Histohematic and blood-brain barriers of the brain. BBB or blood-brain barrier: its structure and significance Penetrates through the GEB into the central nervous system

The blood-brain barrier is present in all vertebrates. It passes between the central nervous and circulatory systems. Next, we will consider in more detail such a term as the "blood-brain barrier": what it is, what tasks it performs.

Historical information

The first evidence that the blood-brain barrier exists was obtained by Paul Ehrlich in 1885. He found that the dye introduced into the bloodstream of the rat got into all tissues and organs, with the exception of the brain. Ehrlich suggested that the substance did not spread to the brain tissue during intravenous administration due to the fact that it had no relationship with them. This conclusion turned out to be incorrect. In 1909, Ehrlich's student, Goldman, determined that trypan blue did not penetrate the brain when injected intravenously, but stained the ventricular vascular plexus. In 1913, he demonstrated that the injected contrast agent into the cerebrospinal fluid of a horse or dog is distributed over the tissues of the spinal cord and brain, but does not affect the peripheral organs and systems. Based on the results of the experiments, Goldman suggested that there is an obstacle between the blood and the brain that prevents the penetration of neurotoxic compounds.

human physiology

The brain has a weight approximately equal to 2% of the total body weight. The oxygen consumption of the CNS is within 20% of the total volume entering the body. The brain differs from other organs in the smallest supply of nutritional compounds. With the help of only anaerobic glycolysis, nerve cells are not able to provide their energy needs. When the flow of blood to the brain stops, after a few seconds, a loss of consciousness occurs, and after 10 minutes, neurons die. Human physiology is designed in such a way that the energy needs of the brain structures are provided through the active transport of nutrients and oxygen through the BBB. The blood vessels of the central nervous system have some structural and functional features. This distinguishes them from the circulatory networks of other systems and organs. These distinctive features provide nutrition, maintenance of homeostasis and excretion of waste products.

Blood Brain Barrier: Physiology

Normal brain activity is possible only under conditions of biochemical and electrolyte homeostasis. Fluctuations in the calcium content in the blood, pH and other indicators should not affect the condition of the nerve tissue. It must also be protected from the penetration of neurotransmitters that circulate in the blood and can change the activity of neurons. Foreign agents should not enter the brain: pathogenic microorganisms and xenobiotics. The structural features of the BBB contribute to the fact that it is also an immunological obstacle, since it is impermeable to a large number of antibodies, microorganisms and leukocytes. Disturbances in the blood-brain barrier can provoke CNS lesions. Many neurological pathologies are indirectly or directly related to damage to the BBB.

Structure

What is the structure of the blood-brain barrier? Endothelial cells act as the main element. The blood-brain barrier also includes astrocytes and pericytes. There are tight junctions between endothelial cells in cerebral vessels. The gaps between the elements of the BBB are smaller than in other tissues of the body. Endothelial cells, astrocytes, and pericytes act as the structural basis of the blood-brain barrier not only in humans, but also in most vertebrates.

Formation

Until the end of the 20th century, it was believed that the blood-brain barrier and its functions were not fully developed in newborns and embryos. This rather widespread opinion was due to several unsuccessful experiments. During the experiments, protein-bound dyes or other markers were injected into embryos and adult animals. The first such experiments were carried out in 1920. The markers injected into the embryos spread throughout the tissue of the brain and spinal cord fluid. This was not observed in adult animals. Some mistakes were made during the experiments. In particular, in some experiments, too much dye was used, in others, the osmotic pressure was increased. As a result of this, partial damage occurred in the vascular wall, as a result of which the marker spread throughout the brain tissue. With the correct setting of the experiment, passage through the blood-brain barrier was not noted. In the blood of the embryo, molecules of such compounds as transferrin, alpha1-fetoprotein, and albumin are present in large volumes. These substances are not detected, however, in the intercellular space of the brain tissue, in the embryonic endothelium, the P-glycoprotein transporter was found. This, in turn, indicates the presence of the blood-brain barrier in the prenatal period.

Permeability

In the process of development of the organism, the improvement of the BBB is noted. For polarized small molecules, such as sucrose and inulin, the permeability of the blood-brain barrier in the newborn and embryo is significantly higher than in adults. A similar effect was also found for ions. The passage of insulin and amino acids through the blood-brain barrier is greatly accelerated. This is probably due to the great need of the growing brain. At the same time, the embryo has a barrier between tissue and cerebrospinal fluid - "belt contacts" between the elements of the ependyma.

Mechanisms of passage through the BBB

There are two main ways to overcome the barrier:

The easiest way to penetrate through the blood-brain barrier is small molecules (oxygen, for example) or elements that are easily soluble in lipid membrane components located in glial cells (ethanol, for example). Due to the use of highly specialized mechanisms to overcome the blood-brain barrier, fungi, bacteria, and viruses penetrate through it. For example, herpes pathogens pass through the nerve cells of a weak organism and enter the central nervous system.

Use of BBB properties in pharmacology

Modern effective medicines are developed taking into account the permeability of the blood-brain barrier. For example, the pharmaceutical industry produces synthetic analgesics based on morphine. But unlike him, drugs do not pass through the BBB. Due to this, medications effectively relieve pain without making the patient dependent on morphine. There are various antibiotics that cross the blood-brain barrier. Many of them are considered indispensable in the treatment of certain infectious pathologies. It must be remembered that an overdose of drugs can provoke serious complications - paralysis and death of nerves. In this regard, experts strongly do not recommend self-treatment with antibiotics.

Drugs passing through the BBB

The blood-brain barrier is selectively permeable. So, some of the biologically active compounds - catecholamines, for example - do not pass the BBB. However, there are small areas near the pituitary gland, pineal gland, and a number of areas of the hypothalamus where these substances can cross the blood-brain barrier. When prescribing treatment, the doctor takes into account the characteristics of the BBB. For example, in practical gastroenterology, the permeability of the barrier is taken into account in the process of assessing the intensity of the side effects of certain drugs on the digestive organs. In this case, they try to give preference to those drugs that pass through the BBB worse. As for antibiotics, among those that penetrate well through the barrier, the drug "Nifuratel" should be noted. It is also known under the name "Macmirror". Well overcome the BBB prokinetics of the first generation. These, in particular, include such means as Bimaral, Metoclopramide. The active substance in them is bromopride.

The BBB and drugs of the next generations of prokinetics pass well. Among them are such medicines as "Motilak", "Motilium". The active substance in them is domperidone. Worse, drugs such as Itomed and Ganaton penetrate the blood-brain barrier. The active ingredient in them is itopride. The best degree of passage through the BBB is observed in such medicines as Ampicillin and Cefazolin. It should also be said that the ability to penetrate the blood-brain barrier in fat-soluble compounds is higher than that of water-soluble ones.

An increasing number of diseases are explained by scientists as a violation of the functions of the blood-brain barrier (BBB). Its pathological permeability develops in almost all types of CNS pathology. On the other hand, to ensure the penetration of certain drugs into the brain, overcoming the BBB becomes a priority. Techniques that make it possible to purposefully overcome the protective barrier between the bloodstream and brain structures can give a significant impetus to the treatment of many diseases.

In one of his famous experiments with dyes, the now well-known scientist Paul Ehrlich discovered at the end of the 19th century an interesting phenomenon that occupies the minds of scientists to this day: after introducing an organic dye into the blood of an experimental mouse, observing cells of various organs under a microscope, including and cells related to the organs of the central nervous system, Ehrlich noted that the dye penetrated into all tissues, with the exception of the brain. After the scientist's assistant injected the dye directly into the brain, the picture observed under the microscope was exactly the opposite: the substance of the brain was stained with a dark violet-blue dye, while no dye was found in the cells of other organs. From his observations, Ehrlich concluded that there must be a barrier between the brain and systemic blood flow.

Half a century after Paul Ehrlich's discovery, with the advent of more powerful microscopes that allowed objects to be observed at 5,000 times the magnification of Ehrlich's microscope, the blood-brain barrier was indeed identified. It lies in the walls of a multi-kilometer network of blood vessels that supplies each of the hundreds of billions of nerve cells in the human brain. Like all blood vessels, cerebral vessels are lined internally with endothelial cells. However, endotheliocytes, which are part of the neurovascular unit of the brain, adjoin each other more densely than throughout the rest of the vascular bed. Intercellular contacts between them are called "tight junctions" (tight junctions). The ability to form a compact unfenestrated monolayer, the expression of highly specialized transport molecules and cell adhesion proteins allow endotheliocytes to maintain a low level of transcytosis. Also, the endothelium is under the influence of regulation by pericytes, astrocytes, neurons, and extracellular matrix molecules, which makes it clear that the BBB is not just a layer of endotheliocytes, but an active organ that includes different types of cells. Such interaction of cells, which provides a barrier function, preventing the free movement of liquids, macromolecules, ions, explains why neither Paul Ehrlich's dye nor some drugs can penetrate from the blood into the brain tissue.

Even before the presence of the BBB became clear, doctors and scientists were aware of its significance. And interfering with the functioning of this barrier was considered a bad idea. Over time, this idea changed, as the BBB turned out to be a highly active structure. Cells on both sides of the barrier are in constant contact, mutually influencing each other. A variety of intracellular molecular signaling pathways determine the capacity of the BBB in relation to different types of molecules (here I would like to recall the Wnt signaling pathway, which coordinates many processes associated with cell differentiation and is also involved in maintaining the integrity of the BBB). Leukocytes, for example, long thought to be too large to cross the BBB, in fact cross it by performing "immunological surveillance." Microscopic equipment and microscopes themselves do not stop in development even now, they are constantly becoming more complex and opening up more and more opportunities for visualizing the finely arranged structures of a living organism. For example, the use of a two-photon microscope makes it possible to observe the living tissue of the cerebral cortex at a depth of about 300 microns, which was carried out by Maiken Nedergaard, MD, from the University of Rochester. She performed the following manipulations: a part of the mouse skull was removed, then a dye was injected into the bloodstream, which made it possible to observe the BBB in action in real time. The researcher was able to track how individual cells moved from the bloodstream through the capillary wall - through the very layer of endothelial cells that was considered impermeable to them just 20 years ago.

Before the two-photon microscope was built, researchers used classical methods: for example, they observed dead tissue cells through a microscope, which did not give much explanation about the functioning of the BBB. It is valuable to observe the work of the BBB in dynamics. In a series of experiments, Nedergaard and her colleagues stimulated a specific group of nerve cells, which revealed the incredible dynamism of the BBB: blood vessels surrounding neurons dilated when nerve cells were stimulated, providing increased blood flow, as the stimulated neurons began to propagate an action potential; with a decrease in irritating impulses, the vessels immediately narrowed again. Also, when assessing the functions of the BBB, it is important to pay attention not only to endotheliocytes, but also to the already mentioned astrocytes and pericytes, which surround the vessels and facilitate the interaction between blood, endothelium, and neurons. The microglial cells circulating around the cell should not be underestimated, since defects in their functions can play an important role in the occurrence of neurodegenerative diseases, because in this case, the immune defense of the BBB is weakened. When endothelial cells die, either naturally or as a result of damage, “gaps” form in the blood-brain barrier, and endothelial cells are not able to immediately close this area, since the formation of tight junctions takes time. This means that endotheliocytes in this area must be temporarily replaced by some other type of cell. And it is the microglial cells that come to the rescue, restoring the barrier until the endothelial cells are fully restored. This was shown in an experiment by Dr. Nedergaard's team, when 10-20 minutes after damage to a mouse brain capillary by laser beams, microglial cells filled the damage. For this reason, one of the hypotheses by which scientists are trying to explain the occurrence of neurodegenerative diseases is the malfunction of microglial cells. For example, the role of BBB disorders is confirmed in the development of multiple sclerosis attacks: immune cells migrate in large numbers to brain tissues, triggering the synthesis of antibodies that attack myelin, as a result of which the myelin sheath of axons is destroyed.

The pathological permeability of the BBB also plays a role in the onset and course of epilepsy. It has long been known that epileptic seizures are associated with a transient violation of the integrity of the BBB. True, until recently it was believed that this is a consequence of epileptic seizures, and not the cause. But with the receipt of new research results, this point of view has gradually changed. For example, according to the laboratory of the University of Amsterdam, the frequency of seizures in rats increased according to the opening of the BBB. The more pronounced was the violation of the barrier, the more likely the animals developed the temporal form of epilepsy. These data also correlate with the results obtained in the Cleveland Clinic (USA) when conducting tests on pigs, as well as on the example of humans: in both cases, seizures occurred after the opening of the BBB, but never before.

Scientists are also engaged in the relationship between the functioning of the BBB and Alzheimer's disease. For example, it was possible to identify two BBB proteins that probably play a role in the development of this disease. One of these proteins, RAGE, mediates the penetration of beta-amyloid molecules from the blood into the brain tissue, and the other, LRP1, transports them out. If the balance in the activity of these proteins is disturbed, characteristic amyloid plaques form. Although the application of this knowledge to therapy is only in the future, there are promising results: in a mouse model, it is possible to prevent the deposition of beta-amyloid by blocking the gene responsible for the synthesis of RAGE proteins in endothelial cells. It is possible that drugs that block the RAGE protein, which are already being developed, will have a similar effect in humans.

In addition to the problem of restoring the integrity of the BBB, another problem associated with its functioning is, as already mentioned, the transfer of drugs through the barrier between the bloodstream and the brain. The metabolism carried out through the BBB is subject to certain rules. In order to cross the barrier, a substance must either not exceed 500 kDa in mass (most antidepressant, antipsychotic and hypnotic drugs correspond to this parameter), or use natural mechanisms for the BBB transition, as does, for example, L-dopa, which is a precursor of dopamine and is transported through the BBB with a special carrier; or the substance must be lipophilic, since the affinity for fat-containing compounds ensures passage through the basement membrane. 98% of drugs do not fulfill even one of these three criteria, which means they cannot realize their pharmacological effect in the brain. The technologists unsuccessfully try to implement the above criteria during the development of dosage forms. Although fat-soluble forms easily penetrate the BBB, some of them are immediately excreted back into the bloodstream, others get stuck in the thickness of the membrane without reaching the final goal. In addition, lipophilicity is not a selective property of BBB membranes, and therefore such drugs can practically indiscriminately pass through the membranes of cells of any organs of the body, which is also, of course, a minus.

Ways to overcome the blood-brain barrier

The real breakthrough was the use of a surgical method to overcome the BBB, developed by a neurosurgeon from the University of Texas at Dallas. The method consists in the introduction of a hyperosmolar solution of mannitol into the artery leading to the brain. Due to osmolar action (the amount of a dissolved substance in a hyperosmolar solution of mannitol exceeds that inside endothelial cells, therefore, according to the law of osmosis, water moves towards a higher concentration of a dissolved substance), endotheliocytes lose water, shrink, tight contacts between them break, and a temporary defect is formed in BBB, which allows drugs injected into the same artery to pass into the brain tissue. Such a temporary opening of the BBB lasts from 40 minutes to 2 hours, after which the restoration of endotheliocytes and contacts between them occurs. This technique is life-saving for patients diagnosed with brain tumors, when the tumor responds well to chemotherapy, but only if the chemotherapy drug reaches the brain tissue and accumulates in the area of ​​infiltration of malignant cells in the required concentration.

This is just one of the ways to overcome the BBB. There are no less interesting ways, they are overviewed in the diagram below. I hope that after reading them, someone will want to delve into the topic in order to understand the possibilities of manipulating the blood-brain barrier and how exactly control over its functioning can help in the fight against various diseases.

Sources:
Engaging neuroscience to advance translational research in brain barrier biology - the full text of the article, excerpts from which were used in the post, about the participation of the BBB in the development of various diseases and ways to overcome it
J. Interlandi Wege durch die Blut-Hirn-Schranke, Spektrum der Wissenschaft, spezielle Auflage, 2/2016
Blood-Brain Barrier Opening - an overview of ways to open the BBB
Endothelial progenitor cells in the development and restoration of the cerebral endothelium - on the formation and modeling of the BBB

M.I. Savelyeva, E.A. Sokova

4.1. OVERVIEW OF DRUG DISTRIBUTION AND RELATIONSHIP WITH PLASMA PROTEINS

After gaining access to the systemic circulation through one of the routes of administration, xenobiotics are distributed in organs and tissues. A series of physical and physiological processes that occur simultaneously depend on the physicochemical properties of drugs and thus form different ways of distributing them in the body. Examples of physical processes are the simple dilution or dissolution of a drug in intracellular and extracellular fluids. Examples of physiological processes are plasma protein binding, accessibility of tissue channels, and drug penetration through various body barriers. The following factors may influence drug distribution:

blood flow;

The degree of binding to plasma proteins;

Physico-chemical features of preparations;

The degree (depth) and extent of drug penetration through physiological barriers;

The degree of elimination, due to which the drug is continuously removed from the body, and which competes with the phenomenon of distribution.

blood flow

blood flow- the volume of blood reaching a certain area in the body per unit of time. The ratio of volume / time and the amount of blood flow in different areas of the body differ. Total blood flow is 5000 ml/min and corresponds to cardiac capacity at rest. Cardiac capacity(minute volume of the heart) - the volume of blood pumped by the heart in one minute. In addition to cardiac output, there is such an important factor as the volume of blood in various parts of the systemic circulation. On average, the heart contains 7% of the total blood volume, the pulmonary system - 9%, arteries - 13%, arterioles and capillaries - 7%, and veins, venules and the entire venous system - the remaining 64%. Through the permeable walls of the capillaries, the exchange of drugs, nutrients and other substances with the interstitial fluid of organs / tissues occurs, after which the capillaries merge with venules, which gradually converge into large veins. As a result of transcapillary exchange, the drug is transported through the capillary wall into the tissue due to the difference in pressure (osmotic and hydrostatic pressure) between the inner and outer parts of the capillary or concentration gradient. The delivery of a xenobiotic to certain areas of the body depends on the rate of blood flow and the site of administration of the drug.

Blood flow is the main factor in the distribution of drugs in the human body, while the concentration gradient plays an insignificant role (or does not participate at all) in the mass delivery of the drug to organs and tissues. The blood flow significantly determines the rate of drug delivery to a certain area of ​​the body and reflects the relative growth rate of the xenobiotic concentration, at which an equilibrium is established between the organ/tissue and blood. The amount of drugs stored or distributed in the tissue depends on the size of the tissue and the physicochemical characteristics of the drug, the separation factor between the organ/tissue and blood.

A phenomenon that restricts blood flow(perfusion-limited distribution; transmission-limited phenomenon; permeability-limited distribution) - dependence of transcapillary exchange

and storage of the drug in the tissue from the physico-chemical characteristics of the drug.

Perfusion-limited transcapillary drug exchange

In order to differentiate between the two types of distribution, suppose that the capillary is a hollow cylinder with length L and radius r , in which blood flows at a speed ν in a positive direction X. The concentration of the drug in the tissue around the capillary - c cloth, and the concentration in the blood C blood. The drug passes through

capillary membrane due to the concentration gradient between blood and tissue. Consider a section or segment of a direction between X and x+dx, where is the difference in drug flow mass between the start and end of the segment dx equal to the mass flow through the capillary wall. We write the equality in the following form (4-1):

then equation (4-4) will take the form:

The mass flow through the capillary wall into the tissue is j fabric in expression

the net mass of the flow leaving the capillary at a certain length L(4-6):

Having made the transformation of equation (4-6) using equation (4-5), we obtain:

Let's find the capillary clearance:

Capillary clearance is the volume of blood from which a xenobiotic spreads into the tissue per unit time. Extraction ratio (extraction ratio) distribution:

Equation (4-9) can be converted:

Equation (4-10) shows that the recovery ratio expresses the balancing fraction between the concentration of the drug in the tissue, arterial capillaries, on the venous side of the capillaries. Comparing equations (4-5) and (4-10) we find that capillary clearance is equal to blood flow times the recovery ratio.

Consider a diffusion-limited distribution (or a permeability-limited distribution). At Q>PS or C artery≈ C vein

the drug is slightly lipophilic and the recovery ratio is less than one, and the distribution of the drug is limited by very rapid diffusion through the capillary membrane. Let us determine the mass transfer of the drug into the tissue:

The driving force for the transfer of the xenobiotic to the tissue is the concentration gradient. Consider a perfusion-limited distribution (or a blood-flow-limited distribution). At Q or C vein≈ C tissue drug concentration in tissue is in equilibrium

with the concentration of the drug on the venous side of the capillaries, and the drug is very lipophilic. The recovery ratio is equal to or close to unity, and therefore the absorption of the drug by the tissue is thermodynamically much more favorable than its presence in the blood, and the distribution is limited only by the rate of delivery of the drug to the tissue. Once the drug reaches the tissue, it is immediately absorbed. Let us determine the mass transfer of the drug into the tissue:

Binding of drugs to proteins

The binding of drugs to plasma proteins significantly affects their distribution in the body. Small drug molecules associated with proteins can easily penetrate barriers. In this regard, the distribution of the protein-bound xenobiotic will differ from the distribution of the unbound drug. The interaction of drug functional groups with membrane or intracellular receptors can be short. Protein binding not only affects the distribution of the drug in the body, but also affects the therapeutic outcome. Therefore, it is necessary to use the concentration of free drug in plasma for pharmacokinetic analysis, regulation of dosing regimen and optimal therapeutic effect.

The protein binding of drugs used together with other drugs may differ from drugs taken alone. Changes in protein binding are the result of the substitution of one drug for another in combination with plasma proteins. Similar substitution can also occur at the cellular level with other proteins and tissue enzymes. Substitution causes an increase in the free fraction of the drug in plasma and its accumulation at receptor sites in proportion to the concentration of the drug. It is important to adjust the dosing regimen of drugs when they are co-administered. Altering the protein binding of drugs is an important issue, especially for drugs with a narrow therapeutic window.

Plasma proteins that are involved in the interaction between the protein and the drug

Albumen- the main protein of plasma and tissues responsible for binding to drugs, which is synthesized exclusively by hepatocytes of the liver. The molecular weight of albumin is 69,000 Da; half-life is approximately 17-18 days. The protein is mainly distributed in the vascular system and, despite the large molecular size, can additionally be distributed in the extravascular zone. Albumin has negatively and positively charged regions. The drug interacts with albumin due to hydrogen bonds (hydrophobic binding) and van der Waals forces. Some factors that have a significant effect on the body, such as pregnancy, surgery, age, ethnic and racial differences, can affect the interaction of drugs with albumin. The kidneys do not filter albumin, and therefore drugs that bind to albumin are also not filtered. The degree of binding affects not only the distribution of the drug, but also the renal elimination and metabolism of the drug. Only free drug can be taken up by liver hepatocytes. Therefore, the greater the percentage of protein-bound drug, the lower the hepatic absorption and rate of drug metabolism. As mentioned earlier, the degree of drug binding to plasma albumin can also be significantly altered by the administration of other drugs that replace the main drug, resulting in an increase in free drug plasma concentration.

Other plasma proteins are fibrinogen, globulins (γ- and β 1 -globulin - transferrin), ceruloplasmin and α- and β-lipoproteins. Fibrinogen and its polymerized form fibrin are involved in the formation of blood clots. Globulins, namely, γ-globulins, are antibodies that interact with certain antigens. Transferrin is involved in the transport of iron, ceruloplasmin is involved in the transfer of copper, and α- and β-lipoproteins are messengers of fat-soluble components.

Estimation of protein binding parameters

The binding of drugs to plasma proteins is usually determined in vitro under physiological conditions of pH and body temperature. Methods of determination - equilibrium dialysis, dynamic dialysis, ultrafiltration, gel filtration chromatography, ultracentri-

fusion, microdialysis, and several new and rapidly developing methodologies for high throughput experiments. The goal is to evaluate the concentration of the free drug in equilibrium with the protein-drug complex. The chosen methodology and experimental conditions should be such that complex stability and equilibrium are maintained and free drug concentration is not overestimated due to too rapid degradation of the complex during measurement. After that, most of the drug-protein complexes are held together by weak chemical interaction, electrostatic type (van der Waals force), and hydrogen bonding tends to separate at elevated temperature, osmotic pressure and non-physiological pH.

The usual method of dialysis of plasma, or a protein solution with a pH of 7.2-7.4, is not effective at various concentrations of the drug. The mixture after dialysis becomes isotonic with NaCl [at 37°C through the dialysis membrane with molecular contractions of approximately 12,000-14,000 Da against an equivalent volume of phosphate buffers (≈67, pH 7.2-7.4)]. The dialysis membrane in the form of a bag containing the protein and the drug is placed in a buffer solution. The prefabricated modified version of the bag has two compartments that are separated by a dialysis membrane. Equilibrium of the free drug passing through the membrane is usually reached in about 2-3 hours. The concentration of the free drug is measured on the side of the buffer, i. outside the bag or compartment, separated by a membrane, which should be equal to the concentration of the free drug inside the bag or compartment; the concentration of the free drug in the bag must be in equilibrium with the drug attached to the protein. In dialysis, an albumin solution or a pure plasma sample containing albumin is used. The drug binding parameters are the free fraction or associated constant, which can be determined using the law of mass action:

where K a- association constant; C D- concentration of free drug in molecules; C Pr- concentration of protein with free attachment sites; CDP- concentration of the drug-protein complex; k 1 and k 2 - level constants of direct and reverse reactions,

respectively. Reciprocal bonds are permanent and are known as dissociation constants (4-14):

The value of the associated constant K a represents the degree of protein binding of the drug. Drugs that bind extensively to plasma proteins usually have a large association constant. Based on equation (4-14), the concentration of the drug-protein complex can be determined:

If the concentration of total protein (C) at the beginning of the experiment in the tube is known, and the concentration of the drug-protein complex (C) is experimentally estimated, then the concentration of free protein can be determined. (C Pr), in equilibrium with the complex:

Replacing Equation (4-15) with Equation (4-16) for C Pr leads:

Let's transform the equation (4-18):

When establishing CDP/ With PT(number of moles of attached drug per mole of protein for equilibrium) is equal to r, i.e. r = CDP/ C PT , then equation (4-19) will change:

When multiplying equation (4-20) by n(n is the number of attachment sites per mole of protein), we obtain the Langmuir equation:

Langmuir equation (4-21) and graph r against C D results in a hyperbolic isotherm (Figure 4-1). Simplify equation (4-21). Let's take Langmoor's equation (4-21) in reverse form. The double reciprocal equation (4-22) shows that the plot of 1/r vs 1/C D is linear with a slope equal to 1/nK a and the intersection point along the y-axis 1/ n(Figure 4-2):

Rice. 4-1. Langmoor isotherm. On the y-axis - the number of moles of the drug attached per mole of protein; on the abscissa axis - the concentration of the free drug

By transforming equation (4-21), two versions of the linear equation can be obtained:

The Scatchard plot describes the relationship between r/C D and r as a straight line with a slope equal to the associative constant K a(Fig. 4-3). Intersection point with axis X is equal to the number of connected sections n, the point of intersection with the axis at is equal to pK a ..

In addition, equation (4-21) can be rearranged to provide a straight-line relationship in terms of free and bound drug concentrations:

Rice. 4-2. Dual reciprocal Klotz plot

Equation (4-21) shows the relationship between reciprocal r(moles of bound drug per mole of protein) and C D

Rice. 4-3. Line plot of CDP/CD (ratio of bound sites to free drug) vs. CDP (concentration of bound drug)

(concentration of free drug). Intersection point with axis at is the reciprocal of the number of bound sites per mole of protein, and the ratio of the slope to the point of intersection at- associative equilibrium constant.

Schedule c dp / c d against c dp -

a line with a slope equal to -K a and an intersection point along the y-axis nKC PT . This equation is used when the protein concentration is unknown. The K a estimate is based on the drug concentration measured in the buffer compartment. Determination of the protein-bound drug is based on the assessment of the free fraction

The Scatchard plot (Figure 4-4) is a straight line (for one type of connected parcels).

Langmoor's equation for several types of connected parcels:

where n 1 and K a1 - parameters of the same type of identically connected sections; n 2 and K a2 - parameters of the second type of identically connected sections, and so on. For example, an aspartic or glutamic acid residue, -COO - , may be one type of attachment site, and -S - a cysteine ​​residue or -NH 2± - histidine residue - the second type of attachment site. When a drug has an affinity for two types of binding sites, then the plot

Rice. 4-4. Scatchard Plot

Scatchard r/D against r represents not a straight line, but a curve (Fig. 4-5). Extrapolating the start and end line segments of the curve results in straight lines that fit the equations:

Rice. 4-5. Scatchard Plot

The Scatchard plot represents the protein binding of two different classes of regions. The curve represents the first two elements

equations (4-26), which are defined as straight lines - continuations of the linear segments of the initial and final parts of the curve. Line 1 represents high affinity (affinity) and low capacity of the binding sites, and line 2 - low affinity and high capacity of the binding sites.

When the affinity and capacity of two bonding sites are different, then the line with the larger intersection point at and smaller intersection point X defines a high affinity and low site capacity, while a line with a smaller intersection point at and larger point of intersection X determines the low affinity and high capacity of the binding sites.

4.2. PENETRATION OF DRUGS THROUGH HISTOHEMATIC BARRIERS

Most drugs after absorption and entering the blood are distributed unevenly to different organs and tissues and it is not always possible to achieve the desired concentration of the drug in the target organ. Significant influence on the nature of the distribution of drugs have histohematic barriers that occur in the way of their distribution. In 1929 Academician L.S. Stern for the first time at the International Congress of Physiology in Boston reported on the existence of

the body of physiological protective and regulating histohematic barriers (HGB). It has been proven that the physiological histohematic barrier is a complex of the most complex physiological processes occurring between blood and tissue fluid. GGB regulates the flow of substances necessary for their activity from the blood into organs and tissues and the timely excretion of the end products of cellular metabolism, ensures the constancy of the optimal composition of the tissue (extracellular) fluid. At the same time, HGB prevents the entry of foreign substances from the blood into organs and tissues. A feature of GGB is its selective permeability, i.e. the ability to pass some substances and retain others. Most researchers recognize the existence of specialized physiological HGB, which are important for the normal functioning of individual organs and anatomical structures. These include: hematoencephalic (between the blood and the central nervous system), hematoophthalmic (between blood and intraocular fluid), hematolabyrinthic (between blood and labyrinth endolymph), barrier between blood and gonads (hematoovarian, hematotesticular). The placenta also has "barrier" properties that protect the developing fetus. The main structural elements of histohematic barriers are the endothelium of blood vessels, the basement membrane, which includes a large number of neutral mucopolysaccharides, the main amorphous substance, fibers, etc. The structure of HGB is determined to a large extent by the structural features of the organ and varies depending on the morphological and physiological characteristics of the organ and tissue.

Penetration of drugs across the blood-brain barrier

The main interfaces between the CNS and the peripheral circulation are the blood-brain barrier (BBB) ​​and the hematoliquor barriers. The surface area of ​​the BBB is approximately 20 m 2 , and thousands of times greater than the area of ​​the hematoliquor barrier, so the BBB is the main barrier between the CNS and systemic circulation. The presence in the brain structures of the BBB, which separates the circulation from the interstitial space and prevents the entry of a number of polar compounds directly into the brain parenchyma, determines the characteristics of the drug therapy.

PII neurological diseases. The permeability of the BBB is determined by the endothelial cells of the brain capillaries, which have epithelial-like, highly resistant tight junctions, which excludes paracellular pathways for fluctuations of substances through the BBB, and the penetration of drugs into the brain depends on transcellular transport. The glial elements that line the outer surface of the endothelium and, obviously, play the role of an additional lipid membrane, are also of some importance. Lipophilic drugs mostly easily diffuse through the BBB, in contrast to hydrophilic drugs, the passive transport of which is limited by highly resistant tight junctions of endothelial cells. The coefficient of solubility in fats is of decisive importance in penetration through the blood-brain barrier. A typical example is general anesthetics - the speed of their narcotic effect is directly proportional to the coefficient of solubility in fats. Carbon dioxide, oxygen and lipophilic substances (which include most anesthetics) easily pass through the BBB, while for most ions, proteins and large molecules (for example, mannitol) it is practically impermeable. There is practically no pinocytosis in the capillaries of the brain. There are other ways of penetration of compounds through the BBB, indirectly through the receptor, with the participation of specific carriers. It has been shown that specific receptors for some of the circulating plasma peptides and proteins are expressed in the brain capillary endothelium. The peptide receptor system of the BBB includes receptors for insulin, transferrin, lipoproteins, etc. The transport of large protein molecules is ensured by their active capture. It has been established that the penetration of drugs and compounds into the brain can be carried out by active transport with the participation of active “pumping in” and “pumping out” transport systems (Fig. 4.6). This makes it possible to control the selective transport of drugs through the BBB and limit their non-selective distribution. The discovery of "pumping" transporters - glycoprotein-P (MDR1), transporters of the family of proteins associated with multiple drug resistance (MRP), breast cancer resistance protein (BCRP) has made a significant contribution to understanding the transport of drugs through the BBB. P-glycoprotein has been shown to limit the transport of a number of substances into the brain. It is located on the apical part of endothelial cells and carries out the excretion of predominantly hydrophilic cations from the brain into the lumen of the vessels.

Rice. 4.6. Transporters involved in the transport of drugs through the BBB (Ho R.H., Kim R.B., 2005)

new drugs, for example, cytostatics, antiretroviral drugs, etc. The importance of P-glycoprotein in limiting the transport of drugs through the BBB can be demonstrated using the example of loperamide, which is a potential opioid drug by its mechanism of action on gastrointestinal tract receptors. However, there are no effects on the central nervous system (euphoria, respiratory depression), since loperamide, being a substrate of P-glycoprotein, does not penetrate the central nervous system. In the presence of an inhibitor mdrl quinidine, the central effects of loperamide increase. Transporters from the MRP family are located either on the basal or apical part of endothelial cells. These transporters remove glucuronated, sulfated or glutathione drug conjugates. In the experiment, it was found that the multidrug resistance protein MRP2 is involved in the functioning of the BBB and limits the activity of antiepileptic drugs.

Some members of the organic anion transporter (OAT3) family are expressed in brain capillary endotheliocytes, which also play an important role in the distribution of a number of drugs in the CNS. Drug substrates of these transporters are, for example, fexofenadine, indomethacin. Expression of isoforms of polypeptides transporting organic anions (OATP1A2) in the BBB is important for the penetration of drugs into the brain. However, it is believed that the expression of "pumping out" transporters (MDR1, MRP, BCRP) is the reason for the limited pharmacological access of drugs to the brain and other tissues, when the concentration may be lower than that required to achieve the desired effect. Significant

the number of mitochondria in the endothelium of the brain capillaries indicates the ability to maintain energy-dependent and metabolic processes available for active transport of drugs through the BBB. In the endothelial cells of the brain capillaries, enzymes were found that are capable of oxidizing, conjugating compounds to protect the cells themselves and, accordingly, the brain from possible toxic effects. Thus, there are at least two reasons that limit the flow of drugs into the CNS. First, these are the structural features of the BBB. Secondly, the BBB includes an active metabolic system of enzymes and a system of “pumping out” transporters, which forms a biochemical barrier for most xenobiotics. This combination of physical and biochemical properties of the BBB endothelium prevents more than 98% of potential neurotropic drugs from reaching the brain.

Factors affecting drug transport to the brain

Pharmacodynamic effects of endogenous substances and diseases affect the functions of the BBB, leading to changes in drug transport to the brain. Various pathological conditions can disrupt the permeability of the blood-tissue barriers, for example, with meningoencephalitis, the permeability of the blood-brain barrier sharply increases, which causes various kinds of violations of the integrity of the surrounding tissues. An increase in BBB permeability is observed in multiple sclerosis, Alzheimer's disease, dementia in HIV-infected patients, encephalitis and meningitis, high blood pressure, mental disorders. A significant number of neurotransmitters, cytokines, chemokines, peripheral hormones, exposure to active forms of O 2 can change the functions and permeability of the BBB. For example, histamine, acting on H 2 receptors facing the lumen of endothelial cells, increases the permeability of the barrier for low molecular weight substances, which is associated with a violation of tight junctions between epithelial cells. The permeability of histohematic barriers can be changed directionally, which is used in the clinic (for example, to increase the effectiveness of chemotherapeutic drugs). A decrease in the barrier functions of the BBB due to a violation of the structure of tight junctions is used to deliver drugs to the brain, for example, the use of mannitol, urea. Osmotic "opening" of the BBB makes it possible to provide patients with primary lymphoma

brain and glioblastoma increased transport to the brain for a limited period of time of cytostatics (eg, methotrexate, procarbazine). A more gentle method of influencing the BBB is its "biochemical" opening, based on the ability of prostaglandins, inflammatory mediators, to increase the porosity of cerebral vessels. A fundamentally different possibility of increasing drug delivery to the brain is the use of prodrugs. The presence in the brain of specific transport systems for the delivery of its life-support components (amino acids, glucose, amines, peptides) allows them to be used for the purpose of directed transport of hydrophilic drugs to the brain. The search for means for the transport of polar compounds, characterized by low permeability through the BBB, is constantly expanding. Promising in this regard may be the creation of transport systems based on natural cationic proteins - histones. It is believed that progress in the field of creating new effective drugs can be achieved by improving the methods for selecting promising chemical compounds and optimizing delivery routes for peptide and protein drugs, as well as genetic material. Studies have shown that certain nanoparticles are able to transport compounds of a peptide structure (delargin), hydrophilic substances (tubocurarine), drugs pumped out of the brain by P-glycoprotein (loperamide, doxorubicin) to the brain. One of the promising directions in the creation of drugs that penetrate the histagematic barriers is the development of nanospheres based on modified silicon dioxide, capable of providing effective delivery of genetic material to target cells.

Transport of drugs across the hematoplacental barrier

The earlier assumption that the placental barrier provides natural protection of the fetus from the effects of exogenous substances, including drugs, is true only to a limited extent. The human placenta is a complex transport system that acts as a semi-permeable barrier separating the mother from the fetus. During pregnancy, the placenta regulates the exchange of substances, gases, endogenous and exogenous molecules, including drugs, in the fetal-mother complex. A number of studies have shown that the placenta morphologically and functionally plays the role of an organ responsible for the transport of drugs.

The human placenta consists of fetal tissues (chorionic plate and chorionic villus) and maternal (decidua). The decidual septa divide the organ into 20-40 cotyledons, which represent the structural and functional vascular units of the placenta. Each cotyledon is represented by a villous tree, consisting of the endothelium of the fetal capillaries, villous stroma and trophoblastic layer, washed by the mother's blood, located in the intervillous space. The outer layer of each villous tree is formed by a multinucleated syncytiotrophoblast. The polarized syncytiotrophoblastic layer, consisting of a microvillous apical membrane facing the mother's blood, and a basal (fetal) membrane is a hemoplacental barrier for the transplacental transport of most substances. During the course of pregnancy, the thickness of the placental barrier decreases, mainly due to the disappearance of the cytotrophoblastic layer.

The transport function of the placenta is determined mainly by the placental membrane (hematoplacental barrier), having a thickness of about 0.025 mm, which separates the circulatory system of the mother and the circulatory system of the fetus.

Under physiological and pathological conditions, placental metabolism should be considered as an active function of the placental membrane, which selectively controls the passage of xenobiotics through it. The transfer of drugs across the placenta can be considered based on the study of the same mechanisms that function when substances pass through other biological membranes.

It is well known that the placenta performs numerous functions such as gas exchange, transport of nutrients and waste products, production of hormones, functioning as an active endocrine organ vital for a successful pregnancy. Nutrients such as glucose, amino acids and vitamins pass through the placenta by special transport mechanisms that occur in the maternal apical membrane and fetal basement membrane of syncytiotrophoblast. At the same time, the removal of metabolic products from the fetal circulatory system through the placenta into the mother's circulatory system also occurs through special transport mechanisms. For some compounds, the placenta serves as a protective barrier for the developing fetus, preventing the entry of destructive

personal xenobiotics from mother to fetus, while for others it facilitates their passage both to the fetus and from the fetal compartment.

Transport of drugs in the placenta

Five mechanisms of transplacental exchange are known: passive diffusion, facilitated diffusion, active transport, phagocytosis and pinocytosis. The last two mechanisms are of relative importance in the transport of drugs in the placenta, and most drugs are characterized by active transport.

Passive diffusion is the dominant form of metabolism in the placenta, which allows the molecule to move down the concentration gradient. The amount of drugs moving through the placenta by passive diffusion in any period of time depends on its concentration in the mother's blood plasma, its physicochemical properties and the properties of the placenta, which determine how quickly this happens.

The process of this diffusion is governed by Fick's law.

However, the rate of passive diffusion is so low that the equilibrium concentration in the blood of the mother and fetus is not established.

The placenta is like a bilayer lipid membrane and thus only the non-protein bound fraction of drugs can freely diffuse across it.

Passive diffusion is characteristic of low-molecular, fat-soluble, predominantly non-ionized forms of drugs. Lipophilic substances in a non-ionized form easily diffuse through the placenta into the blood of the fetus (antipyrine, thiopental). The rate of transfer through the placenta depends mainly on the concentration of the non-ionized form of a particular drug at a given blood pH, lipid solubility, and the size of the molecules. Drugs with a molecular weight > 500 Da often do not completely cross the placenta, and drugs with a molecular weight > 1000 Da penetrate the placental membrane more slowly. For example, various heparins (3000-15000 Da) do not cross the placenta due to their relatively high molecular weight. Most drugs have a molecular weight > 500 Da, so the size of the molecule rarely limits their passage through the placenta.

Basically, drugs are weak acids or bases and their dissociation occurs at a physiological pH value. In the ionized form, drugs usually cannot pass through the lipid membrane.

placenta. The difference between fetal and maternal pH affects the fetal/mother concentration ratio for the free drug fraction. Under normal conditions, the fetal pH is practically the same as the maternal pH. However, under certain conditions, the pH value of the fetus can significantly decrease, resulting in a decrease in the transport of essential drugs from the fetus to the maternal compartment. For example, a study of placental transfer of lidocaine by the MEGX test showed that the concentration of lidocaine in the fetus is higher than in the mother during childbirth, which may cause undesirable effects in the fetus or newborn.

Facilitated diffusion

This transport mechanism is typical for a small number of drugs. Often this mechanism complements passive diffusion, for example in the case of ganciclovir. Facilitated diffusion does not require energy, a carrier substance is needed. Usually, the result of this type of transport of drugs through the placenta is the same concentration in the blood plasma of the mother and fetus. This transport mechanism is specific mainly for endogenous substrates (eg hormones, nucleic acids).

Active drug transport

Studies of the molecular mechanisms of active drug transport across the placental membrane have shown its important role in the functioning of the hematoplacental barrier. This transport mechanism is typical for drugs that have a structural similarity with endogenous substances. In this case, the process of transfer of substances depends not only on the size of the molecule, but also on the presence of a carrier substance (transporter).

Active transport of drugs across the placental membrane by a protein pump requires energy expenditure, usually due to ATP hydrolysis or the energy of the transmembrane electrochemical gradient of Na+, Cl+ or H+ cations. All active transporters can work against the concentration gradient, but can also become neutral.

Active drug transporters are located either on the maternal part of the apical membrane or on the fetal part of the basement membrane, where they transport drugs to the syncytiotrophoblast

or from it. The placenta contains transporters that facilitate the movement of substrates from the placenta into the maternal or fetal circulation (“pumping”), as well as transporters that move substrates both into and out of the placenta, thus facilitating the transport of xenobiotics into and out of the fetal and maternal compartments (“ pumping in"/"pumping out"). There are transporters that regulate the movement of substrates only into the placenta ("pumping").

Research of the last decade has been devoted to the study of "pumping transporters" as an "active component" of the placental "barrier". It is P-glycoprotein (MDR1), a family of multi-drug resistance-associated proteins (MRP) and breast cancer resistance protein (BCRP). The discovery of these transporters has made a significant contribution to the understanding of transplacental pharmacokinetics.

Glycoprotein-P, a transmembrane glycoprotein encoded by the human multidrug resistance gene MDR1, is expressed on the maternal side of the placental membrane of syncytiotrophoblast, where it actively removes lipophilic drugs from the fetal compartment due to the energy of ATP hydrolysis. Glycoprotein-P is an "exhaust" transporter, actively removing xenobiotics from the fetal circulation into the mother's circulation. P-glycoprotein has a broad substrate spectrum, carrying lipophilic drugs, neutral and charged cations that belong to various pharmacological groups, including antimicrobials (eg, rifampicin), antivirals (eg, HIV protease inhibitors), antiarrhythmic drugs (eg, verapamil) , antineoplastic (for example, vincristine).

In the apical membrane of syncytiotrophoblast, the expression of three types of "pumping" transporters from the MRP family (MRP1-MRP3), which are involved in the transport of many drug substrates and their metabolites, was revealed: metatrexate, vincristine, vinblastine, cisplatin, antiviral drugs, paracetamol, ampicillin, etc.

High activity of the ATP-dependent breast cancer resistance protein (BCRP) was found in the placenta. BCRP can activate the resistance of tumor cells to anticancer drugs - topotecan, doxorubicin, etc. It has been shown that

placental BCRP limits the transport of topotecan and mitoxantrone to the fetus in pregnant mice.

Organic cation transporters

The two organic cation transporter (OCT2) is expressed in the basement membrane of syncytiotrophoblast and transports carnitine across the placenta from the maternal circulation to the fetal circulation. Drug substrates for placental OCT2 are methamphetamine, quinidine, verapamil, and pyrilamine, which compete with carnitine, limiting its passage through the placenta.

Monocarboxylate and dicarboxylate transporters

Monocarboxylates (lactate) and dicarboxylates (succinate) are actively transported in the placenta. Monocarboxylate transporters (MCTs) and dicarboxylate transporters (NaDC3) are expressed in the placental apical membrane, although MCTs may also be present in the basement membrane. These transporters are driven by an electrochemical gradient; MCTs are associated with the movement of H + cations, and NaDC3 - with Na + . However, information on the potential effect of these transporters on the movement of drugs across the placenta is scarce. Thus, valproic acid, despite the obvious risk of toxic effects on the fetus, including teratogenicity, is often used to treat epilepsy during pregnancy. At physiological pH, valproic acid easily crosses the placenta and the fetal/mother concentration ratio is 1.71. Studies by a number of authors have shown that there is an active transport system for valproic acid. This transport system includes H + cations - bound MCTs, which cause a high rate of movement of valproic acid to the fetus through the placental barrier. Although valproic acid competes with lactate, it turned out that it is also a substrate for other transporters.

Thus, for some compounds, the placenta serves as a protective barrier for the developing fetus, preventing the entry of various xenobiotics from mother to fetus, while for others it facilitates their passage both to the fetus and from the fetal compartment, generally functioning as a xenobiotic detoxification system. . The leading role in the process of active trans-

The port of the drug through the placenta is carried out by placental transporters with substrate specificity.

At present, it is quite clear that understanding and knowledge of the role of various transporters in the movement of drugs across the hematoplacental barrier is necessary to assess the likely effects of drugs on the fetus, as well as to assess the benefit / risk ratio for the mother and fetus during pharmacotherapy during pregnancy.

Drug transport across the blood-ophthalmic barrier

The hematoophthalmic barrier (HOB) performs a barrier function in relation to the transparent media of the eye, regulates the composition of the intraocular fluid, ensuring the selective supply of essential nutrients to the lens and cornea. Clinical studies have made it possible to clarify and expand the concept of the hemato-ophthalmic barrier, including the histagematical system, as well as to talk about the existence of three of its components in normal and pathological conditions: iridociliary, chorioretinal and papillary (Table 4.1.).

Table 4.1. Hematoophthalmic barrier

Blood capillaries in the eye do not directly come into contact with cells and tissues. The entire complex exchange between capillaries and cells occurs through the interstitial fluid at the ultrastructural level and is characterized as mechanisms of capillary, cellular and membrane permeability.

Drug transport across the blood-testicular barrier

The normal function of spermatogenic cells is possible only due to the presence of a special, selectively permeable hematotesticular barrier (HTB) between the blood and the contents of the seminiferous tubules. GTP is formed by capillary endothelial cells, basement membrane, seminiferous tubule proper, cytoplasm of Sertoli cells, interstitial tissue and tunica albuginea. Lipophilic drugs penetrate through the GTB by diffusion. Recent studies have shown that the penetration of drugs and compounds into the testicles can be carried out by active transport with the participation of glycoprotein-P (MDR1), transporters of the family of proteins associated with multiple drug resistance (MRP1, MRP2), breast cancer resistance protein BCRP (ABCG2 ), which perform an efflux role in the testicles for a number of drugs, including toxic ones (for example, cyclosporine).

Penetration of drugs through the ovarian hematofollicular barrier

The main structural elements of the ovarian hematofollicular barrier (HFB) are the theca cells of the maturing follicle, the follicular epithelium and its basement membrane, which determine its permeability and selective properties with respect to hydrophilic compounds. Currently, the role of P-glycoprotein (MDR1) has been shown as an active component of HFB, which performs a protective role, preventing the penetration of xenobiotics into the ovaries.

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