Pharmacokinetics Notes PDF

Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli PHARMACOKINETICS GENERAL INFORMATIONS Professor Vercelli will share the exam with professor Genazzani, his part will be longer than hers, so there will be more ques on on pharmacodynamic and fewer ques ons on pharmacokine c The exam consists of several short ques ons and the student is expected to answer in a very short term (2-3 rows) targe ng perfectly the topic and give just some nuances of other informa on. The aims of this course are several: we will deal with pharmacokine cs, absorp on, distribu on, metabolism and elimina on of drugs, from their entry into the body ll the exit through metabolism, we will pursue a compara ve approach, dealing with both human and veterinary medicine PHARMACOKINETICS Both the terms “pharmacokine cs” and “pharmacodynamics” seem to be related to something that is moving but actually these two terms refer to diﬀerent aspect of the drugs, in par cular pharmacodynamics is the discipline of pharmacology mainly related to the study of the mechanisms of ac on of diﬀerent drugs, explaining how a drug can act and can induce a speciﬁc eﬀect. For example, a drug can bind a speciﬁc receptor or induce a varia on of the osmolarity of the blood: both are mechanisms of ac on. The eﬀect is therefore related to what the drug is able to induce. Pharmacokine cs is actually the discipline in pharmacology which studies how the drug can move through the body: how it’s absorbed, the way this drug is able to distribute to the several ssues and organs, how it will be changed according to the metabolism pa erns and how it can be eliminated from the body through urine, feces, sweat. This process can be summarized with the acronym A.D.M.E., administra on, distribu on, metabolism, elimina on. Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli These processes are very complex and begin with the drug’s administra on. A drug can be administrated through oral routes, parental routes (so it can be injected into the body through several diﬀerent routes like intravenous, subcutaneous, intramuscular etc.) and topical routes. Clearly diﬀerent pharmaceu cal prepara ons or formula ons are prepared according to a speciﬁc route of administra on: a pill or a tablet cannot be administrated intravenously, or an injected formula on cannot be taken orally. The pharmaceu cal prepara on or formula on must dissolve to become available and to be exploited by the body, this concept is referred to as “bioavailability" The term absorp on refers to the passage from the of administra on to the systemic circula on. The next step is distribu on: in the systemic circula on the drug is transported either bound to plasma proteins or in the form of free drug. This dis nc on is very important because only the percentage of free drug circula ng in the blood can distribute and reach various ssues and organs. In addi on, not all ssues and organs can be reached at the same me and same manner because several factors can inﬂuence this path. Biotransforma ons, metabolic processes that are necessary to eliminate the drug from the body, occur simultaneously with distribu on (actually a very small amount of the drug is eliminated without undergoing metabolism, unchanged, but the majority of the drug administered is biotransformed and then eliminated). What can happen if a drug will not disappear from the body? Side or toxic eﬀects may occur; biotransforma ons are necessary to eliminate the drug and prevent such situa ons. Toxicokine c will also be a main topic of this course in order to understand how the diﬀerent toxic metabolites or toxic compounds can aﬀect the body. A er biotransforma ons, the drug is excreted through urine, feces and other pathways with a process known as elimina on. Elimina on occurs through urine, so it is important to underline the importance of kidney func on, as kidney malfunc on can slow down elimina on and lead to toxic eﬀects. A few drugs are eliminated through feces but this is not a direct elimina on into the bowel: the drug is ﬁrst processed in the liver, excreted into the bile and the bile is then released into the bowel. PERMEATION OF A DRUG How a drug can be absorbed, distributed, excreted or re- absorbed? The most important concept is that the drug must be able to cross the cell membrane, which is composed of a phospholipid bilayer. Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli The concepts of “hydrophilicity” and “lipophilicity” are very important: a lipophilic drug, being similar to the phospholipid membrane, can easily pass through, can be absorbed very quickly and the distribu on in all ssues and organs can be very rapid: on the other hand, hydrophilic drugs may face a lot of diﬃcul es, the absorp on and distribu on will not be so eﬀec ve. This certainly represents a limita on in the use of certain drugs. For example, an an bio c drug should have a systemic eﬀect, but if this drug is not lipophilic, nothing will happen in a systemic way. Considering a pa ent is aﬀected with pneumonia, the an bio c must be well absorbed and well distributed in order to reach very easily and quickly the site of inﬂamma on (the lungs). If this drug is not well absorbed due to a hydrophilic nature the drug will remain in the bowel so there won’t be a systemic eﬀect, the drug will never reach the lungs and will stay in the bowel, so 2 diﬀerent scenarios are present: this drug won’t be used to treat pneumonia or this this situa on can be exploited in order to produce drug that have to stay in the bowel. HOW CAN A DRUG PASS THE MEMBRANE? A drug can pass through the membrane via mul ple mechanisms that are naturally present in the body and are u lized to regulate the concentra on of nutrients like fructose or xenobio c drugs (xenobio cs exploit the same mechanisms). These mechanisms are:  Paracellular Transport: This occurs when a drug passes through intracellular gaps between cells, such as in the vascular compartment of the endothelium and in the central nervous system (CNS). This pathway does not allow the entrance inside the cell, but permits passage between cells. It is rarely exploited therapeu cally.  Ac ve and Passive Mechanisms: Drugs can enter cells and reach diﬀerent receptors or intracellular organelles to induce eﬀects via ac ve or passive transport. Passive Diﬀusion: This mechanism does not require energy and it is concentra on dependent. It can be exploited by medium to small molecules, including ions. The lipophilic nature of a molecule is crucial for passive diﬀusion; if a molecule is lipophilic, it can pass easily through the membrane; if not, it remains outside. Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli Unionized Molecules: The most lipophilic forms of molecules can easily pass the cell membrane. If a drug is ionized, it becomes more hydrophilic and ﬁnds it diﬃcult to cross the membrane. Facilitated Diﬀusion: This mechanism also does not require energy; the driving force by the electrochemical gradient can be mediated by protein carriers. This mechanism can facilitate entry and exit in both direc ons. o Ac ve Transport: In this case, energy is required (ATP usage). Ac ve transport can move drugs against their concentra on gradient. Many ac ve transport mechanisms in the body are vital for protec ng sensi ve areas, such as the brain, and for facilita ng drug elimina on from ssues to the bowel. Endogenous compounds like sugars, amino acids, and vitamins typically u lize this mechanism.  Endocytosis: includes several mechanisms aimed at incorpora ng diﬀerent molecules into the cell. Depending on the size of the molecules, we can refer to endocytosis for large molecules or Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli pinocytosis for small molecules. This mechanism is naturally present and is exploited for the uptake of hormones, immunoglobulins, and vitamins into cells. FACTORS AFFECTING DRUG ABSORPTION The primary factors inﬂuencing a drug's absorp on include its lipophilicity or hydrophilicity. Addi onally, ioniza on can aﬀect the lipophilicity of the molecule, as the ionized form is more hydrophilic and has diﬃculty passing through membranes. The degree of ioniza on depends on the drug's pKa and the pH of the surrounding medium.  pKa: This is the constant of dissocia on, which varies according to the molecule's nature (weak acid or weak base). The characteris cs of the drug can change with the pH of the medium. For example, an -inﬂammatory drugs (such as Ibuprofen) are weak acids. When administered orally, they reach the stomach, where the pH is low (about 1.4). In this environment, the weak acid remains unionized, making it lipophilic and allowing it to easily cross the membrane and distribute to various ssues to induce its eﬀects, such as an -inﬂammatory and pain-relieving ac ons. Similarly, paracetamol can also easily pass through the membrane and reduce body temperature.  pH: Depending on the medium's pH, the drug can become dissociated (ionized) or remain unionized. These eﬀects are summarized by the Henderson-Hasselbalch equa ons, which indicate that if a drug is a weak acid, it will be characterized by its dissocia on constant. Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli [The professor said that it is not necessary to remember the formula; she will not ask it on the exam, but it is very important to understand the correla on between pH and the state of dissocia on.] Non-Steroidal An -Inﬂammatory Drugs (NSAIDs) show diﬀerent behaviors depending on the pH of the gastrointes nal tract. A er passing through the stomach, the drug reaches the bowel, where the pH is higher. In the duodenum, dissocia on can s ll occur, but as the pH increases dras cally (reaching 6, 7, or even 8), the behavior of the drug changes. Diﬀerent species have diﬀerent characteris cs: humans and carnivores typically have one highly acidic stomach, while ruminants have four stomachs with varying pH levels, monogastric herbivores have an alkaline environment throughout their gastrointes nal tract, inﬂuenced by their herbivorous diets, which can also impact the pH of the gastrointes nal tract. ROUTES OF ADMINISTRATION OR EXPOSURE Various routes of administra on are available for diﬀerent xenobio cs:  Oral Route: This is the most comfortable route and is easily exploited in both human and veterinary medicine. The gastrointes nal tract can be used to administer drugs but can also expose animals to toxic xenobio cs. Another administra on route is through the rectum; despite appearing local, the eﬀects can be systemic due to signiﬁcant absorp on.  Parenteral Route: This includes several types of injec ons (intravenous, intra-arterial, intramuscular, subcutaneous, etc.). In this case, absorp on is nearly complete, leading to systemic eﬀects.  Topical Route: Drugs are applied directly to the skin (creams, ophthalmic administra on on the eye surface). This route typically induces local eﬀects; however, any systemic absorp on can occur. For systemic eﬀects, this route is not ideal. Examples include the cutaneous applica on of creams or the vaginal and uterine administra on of drugs, par cularly in food-producing animals (sheep, cows). Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli ORAL ROUTE The oral route is the most familiar to us, in veterinary medicine is not so easy. Enteric absorp on may be limited not only by pKa and pH but also due to simultaneous biotransforma ons and the presence of drug transporters in the enterocytes in the internal surface of the bowel, which can limit the absorp on rate. All ingested xenobio cs must cross the enteric barrier to reach the liver, where various biotransforma ons occur: the body's goal is to eliminate the drug. If a drug is highly lipophilic, the liver must undergo several transforma ons to convert it into a more hydrophilic form. This change prevents the drug from crossing membranes and allows it to remain trapped in urine or bile for elimina on. Lipophilic drugs can remain in the body for extended periods, undergoing recircula on. They may be reabsorbed from the bowel or stored in fat, re-entering the bloodstream and ssues. Speciﬁc formula ons have been inves gated in order to provide the best performance of a drug according to the route of administra on. Common formula ons for the oral route include pills, tablets, and syrups (suitable for children). Some pills are coated to control absorp on at the correct site. For example, a pill may have a coa ng designed to delay absorp on; once the outer layer is disrupted by the gastric pH, the core (which may contain more basic molecules) can be absorbed later in the bowel, where the pH is higher. This strategy allows for a dual eﬀect with the same administra on. Coa ng is necessary to protect the molecule from the gastric environment, which may degrade it before it can pass the membrane and induce eﬀects. For instance, proton pump inhibitors like lansoprazole and pantoprazole must remain intact un l they reach the duodenum to eﬀec vely reduce gastric pH by inhibi ng proton pumps. In veterinary medicine, oral administra on may be diﬃcult. Side eﬀects such as sialorrhea (excessive produc on of saliva) may occur when a tablet has a bad taste: this can dissolve a por on of the drug before inges on. While dogs and cats may be familiar to oral administra on, Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli other species (e.g., snakes and chameleons) can found it constraining; for python some sort of blunt needle is used. Oral administra on can also include the transfer of drugs from a mother to her puppies through milk, this can represent a risk because some unmodiﬁed or unmetabolized drugs can be excreted in body ﬂuids (like milk) and reach puppies through milk consump on. PARENTERAL ROUTES Parenteral administra on involves injec ng drugs into the body, which can include various routes such as intravenous, intraosseous, or intramuscular. Depending on the injec on technique, diﬀerent ssue layers can be accessed, including:  Intravenous Administra on: allows the direct administra on of a drug into the bloodstream and it is also very useful for blood collec on.  Intramuscular Injec ons: penetrate the muscle  Intradermal Injec ons and subcutaneous: reach the ﬁrst layers under the skin's surface. Intravenous Administra on This type of injec on permit to administer the drug directly into the blood stream and it can be eﬀec ve in a very short me. Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli This type of administra on provides rapid eﬀects, but it carries risks. In fact if concentra ons are incorrect the direct administra on into the bloodstream can lead to toxic or lethal eﬀects, because there is no way to remove the drug once injected, the only change may be to administer an antagonist drug. In this case, there is no absorp on because the drug is delivered directly into the bloodstream. This route is preferable for very irrita ng drugs that would cause pain or ssue damage if administered intramuscularly. Blood can buﬀer both acidic and alkaline solu ons: it preferable to make slow injec on or infusion in order to give the blood me to buﬀer the solu on and avoid blood pressure imbalances. Intravenous administra on is commonly used in human medicine: veins in the arms or legs are the most u lized. In animals, several superﬁcial veins can be used for drug administra on and blood collec on. For food-producing animals (e.g., ca le, sheep, and horses), the jugular vein is commonly accessed, and in cows, the caudal vein (located under the tail) can also be u lized for both blood collec on and administra on. Intramuscular Administra on Intramuscular injec on is generally easier to perform than intravenous injec on because even an untrained person can administer an intramuscular injec on. In contrast, intravenous administra on requires knowledge of anatomy and the loca on of veins. Absorp on typically occurs because the drug is administered into a speciﬁc ssue rather than directly into the bloodstream, allowing the drug to cross the membrane and enter the bloodstream. The me required for absorp on can vary depending on the formula on. All formula ons are liquids, which can be categorized into two main types:  Aqueous formula ons  Lipid formula ons The formula on consists of excipients, vehicles, and the ac ve drug molecules, which can be protected in various ways. The formula on must be compa ble with the ssue. For instance, if a formula on contains fats, it may not be suitable for intramuscular administra on, as it could remain in the muscle and slow down drug absorp on. On the contrary, a water-based formula on is more suitable, as it will not damage the ssue and will have a similar pH, allowing a be er absorp on of lipophilic molecules. Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli Injectable drugs administered into the muscle using aqueous formula ons are absorbed more rapidly and eﬀec vely. The drug molecules can be either lipophilic or hydrophilic. A lipid vehicle may cause local irrita on, which is not necessarily a side eﬀect but can signiﬁcantly slow down drug absorp on. This could be beneﬁcial for depot administra on, as the drug will remain in the muscle longer, allowing for controlled release. Formula ons speciﬁcally labeled for intramuscular administra on should not be used for intravenous injec on, as the vehicles may not be suitable for that route. For example, while blood can buﬀer certain solu ons, infusing oil into a vein can cause embolism, poten ally leading to death. Vehicles are chosen to be suitable for the administra on site to either increase or slow down absorp on and to minimize irrita on at the injec on site. Switching from intramuscular to intravenous administra on with the same formula on can lead to signiﬁcant side eﬀects or even death. In human medicine, intramuscular administra on is typically performed in the muscles of the legs or upper arm. In veterinary medicine, it is common to use the neck or leg muscles of cows and horses, as well as muscles in rep les such as chameleons. Subcutaneous Administra on Subcutaneous administra on is technically the easiest way to administer a drug and can be performed by almost anyone, because it does not require targe ng a speciﬁc vein or anatomical district. The subcutaneous layer is the ﬁrst layer under the skin's surface, making it easily accessible. This area is also considered safe because it contains no major nerves or blood vessels, and this minimizes the risk of hemorrhage or damage to the nervous system. The pH of the solu on is very important. Historically, subcutaneous administra on has been misused for illegal ac vi es, such as administering hormones in bovines to promote growth; these prac ces were banned in Europe. Parissi Elena/Curren Simone 02/10/2024 Prof. Cris na Vercelli OTHER ROUTES OF ADMINISTRATION Other routes of drug administra on include:  Intraperitoneal: This is commonly used for laboratory animals, such as mice or rats.  Intracardiac: This route is o en employed in human medicine during emergencies and in veterinary medicine for euthanasia.  Intra-ar cular: Typically used for steroid administra on in both human and veterinary medicine.  Intrathecal: This limited procedure is used for speciﬁc purposes, such as administering contrast medium for certain exams (e.g., CT or MRI) or anesthe cs, like epidural administra on.  Inhala on: This method is eﬀec ve for administering drugs via aerosols or inhala onal anesthe cs. However, it can also facilitate the absorp on of toxic substances, such as car emissions or heavy metals.  Dermal administra on: Direct applica on to the skin.  Mucosal administra on: This can include ocular, intramammary, and intravaginal routes. Martina Giobergia/Luca Gastaldi 09/10/2024 Prof.ssa Vercelli DISTRIBUTION OF DRUGS VOCABULARY FROM LAST LECTURE. - ABSORPTION: transport of the drug from the site of administration into the bloodstream. - SITES OF ADMINISTRATION: can be enteric (oral and rectal), nasal (e.g. aerosol), parenteral (intramuscular, intravenous, intra dermal, subcutaneous, etc…) or topical. With the topical route, it is not expected to have absorption: sometimes you could exploit this route of administration to have a kind of absorption, but it is not expected to have a huge extent. Moreover, the intravenous route of administration bypasses the absorption. - LIPOPHILIC= term referred to a molecule able to pass membranes very easily. The lipophilicity can depend on the size, the shape and the charge of the molecule: if the molecule is able to stay in a unionized form, it will be lipophilic, otherwise, if it ionized, it will not be lipophilic. DISTRIBUTION The second step of kinetics is the distribution; we can define it as the passage of the molecule of xenobiotic from the bloodstream into the tissues. In the systemic circulation, the drug can be found in different conditions: it can be bound to plasma proteins (as showed in the picture), to red or white blood cells, or even transported by the lymph (it is an exceptional way, important for some animals that have a huge amount of lymph in the organism). PLASMA PROTEIN BINDING The binding with a plasma protein is allowed to the fact that several chemical bindings can occur and, in order to remain in a therapeutic condition, this kind of binding must be reversible. Otherwise, we are in the toxicological point of view: the drug will never leave the body, but it will remain inside it. This fact is not acceptable from a therapeutic point of view but can happen when we speak about toxics. Moreover, these bindings must be weak bonds, like hydrogen bonds, Van Der Waals forces and ion-ion interactions. They are all easy to break down and it’s easy for the drug to be freed. The term “plasma proteins” includes several proteins such as albumins, globulins, peptide hormones and others. The ability of a xenobiotic to specific bind albumins or other protein carriers can vary a lot: albumins, or globulins as well, can actually bind a-specifically a lot of substances, but only a few substances can bind specifically. Martina Giobergia/Luca Gastaldi 09/10/2024 Prof.ssa Vercelli Also, the strength of this binding is not always the same: the affinity between the plasma protein and the drug can be a conditioning factor of the duration of the relationship. o Some compounds like Sulfonamides, Warfarin and Cefovecin are characterized by a very high specific binding (measuring this strength from 1 to 100%, they reach up to 98-99%). This means that almost the entire dose administered can bind specifically to plasma proteins. ▪ Sulfonamides are a category of antimicrobial drugs with a specific mechanism of action: they are bacteriostatic, so they stop the replication of bacteria. If they are co-administered with Trimethoprim (another class of antimicrobial drugs), the final effect is bactericidal; the combination can improve the final effect. ▪ Warfarin is a drug containing Coumadin, which is able to avoid the clotting of the blood since it stops the action of the platelets. It is administered to have a fluid blood, in order to avoid thrombus formation. In veterinarian medicine, Warfarin and other anti-coagulants are illegally used in order to induce toxicity in some animals. ▪ Cefovecin is an antibiotic drug belonging to the class of cephalosporins and it is characterized by a very high binding and a long half time (time requested to half the maximum concentration of the drug). o Some other substances have a very low relationship with plasma proteins: between them, we can mention Paraquat (herbicide) or Digoxin (a cardiac drug). RATIONAL OF THE BINDING It is quite normal that molecules bind a plasma protein in order to be transported all over the body, so xenobiotics just exploit the same mechanism of transport. In other words, the rational behind this concept is that molecules exploit plasma protein to circulate, and, if they are lipophilic, they can easily pass membranes and enter in different tissues (thanks to their chemical features). An important concept is that only the fraction which is unbound can actually pass from the bloodstream to tissue, because the fraction bound is limited in its movement. As a consequence, the bound fraction is a sort of storage because the drug cannot pass membranes and distribute to tissues. For example, Sulfonamides, which have a very high percentage of binding, will stay for a long period in the bloodstream, but only the unbound fraction will pass membranes, distribute into the tissues, reach its receptors and induce an effect. We can resume saying: Fu is the fraction of the drug unbound into the plasma and it’s equal to the concentration unbound divided by the total amount of the drug that has been preset into the bloodstream. Martina Giobergia/Luca Gastaldi 09/10/2024 Prof.ssa Vercelli According to this, the binding is the function of affinity between the protein and the drug. If the drug is very strongly bound to the protein, its release into the tissues will be slowed down; the drug will stay for a long period in the bloodstream, it will not pass membranes nor going into tissues. This is not always a side effect: sometimes we want to obtain a slow release of the drug for therapeutic purposes, as a sort of storage if you don’t want to give a huge shot of drug just in one minute. CO-ADMINISTRATION OF TWO LIPOPHILIC DRUGS If I co-administrate two lipophilic drugs characterized by a high percentage of binding, what can happen? This is the case of Sulfonamides and Warfarin and represents a clinical issue. If Sulfonamides are administered in a patient that has already been treated with Warfarin, Sulfonamides will displace very easily Warfarin because they have even a higher affinity to plasma proteins. From a clinical point of view, since Warfarin will be found free in huge amounts in the blood, hemorrhage could happen (it stops the clotting). PLASMA PROTEIN DRUG CARRIERS Albumins are the most important family of plasma proteins: they are present in 3 to 4 g/dl and are almost the 71% of all plasma proteins. They are mostly responsible for the maintenance of the oncotic pressure (= maintaining water balance in plasma) but also have the duty to transport different endogenous compounds all around the body (amino acids, bilirubin, hormones, etc…). Albumins are characterized by non-specific binding with mainly weak acid drugs but can also bind neutral drugs or weak bases (with less capability though, because other proteins do). The specificity is related to only certain types of drugs, and it is possible thanks to the presence, on their surface, of some pockets that are specific sites for specific drugs. However, this specific binding can occur only in a few cases. Alfa 1-acid glycoprotein is expressed in acute phases of inflammation and can host mainly basic drugs like Propranolol, a beta blocker drug that slows down the heart rate, or Lidocaine, a basic drug used to induce a local anesthetic effect. Lidocaine can also be administered intra venously to slow down the heart rate. Lipoproteins are a specific category that can be found in small amounts in the plasma. They play an important role in a physiological condition: they are responsible for transporting lipid soluble endogenous compounds like cholesterol or triglycerides. They also bind a-specifically highly lipid soluble xenobiotics. Globulins transport endogenous compounds like metals (e.g. iron). Even some hormones can bind globulins: for example, cortisol is specifically transported by the transcortine (a globulin). FACTORS AFFECTING THE EXTENT OF A PROTEIN-DRUG BINDING Some factors can interfere with the transport of a drug, altering the clinical response: Martina Giobergia/Luca Gastaldi 09/10/2024 Prof.ssa Vercelli 1) The age: older people have lower level of plasma albumin compared to an adult subject. 2) Specific conditions: alfa 1-acid glycoproteins are produced during acute phases of inflammation, so in case of very severe inflammation, their levels can be very high. The administration of certain drugs that specifically bind this class of proteins can be influenced by a higher concentration of alfa1-acid glycoproteins in the plasma. BARRIERS Some tissues are protected from the external environment by barriers: this is the case of the brain, that is protected by the Blood-Brain-Barrier; it is characterized by different layers composed by endothelial cells, joined together by tight junctions, and by glial cells. This barrier exists in order to protect the brain from the action of every molecule that tries to enter the brain; these molecules must be carefully evaluated before entering. If we want to administrate a drug that has to reach the brain, we need to enhance the drug’s capability to cross the BBB. Actually, the BBB is well crossed by lipophilic drugs like sedatives and anaesthetics. But also, alcohol and caffeine are able to pass the BBB. The main mechanism of protection performed by the Blood-Brain-Barrier is represented by the protein PGP, an efflux pump that is able to selectively extrude different molecules that try to cross the BBB (xenobiotics). It is also present in intestinal walls. FACTORS AFFECTING THE FUNCTIONING OF THE BBB. Some drugs can induce very severe side effects if they pass the BBB due to: 1. Genetic polymorphisms: Collies dogs and breeds related to them have a lower expression of of PGP genes due to a mutation. As a consequence, the BBB is not able to throw away drugs that are crossing the membrane and it can lead to neurological side effects like seizures, depression of the CNS until coma. This happens if these dogs are administered with Ivermectin, an anti-parasitic drug, or Loperamide, the active principle of Imodium. Loperamide is an opioid which usually lacks any effect on the CNS but, in dogs having this mutation, it causes depression, difficulty in breathing and so on. 2. Age: the BBB is more permeable in newborn babies so the poisoning with methyl mercury causes a severe condition known as the Minamata disease, which affects the CNS of babies. The cause of Minamata disease is the depo of methyl mercury in the CNS tissue due to a contamination of fish ingested by the mother but it can also occur in adult people eating a lot of big fish, since they accumulate a lot of heavy metals. In fact, mercury is characterized by a step-by-step biotransformation that induces a higher bioavailability: it can enter easily in cells, and it can be stored inside them for a long period. 3. Inflammation: when an individual has meningitis, an inflammation that directly affects the CNS, the BBB becomes less impermeable and cannot exert its protective effect anymore. As a result, several drugs that usually don’t penetrate the brain (like certain antibiotics) now can easily pass. Other barriers exist in our body: the mammary barrier, testis barrier, prostate barriers. But they don’t have so much impact on a therapeutic point of view. Martina Giobergia/Luca Gastaldi 09/10/2024 Prof.ssa Vercelli TISSUE DISTRIBUTION AND SITES OF ACCUMULATION Besides the affinity to plasma proteins and the percentage fraction unbound, another factor that can affect the distribution of the drug is the perfusion of different organs, so the quantity of blood flow reaching that organ per unit of time. The drug will be distributed very fast in organs with a high perfusion rate (SNC, kidneys, heart, lungs) while in muscles and skin, which have an intermediate perfusion rate, the drug will distribute a little bit slower. In teeth, ligaments, tendons that have a slow perfusion rate the xenobiotic will distribute with a low speed and a low concentration. The perfusion rate is a limiting factor not only for distribution but also for re-distribution. If a drug is able to distribute in different tissues, it will reach its site of action, which can be also a site of accumulation: - Kidneys and liver are the three organs responsible for biotransformation and elimination of drugs; sometimes, they can also host storages of the drug or metabolites inside, for example heavy metals. - Adipose tissue has a low perfusion rate and can host very lipophilic drugs: it represents a depo tissue for organochlorines, avermectins or pyrethroids. These drugs remain for a long time inside the fat and are released in a long period. - Muscles and myocardium host different glycosides, such as Digotoxin which binds to specific membrane ATPases. That’s why it has effect on the heart rate: the cardiac tissue has different membrane ATPases. - Lungs are a depo for Paraquat, a drug used for illegal purposes to induce toxicosis in animals. - Hair and horns are the target of all compounds that have a -SH group in their structure. - Bones and teeth are targeted by tetracyclines (antibiotics) which have a high affinity for calcium. REDISTRIBUTION After the administration, the xenobiotic passes from the site of administration to the bloodstream and, from there, it enters inside different tissues. The driving forces of this movement are: - the bond to plasma proteins - the concentration of the drug inside the bloodstream - the unbound fraction (the only one able to reach tissues) - the affinity that the drug has both for plasma proteins and tissues. According to all these factors, the unbound fraction of the drug can induce a first distribution in organs with a very high perfusion rate (brain, heart, kidneys); the portion which remains in the bloodstream is actually the part bound to proteins: for example, if a xenobiotic is characterized by a 70% of plasma protein it means that a 70% will be bound to plasma proteins and a 30% will be the unbound fraction (able to distribute). The 70% bound to plasma proteins now represents the entire concentration available in the plasma and is divided again and again: the 70% of this 70% remains bound and the 30% is freed. Martina Giobergia/Luca Gastaldi 09/10/2024 Prof.ssa Vercelli It happens continuously with a dynamic equilibrium: what I administered once is distributed but at the same time a certain part is biotransformed by the liver and eliminated. The concentration gradually slows down. All this happens at the same moment inside the body: the blood reaches very easily organs with a higher perfusion rate, creating a difference of gradient so the drug will pass again into the blood. Then, it will reach organs with an intermediate rate of perfusion and, once again, a gradient will push the blood to organs with a low perfusion rate, like the fat. The fat is characterized by a low perfusion rate not only on entry but also in exit: for this reason, it is a depo of the drug that will be released very very slowly. APPLICATIONS OF REDISTRIBUTION Redistribution has very important clinical repercussions on patients let’s analyze the pathway of anesthetics like ketamine (a very lipophilic drug), which can easily cross the BBB. After an intravenous administration, ketamine is immediately available in the circulation and reaches its target: the brain. Here, it binds to NMDA receptors and can induce an anesthetic status in the patient; this status lasts just for half an hour/ 40 minutes because, thanks to the lipophilicity of the drug and the high perfusion rate of the brain, once entered into the brain, the difference of gradient pushes the xenobiotic again into the bloodstream. Ketamine will contemporaneously, but more slowly, reach organs with an intermediate perfusion and, at the same time, a quote will be biotransformed by the liver and in part eliminated. As a consequence, the initial concentration of ketamine gradually decreases and the one remaining in the plasma is fractionated again (portion bound and unbound). Lastly, ketamine will go to organs with a low perfusion rate with a low concentration but, since it has a high affinity with the fat, it will stay there for a long period and will be released very slowly into the bloodstream. This generates a continuous redistribution. The anesthetic effect will last only for a short period of time but, thanks to the redistribution, there will be a sedative effect (sedative is less than anesthetic effect) that can last for 24 hours (because the drug is stored into the fat and released very slowly and in little amounts). As long as ketamine will be found in the blood (even if less concentrated), it will be able to reach its receptors in the brain and induce a sedative effect. The patient will not be asleep, but his functions will be slowed down, and he will not be able to perceive the external environment correctly. Again, this is a dynamic equilibrium among different tissues and several limiting factors are present (binding to the plasma proteins, perfusion of different organs). ⮚ The mechanism of redistribution can be exploited to extend the duration of some drugs: in fact, a lot of anti-parasitic drugs exploit this kinetic effect thanks to the fact that the xenobiotic is accumulated in the adipose tissue (e.g. spot-on formulations are applied once in a month but they are able to protect the animal for the entire period). In humans, it is not something that can be exploited in the same way as in animals because we change a lot our body weight during the same season or in a lifetime. Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli BIOTRANSFORMATIONS AND CYTOCHROME P450 DEFINITIONS AND MOST IMPORTANT TOPICS OF THE PREVIOUS LESSONS: -absorption is the passage of the drug from the point of administration (it could be the point of injection or the storage after oral administration) to the bloodstream. -distribution is the passage of a drug from the bloodstream to the target tissue. -redistribution is a kinetic process characterizing very little liposoluble drugs that can pass easily the membranes and reach the hyperfused organs, for example, the brain. Then there is another passage, due to the different gradient of concentration, to the medium-perfused organs like the muscle. And then another passage back to the bloodstream and once again back to other tissues that are less perfused, so they can be reached by the bloodstream in a very late time and with a low concentration of the drug. An example of low-perfused organs is the adipose tissue. Clinical implications of redistribution: -ketamine is a dissociative anesthetic, a drug that will take longer to be fully metabolized by the organism, so the effect will last longer. The final effect is anesthetic, but then thanks to redistribution, the drug is not metabolized, it's not very fast eliminated, so it can remain inside the body for a longer period and this can influence the time of recovery and the patient will remain sedated for a very long period. -use of antiparasitic drugs in dogs, cats.. these drugs are administered in spot-on formulations that contain inside drugs that are lipophilic, and aim to prolong the action of the drug. The drug is absorbed and stored in the fat under the skin and released short by short in time. So, this is a way of exploiting redistribution for a therapeutic purpose. Routes of administration of drugs -Enteric: oral and rectal -Parenterals: intramuscular, intravenous, intra, subcutaneous, directly in the abdomen, intracardiac and so on. Oral administration and Henderson-Hasselbalch equation I'm administering an acidic drug, for example aspirin, which is acetylsalicylic acid. So in the stomach it’s ionized, because it's a weaker acid than the stomach, and it’s well-absorbed, infact the Inside the stomach environment it can remain in the ionized form, this is the place where absorption of aspirin takes place. This is the same with other NICDS drugs, so other non-steroidal anti-inflammatory drugs. On the opposite, if I am administering a weak basic drug it will be better absorbed in the bowel because the pH is higher compared to the stomach environment. So, there is an absorption in the very first part of the oral mucosa before the stomach,so before the drug can enter inside the systemic circulation, but it is mainly related to an inactivation of the drug. The name of this kinetic process is the first passage/message effect, so the drug administered through our route is immediately absorbed in the first part of the mouth of the oral mucosa and cannot reach the stomach. But due to anatomical considerations, this first passage effect is responsible for carrying the drug directly in the liver, bypassing the systemic circulation and reaching the liver without being absorbed in the bloodstream, so the drug is immediately disrupted and eliminated. Therefore this is something that we don't want to achieve due to the fact that it implies a loss of the total amount of the drug. This is the case, for example, of enalapril. In order to control blood pressure, hypertension, this kind of drug can be administered, but due to the first passage effect, if you administer 100% of enalapril, after this passage you cannot see anything, from 5 to 51% of the drug is available in the body. So, this is enough in order to achieve a therapeutic effect, but we must take care, when we dose the drug, that a huge amount, a huge percentage of the initial dose is lost due to this first passage effect. Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli Start of the new lesson PHARMACOKINETICS If you remember well, in the first lecture, we said that kinetic processes encoded four different passages: absorption, distribution, metabolization and elimination. So, today we need to speak about the metabolization of the different compounds because biotransformations are naturally present inside the body, and they’re something related also to the evolution of the organisms on Earth because even very simple and ancient organisms possessed a basic way of biotransformation. According to the evolution of the human species, the capacity of the liver to biotransform the different endogenous and exogenous compounds changed a lot, so also xenobiotics. Normally speaking, the biotransformations are processes related to the fact that the body wants to eliminate certain substances, like hormones or vitamins, that are normally present inside the body and that are necessary to allow physiological processes, but have to be stopped and eliminated once they finish their action. The same mechanisms are also exploited by the body to eliminate a huge amount of xenobiotics, which means something foreign from the body, something that belongs to the external part of the body. That’s why the same biotransformations can be responsible of different detoxifying processes, for example, those related to environmental pollutants, pesticides or drugs, and also drugs ,in a certain concentration, can exploit a toxic effect. Biotransformations are the main characters that can play this kind of transformation in order to allow the body to eliminate the substances that are no more functional to the physiological processes. Sometimes we have to take into consideration also feed additives or substances that can be carried on with feed and food. So, the biotransformations are responsible for first endogenous compounds transformations, for example, steroid hormones, thyroid hormones and so on. The same modifications can occur in different substrates. If a drug, a substance, a xenobiotic, is lipophilic, it means that it will stay for a long period inside the body, in order to be eliminated, this drugmust be biotransformed in a more hydrophilic compound. So, the final aim of all the biotransformations that we will see today is to transform a lipophilic substance into a more hydrophilic substance. In this way, the drug can be eliminated, excreted, at first through bile or urine and, of course, it is expected to terminate the bioactivity. So, it is expected that a drug that is more hydrophilic has no more efficacy in the body and cannot exploit any effect into the body. Actually, this is true for the 90%-95% of the cases, but sometimes there are some exceptions. Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli DRUG-METABOLIZING ENZYMES (DMEs) How can the biotransformations occur? Biotransformations are enzymatic processes that can occur in different parts of the body, mainly in the liver, but they are also virtually in all organs and tissues. These biotransformations, enzymatic pathways, can limit the action of the different compounds, for example the glycoprotein B in the brain barrier is also expressed in the bowel. So we have to consider that sometimes, in order to achieve a therapeutic effect, there are several limitations. One can be the presence of a very strong and early enzymatic process that can limit the biological activity, so a very early biotransformation, and, in accordance, we can also have the presence of PGP in some districts of the body. The expression of these enzymatic pathways can vary a lot among the different organs and tissues. According to the highest concentration and capability to perform biotransformations, enzymes can be found in the liver. We can also identify different enzymatic processes related to biotransformations in the lungs, but also in the brain, kidney, and virtually in all tissues, with the only exception of corneous tissues, so nails, nails and hooves, where there is no possibility to have these enzymes, this kind of biotransformation cannot take place there. Some species-specific differences exist between humans and animals. The most evident difference is related to the fact that in ruminants, there is a bowel, a stomach and a bowel environment that is really characterizing and can distinguish ruminants from the other animals and from humans. Infact in ruminants, in the bowel, there are some specific microorganisms of these animals that can drive different biotransformations that cannot be ruled out in different species. This is because the microorganisms contained inside the intestine and in the rumen represent the specific microbiota and are able to produce enzymes and perform biotransformation by their own. According to the fact that inside these organs there’s an anaerobic condition, only two different chemical reactions can take place: one is hydrolysis and the other one is reduction. We will see later that according to the different metabolic pathways, also oxidation can be encoded, but not in these species and not related to the presence of the microbiota. XENOBIOTIC-METABOLIZING ENZYMES ( XMEs) The different enzymes related to the biotransformations usually are not so selective, so the same enzyme can biotransform different substrates and the substrate affinity is calculated in kinetics using the Km constant of affinity of the different substrates. This is a general concept, the same enzyme can transform several substrates. What are the implications of biotransformations? The first goal is to perform a transformation from a lipophilic drug into a hydrophilic drug, and this is the most important way to interrupt the biological action of a drug and to eliminate it through urine and bile, for example. So the first effect is to induce a stop of the drug effect. On the other hand, we can also have alteration or interference of the drug persistence inside the body, because if we are in presence of detoxifying processes, enzymatic processes, of course the drug will be eliminated from the body. This is the general rule, but there are some exceptions. For example, we will see later that the biotransformation processes can be divided in phase one and phase two, also phase three occurs sometimes, where the drug that was previously Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli biotransformed and inactivated is activated once again, so the drug can persist inside the body and can be active once again. Another thing about drug effect and drug persistence together is the possibility to have systemic biotransformation, this is the case of the first passage effect: very early biotransformation that can inactivate the huge amount of the dose that was administered, limiting the presence of the drug inside the body and the final effect. Or we can also have the transformation of the drug into a metabolite that is still active as the parental drug, so as the drug that was administered. This is the case, for example, of ambrofloxacin and ciprofloxacin. Ambrofloxacin is an antibiotic drug belonging to the class of fluoroquinolones, and it is metabolized in the liver, its first metabolite is called ciprofloxacin, which is active as well, so the metabolite has the same activity as the parental drug. For human beings, ciprofloxacin is labelled, specifically without name, it’s a chyproxene in Italy, and it is administered like the parental drug to achieve antibiotic effect. So the biotransformations can be responsible normally of a metabolization of the different drugs, and this is the step before the elimination, but there are some exceptions related to an initial inactivation, or maybe a further activity, or the possibility to have the formation of metabolites that are still active and can be exploited to achieve a therapeutic effect. Phase one and phase two is a way of dividing these processes that can happen simultaneously on the same molecule or one subsequent to the other. PHASE ONE Some polar groups, for example OH, COH, NH2, can be introduced, so it can be attached on the molecule, and this may happen through the oxidation process, or these polar groups can be unmasked. They are still present inside the molecule, but thanks to hydrolysis, they are unmasked and the transformation of the molecule occurs in this way, then the molecule is transformed and can be eliminated, if it's sufficiently hypophilic, it can be eliminated through urine or bile according to the pH. Otherwise, the same drug can also undergo to phase two process. PHASE TWO In this case, polar groups can be conjugated on the molecule. This operation is driven by transferases, specific enzymes that are able to bring specific groups that can be glutathione, acetyls, amino acid, glucuronic acid and attach them to their compounds. Both phase one and phase two are normally expressed in the body, so these kind of reactions can occur naturally to transform hormones, for example, and the same processes are also exploited to biotransform and eliminate xenobiotics. PHASE THREE It’s a specific process that can occur only after phase two. This means that compounds that already have been conjugated with, for example, glutathione or glucuronic acid, are present inside the bile, so they are excreted into the bowel. When these conjugated molecules arrive in the bowel, the microbiota is responsible for the hydrolysis that can cut the conjugated substrate from the conjugation. So for example, glucuronic acid is released and the parenteral drug is present once again. The parenteral drug can remain inside the bowel and can be eliminated if it is not lipophilic or in general we have to consider the Anderson-Hasselbalch equation, infact maybe that drug cannot be reabsorbed due to the pH of the environment. In these cases, the drug, even if it is free from the conjugation, will be eliminated. Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli Otherwise, if the molecule is lipophilic enough, also related to the specific pH of the bowel, the drug can be reabsorbed and put again in the bloodstream. This implies the fact that the drug is active once again, so it's not eliminated and can achieve, once again, a therapeutic effect or a side effect if this drug is a toxic. This is the hepato-enteropathic cycle. The main site is the liver, but enzymatic processes can also occur also in lungs, in the brain and in the bloodstream. To re-enter the cycle, it has to be transformed in the liver to enter the bile, because the way to reach phase three is through bile, so we are inside the bowel. To summarise, during phase one, only oxidation, hydrolysis, and reduction can occur. In phase two, we have the addiction of some polar groups to the substrate, it’s a metabolic process that implies consumption of energy. It can occur alone or after phase one,independently from the BKA, the concept of dissociation of the drug. Hydrolysis is the only enzymatic process that can occur in a phase three process, and it is responsible for the detection of the substrate from the conjugation. Reabsorption is mainly related to the BKA of the drug and the pH of the environment. It depends if the drug is lipophilic enough, but also is related to the fact that if the drug is ionized. If the drug is lipophilic enough and ionized, it can pass back and come back to the different membranes and reach the bloodstream. This kind of drug, even an acidic drug, can undergo this kind of hydrolysis. If the substrate is, for example, a weak acid, the BKA is fighting against the pH of the bowel environment, so the drug will not be reabsorbed. Actually, even if phase three can occur, the atheropathic cycle cannot occur because the drug will be not reabsorbed. Just a scheme to resume what we already said: the exanthobiotic can undergo to phase one. A first metabolite is produced, and it can be, once again, conjugated with something, and we are speaking about phase two. The final aim is to transform a lipophilic compound into a hydrophilic compound because only a hydrophilic compound can be eliminated, otherwise, the drug will stay inside the body. SUBCELLULAR LOCALIZATION Phase one enzymes are mostly found in the smooth endoplasmic reticulum, while phase two reaction can happen in the cytoplasm because the molecular weight of these substances is quite huge, usually, we will find there the different enzymes and also the different conjugative agents. The most important exception related to the localization of the different enzymes is related to the glucathione transferases that is present in the SER as well. What about the studies related to the different way to understand the localization and the action of the different enzymes? There are a lot of sophisticated techniques, mainly molecular! that permit us to understand the different presence of the different enzymes in different parts of the body and so on. At the very beginning the technique was quite simple and started with the collection of the liver of different animals. These livers were cutted into small pieces and sonicated, then a first centrifugation occurred, and then a universal centrifugation with different substances was performed in order to divide the different components of the cells. This kind of technique permitted to create a virtual structure that is a microsome. Microsomes are heterogeneous artifacts containing different organs and vesicles holding cytosols, and they were the first structures that have been studied in the past in order to understand the different enzymatic families, to quantify the enzymes and to understand how they work. The same technique was applied also for other tissues. That's why we know that this kind of biotransformation can occur mainly in the liver, but also other tissues, for example the lungs, but they are less important according to the quantity and to the quality of the different biotransformations. Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli OXIDATIONS We can have two different types of oxidation: - Non-microsomal oxidation that are less important quantitatively and qualitatively and are mainly responsible for the transformation of amines including also the neurotransmitters, for example, epinephrine, and alcohols. - Microsomal oxidations are the most important route of biotransformation. For example there an hydroxylation or an epoxidation. According to the impact in human and animal health this heterocyclic epoxidation represents a weird case of biotransformation. Aaflatoxin B1 is a substance created by the action of aspergillus flavus, that is a fungi that can parasitize several plants, cereals, food and feed, and so on, so this substance can be accidentally ingested by humans and animals. According to a tentative of the liver to detoxify this substance, this aflatoxin B1 undergoes to microsomal oxidation, and actually this biotransformation is not able to transform this drug in a less toxic compound because an epoxide is generated, as you can see here. So this triangle is the epoxide. Epoxides are very reactive, so they are not stable molecules and they can react easily with a lot of other structures. In this case, aflatoxin B1 epoxide can react with guanine nucleobase in the DNA leading to modification of the DNA and leading to a carcinogenic effect: this is proven in animals and in humans, where the most severe consequence is the identification of a hepatocineral carcinoma, a very malignant tumor of the liver. This is an example when a biotransformation is not able to detoxify a compound, but actually the metabolize is unstable, very reactive and can cause damages to the host. The same reaction occurs also in animals, but aflatoxin B1 is mainly related to other toxic outcomes for example, teratogenic or mutagenic effects. Teratogenic means effects related to the fetus, so malformations, mutagenics or some mutations can lead to death or cancer. That's why aflatoxin B1 is a substance that is normally checked and controlled in surveillance plan. Some examples that are also related to normal life: benzopyrene is a substance contained into tobacco smoke and according to phase one biotransformations in lungs, is transformed into an epoxide metabolite. The epoxide group is unstable and can bind very easily different structures in the body. The most strong and severe effect is related to the fact that this epoxide can bind one ion nuclear base, leading to a carcinogenic effect, that's why tobacco smoke is related to lung cancer. Then, another biotransformation, is an oxidation in phase one, that is responsible for the transformation from amylofloxacin to ciprofloxacin, because the oxidation can occur. Also in this case, this is not a transformation from a lipophilic to a non-lipophilic compound, we are in an exceptional situation because ciprofloxacin is metabolized,and it’s actually called M1. When you perform a PLC, the first peak of the chromatographic graph is ciprofloxacin, that's why it is called M1. This metabolite is actually powerful as well as amylofloxacin. This is not a detoxifying, it’s not a biotransformation that leads to the formation of a more lipophilic compound, so several following passages of the biotransformation must occur in order to eliminate ciprofloxacin from the body. Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli CYTOCHROME P450 These microsomal oxidations are operated by a specific cytochrome system that is called cytochrome 450. It’s named in this way because in very ancient times, the only way to understand the composition of different samples was to perform spectrometry, so according to the spectrometry of microsomes containing enzymes of the cytochrome 450, the peak of the absorbance was in correspondence of a wavelength of 450 nanometer. Nowadays we have a lot of molecular techniques that permit us to go deeper inside the characterization, so we just maintain the name, but the techniques are better than before and more accurate. We divide the different cytochromes thanks to three different kind of categorization, we rank them in families, sub-families, and isoforms. The different families of CYP are codified and ranked according to the amino acid sequences, if similarity measured at 40% is present the cytochromes are ranked together in the same family. The same concept is behind the division in sub-families, and then we can also subdivide them into different isoforms. The family is always indicated using a number, while the sub-family is identified using a letter, and then the isoform is, once again, identified using a number. We already the catalytic cycle that is related to the different biotransformation in order to restore the enzyme from the beginning, so the system will be restored every time it is used and can start once again. Cytochrome p450 can be divided into families, sub-families, and isoforms. Usually, the different enzymes can biotransform several substrates. Here there are some examples of different cytochromes, you can find the number of the family and the letter related to sub-family, and some examples of substrate that can be specifically biotransformed by these enzymes. These examples are related to the human beings: -Cytochrome-1-A is responsible for caffeine biotransformation. -Cytochrome-2-B, this family is very important because it can transform a lot of toxic compounds. Giorgia Bersani/ Beatrice Mina 23/10/2024 Prof. Cristina Vercelli For example, organochlorines (DDT), was the name of a commercialized pesticide that was really used in the past, also in Piedmont. It’s also the family responsible for the biotransformation of some anesthetics like barbiturates and propofol. -Cytochrome-2-C is responsible for the biotransformation of NSAIDs. Diazepam is the main principle of valium, so benzodiazepine, a tranquilizer. -Cytochrome-2-D, beta-blocker it's a drug acting on the heart in order to achieve an anti-rrhythmic effect. -Cytochrome-2-E, methanol and ethanol, they are short-chain alcohols. -Cytochrome-3-A can bio-transform many drugs. This is a graphical representation of the different families of cytochrome expressed in the liver of human beings. According to this graphical representation, the most important cytochrome family in human beings is represented by 3A, 4, and 5, which are responsible for the bio- transformation in several drugs. So based on these enzymatic pathways, several side effects or just some side effects can be observed. A very specific example is zearalenone, a case related to a possible outcome of side effects. Zearalenone is a substance that can be present in this feed and can be administered to the swine which has a specific capability of performing reduction in phase one of bio- transformations leading to the transformation of the parental compound into different metabolites. The alpha metabolite is responsible of several side effects, many related to the fact that this chemical structure and the bioactive action is really similar to estrogens. So the female or male subject, can have severe side effects related to an impairment of hormone balance. These can cause abortion or a transformation of the second characteristics related to the sex, so also morphological alterations. If something like this occur in the production of swine meat, for example, there’s a reduction of the newborns because the estrogen-like effect, also the fertility of the male subject will decrease the newborn life expectancy. So this really can impact a lot the quality of life of these animals and can have an economic impact. The percentage, the presence of the certain enzymes can decrease along with the life of humans or animals, and this example about zearalenone, which is converted into alpha and beta zearalenone, proposes another difference that can impact the different biotransformations. Infact there are specific biotransformations that can occur only in certain species, and this is related to the fact that, for example, cytoprotein p450 families, subfamilies and isoforms can be present in different amounts in the different species. If we take into consideration, for example, the liver of bovines or the liver of dogs, this distribution of the different cytoprotein families can change really a lot, this implies that the different biotransformations will not be the same according to the species. So a drug that in human is absolutely safe can be toxic for dogs, for cats and so on. For example, in case of toxicity of zearalenone, this is a trouble just for swine and not for other species, or in the fad of toxin B1, the effect is more or less the same according to the toxic effect that can occur in humans and in animals. Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli PHASE II BIOTRANSFORMATIONS BIOTRANSFORMATION REACTIONS Biotransformations are reactions performed mainly in the liver but also in lungs, brain, kidneys, and in other organs or tissues. These kinds of transformations aim to transform very lipophilic compounds into hydrophilic compounds, because only hydrophilic compounds can be eliminated through kidneys and through the urine, or through the liver, in the bile, in the bowel and then through the feces. If we have a very lipophilic drug that can’t undergo extensive biotransformations, we could have redistribution, a kinetic mechanism in which the drug continuously remains lipophilic and can’t be eliminated, thus it will stay longer in the organ and in the body. The table above summarizes the different reactions of phase I and phase II. 1) In phase I there are oxidation, reduction and hydrolysis: they are all degradative reactions that are aimed to introduce some functional groups (-OH, -NH2, -SH, -O-, -COOH); the final aim is to have a more hydrophilic compound. These reactions occur mainly in the microsomal fraction of the cytosol of the cells. The metabolites that are formed are usually smaller, polar or non-polar, and usually inactive; very few compounds that undergo phase I reactions are actually more active than the parental compound. 2) Phase II reactions can happen simultaneously to phase I or immediately after. There are different types of phase II reactions: they are synthetic reactions because something is added to the parental compound, therefore it’s a reaction that implies the synthesis of a new molecule. These reactions consist in a conjugation, which could be with glucuronic acid, sulfate, acetyl, methyl groups or also amino acids. These reactions occur mainly in the microsomal fraction, but also in mitochondria and in the cytoplasmic space. The metabolites are usually larger than the parental compounds, due to the fact 1 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli that the parental compound is linked to something else (glucuronic acid, sulfate, acetyl etc...), and also water soluble and inactive. 3) Also phase III reactions exist: they consist in cutting the conjugates of phase II in order to free the parental compound. Phase III reactions usually happen to conjugates with glucuronic acid and with sulfate groups. PHASE II BIOTRANSFORMATIONS Biotransformations of xenobiotics exploit the same natural pathways that normally the body exploits to eliminate or recycle endogenous compounds. Phase II can happen after phase I or can it be self-standing, therefore happening directly to the xenobiotic. Conjugations can happen with several compounds. The most common pathway is represented in the picture below: the endogenous group, for example glucuronic acid, must be activated (this is an energetic pathway). Then, the active conjugating agent can be added to the parental compound thanks to a specific enzyme, usually a transferase (but it can have a different name according to the specific conjugating agent). This pathway happens with all the different conjugating agents except with amino acids. In case of amino acids conjugation, the substrate, which in this case is the xenobiotic, must be activated first. Then, the active substrate can be bound to the amino acid thanks to a specific transferase, forming a conjugated substrate. To summarize: usually in phase II it’s the conjugating agent that must be activated first, but there is an exception with amino acids conjugations, in which it’s the parental compound that must be activated first. Through both pathways we obtain a final product that is larger than the parental compound, it’s polar, it’s hydrophilic and can be eliminated. GLUCURONIDATION This kind of phase II biotransformation is the most frequent conjugation that exists in mammals (with an exception). The active conjugating agent is called UDPGA (= uridine-diphospho-glucuronic acid); thanks to a specific transferase, UDPGA is conjugated with the substrate. 2 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli Glucuronidation is one of the two phase II reactions that can undergo phase III reactions: a glucuronised compound can be hydrolysed into the bowel, so the parental compound and the glucuronic acid are released; in this case, if the parental compound is lipophilic and therefore can cross the membranes, it can enter once again into the cycle and achieve once again a biological action. According to the localization, the majority of UDPGA-transferases is located in microsomes, and are very close to the cytochrome p450, which is the enzyme responsible for the vast majority of phase I reactions. Due to a very close proximity between these two enzymatic systems, a single xenobiotic can undergo a first phase I reaction through the cytochrome p450 pathway and then, immediately after, this oxidized metabolite can undergo a glucuronidation reaction; all the groups added in phase I can be functional groups able to perform the binding with glucuronic acid. Different glucuronides can be formed: -N, -O, -S. The O-glucuronides (oxidase-glucuronides) can be of “ester” or “ether” nature based on whether the UDPGA binds a carboxylic or hydroxyl groups, respectively. Glucuronidation is the most important phase II biotransformation pathway in mammals, except in cats (and felines in general): cats have a very poor glucuronidation storage, so they can perform a very low quantity of glucuronidation reactions, especially against aromatic compounds. In clinical practice, it means that in case of administration of a xenobiotic that contains aromatic groups in cats, we can have a toxic situation because they are not able to exploit this kind of biotransformation in order to detoxify the drug, hence to allow the elimination. In case of administration in cats of drugs containing aromatic groups, we can have two different scenarios: Acute toxicosis: after one single administration the cat starts to demonstrate toxic effects; Sub-acute toxicosis: more than one administration is required to see side effects. A lot of NSAIDs (non steroidal anti-inflammatory drugs) contain aromatic groups inside, and only new generation NSAIDs have been specifically labeled for cats, because they modified them to contain an aliphatic structure, so that the biotransformation can be allowed in cats too. SULPHATION It’s quantitatively less important than glucuronidation. The active conjugating agent is called PAPS (= phospho-adenosine-phospho-sulfate), and also in this case it’s the conjugating agent that must be activated prior the binding with the substrate. The quantity of this conjugating agent is just ¼ or ⅕ compared to UDPGA; it can be stored or re-synthesised according to the request of the body. The conjugable substrates are -OH, -NH2, -SH. The conjugable groups for this reaction are very similar to those undergoing glucuronidation (with the exception of -COOH). 3 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli GSH CONJUGATION This other kind of conjugation is performed with the conjugating agent called GSH (= gamma-glutamyl-cysteinyl-glycine), which is an endogenous compound (as the previous ones) and is present in the cytosol of hepatocytes. Contrary to all the other conjugating agents, GSH can be activated prior to conjugation or not, meaning that it is able to conjugate appropriate substrates spontaneously; however, this spontaneous linkage happens with a very low rate. The conjugation takes place according to the thiolate anion, and there are some species-specific differences. A specific transferase is present, it is mainly in the cytosol and there are several families. GSH is normally present in order to bind some endogenous compounds, for example steroids. GSH is extremely active in some cells and can exert a protective effect against aflatoxin B1 epoxide (AFB1), which is a toxin synthesized by Aspergillus Flavus (a parasite of some plants). This drug is highly reactive after phase I reaction, because an epoxide is formed and it’s highly reactive against some structures like DNA: the guanine base can be linked to AFB1 and can cause cancer (it’s one of the agents responsible for epatocarcinoma in humans). Glucuronidation is a lacking process in cats. However, mother nature, in order to preserve cats, gave them more biotransformation pathways according to GSH conjugation. So, in felines, there are several subfamilies of transferases for GSH conjugation. The GSH conjugates have two different destinies: ➔ The most common destiny is the elimination through the urinary route; we can appreciate the presence of mercapturic acid in the urine, that is the final step of this kind of biotransformation. ➔ Another way to eliminate the GSH conjugates is through the biliary route and the final production of feces; but this way is quantitatively less important. 4 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli N-ACETYLATION This reaction is very particular both in human and veterinary medicine. The conjugating agent is an endogenous compound called acetyl-coA, which is activated using energy. The specific transferase is called N-acetyltransferase; it has two different isoforms, NAT1 and NAT2, that are both lacking in the dogs. In humans there is not a single rule, because there is genetic polymorphism; normally, we can describe two different populations: - Fast metabolizers: population composed by people who have a normal activity of N-acetyltransferase, so drugs that can undergo this specific route are normally biotransformed, with no side effects; - Slow metabolizers: population composed by people who have a very low activity of N-acetyltransferase, so some drugs can’t be transformed and will stay in the lipophilic form; they can be accumulated inside the body, causing side effects. Another problem in this population is drug-to-drug interaction: high concentrations of certain drugs in the body can fight with other drugs, causing several side effects. This is the case of hydralazine (a tranquilizer), procainamide (a local anesthetic), isoniazid (an anti-parasite), that can stay with an high concentration inside the body and fight with phenytoin (a tranquilizer) and rifampicin (an antibiotic). METHYLATION The endogenous conjugated agent is the activated methionine, while the methyltransferase is the specific transferase responsible for this type of reaction. In some cases, the final product is more lipophilic than the parental compound. Example of the mercury: mercury is biotransformed through this route, but after the addition of the methyl group a more lipophilic compound is obtained, which is called methylmercury. Methylmercury can be stored in very simple organisms, for example bacteria and algae; it was demonstrated that algae can contain large quantities of methylmercury. Algae are eaten by small fish, which are eaten by bigger fish (for example tuna fish or swordfish), and at each step methylmercury can accumulate inside the body. Lastly, big fish are usually consumed by humans and the possible effect of methylmercury is toxicosis. It was demonstrated that in Japan, where a lot of tuna fish is consumed (without processing it, but it doesn’t matter because methylmercury doesn’t disappear with cooking), the accumulation of methylmercury in the human body cause the Minamata disease, a neurological alteration and toxicosis leading to paraesthesia, tremors, seizures (crisi convulsive), also leading to malformations in the fetus, which is why pregnant women must not eat big fish. Another exception is represented by selenium, that according to methylation can form two different compounds: Dimethylselenide, which is a volatile compound that can be eliminated through breathing; if this compound undergoes to a second step of methylation, the final production is Trimethylselenonium, which is eliminated through urine, but before the elimination it can be a very dangerous compound and can cause toxicosis in humans and animals. 5 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli AMINO ACID CONJUGATION In case of amino acid conjugation, it’s the substrate that must be activated first, and then the specific transferase can put the amino acids on the activated substrate. The conjugable groups are mainly represented by hydroxylamines and -COOH. In mammalian species, there is a deficiency of glycine amino acid in cats. PRE- AND POST-SYSTEMIC METABOLISM PRE-SYSTEMIC METABOLISM The first pass metabolism is a pre-systemic metabolism which requires that the drug is not correctly absorbed after oral administration: the drug doesn’t enter inside the systemic circulation because otherwise it is distributed. In the first pass metabolism, the drug is directly conducted through the portal vein into the liver, without being distributed into the tissues. Since it reaches directly the liver, the drug is biotransformed and eliminated almost immediately. In clinical practice, it means that this kind of biotransformation limits the portion of the drug that is available to the body and that can achieve any kind of biological response. So, there is a limitation of the bioavailability: the bioavailability of a drug is the portion of the drug that can be distributed into the tissues and that can achieve a certain biological response. If the drug undergoes to first pass metabolism, it means that only a small quantity of it will be bioavailable, therefore only a small quantity of the drug is present in its active form and so the biological effect will be very limited. However, in some cases, like for pro-drugs, the first pass metabolism is a mechanism used in order to activate the specific drug. A pro-drug can be administered into the body but it must undergo a first 6 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli passage on the way of biotransformation in order to release the active drug. Some drugs are synthesized as pro-drugs and the first pass mechanism is exploited in order to have the release of an active form that can be present in a good concentration in the body and achieve a biological effect. Some specific drugs, for example cyclosporine (an immunosuppressant that is used both in human and veterinary medicine, to control certain diseases caused by an excessive proliferation of the immune system), or penicillin G (the first penicillin synthesized by Fleming in 1938), or propranolol (a beta-blocker, used to induce an effect directly into heart in order to avoid arrhythmias), undergo to an extensive first pass metabolism after oral administration and this can limit a lot the power of this drugs in a clinical scenario. In certain cases, the loss of efficacy due to first pass effect is so high that the oral route becomes unsuitable in order to have a therapeutic effect; so it’s preferable to change the route of administration, using intravenous administration or other routes. POST-SYSTEMIC METABOLISM This is the phase III reaction. This metabolism can affect specific metabolites of phase II reactions: glucurono- and sulpho- derivatives. This specificity is due to the presence of bacterial glucuronidases and sulphatases, so only glucuronide and sulphate conjugates can be transformed by these enzymes. These enzymes, synthesized by commensal bacteria inside the bowel, can cut the linkage between the parental compound and the conjugating agent; the parental compound is released and also the conjugating agent is released. ➔ The conjugating agent can be recycled and used in another biotransformation; ➔ The parental compound, if it’s a lipophilic compound, can be re readsorbed and cause once again some biological effects. The reason behind this specific mechanism is a way of “saving and recycling” high energy consuming endogenous compounds; this is the way steroids, corticosteroids or thyroid hormones are recycled inside the body. When these hormones (that are really energy-wasting hormones due to the very difficult synthesizing pathways) are released and eliminated through different reactions, they can be recycled through this kind of metabolism when they are in the bowel. 7 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli FACTORS AFFECTING BIOTRANSFORMATION There are a lot of different factors that can affect biotransformations. In the graph we can see the most important factors that can influence biotransformations: Age (for example about alcohol dehydrogenase); Gender, which can generate differences in human but also animal species; Pregnancy; Species-specific differences and breeds that can cause genetic polymorphism; Diet; Pathological conditions. Induction and inhibition are two terms related to the possibility to increase or decrease the activity of the enzymatic systems. The different conditions leading to induction or inhibition are very common and can characterize the activity of several drugs both in humans and animals. SPECIES RELATED DIFFERENCES About the differences in phase I enzymes, the most important enzymatic pathway is represented by cytochromes; there are several families and subfamilies of cytochromes. In humans they are more or less the same, according to the different parts of the world (for example some differences exist between european and asians people); in animals the differences are a lot, according to the species. The image is an example of different expression of hepatic cytochrome isoforms in different food producing animals (rabbits, horse, chickens, pigs) compared to rats. It’s important to highlight that usually rats are used as experimental models for humans, to perform pre-clinical trials for humans. Many physicians say that the rat is similar to a human, according to the metabolism and the ways of biotransformation. From a veterinary point of view, this is not true, because rat is a suitable model for experimental purposes only because it’s easy to breed and it’s a fast-rate-growing species. In addition, considering the fact that rats and other animals express different cytochromes (very different compared to humans), the reliability of results obtained in rats is uncertain in human medicine. Therefore, it’s very important to follow each step of the experimental procedures prior reaching the human beings, going from rats to dogs, then to the scimpanzé, and lastly humans. 8 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli According to the differences in the expression of phase I enzymes, the same substrate can be biotransformed by several different cytochromes or be involved in different pathways. The different expressions of the cytochromes can be responsible for species-specific differences and also for different clinical responses. For example, the equines can’t be treated with some antibiotics belonging to the class of ionophores, such as the antibiotic monensin. Since there is a deficiency of cytochrome 3A in equines, this kind of drugs can be toxic to them. The percentage of metabolized monensin between the different species can be observed in the image. This different metabolization of monensin is specifically related to the levels of cytochrome 3A. In chickens, cytochromes 3A are well expressed, so given 100 doses of monensin, the entire concentration is biotransformed and metabolized. Thus, the lethal dose is double, so you have to exaggerate with the administration of monensin prior to reach a toxic level in chickens; Bovines, swines and rats have decreasing levels of metabolization of the same drug; In horses, given the same dose, only 15% of monensin is metabolized. Toxic effect can be reached after a single dose because only a small quantity, of 2-3 mg/kg of the body weight, can be responsible for lethal effect in horses. In conclusion, the different expression of cytochromes can be related to the response of the animal to the dose and thus the possible outcome of side effects. Another example is the presence of cytochrome 1A in cows. Cows can eat a huge amount of cereals containing AFB1 and they won’t have any side effect: this is due to the fact that cows are able to biotransform AFB1 into another metabolite that is called AFM1 (aflatoxin M1), thanks to the strong activity of cytochrome 1A. So, cows are safe, but the AFM1 contained in the milk can lead to toxicosis in the consumer. DIFFERENCES IN REDUCTIVE PATHWAYS: Another species-specific difference is the different sensitivity of Zearalenone. In phase I in swines, Zearalenone is metabolized in alpha-Zearalenone (α-ZEL). This compound has a strong estrogenic-like effect, leading to decreased fertility in female and male subjects and also newborn death. 9 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli The same compound administered to chickens is biotransformed in a different way, in a compound called beta-Zearalenone (β-ZEL), which has a very low estrogenic effect compared to the alpha metabolite. Therefore, in different species different metabolites are formed and different effects are induced. DIFFERENCES IN CONJUGATIVE PATHWAYS: Cats: they are deficient of glucuronidation and conjugation with the glycine amino acid. Dogs: they lack NAT1 and NAT2 genes, so they can’t produce the specific transferases for N-acetylation; it means that drugs that need this pathway are not biotransformed at all, leading to possible side effects. Example: sulphonamides are antibiotic drugs normally used in clinical practice of dogs, cats and humans as well. In dogs, in case of oral administration, the system can compensate by exploiting other routes of biotransformation in order to biotransform sulphonamides. But in case of intravenous administration, a very high concentration of drug is immediately present inside the body, and the lack of NAT1 and NAT2 causes an inadequate biotransformation of sulphonamides. Sulphonamides are mainly eliminated through urine; the presence of high concentrations of them in the urine can cause the precipitation of crystals that remain stuck in glomerulus and in tubules, blocking the functionality of the kidneys and therefore leading to kidney failure. In clinical practice, if the administration of sulphonamides through an intravenous route is necessary, the dog is usually hospitalized in order to administer fluids intravenously for the entire duration of the therapeutic plan (to avoid formation of crystals in the kidneys). Pigs: they lack sulfation pathway, so usually other pathways are used. About phase II enzymes, cats have a small amount of biotransformations, so they can have a lot of side effects after drug administration. A specific alteration is caused by the administration of paracetamol (or acetaminophen, they are synonyms). Paracetamol is a pro-drug: it’s biotransformed in the liver, releasing a first metabolize that is able to achieve a therapeutic effect. In dogs and cats the first biotransformation is a N-acetylation, but both species are deficient of this route: - Dogs are deficient in NAT1 and NAT2; - In cats N-acetylation it’s not the most common biotransformation pathway. Therefore, both of them can be affected by side effects after single administration. However, dogs can find another way of biotransformation through glucuronidation. On the other hand, cats are deficient also in glucuronidation, so there are side effects. The first side effect is methemoglobinemia: hemoglobin is linked with unmet residues, forming Methemoglobin (MetHb), making red blood cells unable to bind oxygen. As a consequence, blood changes color and liver failure can occur. Therefore, cats must not be treated with paracetamol (also because it’s aromatic). 10 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli Paracetamol can be antagonized by N-acetyl-cysteine, which is a specific antagonistic drug to use in case of toxicosis by paracetamol. This can happen in humans too, with dosages above 600 mg/kg: we can use an intravenous infusion of N-acetyl-cysteine to avoid hepatic impairment. These variations in feline species are considered not only in the development of new drugs, but also when feed is created; the different ingredients inside cat feeds are carefully evaluated in order not to impair the liver, due to the fact that a lot of enzymatic pathways are deficient in cats. EFSA (European Authority of Safety) decided to limit the feed additives for cats in two different ways: - The application of an additional safety factor, for example maltol: cats are sensitive to the action of a certain molecule, so a stronger and higher coefficient of safety is added in order to multiply the safety level; - If it’s not possible to establish a safety level, it was decided to completely avoid the presence of certain substances in food additives, for example Cassia gum. BREED RELATED DIFFERENCES Differences of cytochrome expression can be not only related to the species, but also to breed. Two different breeds of cows: Piedmontese and Limousine. Both are breeds used to produce meat and the expression of cytochromes is very different between these two breeds. Also, the different expression of cytochrome 2B11 is appreciable in the canine breeds, for example Beagle and Greyhounds. 11 Giulia Alloatti / Francesca Montuoro 30/10/2024 Prof. Cristina Vercelli AGE AND GENDER RELATED DIFFERENCES AGE: The picture shows the different metabolic ability of a very young calf and an adult cow. Young subjects have a naive enzymatic storage, so they have to acquire new enzymatic competences along the growing process. The highest level of enzymatic activity is demonstrated in adult age, while during elderly the enzymatic activity decreases a lot. GENDER: Male rats and male humans generally demonstrate higher capability of performing phase I reactions, related to the fact the cytochrome p450 is more expressed. But this is not the case in goats, where the situation is the opposite: female subjects have a higher capability to perform phase I reactions. EFFECTS OF DISEASE The vast majority of reactions happen in the liver, but the liver can be affected by some diseases, like hepatitis (inflammation) or cancer. The same can happen to the kidneys, which are responsible for biotransformation and elimination; for example, renal impairment, kidney failure or inflammation. There is a first deficiency of the organ responsible for the biotransformation and elimination, therefore a higher concentration of drug can be stored and accumulate inside the body. In another situation, there could be a decrease in the transcription of different cytochromes produced in hepatocytes, so there will be a small concentration of enzymes related to cytochrome p450. Otherwise, there could be an increase in secretion and in production of certain proteins, for example α1 acid glycoprotein; in this case distribution is affected, and not biotransformations. If there is an increased concentration of plasma proteins, there is a higher concentration of drug bound with the proteins: the fraction that is bound to the proteins can’t be distributed at all, but the free fraction can be. In case of inflammation, which leads to increased production of α1 acid glycoprotein, there will be a higher concentration of drug that is bound to plasma proteins and can’t be distributed in tissues. 12 Translated from Italian to English - www.onlinedoctranslator.com Alice Pezzoli / Elena Capizzi 11/13/2024 Prof. Cristina Vercelli Summary of previous lessons: The routes of administration are: intravenous, oral, intramuscular. For all routes of administration, except intravenous, we must speak of absorption, which is the passage from the site of administration to the blood. Once in the blood, the administered drug must distribute into the tissues where it will be able to reach receptors. The drug is then metabolized in the liver in two phases whose final purpose is to make the drug more water-soluble and therefore easier to eliminate from the body. If the drug remained fat-soluble, it could undergo redistribution and would be difficult to eliminate because it would continue to pass through membranes. Phase 3 reactions, at the intestinal level, break the union between the parent molecule and a variable group; if the parent molecule is fat-soluble, it can then return to circulation. We have seen administration, absorption, distribution and how there are differences in each step that can affect the extent of these processes. If we talk about absorption we may have variations due to the type of administration I perform: - If I give an intramuscular administration, the speed of absorption may be influenced by the liposolubility of the compound and how much blood per minute reaches that muscle area; - If I perfo

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