Appunti Drugs -40-52 PDF

disulphide bonds form between two different chains (interchain) we have the aggregation of different proteins. This phenomena are generally called disulphide exchange. Each one of these modifications can alter the structure, and thus the function, of our protein of interest, which must be kept in it...

disulphide bonds form between two different chains (interchain) we have the aggregation of different proteins. This phenomena are generally called disulphide exchange. Each one of these modifications can alter the structure, and thus the function, of our protein of interest, which must be kept in its native form. They could occur at any step of purification, so both during chromatography and after chromatography (for example oxidization could happen because we have potent oxidizers in solution but also just by contact with air, so it could happen also at the end of the process). To avoid it we perform all the steps at low temperatures or by adding chemical reagents that prevent these modifications. Once we have purified our protein and once we are sure that it was kept at its native form, we have to stabilize the protein. This is performed by adding stabilizing excipients, like human serum albumin. The human serum albumin does not alter the activity of the protein (obviously we have to use a recombinant one because if we used the one extracted from blood directly there would be the risk of transmitting infections from the blood of the donor). It is stable at low pH or at elevated temperature and it is very useful because it could work as an alternative target for proteases that are eventually still present in traces. It is also an effective cryoprotectant and once injected in blood it doesn’t cause anything wrong because we already have it in our blood (if we inject little quantities of it nothing happens in our body). Human serum albumin is the most used stabilizing agent, but we could also use amino acids, like glycine (used for IFN, EPO, factor VIII, …), which reduces the surface absorption of products, inhibits aggregated formation, stabilizes the conformation of the protein and prevents heat denaturation. We could also use polyols (like glycerol, mannitol, sorbitol, PEG and carbohydrates) or surfactants (protein denaturants which reduce the surface tension and increase the solubility of the protein, they avoid the mis-folding of the protein and they reduce the rate of protein denaturation at interfaces). The final step before the lyophilization is the sterilization: we pass the protein solution in a filter with pores of 0.22 micrometres which can get rid of bacterial cells. In this way we are 100% sure that we don’t have any bacteria in the solution. However, we could also have other types of microorganisms, like viruses, which can infect mammalian cell cultures. The filters that we use do not get rid of viruses so we have to change filters and use the ones that have 0.1 micrometres pores. Our sterilized protein then is ready to be lyophilized: lyophilization is the process through which we remove the solvent directly from a solution while in the frozen state, obtaining a powdered product (3% water). It is extremely useful to preserve the biological properties of the product, however it can promote the inactivation of some proteins, so specific excipients are added (e.g. HSA), along with amino acids (e.g. lysine, glycine). The whole process is performed while decreasing the temperature and, while it lowers, it induces the formation of ice crystals, instead the protein will aggregate and separate from the water molecules. The crystalized water is removed, while the temperature is still lowered until molecular mobility ceases (the lowering of the temperature is responsible for an increase in the viscosity of the solution, the mobility ceases around -60° of temperature). Finally, we apply a vacuum which is going to remove all water crystals (through sublimation), other solutes and salts, so that eventually only the protein will remain. After this process, a label is applied on the final product, which contains information on the name, the strength, the batch number, the date of manufacturing, the expiry date and the storage conditions of the drug. First, though, we have to perform a quality control of potency, sterility, final volume, absence of endotoxins or other toxic substances. In this phase, some mis-labelling errors could happen, so we have to pay attention. This process is performed for any lyophilized  compound, with the only difference that, if the compound is intended for laboratory practice, it cannot be used for therapeutical purposes because it will not be as highly purified as biotech drugs (in fact in the vial you can read “for laboratory use only”). Once you have your final lyophilized product, before marketing and using this drug you have to perform other quality control analyses on your drug, because you have to be sure that it is in its entire form, that there are no aggregates, no shorter form of the protein and that it works. So you take one out of the thousands vials that you produce and you test it. What do you test? You test potency (potency must be analysed not only to check if the drug works but also to assess the right dosage of administration of the protein, so we have to check the potency of the drug at a specific concentration) and safety (you have to be sure if there are no potential contaminants in the final product). Which types of contaminants can we find inside our final product? We can find residues of proteins added to remove tags, proteins from the cell culture media (bovine serum/foetal calf serum), endonucleases used to degrade the DNA in microbial cells, … Why do we still find them even if we used different purification steps? There is the possibility that these proteins that we find in the final product had the same size, isoelectric point, hydrophobic level, … than our protein of interest, so even if we purified the solution we still find them in the final product. Obviously, these residual proteins need to be removed because they can be immunogenic, especially if repeatedly administered with our drug. Other impurities may be derived from the modified product, due, for example, to covalent modifications. So some proteins may have a different tertiary structure (wrong folding during the production), so they may also be biologically inactive and thus they could confer to the product a reduced potency. Some modified forms could also be biologically active, but they could have modified pharmacokinetic characteristics. Modified forms may be immunogenic too, so they must be removed. The removal of altered forms of the protein from the product happens usually in the downstream processing, exploiting the different characteristics that these modified proteins have compared to our protein of interest (for example, if the modified form is shorter than the original protein, it can be removed through the size-exclusion chromatography). Different techniques can be used to detect any protein-based impurities that may be present in the product, like non-denaturing electrophoresis, SDS gel electrophoresis, capillary electrophoresis (capillary with a diameter of 20-50 microm; this technique is highly costly so, even if it allows to analyse the product in a fast way, we tend to not use it), peptide mapping, isoelectric focusing, HPLC, mass spectrometry (most effective and precise way to determine the molecular mass of many proteins) and so on. Two-dimensional electrophoresis is really useful to detect protein-based product impurities because it separates proteins based on their molecular weight and isoelectric point, instead an SDS-PAGE only separates protein based on their molecular mass, so if contaminants have the same weight as the product they will go undetected as they will co-migrate with it. So two-dimensional electrophoresis is preferred because it allows to determine product homogeneity (guaranteed by the appearance of a single protein band) and to verify the stability of the product over the course of their shelf life. HPLC can also be used to determine the purity of high and low MM of the product; for what concerns mass spectrometry, we can use a specific type called electrospray mass spectrometry (small, highly charged droplets are formed by electrostatic dispersion of a protein solution through a glass capillary subjected to a high electric field, then protein ions are desorbed from the droplets into the gas phase, assisted by evaporation of the droplets in a stream of hot N2 gas; the last step is the analysis of the protein ions in a mass spectrometer). If we find contaminants we must perform additional purification processes, like chromatography again. How do we test the product potency? Potency is expressed as «units of activity per vial or dose» or «mg» of product. We have different approaches to determine product potency: Bioassays: you reconstitute the protein (just by adding water to the lyophilized product) and then you perform tests to see if the protein still works. For example if we have to test interferons, we can use them in a mouse model infected by a virus and see if the interferon, which works as an anti-viral protein, works. Other than animal models we can also test the efficiency of the protein in single organs, tissues or cell lines. All bioassays are comparative (which means that the product is tested against a standard). The disadvantages are the lack of precision (the test is influenced by the  physiological state of the cell or of the animal), the times of performance (some tests could require weeks) and the high costs (especially if they involve whole animals) Immunoassays: radioactive assay (RIA, quite old) and ELISA are rapid way of testing the potency of the product. They are very effective for the testing for the quantification of the working protein in the final product, but we cannot calculate the quantity of the modified protein in the vial. However, if the quantity of functioning protein calculated with these assays is not the same as the product that we expect to have in the vial we can understand that something is wrong. Other disadvantages involve the fact that these tests are based on the immunological reactivity of the product, which not always correlates directly to the biological activity. The determination of the protein concentration can be assessed also with simpler methods, like absorbance. However, these techniques allow to quantify the total amount of protein present in the vial, without differentiating between working protein, correctly folded protein and modified protein. So ELISA is better because it allows to differentiate between the actual working product and the modified one. But we can use immunoassays also to detect the presence of product-unrelated contaminants (so not the modified forms). The “blank run approach” is performed: we construct a host cell identical in all the characteristics to the cells that we used for the production of the product, except that it lacks the gene coding for our desired protein. The two cell lines will undergo the same processes during the upstream and downstream processing. Finally, the protein are extracted from the cells that do not present our protein of interest are used to immunize horses or other animals. Then, we collect the serum of these animals and purified polyclonal antibodies directed against the protein of that specific cell lines are used in RIA or ELISA approaches against the final product of the cells lines that produced our protein of interest. If these antibodies recognize antigens in this test it means that in the final product we still have some contaminants coming from the proteins of the cell line in which we produced our protein. To verify the protein identity (so to be sure that the final product is actually made of the protein that we wanted to produce and that the primary sequence of the protein is conform to the licensed product specifications and is not modified) we can perform several tests: amino acid analysis, peptide mapping, N- terminal sequencing or analysis of secondary and tertiary structure. Amino acidic analysis is the analysis of the range and quantity of amino acids present in the product just by inserting the latter in an automated amino acid analyser (which is able to study peptides or small polypeptides with a molecular mass lower than 10 kDa). The disadvantages of this technique that limit its usefulness in biopharmaceutical analysis are that it has to be performed in hydrolysis conditions, which could destroy or modify certain amino acid residues (like tryptophan, tyrosine, serine), the method is half-quantitative rather than quantitative and the sensitivity is at best moderate, so low levels of contaminants may go undetected, especially if the product has high molecular mass. However, there is no test that can be considered complete to identify the protein so we still perform this one combined with other test. We can in fact perform also a peptide mapping: it takes advantage of an endopeptidase digestion, so that the protein is cut out in small pieces by enzymes like trypsin, or by chemical agents (the most used is cyanogen bromide, which cleaves the peptide bonds on the carboxyl site of methionine residues). Then the digested protein can be run in bidimensional electrophoresis so we can separate the single polypeptides based on molecular mass and number of amino acids. This allows to evaluate if there is even a single point mutation in the product’s gene that alters the primary structure of the protein, because trypsin won’t be able to digest the protein like it would with the native one so the results of the electrophoresis will be different that the ones expected. Usually single amino acid substitutions, deletions, insertions or modifications can be detected with this method. However we could have a problem with this method, because if a reagent generates only a few very large peptides, a single amino acid alteration in these peptides will be more difficult to detect than if it occurred in a much smaller peptide. Instead, a large number of very short peptides would not allow to resolve all the peptides from each other, so an average of 7-14 amino acids per peptide would be the best for this type of technique. Another test that we can use is the N-terminal sequencing: we have machineries that can sequence proteins and so they allow us to identify the presence of modified forms of the product in which one or more amino acids are missing from the N-terminus (up to the first 100 amino acids from the N- terminus). This sequencing is known as Edman degradation, it is based on the use of phenyl isothiocyanate which can bind to the N-terminal of the protein and allows the identification of the amino acids. The last method that we can use is the analysis of secondary and tertiary structure through X-ray crystallography (which is very complex and highly expensive, so we tend to avoid it for biopharmaceuticals) or through spectroscopic methods (like far-UV circular dichroism, which shows us spectral features related to the structure of the backbone of the protein and can be used to predict the secondary structure of the product). One other step must be performed before the commercialization of the drug, and this step is mandatory if we produced the protein in bacterial cells: we have to check for the presence of endotoxins, like LPS, and other pyrogenic contaminants. LPS can still be present in the final product even if we already filtered the debris of the solution, so we have to check again at the end of the process because no molecule of LPS should be present in the final formulation, since even low dosages of this endotoxin (0.5 nanog/kg of body weight) could still induce adverse medical reactions. LPS is made of a polysaccharidic core and it is bound to a lipidic molecule, the lipid A, which is highly toxic for humans because it is pyrogenic. LPS molecules are present on the surface of Gram- bacteria and they constitute the 75% of their outer membrane surface. It is heat stable so autoclaving the solution will not destroy the endotoxin. To check for the presence of this endotoxin we need animals: the first test that we could use is the pyrogen detection in rabbits. We simply inject the final product in a rabbit and then we check for the increase in body temperature. If it increases of more than 2.65°, the formulation contains some sort of pyrogenic molecule that must be removed; if the temperature increases of 1.15° or below, then the product is safe (no pyrogen inside); if the temperature is in between the two values, then the test must be repeated. This test is very easy and fast, however it is expensive, is involves the handling of rabbits (their excitation or poor handling may affect the results of the test), false positive results may be due to subclinical infections or poor animal health and different colonies can yield variable results. So we can use another test, performed on another animal, which is the Limulus (also called Horseshoe crab), which is called LAL, Limulus amoebocyte lysate. This test is used to detect the presence of LPS, not of any other pyrogenic or endotoxic molecule. This is because this animal presents a very simple blood, made of only one type of cell called amoebocyte, and once the blood is in contact with air it becomes blue, because it contains cupper in a pigment called hemocyanin, which transports oxygen in this animals just like haemoglobin in men. This animals present a very simple immune system, whose action is carried out by these single cells that we find in blood, and these cells, once in contact with LPS, are able to recognize it and eliminate it forming a coagulation of this endotoxin. So the test is performed by collecting the blood of the animal without killing it, then we obtain a lysate of amoebocytes from it in order to use its enzymes with our final product and check for the presence of LPS (if LPS is present, a coagulation cascade will start and so we will observe the formation of aggregates in the final formulation). The advantages are that this test is highly sensitive, it has low costs and it can be conducted in a short period of time (15 to 60 minutes). However, the disadvantage is that is high highly selective only for LPS, and, even if LPS is by far the most likely to be present in a pharmaceutical product, if we have to check also for the presence of other endotoxins we must use the rabbit test. Which other types of contaminants can we find in the final vail? DNA, for example, could still be present in the final product. No more than 10 picograms of DNA per dose can be tolerated by humans, so we must check for the concentration of this molecule before commercializing the drug. The technique that we use is the DNA hybridization (dot blot assay): we isolate the contaminating DNA from the product with a simple phenol/chloroform procedure, we spot this recovered DNA on a nitrocellulose filter, we extract the total DNA of the host cell that we used to produce the protein and we label it, then we hybridize it to our spotted DNA (the one recovered from the final product). The labelled DNA will anneal with any complementary DNA strand onto the nitrocellulose filter, so if we visualize a signal that means that the DNA of the cell line is still present as a contaminant in the final formulation. Then we have to make sure that we don’t have any viral contaminants: viruses can be removed with filtration with 0.1 micrometres filters. Usually this filtration is performed before lyophilization because we know that we have to remove any type of microbe inside our solution (they can cause severe infections, they may be capable of metabolizing the product because they could be able to produce extracellular proteases and microbial-derived substances secreted in the product may be toxic for the patient), however, to make sure that we don’t have any microorganism left (especially viruses, that are smaller) we repeat this process also in the last steps. Prior to that, to check for the presence of viruses, we perform viral assays: they are specific for a kind of virus or for a family of viruses and they could be immunoassays (which are sensitive and inexpensive), virus specific DNA probes (similar to dot-blot assays) or bioassays (in this case we could incubate the final product with the cell lines sensitive to a range of viruses or we could administer the final products to a test animal, and if the animal shows any symptoms then its serum is screened for a range of antibodies recognizing a range of viruses). Lastly, some contaminants may be unintentionally added to the product stream. Metal ions can leach from the product-holding tanks pipework or from chromatographic media, so finished products are often subjected to “abnormal toxicity” or “general safety” tests. These entail parenteral administration of the product to at least five healthy mice, then the animals are placed under observation for 48h and should exhibit no ill effects. The death or illness of one or more animals signals a requirement for further investigations. All of these procedures are a part of the so-called validation studies: validation can be defined as “the act of proving that any procedure, process, equipment, material, activity or system leads to the expected results”. New and older items must be routinely validated, and older ones with increased frequency. Validation of biopharmaceutical aseptic filling procedures is amongst the most critical. Contaminant-clearance validation studies entail spiking the raw material with a known level of the chosen contaminant and subjecting the contaminated material to the complete downstream processing protocol. This allows determination of the level of clearance of the contaminant after each purification step. This is mandatory for biotech drugs, for chemical drugs this complex validation step is not required because they are produced with simple chemical reactions, so the validation is far easier and quicker. In this case, all validation procedures must be carefully designed and fully documented in written format. Once we are sure of what we are dealing with as our final product we wait for the FDA approval; if the FDA approves it the product is licenced, marketed and sold. This in the picture is a recap of all the steps. Remember: different process run at different times equals to different product, even if it is performed by the same pharma company. This doesn’t mean that the protein will be different, but that the total product will be slightly diverse (because maybe the DNA vector that we use for the cloning is a little different, or the host cell has different expression levels, or the fermentation process occurs differently, or the downstream protocol is run in a different way, or the final formulation is different). This is something typical of biotech drugs, chemical drugs are always the same even if the processes are different and run at different times. The paradigm of biopharmaceutical is that the process is the product. THERAPEUTIC HORMONES 1. Insulin: insulin has a lot of functions in the body, the most important is the reduction of glucose in the blood stream. It is a very small protein (6 kDa in weight), it is composed by two chains, which are the A chain (21 amino acids in length) and the B chain (30 amino acids in length), that are kept together by disulphide bonds, which are both intrachain and interchain. The structure and the length of the protein are really important to know because through the different composition we can obtain different types of insulin. Physiologically, it is produced as a pre-proinsulin, which is a single polypeptide molecule which contains the two chains (A and B) bound together by a linking peptide called C chain. Then the disulphide bonds are produced and the C chain is digested, so that the protein is only made by the A and B chains. This physiological process can be reproduced in vitro to produce recombinant insulin. Moreover, we know that native insulin molecules are kept together by zinc atoms, forming multimers, in particular hexamers, which determine how quickly the insulin is going to be absorbed into the tissues, and this is really important for a pharmacological application. In the past, insulin was obtained by pigs, and about 70 pigs per patient per year were needed for the production of this protein for diabetic patients. Porcine insulin differs of only 1 amino acid from the human one, however it was immunogenic (the bovine insulin differs of 3 amino acids so it’s even more immunogenic than the porcine one). So in order to facilitate the treatment of diabetic patients, the recombinant insulin was first produced and marketed in 1982, becoming the first marketed biotechnological drug. The main advantages is that we can produce huge amounts of the protein in this way, with a low grade of immunogenicity, however the disadvantage is that E. Coli is not able modify the eukaryotic pre-mRNAs nor it can operate post translational modifications of the protein. Anyway, with biotechnological approaches and with recombinant DNA techniques, it is possible to produce insulins with altered pharmacokinetic properties (faster- or slower-acting) and to select insulin with higher receptor affinities (ensuring economic benefits from using smaller quantities of insulin per therapeutic dose). How do we produce insulin? The first method is the one of insulin crb: we have two cell lines of production (of E. coli), in one we clone the gene of the A chain, in the other one we clone the gene of the B chain. We make these cells express the two chains, we purify them through chromatographic steps and then we incubate both chains under appropriate oxidizing conditions to promote the formation of interchain disulphide bonds. In the second method, on the other hand, we produce the whole molecule in a single cells line (insulin prb): we insert the gene encoding for human pro-insulin in recombinant E. coli, we purify it from the molecules of the bacteria and then we remove the C peptide in vitro with a proteolytic excision. This second method is really easy and we use HPLC chromatography to purify it in the “clean-up process” (this chromatography method yields a final production of approximately 99% purity in 1 hour, keep in mind that the loaded product is 92% pure), achieving low levels of impurity to avoid any significant immunological response. The engineering of a protein allows us to modify the protein if we need it: we can change the amino acidic chain to obtain different types of insulin with different properties. The different types of insulin can be divided based on how much time they spend in the circulation: if we create a graph comparing the plasma levels of the different types of insulin we can see that there are some types of insulin that remain in blood for a longer time (multiple hours, almost a day) than others. In fact, we can have rapid-acting insulins (Humalog, novolog and apidra; they are rapid acting and they are rapidly eliminated from blood), short acting insulins (regular one), intermediate (NPH) and long-acting ones (glargine, detemir; long onset of the therapeutic effects but they last for a longer time). Regular insulin was the first one produced: it is identical to the physiological one, however its curve of duration in the blood circulation doesn’t identically overlap with the physiological insulin (it lasts for a longer time), so this means that there is a delay in the onset of insulin in the blood, in fact after injection we have the so called “lag phase”, which lasts for about half an hour/ 1 hour. After the lag phase, the insulin reaches its peak after 2-3 hours and it is cleared within 8 to 1 hours (so it doesn’t last for the whole day). This means that patients need to inject this type of insulin at least 30 minutes/60 minutes before a meal. Physiologically, zinc atoms keep together single molecules of insulin, which are called monomers, and usually physiologic insulin is present in groups of six monomers, so it forms hexamers. For regular insulin it is the same (because it is associated to low zinc concentrations, around 0.5-0.7%), that’s why it takes a while until it is absorbed, because for insulin to be absorbed it has to be in a monomeric form. Regular insulin exists in preparations as a mixture of monomer, dimer, tetramer and zinc-insulin hexamer and, since absorption must occur under monomeric form, the other forms slow the rate of absorption. The second insulin that was produced was NPH (neutral protamine Hagedorn): it is an intermediate acting insulin that takes about 2 to 3 hours to reach the maximal concentration in the blood and then its action lasts up to 12 hours. It contains protamine, a protein contained in fish, that is able to delay the absorption of this type of insulin so that one injection can last for a longer time compared to regular insulin. Why do we need these different types of insulin? Because we have to mimic the physiologic levels of insulin, so it’s really important to maintain also a basal level of insulin, instead of having to inject regular insulin multiple times in a day. Sometimes, the risk for a diabetic patient is the nocturnal hypoglycaemia, in fact some insulins cannot be administered before the night because if the insulin is too high in blood during sleep time the patient could suffer from hypoglycaemia. NPH is one of these: the peak of action is after 5 to 7 hours after administration, so we have the risk of nocturnal hypoglycaemia if we administer it at bedtime. Moreover, even if it lasts longer, the duration of its action is not long enough to cover insulin requirements for the whole day with a single injection. On top of that, the action profile depends on the dose and it is highly unpredictable also because we have a variability of absorption that depends on site, on exercise and on the variation in mixing of suspension. For this reason, some devices have been invented to inject amounts of insulin repeatedly and directly in the body of the patient, and they are called insulin pumps. For these reasons we can understand why we need insulin analogues even if we have insulins with improved characteristics: insulin therapy is supposed to mimic physiological insulin secretion (both basal and bolus), so preparations providing bolus (meal-related) insulin requirements should have a rapid onset, rapid peak and short duration, while those providing basal requirements should have low basal concentration, without a peak, for a long duration. Regular insulins don’t mimic physiological insulin secretion, they often result in mismatches between requirement and availability and this results in an inadequate glycaemic control and late hypoglycaemia. So different mixtures of short and long- acting insulins are now marketed as products. Insulin analogues are called analogues because they are slightly different from the normal insulin, some amino acids have been changed in order to alter its pharmacokinetic profile but not its pharmacodynamic one (in the previous cases the sequence of the insulin was the same but we used additional proteins to prolong the action). The analogues can be divided into short acting ones and long actin ones: in the short acting ones we have Lispro, Aspart and Glulisine. Their onset is after 5 to 10 minutes, their peak is reached after 30 to 90 minutes and the elimination is usually in 4-6 hours (curve of duration in the blood stream really similar to the normal physiological insulin). This means that these analogues can be injected 5 minutes before a meal, and this makes the life of the diabetic patient much easier. Lispro is an analogue where the second last and the third last amino acids on the B chain have been switched (they are a proline and a lysine, that’s why it is called Lispro) and this is enough to induce a very rapid absorption, because this alteration decreases the propensity of the individual insulin molecules to self-associate, so preventing aggregation and reducing the formation of hexamers. Lispro is produced as pro-insulin in E. coli. In Aspart, we have the substitution of proline with an asparagine in the B chain (position B28). This type of analogue is produced in a modified Saccharomyces cerevisiae strain, so in yeast. Again, the modification induces a lower tendency to aggregate in hexamers and so the peak time is reached I less time compared to regular insulin. The Glulisine has a substitution with a lysine instead of an asparagine and glutamic acid instead of a lysine. In this way we introduce two charges, one positive and on negative, and in this way we avoid the association in hexamers (slight decrease in the isoelectric point compared to normal human insulin). This happens again in B chains because B chain is really important for the aggregation of the monomers. So with these small changes we change the pharmacokinetics of the protein, ensuring a quick absorption from the subcutaneous site to the bloodstream. There have been other attempts to produce short-acting insulin analogues, however most of them were not approved because it induced tumors in the mammary glands in rats. Instead, the long-acting insulin analogues are used to guarantee a basal level of insulin for a very long time. The first one produced was the Glargine, which presents a substitution in position 29 on the A chain of an asparagine with a glycine and in chain B it has the addition of two arginine residues which raise the isoelectric point of the protein (because the arginine is positively charged), lowering its solubility. It is produced as pro-insulin in E. coli, the purified product is formulated at pH4 (it is soluble at pH 4, so it will be in the form of monomers), but upon sub-cutaneous injection the pH increases (up to 7) so that the protein becomes un-soluble and it aggregates and precipitates in the tissue. In this way we have a prolonged duration of release (the longer the absorption time, the longer will last the peak of concentration in the blood). After subcutaneous injection, plasma insulin levels rise slowly to a peak  after 8 to 10 hours, with a duration of action of about 20 up to 30 hours (whole day long duration, so Glargine can be administered also once a day). Another long-acting insulin is the insulin detemir (Levemir): in this case we are in the B chain, B30 threonine is removed, and it is replaced by a chain of fatty acids, which confers the property to the protein to bind to the serum albumin (the pH of the protein remains neutral though). In this way, the release is very slow, so in this case the long duration depends on the release kinetics and not on the absorption. It is produced in Saccharomyces cerevisiae, and it has a shorter duration compared to Glargine, however it is still used a lot (onset in 0.8-2 hours and mean duration of 16-18 hours). The last one is the degludec: it has a fatty acid chain attached again to the lysin at B29 (so again we have the removal of the B30 residue). Thanks to the presence of zinc atoms, it assembles in multi- hexamers and so it takes a lot for the monomers to be absorbed. The degludec’s day to day glucose lowering effect is usually constant during the day, while the glargine one usually varies at different hours, and degludec’s half-life is actually longer (25.4 hours vs 12.1 hours), but they are both really used. Even if these analogues have some particular modifications in the amino acidic chains, the binding affinity to the insulin receptor is always the same, otherwise they could not be used as drugs. New attempts of producing different types of insulins that could be administered through other types of routes, other than the parenteral one, have been made, with the intent of making the life of diabetic patients easier. In fact, insulin must be administered to these patients multiple times during the day, and the sub-cutaneous route makes this process harder because of how impractical it is if used in recurrent deliveries. So, researchers have tried to come up with new formulations for an easier distribution of the drug, that could be compatible with normal life. One example is Exubera, an inhaled insulin, that was however withdrawn from the market in USA in 2007, after one year of marketing. This retirement was due to the fact that the device used for this type of formulation was not comfortable and it was expensive, and some cases of lung cancer have been recorded in ex-smokers after the use of this type of insulin. Moreover, this type of formulation needed patient’s training and pulmonary function tests, which are highly costly. However, the idea of an inhalable insulin was not abandoned, also because we have an history of protein delivery to the lungs routinely without adverse events, of which the main example is Pumozyme (Dornase alpha or DNase, therapy for cystic fibrosis). In 2015 Afrezza, an inhaled insulin for type I diabetes, has been approved in the USA (it is an insulin which must be used in combination with a long-acting insulin; it is not recommended for the treatment of diabetic ketoacidosis, and it cannot be used by patients who smoke), and now many other types of insulins that can be administered by the pulmonary route are in the pipeline. Another type of insulin that was developed is Oralin, but in this case the administration is by oral route (actually, it is buccal, because it is absorbed by the oral mucosa, in the mouth and throat). It has been developed by Generex and it has been approved in Ecuador and India. This type of insulin is formulated with a variety of additives and stabilizers to prevent denaturation on aerosolization and to stabilize aerosol particles (however, the dosage is still different compared to the sub-cutaneous type because we actually don’t know how much of the insulin is absorbed by the mucosa). It hasn’t been approved by the FDA yet, but researchers are trying to formulate it also for diabetes type II. 2. Glucagon: glucagon is synthesized by the alpha-cells of the islet of Langerhans of the pancreas. The major biological actions of this hormone tend to oppose those of insulin, in fact it is only used as a therapy to the major and most frequent complication of insulin administration in diabetic patients, which is hypoglycemia. Glucagon preparations utilized therapeutically were chromatographically purified from bovine or porcine pancreatic tissue (their structures are identical to human), but recently,  recombinant glucagon has been produced by Novo Nordisk in Saccharomyces cerevisiae and by Eli Lilly in E. coli. 3. Growth hormone: it is used to treat dwarfism. It was the second recombinant protein produced and marketed after insulin (1985). Before the production of its recombinant counterpart, growth hormone was purified from the pituitary glands of corpses because this protein, in order to be used as a therapy, must be human. However, extracting it from corpses had a lot of disadvantages: first of all we didn’t have a lot of corpses to purify it from, so the amount of hormone that we could obtain was vey low, moreover we extracted the protein from neuronal tissue, and this could cause the insurgence of infections in the patients, such as the Creutzfeldt-Jakob disease (mad cow disease). So the production of a recombinant growth hormone in E. coli was a really great innovation, because the native and recombinant forms of the molecule are identical. The growth hormone is used for the treatment of dwarfism but also of short stature in general, for the induction of lactation, for the counteraction of ageing, the treatment of obesity, body building and induction of ovulation. Other than the recombinant growth hormone, also an analogue that acts as an antagonist has been produced: it is called Somavert and it is used for the treatment of selected patients suffering from acromegaly. This molecule, commercially called Pegvisomant (Peg stands for pegylated), is able to bind to the receptor for the growth hormone but it does not induce receptor dimerization and thus the JAK/STAT pathway, preventing the expression of the genes regulated by the activation of this receptor (like IGF-1). The main adverse effect of this drug is that it may raise endogenous GH circulating levels because we don’t have any negative feedback regulating its synthesis in the pituitary gland or hypothalamus. The main difference between this analogue and the growth hormone molecule is that it has a substitution of 9 amino acids and it is pegylated, so that we lower the immunogenicity and we raise the half-life of the molecule. The aminoacidic substitutions include the G120K mutation, which is able to increase the affinity of the molecule with the GH receptor, but at the same time it prevents its activation. 4. Gonadotrophins (or gonadotropins): they are really important also in the veterinary field. They are a group of hormones constituted by the gonadotropin releasing hormone, released by the hypothalamus, the follicle stimulating hormone (FSH) and luteinizing hormone (LH), produced by the pituitary gland, they have the gonads as target organs (ovaries and testis), and human chorionic gonadotrophins, released in the placenta. They bind to different receptors but these receptors all share the same subunit. They must be glycosylated, otherwise they cannot function, so they cannot be produced by E. coli. Human gonadotropin is excreted in the urine during pregnancy (used for pregnancy tests), so before being reconstituted in vitro (recombinant gonadotropin), it was extracted from the urine of pregnant women through ion exchange chromatography and gel filtration. This process had a lot of disadvantages, because dealing with urine is very complicated (urine is toxic) and also the quantity of hormone that was extracted was really low. However, even nowadays it’s still available as an extracted protein because the process is cheaper. Menotrophin (human menopausal gonadotrophin) is the name given to FSH-enriched extracts from human urine (2,5 l/dose). Gonadotropins could also be extracted from pituitary glands (especially FSH and LH which are produced from this gland). However, in 1985 the extraction of gonadotrophins from the human pituitary was abandoned because of the risk of the Creutzfeldt-Jacob disease. Now the human chorionic gonadotropin is produced as a recombinant protein, it has the name of Ovitrelle and it was approved in 2001; it is used for the treatment of female infertility due to anovulation and for patients undergoing assistive reproductive technology, in fact it is used to trigger final follicle maturation and luteinization after follicle stimulation. It is produced in CHO cell lines because it has to be glycosylated, it is identical to the human protein and it is purified by multistep chromatography. The number of oocytes retrieved with recombinant hCG was similar to the number retrieved when urinary-derived hCG was used. Sometimes it is used also for the treatment of male infertility in assisted reproductive techniques. For veterinary use, the use of gonadotropins is pretty similar: we use it to obtain an identical offspring from surrogate dams (animals that receive multiple embryos coming from the same donor). So a cow is inseminated from the sperm of a superior bull (selected species of bull, with some particular characteristics), then we recover the embryos produced, we split them and we reimplant them in other cows, which are surrogate mothers, for differentiation, growth and birth. This technique is used to amplify the offspring of a genetically selected sperm donor and it is called embryo transfer; the aims  are to increase the productivity of the superior donors, maximize the use of their valuable semen, transport genetics across long distances and produce identical offspring by embryo splitting (potentially valuable as research animals). If we select females in the offspring we can use them for the production of milk, for the high growth rate (if we use a superior donor with an enhanced growth characteristic) or for the outstanding reproductive capacity. The limitations of this embryo transfer technique is that it is expensive, it takes time and you need to have experienced and trained people to perform it. For the success of this technique we have to induce superovulation in the surrogate mothers, and for this purpose we use recombinant gonadotrophins. Both the donor and the recipients surrogate mothers are synchronized with a treatment with recombinant prostaglandins (to obtain a synchronized the oestrus cycle in all the animals and prepare their uterus to support embryogenesis). The donor is artificially inseminated, then the embryos are recovered (after 68 days a catheter is inserted into the uterus and inflated to prevent retrograde flow of flushing medium) and transferred to the recipient animals (using a transfer pipette). Each of the recipient animal will become pregnant and they will give birth to the offspring that will be identical to the donor. The superovulation is induced in the donor through FSH (it has a short half-life, of about 2 hours; sometimes also another hormone, called PMSG, which stands for pregnant mare serum gonadotrophin, could be used in Europe, which has a long half-life, of about 2 to 4 days); the purpose of the superovulation is to hyper-stimulate ovaries with gonadotropins and provide a higher number of follicles than normal. A typical treatment response would be of 8 to 10 ovulations. 5. Three additional recombinant hormones: they are thyroid stimulating hormone (TSH), parathyroid hormone (PTH) and calcitonin. Thyroid stimulating hormone (TSH) is structurally similar to gonadotrophins, although it functionally targets the thyroid gland as opposed to the gonads. It is produced in CHO cells and it has been approved for medical use as a diagnostic aid in the detection of thyroid cancer/thyroid remnants in post-thyroidectomy patients, so we don’t use it for therapy purposes. Parathyroid hormone (PTH) is a primary regulator of calcium and phosphate metabolism in bones; a truncated version of PTH (of 4 kDa) is produced, it is identical in sequence to the N-terminal residues (1-34 AA) of endogenous hPTH and triggers the same effects. It is produced in E. coli and it is used for the treatment of osteoporosis in postmenopausal women. Finally, calcitonin is a hormone that lowers serum Ca2+ and inorganic phosphate (Pi), it is used clinically to treat hypercalcemia (condition that can occur in come tumors of the bone), associated with some forms of malignancy and Paget’s disease. Salmon calcitonin is 100-fold more potent than the human native hormone (it differs from the human hormone by 9 amino acids), so the recombinant protein that we produce is not human but it is derived from fishes (it could be immunogenic but the treatment with calcitonin is very short so it’s not so problematic). It is reduced in E. coli and the amination is carried out in vitro. CYTOKINES 1. The main cytokine used in therapy is interferon: it is a key molecule in the first line defence against viral infections, it belong to the innate immunity and it is released by the cells upon a specific stimulus. We can have two types of interferon: type I (mainly represented by interferon alpha, produced by virus- infected leukocytes, and beta, produced by virus-infected fibroblasts or epithelial cells) and type II (represented by gamma interferon, produced by T cells and activated NK cells). When a virus infects a cell, interferon is produced: it binds to its receptor on NK or T cells and they will activate an immune reaction. In particular, interferon alpha and beta induce the synthesis of inhibitory proteins, while interferon gamma increase the expression of MHC II molecules on APCs and increase the ability of macrophages to resist to viral infections and to kill other infected cells. All types of interferon increase the expression of MHC II molecules and increase the NK cell activity. Interferon is used in therapy against infection (like from hepatitis virus), cancers or multiple sclerosis. Up until the 1970s, interferon was sourced directly from human leukocytes obtained from transfused blood supplies (species- specific), but only 1% pure (they were extracted as a mixture of IFNs). Moreover, the extraction from blood was really dangerous because of the possible transfer of infection from infected blood to the patient. In the late 1970s, the production of interferon became possible in significant quantities: various cell lines were found to secrete substantial amounts of IFNs (for example Namalwa cell line was able to produce 8 subtypes of IFN-). However, it was still produced in mixtures, so we still weren’t able to obtain single proteins. DNA recombinant technology led to the production of a single IFN subtype in the 1980s. Most interferons have been produced in E. coli, yeasts, and some mammalian cell lines. Most IFNs currently in medical use are produced in E. coli because the majority of human IFNs are not normally glycosylated (mostly IFN-a and IFN-b). IFN-g is glycosylated but the non-glycosylated form displays a biological activity similar to the native protein, so the glycosylation is not mandatory for this molecule to be use in therapeutical applications. However, the production of IFN in recombinant microbial systems means that any final product contaminants will be microbial in nature. So a high degree of purification is required: up to 99% of purity can be achieved with extensive chromatographic purification. The first recombinant INF was produced in 1980 (INF-alpha2a), in 1986 it was approved for the treatment of hairy cell leukemia (Intron A), but it can be used also against viral infections like in hepatitis. PEGylated interferons are now available: the PEG molecule is able to prevent the degradation of the molecule, shielding it from enzymatic degradation, so we have a prolonged half-life (13 hours to 25 hours compared to the 4 hours of the non-pegylated protein) with the same intrinsic biological activity as non-PEGylated INFs. Moreover, the PEGylation lowers the immunogenicity of the protein, allowing multiple and frequent dosing and achieving a higher and sustained interferon concentration in blood. If we administer the interferon with an antiviral chemical drug, we can see that the combination of this molecule with the non-PEGylated molecule reaches the 44% of remission of the HCV infection, while the combination with the PEGylated molecule allows us to reach a remission of 56%, so we obtain better results (both these combination work even better than immunotherapy). We can see that another main advantage is that, with better results, the dosage can be decreased, and this is very important for the adverse reaction profile (the less drug we use the less side effects we could have). We could have two different types of PEGylated interferon: PEG interferon alfa-2b (linear molecule, with a weight of 12 kDa) and PEG interferon alpha-2a (larger and branched molecule, with a weight of 40 kDa). This changes their pharmacokinetics: the volume of distribution of the larger PEGylated interferon is lower than the smaller one (8L vs 20L), in fact, being a larger molecule, it is harder for this interferon to pass through tissues, in fact also the absorption half-life is longer, just like the mean elimination half-life. If we change and we modify the molecule by adding a different type of PEG molecules we can obtain different molecules with different pharmacokinetics characteristics (for example, if we want to have a longer half-life, we will use a bigger PEG molecule, but we have to remember that in this way also the time of absorption will be prolonged, while the volume of distribution will be lower, so some characteristics will have to be “sacrificed”). When talking about recombinant interferons we have to mention Infergen (interferon alphacon-1): it is not a real natural interferon molecule, it is “invented”. This protein is in fact an engineered INF approved for the treatment of hepatitis C, produced in E. coli, with a higher antiviral, anti-proliferative and cytokine-inducing activity than native type I interferons: the aminoacidic sequence presents the most frequently occurring aminoacidic residues in native IFNs-a. So it is not the copy of any other existing interferon, but it still works in a very good way. For what concerns INF beta, it is used for the treatment of multiple sclerosis, it inhibits the production of pro-inflammatory cytokines (like TNFalpha and INF-gamma) and it can be produced in either E. coli or CHO cell lines, because even if it is not glycosylated it yields the same therapeutic efficacy as the glycosylated protein. The recombinant INF-gamma is a particular interferon because it is used against the CGD (Chronic Granulomatous Disease), a rare genetic disorder where there is a defect in the granulocyte response to infections, so granulocytes do not activate properly. This pathology is responsible for the development of a lot of infections and granulomas in the patients and sometimes they could be lethal. It is produced in E. coli, hence it lacks the carbohydrate component, but it still displays the identical biological activity as the glycosylated protein. It is a potent activator or phagocytes, in particular of granulocytes, that’s why it is used against CGD. So interferons are really important for their therapeutic effect against infections, tumors and other types of disorders, however they could be toxic: INF elicit a number of unwanted effects, such as flu-  like symptoms (we naturally produce interferons during an infection, that’s why we have this type of toxicity), which is typical of all types of interferon, but then we could have also more serious adverse effects, like anorexia, insomnia, cardiovascular complications, autoimmune reactions and hepatic decompression (typical of interferon alpha use, but they are rare); hypersensitivity reactions, menstrual disorders, anxiety and depression (typical of the use of interferon beta); heart failure, CNS complications and metabolic alterations (typical of interferon gamma). Generally speaking, we consider interferons safe molecules despite these adverse reactions, because the more serious side effects are usually rare and the flu-like symptoms are not so dangerous, since they usually appear within a few hours (8 for interferon alpha) from administration, but then tolerance is developed within the first week of treatment. We could also have additional interferons, and they are: - Interferon tau: trophoblastin, has similar effects to type I INF, with significantly lower toxicity. It is found in sheep and cattle and it is used for the synchronization of oestrus cycle in the veterinary field. - Interferon omega: it is found in human and pigs but not in rodents, so it cannot be studied in these animals. It is a type I interferon; a recombinant form of feline INF-omega is used for the reduction of mortality and clinical symptoms of parvoviral infections in young dogs. 2. Another type of cytokines that are used for therapeutic purposes are interleukins. They are a group of protein constituted by 33 members, produced by a number of different cells. Most interleukins exhibit paracrine activity, and some displays autocrine activity, through which they regulate a variety of physiological and pathological conditions. Thus far, only three interleukin-based products have gained approval for general medical use, IL-2, IL-11 and IL-1. IL-2 is a single chained polypeptide containing 133 amino acids. It is a glycoprotein, but glycosylation is not required for its biological activity. It is an autocrine growth factor for T cells and it has proven to have beneficial effects on the treatment of some cancer types (through the activation of T cells, whose activation is normally suppressed when the cancer develops). Recombinant IL-2 is produced in E. coli and differs from native human IL-2, since that it is non-glycosylated, lacks an N-terminal alanine and has a replacement of one amino acid. Despite these modification, this product displays the same biological activity as native IL-2. IL-2 is able to activate LAKs (lymphocyte activated killer cells), which are NK cells activated by the binding of this cytokine with its receptor on the surface of these cells. Clinical studies have shown that if we collect NK cells from the patient (ex vivo), we activate them in vitro with the use of IL-2, generating activated LAK cells, these were able to induce a complete tumor regression in 10% of patients suffering from melanoma or renal cancer. This percentage is too low, so in another experimental setting we extract a tumoral biopsy, we isolate TILs from it (tumor infiltrating lymphocytes), we activate these TILs with IL-2 in vitro (these cells are already directed against tumoral antigens) and then we reintroduce them in the patient. In this case 50% of patients presented the regression of the tumor, so the technique worked better. However, this result is still not so encouraging. So they thought about administering IL-2 directly to the patient: ovarian, bladder cancer, non-Hodgkin’s lymphoma and acute myeloid leukemia are at least partially responsive to IL-2 treatment. However, several trials yielded conflicting results, because IL-2 could cause the so-called cytokine storm, causing very serious side effects. For this reason it cannot be administered directly to the patient. This application of IL-2 was extended also to the treatment of infectious diseases, because the IL-2 capacity of stimulating T cell responses against intracellular pathogens can be useful for the treatment of a wide range of infections (like mycobacteria, Listeria and Legionella). However, this applications yielded the same problems are the one against tumors, which are mainly related to the side effects of IL-2. These unwanted reactions are usually related, as we said, to cytokine storms, because usually IL-2 stimulated the production and release also of other cytokines (IL-3, IL-4, IL-5, IL-6, TNF and INFgamma) by the cells that it activates; they are dose-limiting and they may cause cardiovascular, hepatic and pulmonary complications, which usually induce immediate termination of the treatment. However, IL-2 has been used to produce a fusion protein with an antibody (short chain variable fragments, so only the portion of the antibody that recognizes the antigen). Some examples are the