine circulation time by acting as a storage depot or enhancing the cytokine clearance), non-specific binding to plasma proteins (like albumin). Then for what concerns elimination, once the protein is digested by proteases in tissues it is eliminated. Proteic drugs are almost exclusively eliminated by metabolism via the same catabolic pathways as endogenous or dietary proteins, but once the blood stream gets the protein at the level of the kidneys, also the kidneys can eliminate them (but only proteins with a molecular weight lower than 50 kDa can be eliminated through the kidneys). In the kidneys we have three options: in the first case the glomerular filtration is followed by the reabsorption into endocytic vesicles in the cells of the proximal tubule (to be digested, typical of IL-2, GH and insulin); in the second case, glomerular filtration is followed by intraluminal metabolism (so the protein is digested in the lumen first) and reabsorption (of amino acids) into the systemic circulation (typical of angiotensin I and II, glucagon and LH-RH), then the amino acids can be reintroduced in the blood circulation; in the last case, the proteins reach the peritubular blood vessel, they are reabsorbed in the epithelial cells of the lumen and then they are digested as amino acids. In any case, the result of digestion of the drug, so amino acids, is not going to be eliminated (like for chemical drugs, because they are recognized as foreign molecules), but it’s going to be reused (because amino acids are recognized as endogenous molecules). What happens to molecules that have a molecular weight higher than 50 kDa? They are eliminated through proteolysis: their digestion is operated by proteolytic enzymes (such as proteases), which are ubiquitous, so it could potentially happen in any tissue and in any cell (they need to be up taken by cells through specific receptors and then they are digested by intracellular proteases). So we can say that eventually the factors that influence the modality of elimination are molecular weight and the molecule’s physico-chemical properties (size, charge, lipophilicity, functional groups, glycosylation pattern, secondary and tertiary structure, propensity for particle aggregation). It is important to note that if peptides and proteins are administered through the oral route, the gastrointestinal tract is the major site for their metabolism, however we already said that this cannot happen yet because we still do not have oral biotech drugs. Hepatic elimination, which is typical of insulin and tPA, happens in hepatocytes, where the amino acids that are produced by the digestion are going to be reutilized (in the liver we usually have process of metabolism, for biotechnological drugs metabolism and elimination are basically the same thing because they both happen through proteic digestion). However, in order to have the digestion of the molecule, it needs to bind to some receptors present on the surface of the cell (not only in hepatocytes) and then it needs to be internalized by the cell. For this reason we can say that for digestion to happen, a receptor-mediated endocytosis needs to happen. Anyway, since the number of receptors is limited, drug binding and uptake can usually be saturated within therapeutic concentration: the excess of the drug will not be internalized, so it will not be eliminated and it can still act on the target. Immunogenicity could alter the pharmacokinetics of a protein: if we inject the protein directly at the level of the blood stream (intravenously), there will be a lower probability to trigger an immune response; on the other hand, in a sub-cutaneous administration, the probability to trigger the activation of the immune response is higher, because the drug will form a sort of precipitation on the site and its release in the blood stream is going to be slower so immune cells that reside underneath the skin have all the time to get activated and react against the protein; intra-muscularly the absorption is much quicker than in the sub- cutaneous route, so it is less immunogenic. So in general we can say that extravascular injections (s.c > i.m.) stimulate antibody formation more than intravascular applications and it depends on the speed of the absorption. Once antibodies are produced, protein-antibody complexation can slow down if it forms a depot for the protein drug. But immunogenicity can be reduced by chemical modifications: PEGylation is the addition of a non-reactive molecule (PEG, a polymer), which shields the antigen, so the protein won’t be recognized as immunogenic. 2. ANTIBODIES: What about the pharmacokinetics of monoclonal antibodies (so therapeutic antibodies, no therapeutic antibody is a polyclonal one)? It is similar to the pharmacokinetics of the protein drugs (because they are proteins), the only difference is in the size. So the main problem for the antibodies is the passage in the tissues. We have different types of mAb, the first one was produced in the 70’, the technique used was the hybridoma technique and it allowed the production of mouse mAb. Nowadays, mouse mAb are not used as therapeutics anymore, they are just used in laboratory practice, and that’s because now there is the possibility to produce either humanized or human mAb, which are better for therapeutic purposes. The hybridoma technique starts with the immunization of a mouse with the antigen against which we want to produce antibodies, then we take spleen of the mouse, we isolate B cells (which are able to produce antibodies), we fuse these cells with myeloma cell lines (they are cancer cell lines, so they have the ability to duplicate continuously) in the presence of polyethylene glycol (PEG, in this case it is not used for the purposes we explained before, but it just allows the fusion of the two cell lines). The selection of the correctly fused cells happens in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), so only fused cells can survive (because myeloma cells don’t have the enzyme that is able to utilize the HAT component and B cells die because of their short life-span, so only fused cells, which arbour this enzyme, can survive). Then, by making dilutions in 96-well plates, we can have one cell per well, and in this way we obtain multiple wells each one containing a single cell that produces a single type of monoclonal antibody against the original antigen (monoclonal antibodies). The antibodies that we obtain with this technique were once used as drugs, but they were all mouse antibodies (suffix -omab) so they were highly immunogenic, so they were abandoned. So another type of monoclonal antibody was created: the chimeric antibodies (suffix -ximab) were less immunogenic than the murine ones, because the constant portion of the antibody was humanized; however the immunogenicity was still high because the variable portion was still of murine nature (1/3 of the molecule is still murine, so 33%). So the third type of monoclonal antibody generated was the humanized one (suffix -zumab), which just presents 5% (or 10%) of murine sequences in the molecule, so the immunogenicity decreased significantly. However, nowadays we have the possibility to reduce the immunogenicity even more because we can create human antibodies (suffix -umab). So the lower is the amount of the murine portion in the antibody, the lower is the immunogenicity of the molecule. In any case though, even if in different levels, the monoclonal antibody that we use for therapy purposes is going to induce the production of anti-drug antibodies (ADAs), regardless of whether it is non-human or completely of human origin. How can these antibodies bind to therapeutic antibodies? There are different parts of the mAb where the anti-isotype antibodies can bind: first of all, if we have the chimeric mAb, neutralizing antibodies are going to bind to the ligand-binding site, so as a result the patient will not benefit from the therapy (moreover we have changes in the bioavailability, clearance and biodistribution of the drug); then we could have non-neutralizing antibodies that bind in the “hinge” region of the mAb (so the part between the ligand binding site and the Fc domain), and they are non-neutralizing because they bind a region which is not so important for the function of the drug, however in this way we have two antibodies bound together so the molecule becomes bigger and its bioavailability, clearance and biodistribution will be altered; then we could have the formation of immune complexes between the therapeutic antibody and antigens (this is actually not related to the production of antidrug antibodies but it is still very important to note because it’s still a reaction that causes changes in the bioavailability of the drug, so it’s important). So in general immunogenicity is going to alter either the function or the pharmacokinetics of the drug (or both). Immunogenicity also depends on the immune system of the patient: there are more sensitive patient that others, for example patients that suffer from autoimmune diseases are more prone to develop antibodies against therapeutic antibodies, so the genetic of a patient is very important. It also depends on the frequency of administration of the drug. The consequences for patient safety involve hypersensitivity reactions and cross-reactive neutralization of an endogenous protein. Let’s see each type of mAb: 1. The first murine Ab to be approved for clinical use was “Muromonab-CD3” (OKT3), used in organ transplantation and directed against the CD3 antigen of T cells to prevent immune reactions against transplanted organs and thus their rejection. At the beginning it worked, but then because of the formation of antibodies against this mAB its effects vanished. In fact it can induce the production of specific anti- antibodies (so human anti-murine antibodies, which are in short called HAMAs): this induced an immune response with influenza-like symptoms shock. 2. For what concerns the chimeric antibodies, the antibodies that can develop against them are called human anti-chimeric antibodies (HACAs), however we still use this kind of antibodies in the therapy field because their immunogenicity is lower compared to the one of the murine ones, so they work really well until they induce an immune reaction. One example is rituximab, used against rheumatoid arthritis, it is directed against CD20 in B cells. These antibodies are produced with recombinant technology. 3. In humanized antibodies, the murine fraction of 5-10% consists only of CDRs (complementarity- determining regions). Antibodies against these humanized mAb can still be produced and they are called human anti-human antibodies (HAHAs). One example is the alemtuzumab, an anti-CD52 mAb. Also this type is produced with recombinant technology.  4. Human antibodies are instead complete human mAb, the first created was adalimumab (anti TNF- alpha antibody). Despite its nature as being completely human, there are reports describing the occurrence of production human anti-human antibodies (HAHAs, same name as for the humanized ones) in patients. This is due to the immune system of the receiving subject, because even if the immunogenicity is really low we could still have the activation of the immune system. Also this type is produced with recombinant technology. We have also different types of mAb that are modified from the normal mAb to make them less immunogenic or more effective: for example the primatized antibodies are genetically engineered from cynomolgus macaque monkey, they are structurally indistinguishable from human antibodies and so they may be less likely to cause adverse reactions and they are potentially suitable for long-term treatments (an example is lumiliximab, an anti-CD23 antibody). However, the production of antibodies in monkeys is not so easy, moreover it is unethical and they are not approved, at least extensively. We can mention also another type of antibody, the bi-specific antibody, which is particularly useful against tumoral cells: a characteristic of cancer cells is their ability to perform immune-escape, so they won’t be attacked by T cells anymore. If we use bi-specific antibodies, which are structured in order to have one Fab portion directed against a tumoral antigen and the other directed against an antigen of T cells (like CD3, especially in CD8+ cells), we can make T cells become closer to tumoral cells and in this way the probability that they are going to activate against them is higher. If we then consider the fact that the Fc portion of the mAb is going to recruit also NK cells (or macrophages), the final action against the tumor is going to be amplified by the contemporary activation of the T cell. This is really revolutionary because usually infiltrating T cells, once they get activated, become anergic so they cannot get activated against the tumor anymore, while circulating T cells are still able to act against the tumor but they cannot reach it and infiltrate it because the tumor can escape their action. So by bringing T cells and tumoral cells very close we can induce the activation of CD8+ cells against the tumor. Moreover, in this type of antibody, the two binding sites can significantly increase affinity or internalization rates of particular antigens on a cell's surface by binding to two different epitopes on an antigen, and they usually have a higher cytotoxic potential to bind to antigens with low expression level. With the development of antibody engineering, many types of bispecific antibodies have been designed to overcome short half-life, immunogenicity and side-effects caused by cytokine liberation. They include trifunctional antibodies, chemically linked Fabs, various types of bivalent and trivalent single chain variable fragments (scFvs), and fusion proteins mimicking the variable domains of two antibodies. The furthest developed of these newer formats are the bispecific T cell engagers (BiTEs) and mAb2's, antibodies engineered to contain an Fcab antigen-binding fragment instead of the Fc constant region. Why are scFvs so innovative? The part of the antibody which recognizes the antigen is the variable portion, which is made of light chains and heavy chains. The heavy chains have one variable domain (VH) and three constant domains (Ch1, Ch2, Ch3), while the light chains have one variable domain (VL) and one constant domain (CL). If we link together the variable domains of these two chains by a short peptide linker, we can create a molecule that only contains the light and heavy chains of the variable portion and that can recognize the antigen. This is an advantage because the resulting molecule is very small, much smaller than a whole antibody, and so these scFvs could possibly enter the cell and target intracellular antigens, like viral ones (something that an antibody cannot do because of its size; called intrabodies). So the sequence of the gene of the intrabody can be inserted in a vector (very difficult thing to do with a whole antibody, which is encoded by very long and complex sequences), which is going to be transfected inside our cell of interest, then the intrabody will be expressed inside it and it is going to target antigens found inside it (for example in the ER, which is pretty simple to induce because antibodies are normally produced there so you don’t need any particular folding or stability characteristic, or in the cytoplasm or in the nucleus if the target is located there, and in that case a correct folding of the intrabody is required). Antibodies may have different kind of actions inside the body: - one of the most important is the ADCC (antibody-dependent cellular cytotoxicity): macrophages, dendritic cells and NK cells can recognize the Fc portion of antibodies with their FC receptor, once they bind it they get activated and they also start to produce cytokines to activate T cells, so ADCC reaction is really important because it is the first reaction for any type of antibody to trigger the response against particular types of cells, especially tumoral ones. So this is how any type of anti-tumoral antibody works. The ADCC is a sort of “unspecific” reaction because it is something that happens independently from the specificity of the antibody towards the target. For this reason it is only used for anti-tumoral therapies: if we use an antibody against an autoimmune disorder we need to modify it to avoid the activation of the ADCC because we don’t need a further activation of the immune system, it is already too activated. - Then we have the complement-dependent cytotoxicity (CDC), which is also related to the recognition of the Fc portion of the antibody, so it’s again independent from the recognition of the specific antigen. - Neutralization of exotoxins and viruses is instead correlated to the ability of the antibody to recognize specific targets, in this case derived from microorganisms. - Prevention of bacterial adherence to host cells. - Membrane attack complex (MAC) resulting in cytolysis. - Agglutination of microorganisms. - Immobilization of bacteria and protozoa. - Opsonization. In general, the activity due to the specific binding is mainly of blockage of ligands and prevention of their interaction with the receptor. If we have a tumor cell we can use many strategies: we can use the naked antibody, or we can add some radioactive ligands (which are in this way brought in proximity of the tumoral cell) by either using a streptavidin molecule (that binds to radioactive molecules on one side and to the Fc portion of the antibody on the other side) or a bispecific antibody (that can directly bind one Fab portion of the mAb) or directly binding it to the antibody (radioimmunoconjugate); then we could use bispecific antibodies that bind, other than to the tumoral cell, also to NK cells or CD8+ cells; or we could use scFvs bound to the surface of a liposome containing, for example, a drug; then we could use the scFv bound to an enzyme that is able to transform a pro-drug into an active drug; or the antibody could be bound to an immunotoxin, or to a cytokine (for example a pro-inflammatory one to trigger the activation of immune cells). In any case, we can increase the reactivity against the tumoral cell by bringing a toxic molecule, a drug or immune cells next to it. Out of all these strategies that we talked about, the pro-drug one is the one of the most used: it is called antibody-directed enzyme prodrug therapy (ADEPT), after tumor localization and deactivation or clearance of the enzyme from blood and other normal tissue, a prodrug is administered to the patient, and it is going to be converted into a toxic chemotherapeutic by the pre-targeted enzyme at the tumor site. Then another innovative strategy is the Potelligent technology one: we know that antibodies have carbohydrates inside, in particular they have a fucose that, if removed, can increase the ADCC activity. In fact, it was demonstrated that the mechanism behind the enhanced ADCC of a low/no-fucose antibody was its increased affinity to FcγRIIIa (CD16), the major Fc receptor for ADCC in humans. So, this technology dramatically enhances the potency and efficacy of antibodies. Clinical results with Potelligent-enhanced antibodies are expected to show higher efficacy in human patients when compared with antibodies that have not been enhanced. So by increasing Fc receptor binding it overcomes the problem of low clinical responses due to genetic differences in the Fc receptor; moreover, it lowers the effective therapeutic dose of mAb, so you can deliver much more with much less. Potelligent Technology creates antibodies that are expected to be proven safe and well tolerated with no immunogenic concerns and it also reduces the costs of production. When can we use a therapy with antibodies? First of all, since the main use of antibodies as therapeutics is to trigger an immune response (especially against cancer or against microorganisms), the patient must not be immune suppressed, so his immune system must be intact. Moreover, the antibody that we choose to use should have a high serum stability (The concentration of mAbs in serum can be measured by ELISA, electrophoresis on polyacrylamide gels (PAGE) and FACS) and it must be highly specific against a specific antigen expressed exclusively on cancer cells, so that cross-reactivity is limited to the minimum. The best solution would be to produce an antibody against a unique target, but which should also be highly expressed (for example, fibroblast activating protein-FAP-, expressed in tumor-associated fibroblasts). Moreover, the antigen must be present in a homogeneous target population and not show heterogeneity. One important phase in the pharmacokinetics of antibodies is their catabolism, which is actually different from the protein one. There is a specific receptor, called Fc receptor of neonates (because it is highly expressed in the bowel of neonates), which is almost ubiquitous and can bind the Fc portion of any antibody. Once bound, the antibody is going to be internalized in the cell inside vesicles, together with unbound molecules of the same mAb. Then these vesicles will fuse with lysosomes which will digest their content. However, the lysosome enzymes cannot digest the bound antibodies, but just the unbound ones. In this way the bound antibody can be released again in the blood stream once the vesicle is fused back to the membrane of the cell. This particular catabolism is responsible for the long half-lives of the three IgG subclasses. These receptor is mainly present in endothelial cells, however it is also expressed in the bowel, especially of neonates, to transport antibodies from the lumen of the intestine to the blood vessel (which otherwise cannot pass the intestinal mucosa easily). As we said, this binding induces a longer half-life of mAb, however since it involves a receptor, we know that it could get saturated. So, as a consequence, the higher is the serum concentration, the shorter will be the half-life of the antibody: at high concentration Fc receptors are saturated more rapidly and so there would be a lot more unbound Abs (IgG molecules) which are going to be digested. The mAb are administered through the parenteral route (intra-venously, subcutaneously, like adalimumab, and intra-muscularly, like palivizumab). From the subcutaneous and the intramuscular route they enter the lymphatic system and thanks to it they can reach the venous system. This is different from proteins, which can still pass the cells through the inclusion in vesicles or by infiltrating in the junctions and spaces in- between cells, antibodies reach the blood stream through the lymphatic circulation, because they are really big molecules. However, since the flow rate of the lymphatic system is relatively low, the resulting time of the maximum concentration (Tmax) of mAbs in the blood is after 1 to 8 days (and not after hours like for proteins!). The major limitation of the subcutaneous and intramuscular route of administration is that the maximum volume we can inject is 2.0-2.5 ml for the subcutaneous administration and 4.5-5.0 ml for the intramuscular one. This is something which is true for any kind of drug, so not only for mAb, but it is a problem specifically of monoclonal antibodies because the solubility of IgG is 100 mg/ml, so this means that the maximum subcutaneous and intramuscular doses are 200-250 and 450-500 mg/ml, respectively. For intravenous administration there is no limitation of volume, you can administer as much drug as you want (obviously, in a dose that is coherent with the therapeutical window of the drug). Oral administration as the same problems as for proteins. The distribution of mAb is very poor: they cannot pass the tissue easily due to their high molecular mass (MM) and to their hydrophilicity/polarity, so they have to use the lymphatic system. Hence, the transport happens through convention (transport of molecules within a fluid, so the mAb go from blood to the interstitial fluids of the tissues and from the interstitial flids to blood, via the lymphatic system, so they always take advantage of fluids) and through endocytosis which could be receptor-mediated (Fc receptor) and non-receptor-mediated (the endocytosis could be a phagocytosis or a pinocytosis; remember that the difference between the two is that pinocytosis is the internalization of molecules in a liquid phase, while phagocytosis is the internalization of solid material of a big molecular mass). The binding of the mAb to the target could be either specific (if it happens through the Fab portion) or non- specific (if it happens through the Fc portion), in any case we have to remember that the mAb preferentially binds to soluble antigens, while the physiological ligand of the antigen (for example a receptor, if we think about a cytokine) preferentially binds to the ligand on the cell surface. This just depends on the kinetics of the binding. Obviously, if the antigen could only be present on the surface of the cell, the antibody directed against it will still bind to it (same if the ligand is only available in a soluble form), so this high competition with the endogenous antigen ligand is only in the case in which the antigen can be present in both forms. How can the antibodies be eliminated? They are proteins, so they can be digested through proteolysis (but we have to remember that the protective mechanism of the Fc receptor against catabolism is responsible for the long terminal half-lives, for all IgGs except IgG3). However, some types of mAb are degraded in a more rapid way than others, in particular the murine mAb is degraded in few days, while the human is degraded in a few weeks (so the degradation is faster for the antibody that contains a higher percentage of murine chains: murine < chimeric < humanized < human). It also depends on the low affinity of the Fc receptor to non-human antibodies, so the longer half-lives are usually typical of fully human antibody (this is another reason why they are extremely useful). Antibody fragments (especially the intrabodies) show a very short circulation period, they are degraded and digested faster than a normal size molecule. To prevent it we PEGylate the scFv, because PEG can mask the cleavage site of the molecule so it efficiently avoids its degradation by proteolytic enzymes. This is a list of the half-lives of many antibodies (however we can see that there are some molecules reported, Alefarcept and Etanercept, which are recombinant receptors, and they are in this list because they have a similar action as antibodies, they are directed against the TNF). Adalimumab (the first fully human antibody produced) has a half-life of 14 days, while Gentuzumab has a half-life of 66 days (that can vary of 34 days). The recombinant receptors have a half-life that is tendentially shorter than the one of antibodies (for example Etanercept has a half-life of almost 3 days). The binding to the antigen with the Fab region is IRREVERSIBLE. For example when the mAb/antigen complex is located on the surface of a cell, it will be internalized as a whole and degraded. Moreover, if there is an immune reaction against the mAb, the anti-idiotype antibodies are going to be observed after 1 to 2 weeks. As we already said, the adverse effects correlated to the immunogenicity of the mAb depend on: the type of mAb (the lower the extent of the humanization, the more anti-idiotype antibodies are going to be formed), the frequency (multiple dosing often results in anti-idiotype antibodies formation), route of administration (subcutaneous administration is more immunogenic than the intramuscular or the intravenous one, or the reasons we already said for proteins), patient’s genetics (patients with an autoimmune disease are more likely to produce an immune response). Another important aspect is the drug interaction: we know that chemical drugs can interact with each other, because they are all metabolized in the liver by the same enzymes (P450) and so at that level drugs could interfere with one another. This problem does not exist with biotechnological drugs, neither with proteins nor with mAbs. This is because they are proteic molecules, so this means that they are not metabolized exclusively in the liver, but in any tissue, and by multiple types of enzymes (which are all proteases but of different types). So this means that, at low doses, you can administer both biotechnological antibodies and chemical drugs (moreover, you can also administer two biotech drugs together). In this table we can see a comparison between small molecules and mAb: small molecules can easily pass through tissues while mAb cannot; the binding between a small molecule and its target usually implies distribution (and during distribution the drug is present both in the plasma and in the tissues), instead the binding between mAb and its target implies clearance (the interaction of the antibody with the target “clears out” the drug from the circulation); the degradation of small molecules is metabolic, while the one of mAb is proteolytic; for small molecules renal clearance is really important, while for mAb it is uncommon, it rarely occurs just for small peptides; the unbound concentration (drug that has not reached the target) of small molecules could still have an effect on targets of other tissues, whereas for mAb the unbound concentration doesn’t have an effect on targets on other tissues (also because usually mAb have only one specific target), however it could still cause the activation of the immune system (so in this case the side effects are due to this activation, not to an unspecific action of the drug); finally, the pharmacokinetics is linear and independent from dynamics for small molecules, while it is non-linear (you cannot predict exactly how the pharmacokinetics will be) and often dependent on pharmacodynamics and the production of anti-idiopathic antibodies. NUCLEIC ACID BASED DRUGS The main purpose of the use of nucleic acid-based drug is to prevent the mRNA translation, to reduce or prevent the synthesis of the protein encoded by that mRNA, which could be overexpressed or with an altered function. The translation can be inhibited simply by creating a small nucleic molecule (15-25 nucleotides) that is able to bind to our target mRNA. Before the development of this technique, to inhibit the action of an altered protein, we used traditional drugs, however if we block the action of the protein directly by inhibiting its production it should be more efficient. The first nucleic acid-based drug that was developed was the ASO (antisense nucleotides), a single-strand antisense molecule of a short length (15-25 bp), usually of DNA but it could also be of RNA, that is complementary to a portion of the mRNA and binds to it blocking its translation. The reduction of target mRNA is measured on the amount of this mRNA and confirmed by Western blot analysis of the encoded protein. The problem with this approach is that DNA and RNA molecules are digested, inside our body, by nucleases. That’s why the first attempt of producing this kind of nucleic-based drugs led soon to the generation of modified antisense oligonucleotides: they had the substitution of a sulphur atom instead of an oxygen in the phosphate group (obviously, an oxygen that doesn’t participate to the phosphodiester binding, so in a non-bridging oxygen), and this sulphur atom was able to prolong the half-life of the oligonucleotide because it avoided the action of nucleases (it also increased non-specific plasma protein binding). This type of oligonucleotide is called phosphonothioate antisense oligonucleotide (PS), however their half-life still wasn’t long enough to have a good therapeutic result. So these were the first-generation PS, and rapidly a second generation was produced: they are called 2’MOE (methoxyethyl) and they present the addition of a methoxyethyl group at 2’ level. In this way the half-life was greatly increased, without changing the ability of these molecules to bind to their target mRNA. This means that just by making some chemical modification to the molecule we can increase their stability without altering their complementarity and their affinity to the target. The prevention of the translation of the target mRNA occurs in two different manners: in the first case the binding of ASOs to the target mRNA induces the digestion by the RNase H, so the mRNA is rapidly degraded and its translation is impossible; the second way consists in a simple steric block of the translation, because the antisense oligonucleotide just obstructs the normal ribosomal activity. However, 2’MOE ASOs do not recruit RNAse H, so their antisense effect is limited to prevention of translation. In the past 7 years, over 100 ASOs have been tested in Phase I clinical trials, a quarter of which have reached Phase II/III. After the second generation, a lot of other modifications of ASOs have been produced and tested, and they all belong to the third generation ASOs. For example, the morpholinophosphoroamidate (MF) and non-ionic DNA analogues (DNA usually has a negative charge, these types of oligonucleotides have no charge) are highly resistant to nucleases. Which are the FDA-approved oligonucleotide drugs? Until now they are three: fomivirnes (1998-2001), mipomersen (2013), pegaptanib (Macugen, anti-vascular endothelial growth factor, so anti-VEFG, RNA aptamer, so it is actually a different molecule). Fomivirsen is a 21-nucleotide anti-sense molecule used to target the mRNA for the major immediate-early transcriptional unit of cytomegalovirus (CMV). CMV is in fact able to cause an infection in the eye (retinitis), especially in immune suppressed patients. So fomivirsen was used as a therapy for local treatment of CMV retinitis in AIDS patients. This drug worked, but then new drugs against AIDS were invented and these drugs were able to prevent this type of infection, the treatment wasn’t needed anymore. So, despite the initial enthusiasm and unmet clinical need in the late 1990s, the drug was withdrawn by the FDA in 2001. The EMA followed in 2002, when the manufacturer voluntarily withdrew the drug from the market due to low demand. Fomivirsen was used with a 4-week induction phase, with a single injection every other week (so two doses every induction phase), followed by a maintenance phase, in which a single injection is administered every 4 weeks. The only adverse effect that it could cause was ocular inflammation (uveitis). Nevertheless, the success of fomivirsen provided proof-of-concept of the clinical promise of treatments based on antisense oligonucleotides, which was valuable for the next wave of antisense drug approvals, beginning in 2013 with the FDA approval of mipomersen. It is a 20-nucleotide anti-sense molecule with each inter-nucleotide linkage chemically modified as a PS diester and with a 2’-O methoxyethyl sugar. It is targeted against the mRNA of ApoB-100 (apolipoprotein B), for the treatment of homozygous familial hypercholesterolemia (HoFH). Apolipoprotein B is a part of the LDL lipoprotein, so the bad cholesterol, so by reducing the expression of this protein we reduce the formation of LDL. The treatment involved the subcutaneous injection of the dug once a week: this is a real innovation because fomivirsen was used locally, instead mipomersen can be used systemically (because from the subcutaneous administration it can reach every tissue). In the skin it forms a depot that allows the slow release of the drug (which actually has a short half-life because of its nature, even if it is modified). Adverse effects involve erythema or pain at the injection site (subcutaneous), flu-like symptoms, increase in liver fat content in some patients. Pegaptanib (Macugen) is particular and different from the others because it is not a linear molecule, but it has a complex tertiary structure. It is in fact a 27-nucleotide RNA aptamer (and aptamers are oligonucleotides that share some attributes of monoclonal antibodies due to a complex 3D structure, in fact they usually recognize their target through their shape, not through complementarity), it has nucleotides annealed with hydrogen bonds and it has a 2’-O methyl modification. It targets the mRNA of VEGF; it was approved by FDA in 2004 for the treatment of age-related macular degeneration, a condition that could lead to blindness. It is injected locally (in the eye, intravitreal injection) once every 6 weeks (it is a treatment that doesn’t need to be repeated very frequently, also because of the site of injection). Adverse effects involve pain at the site of injection, sudden vision problems and headaches. This is the first type of oligonucleotide drug that was invented, but we could have also other molecules that can work to prevent the translation of mRNA, and they are the RNAi (interference). They are double stranded RNA molecules and they mainly divide into two categories: miRNA and siRNA. These two types of RNA are produced from the region of the genome called non-coding DNA, which represents the majority of the genome, it doesn’t encode for any protein but it actually encodes for RNA molecules with regulatory activities. The amount of non-coding DNA increases in more evolved species; in vertebrates, and in particular in mammals (especially in men), we have the highest amount. This is because the species that are high in the evolution scale have a fine regulation of gene transcription, which is what makes them more evolved. In the group of non-coding RNA (ncRNA) we actually have a lot of different molecules, like rRNA, tRNA, snRNA and snoRNA, other than RNAi, but this last class is the only one used for therapeutic purposes. RNAis are short non-coding RNA sequences involved in the regulation of expression, in fact both miRNA and siRNA, even if with different approaches, induce the block of the translation of specific mRNAs preventing the production of the protein for which they encode, so the purpose is gene silencing. 1. siRNA (19-24 nucleotides): they are transcribed from non-coding regions from RNA polymerase II, they are produced in the form of dsRNA or shRNA (short hairpin RNA). This precursor is then released in the cytoplasm, where it is digested by an enzyme called DICER, which produces a double stranded siRNA (also called siRNA duplex or siRNA-siRNA). Of this duplex, only the antisense strand is used as a guide strand, in fact it is incorporated in the enzymatic complex RISC-AGO2, while the sense strand (also called passenger strand) will be discarded. The guide strand of siRNA, once incorporated in the RISC complex, will bind to the target mRNA because of its complementarity with it, and thanks to this binding the enzymatic complex is going to induce a cut in the target mRNA, in a region called seed region, inducing its cleavage and its subsequent degradation. 2. miRNA (20-23 nucleotides): miRNAs are actually transcribed from introns (so they are still encoded by non- coding RNA, but from the one contained inside genes!) by RNA polymerase II. They are transcribed first in the form of pri-miRNA, a precursor, which is then transformed in pre- miRNA from an enzyme called  Drosha-DGC8, inside the nucleus. Then the pre-miRNA is exported in the cytoplasm (thanks to exportin 5), where the DICER is going to transform it into a double stranded miRNA molecule (miRNA duplex or miRNA-miRNA). Even if the process is quite the same of the siRNA one, we have a difference between these two molecules: siRNA have a complete complementarity with the target mRNA, so also the duplex will be a linear double strand molecule, while miRNA do not have a precise complementarity, and we can understand it from the fact that their duplex contains a sort of “hole” inside it (region in which the nucleotides do not pair with each other). Again, only one strand will be the one used for the gene silencing (the other one is digested), and, incorporated in the RISC-AGO2 complex, it will bind either to the regulatory regions of the mRNA molecule (5’-UTR and 3’-UTR) or to the ORF (open reading frame, so the encoding region). This is because the miRNA doesn’t have a specific complementary to the coding region of the mRNA, so they can target a lot of parts of the mRNA molecule. Finally, the miRNA could also go back inside the nucleus and, always bound to the RISC-AGO2 complex, bind to the promoter region of genes (so of DNA). More than 500 miRNAs are encoded in the human genome, so they constitute the largest gene family; it has been estimated that more than 30% of protein-coding genes can be regulated by these miRNAs. Both miRNA and siRNA are molecules that are normally produced by the cell but they could also be injected inside a cell (or they could be expressed by it), like ASO molecules. What is the seed region? It is a short sequence present on the target mRNA that is usually perfectly complementary to the siRNA, while we don’t have the same level of complementarity between this region and miRNAs. So, if the molecule that binds to the seed region is perfectly complementary (usually siRNA, full match), the mRNA will be degraded through the action of the RNase H; if the molecule that binds the seed region is not perfectly complementary (usually miRNA, partial match) the mRNA will not be degraded, but there will just be a physical block of the translation. In both cases, the result is the repression of the translation and the mRNA will not be translated. However, these different levels of complementarity are index of the fact that siRNA will eventually have a single target (they are complementary to only one mRNA because they have the same sequence), while miRNA have multiple targets (because they are not fully complementary to any of them, so they could potentially target a lot of different mRNAs). We already said that these RNAi are physiologically produced by our cells, in particular miRNAs are the most represented ones. They have a lot of physiological roles, that they carry out through their regulatory action, for example they are involved in organ and tissues development, in stem cell differentiation and maturation, in cell growth and survival, in metabolic homeostasis, oncogenic malignancies and tumor formation (because it can regulate the expression of genes involved in the growth of the cell, so they are unfortunately also involved in these pathologic activities), viral infection and epigenetic modification. But we can use them also as therapeutics. In this sense we can consider them as both New Chemical Entities (NCEs), because they can be chemically synthetized, and New Biological Entities (NBEs), because they have a highly specific target and they have a complex modality of action. So they are different from the other biotech drugs that we talked about, which are proteins and cannot be chemically synthetized. Another big difference is that one molecule of RNAi can target one mRNA but then it can be recycled and target another mRNA molecule, so this means that RNAi based compounds can be used for several rounds of cleavage (obviously it also depends on the concentration because the lower will it be, the less frequently this phenomenon will happen, but at the same time the lower the dosed the less the side effects). So which type of RNAi have been used in the therapeutic field? Systemic administration: this type of administration is generally preferred for any type of drug because in this way it can easily reach every tissue. However, nucleic drugs should be modified prior to a systemic administration because their stability inside our body is very low. The first example is the one of Patisiran: it is a siRNA that targets the mRNA of a protein called transthyretin, that, when aberrantly produced, causes the accumulation of amyloid fibrils in different organs, determining the appearance of a pathology called Familial Amyloidotic  Plyneuropathy (FAP). The siRNA is encapsulated in a lipid nanoparticle formulation (LNP) to be protected from degradation so that the drug can be administered systemically. This drug is currently in phase III trial and it’s being used in infusions every three weeks. The second example is the ALN-PCS02, used for the treatment of coronary heart disease, in particular for the reduction of LDL. In fact this drug targets the mRNA of the pro-protein convertase subtilisin/kexin type 9 (PCSK9), but it is still in phase I. Another siRNA used for the same purpose is the TMK-ApoB, which targets the apolipoprotein B (ApoB) to reduce the production of LDL. However, this drug was halted in 2010 due to unexpected immune stimulation (so not every nucleic drug, even if it works, eventually reaches the approval because there could also be some problems). Both these drugs are encapsulated in LNP like in the first example. Another example is the one of siRNA used against cancer, in particular for the treatment of hepatocellular carcinoma (TKM-PKL1, encapsulated in LNP, targets polo-kinase1, so PLK1) and of neuroendocrine tumors (Atu027, encapsulated in lipoplex nanoparticles, targets protein kinase N3, so PNK3). They are both in phase II. We have then another siRNA, the naked siRNA 2’-O methylated QPI-1002, with a modification that is usually applied to ASOs but that prevents the digestion of the molecule without the need for liposome nanoparticle encapsulation. This siRNA targets p53 in the treatment of acute renal failure, it is in phase II currently. Local administration: the local administration is preferred when the tissue that the drug needs to target is easily accessible from the outside. This is because the systemic administration, even if it is generally always preferred, cannot always be achieved with biotech drugs (especially with molecules so unstable like RNAs), so in that case we prefer to use local administration. One example of this administration is Bevasiranib, a siRNA targeting VEGF for the treatment of the age-related macular degeneration; however, it was discontinued in phase III clinical trials. Other local administration examples could be the ones of siRNA for the treatment of diabetic retinopathy, targeting fibronectin, laminin and collagen IV, of ocular neovascularization, targeting VEGF and of glaucoma (targeting myocilin). We can see how all of these examples involve a retinal administration, and we already saw another nucleic drug used for the treatment of macular degeneration that was applied by this route, so in general we can say that this route of administration is optimal for this type of drugs. Could miRNA and siRNA be toxic? We know that RNA and DNA molecules cannot be administered naked, at least not as they are, they need to be at least chemically modified. Another expedient could be to encapsule them in lipid nanoparticles and polymeric nanoparticles. These complexes are then internalized by the cells through endocytosis. If the endosomal vesicle doesn’t break (no endosomal escape), once the nanoparticles disrupt, the RNAi is released outside of the nanoparticle complex but still inside of the vesicle, where we can usually find toll-like receptors (which were previously on the part of membrane that produced the endosomal vesicles). The binding of the RNAi molecule to the toll- like receptors (especially TLR7 and 8) activates them because the molecule is recognized as exogenous. In this way we could have an unwanted immune response. Otherwise, if the endosomal vesicle breaks (endosomal escape), the RNAi, let out from the nanoparticle complex, is released in the cytoplasm of the cell, but the same will happen with the residual nanoparticles, and especially if they are polymer nanoparticles, they induce the release of cathepsin B and of IL-1b (responsible for fever) and the triggering of inflammation and apoptosis of the cell. So RNAi could be toxic for our body: even if the benefits that these drugs give are still much higher than the risks, some side effects could still manifest. Another aspect to consider is that the toxicity can also derive from the hybridization on the target: if miRNA could have multiple targets, how are we sure that the miRNA that we are using as a therapeutic agent is not actually silencing genes which are not our target? This is the risk of having off-target effects. This risk could be avoided by using a drug with a higher specificity and selectivity for our target, for example siRNA have a higher complementarity for their target compared to miRNA, so using the short interference instead of the microRNA could be a solution. But if our final goal is just to reduce the expression of a protein, and not of totally silencing a gene, how can we know that our RNAi is just decreasing the expression and not preventing it completely? This is a risk called exaggerated pharmacology, and it should be avoided. So in conclusion, the toxicity of RNAi could be hybridization-dependent, and in that case it depends on the molecule itself, which could cause exaggerated pharmacology (the result is an effect much bigger than the one we wanted, so it has a negative impact to the cell) or off-target effects; or it could be hybridization- independent, and in that case it is caused by the chemical modification that we make on the drug to improve its stability or by the delivery agents. Lipid based vehicles (like LNP), cause infusion-related reactions, activation of the complement, and other reactions that we already saw. A lot of RNAi are currently in clinical trials (especially for the treatment of cancer), a lot of them are in phase III trials and one has recently been approved (for the treatment of transthyretin mediated amyloidosis). Also an aptamer called “aptamer-antidote” has been approved against factor IX (coagulation cascade) for the treatment of percutaneous coronary intervention. Other than RNAi and ASOs, other nucleic-based molecules are being tested right now (like shRNA). Moreover, exogenous miRNA can be used also to replace their physiological counterpart: if one pathological condition is given by the alteration on the expression or on the function of a particular miRNA, we administer an exogenous miRNA with the same sequence and function and we can counteract the pathologic condition. In some other cases, especially in cancer, miRNA have been found to have an altered action, so they are used as targets instead of drugs. What about the pharmacokinetics of RNA/DNA-based drugs? For what concerns the administration, they are mostly delivered locally but they could also be delivered systemically, in particular by intravenous and subcutaneous route. The non-parenteral administration could be only made possible with the aid of new formulations. They are rapidly distributed, they have a short half-life because they are degraded by nucleases, but we can modify them to avoid this problem (for example the 2’-MOE can make them last for 10 to 30 days). They usually have a high binding affinity for plasma proteins. After intravenous administration, the greatest accumulation of ASOs occurs in the liver and kidneys (so this could also be used to possibly target these organs), however it is difficult to find them in urine or in the faeces because they are degraded in liver and kidney cells (so you could find traces of the drug); if they are injected subcutaneously they form a depot and they are accumulated in the skin and they are very slowly released to the blood stream and also slowly digested. It would be possible to create new formulations for the gastrointestinal delivery of these drugs (like suppositories, which cover less surface area, enemas, gels and foams, which can reach the sigmoid colon, for the rectal delivery, and capsules and tables for the oral delivery). However, this is just for a local use in the gastrointestinal tract (for example the use of capsules and tables could allow a regional release of the drug in the colon, like in the treatment of Chron’s disease). The ocular delivery is still the most efficient for the local administration until now (with intravitreal injections, but an alternative is low current iontophoresis). Another delivery could be the pulmonary one, through single-dose nebulizers, metered- dose inhalers, and dry powder inhalers, but anyway the aim is always to locally treat pulmonary pathologies. The delivery to the brain is extremely difficult because of the blood-brain barrier, however, since the BBB is only permeable to lipophilic molecules of a molecular weight lower than 600 Da, a possible way of administration could be the use of lipidic nanoparticles, or a continuous intracerebral infusions with a mini-osmotic pump, or we simply create conjugates of streptavidin as carrier to transport ASOs after systemic administration. Another way of delivery could be through the skin, for example by using some chemical modifications of ASOs (alteration in the thermodynamic properties of ASO through a hydrophobic counter cation, like benzalkonium; chemical elimination of the anionic backbone charges of the ASO, to increase the penetration in the skin), or by the use of ultrasound-induced sonophoretion and iontophoresis or by gene-gun, or finally by topical formulations containing permeation agents (however, all of these are still under study, because we are trying to administer biotech drugs with the techniques that we use for chemical drugs). For what concerns the parenteral administration, with the use of liposomes we can prevent the degradation and prolong the half-life of the nucleic molecule. Given the excellent solution stability and solubility, simple aqueous formulations are generally administered by slow infusion, rather than intravenous bolus to control pick plasma levels