Appunti Drugs-8-12.pdf

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 elimina...

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