Interest in medical and biological chemistry of polymers caused by the need to have many areas of pharmacology and medicine.
The most promising in this regard is to provide a macromolecular systems for the controlled release of biologically active substances (BAS), mainly — different drugs.
All currently used drugs consist of two major components — BAA and components of the dosage form. Conventional low molecular weight drugs are not designed for a long stay in the body; they are rapidly eliminated or metabolized. Therefore, to achieve therapeutic effect resorted to multiple receiving medication, whereby its concentration in the body changes continuously — from large to very small at the beginning before re-admission. The role of the components of the dosage form is precisely to ensure favorable conditions for the operation of UAS. Unfortunately, until recently used dosage forms (tablets, ointments, capsules, solutions) are not optimal in terms of the functions they perform. They do not provide a long and uniform feed into the bloodstream ALS and practically do not contribute to their delivery to the target organ. In the body of ALS are distributed according to their physico-chemical properties, and only in rare cases, the affected organ misses about 10% of the injected often quite expensive and toxic to normal tissues, drugs.
In the 70s, based on synthetic polymers has been proposed and implemented a new approach, which provides a solution to this problem. One of the first such systems were ocular medicinal films based on a copolymer of acrylamide, N-vinylpyrrole-don and ethyl acrylate containing various ALS: antibiotics, sulfonamides, vitamins, anesthetics, etc. When introduced into the conjunctival cavity of the eye, such films are gradually dissolved, highlighting contained therein ALS. The dissolution rate can be controlled by varying the copolymer composition. As a result, the concentration of ALS therapeutic, e.g., sodium sulfapiridazina in the eye cavity was maintained for a half day, whereas if a drug solution is the no more than three hours.
A further task was to create systems that operate on the principle of feedback and can, in accordance with the requirements of the internal environment to ensure long-uniform supply of ALS in an active form in the target organ. The systems operate on the principle of feedback and to some degree simulate some functions of the body, it should be attributed to the most universal and physiologically optimal. Problems of designing such systems are considered in this review.
Polymeric hydrogels — a porous, highly swellable but not water soluble materials. Typically they are prepared by polymerizing water-soluble unsaturated compounds in the presence of a bifunctional crosslinking agent. The water content in the equilibrium swollen hydrogel is from 10 to gt; 95%.
The first mention of the use of hydrogels in medicine refers to the 60 th year when the hydrogels based poligidroksietil methacrylate were used to create soft contact lenses. In the 70s it was suggested to use hydrogels as inert matrix for the controlled release of ALS. Thus, contraceptives based on progesterone dispersed in a hydrogel upon single application provides the necessary effect for two months.
In such devices, ALS is not associated with a hydrogel chemical bonds, and the rate of discharge is determined by the nature and structure of the hydrogel. Adjusting the rate of release of ALS in the synthesis of the hydrogel is achieved by varying the nature of the water-soluble monomer or ratio between the monomer and crosslinking agent. The most simple and versatile device for the controlled release of ALS through a feedback mechanism is a rigid container with holes filled with the hydrogel, swollen in a solution of ALS. When conditions change, the hydrogel or reduces the degree of swelling or partially destroyed. As a result, the drug solution container appears that quite readily diffuses into the environment. It is obvious that the main task is to synthesize suitable for each case of the hydrogel, in which small changes in the parameters of the environment should take place first order phase transition, accompanied by a significant change in the volume.
One of the main parameters that change in pathological conditions is the temperature. Therefore, in many cases, for the controlled release of ALS should be used based hydrogel polymers having a lower critical temperature mixing (NCTS). Phase separation of such polymers in solution at higher temperatures due to the transition of the conformation of macromolecules in the swollen globule conformation compact coil. This transition is accompanied by a sharp decrease in the size of macromolecules, and in relation to the cross-linked polymer system — a sharp decrease in their volume. A large number of polymers, mainly N-substituted polyacrylamide, NCTS which lies in a temperature of 9 to 85C.
At temperatures below the LCST drug (e.g. analgesic) is practically not released. When the temperature rises above 32 ° C, the gel collapses and the drug release rate is increased. Reducing the temperature, for example, by the action of the released drug, leads to the fact that its rate of discharge falls to almost zero. This device can operate repeatedly until the complete exhaustion initially introduced into a drug. As the basis of the phase separation of the polymer solutions are hydrophobic interactions, the LCST polymer can be easily adjusted using the copolymerization reaction. Thus copolymerization with a hydrophobic monomer reduces CNTS, while the use of hydrophilic monomers — its rise.
Similar actions and pH-sensitive hydrogels, which are typically synthesized by polymerization of ionic monomers in the presence of a crosslinking agent. Available today set of monomers with functional groups of an acidic or basic nature allows widely vary the degree of swelling of hydrogels, i.e. the parameter that defines a possible change in volume of the hydrogel when changing the pH of the surrounding medium. Hydrogels containing acidic groups such as polyacrylic, polymethacrylic acid, styrene sulfonic acid and the like, are swellable in alkaline medium, but the acid collapse. At the same time, hydrogels with basic groups, in contrast, swell in acidic environments and dramatically reduce its volume with increasing pH. These hydrogels are crosslinked poliaminoetilmetakrilat, polyvinyl, etc. From the viewpoint of practical use there are two possible ways of using pH-sensitive hydrogels. In the first case, for example, in the treatment of wounds, the wounds on the surface of the dried powder core coated hydrogel comprising ALS. If the blood pH (7.4) was virtually no drug release from the hydrogel, based on copolymer of methyl methacrylate and dimetilaminoetilmetakrialata. In the pathology of which is accompanied by a decrease in pH to 5.0, the hydrogel swells, highlighting therein medicine. Such hydrogels are suitable for a single allocation of ALS.
If desired pulsatile secretion ALS, it is possible to use the device described above. In this case it was used the hydrogel based on polyacrylic acid, and as a model of ALS — kanamycin. At pH 7.4 the container wall kanamycin prevented allocation of swollen hydrogel, and below pH 5.0 the drug release rate increased significantly. In the reverse change in pH (restoring physiological values) system «off» release of the drug.
It is much more complex polymer systems have been used for the controlled release of insulin. Since the increase in the concentration of blood glucose is the principal stimulus of insulin secretion by the pancreas, with reference to the problem of treating diabetes promising are glucose -sensitive polymer systems.
The principle of creating such systems, first formulated in 1979, was based on the use of lectins — proteins capable of selectively and reversibly bind carbohydrates. The polymeric membrane permeable to glucose and insulin, but impermeable to the lectin, lectin complex is placed with pre-synthesized carbohydrate and insulin derivatives. Glucose appearing in the environment permeates through the membrane and are replaced by an insulin derivative of its complex with Con A. An experimental performance expressed the idea was tested by the authors on the example of insulin derivatives and maltose. The biological activity of these derivatives, estimated by their ability to lower the concentration of glucose in the blood of animals when administered subcutaneously, averaged 90% of the activity of native insulin. The synthesized derivatives form a sufficiently stable complexes with Con A, and their binding constants with Con A were close to the binding constants of glucose to Con A. The latter determines the main disadvantage of this system is the replacement of the glucose insulin derivative in its complex with Con A and the allocation of its environmental Wednesday occurred in virtually all glucose concentrations.
Was subsequently synthesized a large number of different carbohydrate derivatives of insulin, the biological activity of which was 82 — 88% of the activity of native insulin. By pharmacodynamic characteristics of these compounds do not differ from the native insulin and perfectly acceptable dependence of the rate of release of insulin on the glucose concentration. Animal experiments, although confirmed the results obtained in vitro, but identified a new problem: increase of reliability. The authors note that in some animals in 2 days after implantation developed a severe form of hypoglycemia caused by the destruction of the membrane and the release of huge amounts of insulin.
Therefore, further development work has been focused mainly on the improvement of the system to improve its reliability. For example, instead of the Con A solution was proposed to use the protein immobilized on Sepharose beads or Con A, carbodiimide-crosslinked, and as a material for the polymer membrane — cellulose acetate, polycarbonate, or polyvinylidene fluoride. The binding constants of glucose-insulin Rowan immobilized or crosslinked Con A had similar values in the 3 — 4 times higher than the binding constant of glucose lectin. These systems began to secrete insulin when glucose concentrations above 1 mg / mL, and the response time was determined by the nature of the membrane. Of the membranes based on cellulose acetate begins to secrete insulin at 2 hours after the addition of glucose, and from a polycarbonate membrane — half an hour. Even shorter induction period (10 minutes) was achieved for the membranes derived from polyvinylidene fluoride.
At the heart of the creation of another type glyukozochuvstvitelnyh hydrogel is the ability of glucose, like other diol to form complex compounds with borates. Initial hydrogel was obtained by treating a copolymer of N-vinylpyrrolidone with train akrilamidofenilbornoy acid solution of a polymer containing hydroxyl groups, for example polyvinyl alcohol solution. When glucose in the environment of the inter-relationships of macromolecular destroyed and replaced with a boron bonds glucose polymer; the degree of swelling of the hydrogel increased, and if the hydrogel was initially filled with insulin, its rate of release is also increased.
With regard to the controlled release of insulin quite promising is the use of membrane technology. The operating principle of one such system is shown in Fig. 3. Reservoir with a saturated solution of insulin closed hydrogel membrane amine based polymer, the amount of which was immobilized glucose oxidase — an enzyme which selectively catalyzes the oxidation of glucose to gluconic acid.
Glucose appearing in environment, diffused into the hydrogel, an enzyme glucose oxidized to form the acid and lowering the pH leads to ionization of the amine groups of the hydrogel. As a result of electrostatic repulsion of charged amino degree of swelling of the hydrogel increased insulin diffused through the membrane into the environment.
While these systems were further improved significantly, their main disadvantage insulin secretion under any, even very low concentrations and physiologically acceptable glucose persists. It is recalled that in vivo glucose threshold for insulin secretion is 80 — 100 mg / 100 ml of blood amaksimalnaya speed is achieved at a glucose concentration of 300 — 500 mg / 100 ml.
To solve this problem quite difficult (creating a system that responds to a specific concentration of glucose) has been suggested to use a fundamentally different polymer system — hydrogels based on copolymers of acrylamide (AA) with an unsaturated glucose derivative — N- (2-D-raio-goats) acrylamide ( GAA), crosslinked Con A.
Hydrogels are synthesized co-polymerization with AA GAA and subsequent mixing of aqueous solutions of the copolymers obtained with Con A. When studying the interaction of hydrogels with glucose solutions of various concentrations was found that there is a threshold concentration below which any apparent changes to the hydrogel occurs. When glucose concentrations above the threshold observed rapid transition of the hydrogel in a soluble state, — complex [Con A-GAA] decayed to form a complex [Con A-glucose] and water-soluble macromolecular copolymer GAA and AA. The higher the content GAA units in the copolymer, the higher the threshold concentration. Thus, for the maintenance units GAA 4.92; 10.14 and 14.45 mol.%, The threshold concentration of glucose at which the destruction of the hydrogels was 180, 380 and 565 mg / 100 mL, respectively. At the same time, the threshold concentration of glucose was even slightly higher than would be expected based on the values of the constants of [Con A glucose] (3.7 x 10 (3) L / mol) and [Kohn GAA] (1100 l / mol). Priudalenii glucose from the dialysis solution by complex [Con A-glucose] destroyed, which led to the reverse transition from a copolymer solution in the hydrogel. The newly formed hydrogel according to its physical and mechanical properties are not different from the original and could be reused.
From the standpoint of controlling insulin secretion when the glucose concentration specific interest are the mixtures of copolymers with different contents of GAA. Fig. We used copolymers containing 4.92 GAA, 10,14i 14.45 mol.%. The number of each type of hydrogel was 1 ml; each hydrogel was introduced 1.3 mg of insulin. The vessel was then placed in a beaker with 10 ml of buffer solution at pH 3.5. Diffusion of insulin from the vessel in the absence of glucose did not exceed 15 mg per 10 hours. By adding glucose to the buffer solution was observed its diffusion into the vessel with a hydrogel at a concentration above the threshold for each type of the hydrogel is its transition to a solution with the release of insulin contained therein. Such a system is easy to «tune» to allocate the required amount of insulin for a given glucose concentration, ie in this case we can talk about creating, even in the zero-order approximation, synthetic model of the pancreas that can effectively replace one of its functions, namely to allocate insulin response to rising levels of glucose.
Heat activated areas of transport ALS
This approach is most promising, mainly due to the very high selectivity of such a system. Its disadvantages include extreme complexity, since in each case it is necessary to find out a compound damaged organ can be used as a ligand, found or synthesized material capable of selectively reacting with the ligand, and then attach a substance to the drug without altering the activity of both components .
The second approach — a modification of drug substances, which are the building blocks of the lesion. For example, thrombolytic enzyme can be modified fibrinogen — a protein that is the basis of a blood clot. In this case, fibrinogen acts as a Trojan horse that brings the clot enzyme destroys it. This approach is relatively simple, but, unfortunately, has a special character.
The third group of methods comprises administering to a drug ferromagnetic substances, followed by application of a magnetic field on the diseased organ. However, this method is not widely spread, since the use of such drugs is a serious problem removing particulate matter from a ferromagnetic body.
Finally, a fourth approach is based on the use of polymeric systems that modify its properties at the ambient temperature, i.e. water-soluble polymers with LCST. Introducing into the circulatory system of such a polymer is immobilized on the drug and the target organ by heating to a temperature above the LCST, the polymer concentration can be expected and the associated drugs in the body.
The biological activity of the polymeric derivatives of ALS was studied on the example of two systems: trypsin and horseradish peroxidase immobilized at the N-IPAA. Immobilization of the enzyme in an amount of 1 — 5 molecules per polymer molecule with MR 150000 — 200000 did not alter this LCST polymer.
The results of studying the activity of immobilized enzymes at different temperatures are shown in Fig. 6. It can be seen that if the activity of native trypsin with increasing temperature in the range 10 — 40 ° C is continuously increased, then immobilized on N-IPAA trypsin in the region above the NCTS activity ceased depend on temperature. Reduction of immobilized trypsin activity relative to native temperature above LCST was reversible and when the solution is cooled trypsin activity was completely restored.
Qualification conformational behavior of macromolecules held by circular dichroism revealed that the degree of helicity of native and immobilized trypsin at temperatures below the LCST is practically identical (10,2 ± 1,3 and 9,6 ± 1,2%, respectively), and the activity of trypsin, polyacrylamide immobilized on a model that does not have the NCTS is identical to the native enzyme activity throughout the temperature range studied. Therefore, the decrease trypsin activity immobilized on a poly-N-IPAA, when the temperature rises above the LCST due to water repellent polymer carrier. Confirmation of this conclusion was immobilized on the behavior of the same polymer, horseradish peroxidase, which is more than trypsin sensitive to hydrophobic inactivation. The activity of this enzyme by heating above NCTS dropped sharply and is 5 — 10% of the activity of the native enzyme at the same temperatures.
Installation for the transport of the substance consists of two separately termostatiruemyhsosudov (A and B) 1.5 ml. Each receptacle formed a fibrin clot 0.5 ml and the system was filled with 10 ml of either native or immobilized trypsin (enzyme concentration was 0.03 mol). When using samples I or II Ado heating vessel 38 ° C did not alter the observed pattern; and using a sample with NCTS III 37,2 ° C there was a dramatic acceleration of the rate of hydrolysis of the clot in the vessel A — full dissolution time decreased to 1 hour. B In a vessel heated to a temperature of only 36 ° C, dissolution of the clot does not occur even in 15 hours.
It is obvious that the only reason for the observed effects are likely immobilized enzyme concentration in the vessel A through the provision of a separate phase of the polymer and associated trypsin. If the clot dissolution vessel A in the vessel was cooled to 36 ° C, and the vessel B was heated to 38 ° C, all the immobilized trypsin is passed into the vessel B and the clot in the vessel is dissolved within 1 hour.
A polymer hydrogel for directions TRANSPORT POLYPEPTIDES
Enough of the original is the idea of using polymer hydrogels modified with proteolytic enzymes and inhibitors biospecific ligands to improve stability ALS polypeptide to proteolytic enzymes for targeted transport of ALS in a certain area of the body. The essence of this idea for the case of oral administration, insulin is illustrated in Fig. A polymer hydrogel modified by ovomucoid (protein of duck eggs), which molecule consists of two parts: the polypeptide and polysaccharide. The protein portion of the molecule ovomucoid provide inhibition of proteolytic enzymes, and its polysaccharide portion interacting mechanism biospecific binding to lectins contained in the walls of the small intestine, to ensure adhesion of the hydrogel particles to the walls of the small intestine.
After injection of a solution of native insulin concentration in the blood is first increased, reaching a maximum of one and a half hours and then begins to decrease. A similar pattern was observed with oral administration of insulin as part of a modified hydrogel. As expected, the oral administration of the drug based on a hydrogel modified with ovomucoid remote polysaccharide portion, does not alter the concentration of insulin in the blood, although such a protective effect against ovomucoid derived proteolytic enzymes was maintained. This means that determines the role of insulin in the penetration through the mucosa does belong biospecific interactions lectin-polysaccharide.
Insulin enters the bloodstream in an active state, leading to reduction in blood glucose concentration. The efficacy of the orally administered drug insulin is not much inferior to subcutaneous insulin solution. The synthesized drugs are active with respect to animals with experimental diabetes. Established and versatile system has provided protection of polypeptide drugs (except insulin, glucagon were examined, calcitonin and growth hormone), from the aggressive action of environment (proteolytic enzymes of the digestive system) and a controlled release of the drug in the desired location, in this case in the small intestine.
Polymeric materials with higher hemocompatibility
The main problem in creating hemocompatible polymers consists in preventing thrombus formation on the surface of the polymer. Thrombus formation — a natural protective reaction of the organism to the introduction of a foreign body, the end result of which is the conversion of soluble blood protein fibrinogen to insoluble fibrin clot. Unfortunately, conducted over the past decades, numerous studies and did not answer the question, what properties should have a polymer to be hemocompatible.
The variation of these properties as physico-chemical parameters, chemical composition, degree of «smoothness» of the surface and wetting it with saline electrochemical characteristics, surface free energy, etc., did not lead to a decisive success.
More encouraging results have been obtained using polymers that have been surface modified ALS, have a positive effect on processes of thrombogenesis and / or fibrinolysis, such as heparin or fibrinolytic enzymes. Some of these materials have already found application in clinical practice for creating products for short-term contact with blood. However, it is clear that the action of the substances present in the blood of ALS should be immobilized inactivated, ie modification of the polymers are unlikely to be of interest for long-term (ten months) contact with blood, especially if it is a sufficiently large prostheses.
In this regard, special attention is given to improving hemocompatibility approach based on using the principle of biospecific chromatography. It is well known that pre-coating the surface of the polymer main blood protein serum albumin (SA) results in a marked inhibition of thrombus formation. However, it is clear that no matter how firmly attached either to the polymer, albumin, it is foreign to this organism and during long-term implantation is required to be destroyed by enzymes blood.
In order to create self-renewing coatings were asked to use a modification of the polymer surface ligands for biospecific sorption SA. To this end, the surface of the polymer grafted copolymer thin layer of hydrophilic acrylic amide tsetilmetakrilatom. CA tsetilmetakrilatom forms a sufficiently stable complexes with a dissociation constant of 5 x 10 5 M. Upon contact with the blood of the polymer the surface is coated with albumin automatically, and if the adsorbed albumin is denatured, its interaction with tsetilmetakrilatom attenuated and denatured protein from the blood is replaced by the native . In other words, the material may be regarded as analogous, though very remote, self-renewing living tissue. The study of hemocompatibility synthesized materials was performed on dogs using polyethylene arteriovenous grafts with a diameter of 2 mm. It was found that if all of the 10 catheters unmodified polyethylene thrombosed the first 7 — 10 minutes of contact with blood, that 9 of the 10 modified catheters remained free after 14 days.
It is much more efficient compared to the immobilization of albumin, a surface modification fibrinolytic enzymes — catalysts for hydrolytic cleavage of fibrin clots. However, in this case, there is a problem, especially with the rapid inactivation of the immobilized enzyme inhibitors present in the blood. Biospecific chromatography method and provide a positive effect here. Like most proteolytic enzyme plasmin, the active fibrinolytic enzyme most blood is in the form of inactive precursor — plasminogen. In the formation of fibrin blood clots in the activation of plasminogen by the action of specific activators and blood appears active plasmin destroys these clots. Conversion of plasminogen to the active enzyme can be carried out autocatalytically, especially at high concentrations it. It was necessary to choose a rather stable in real flow low molecular ligand capable of reversibly bind plasminogen. As such a ligand capable of reversibly binding plasminogen, was used N-methacryloyl-L-lysine — unsaturated derivative of L-lysine:
The surface of the source material is modified with a thin layer of a hydrophilic polymer with immobilizovanym lysine derivative. The layer of hydrophilic polymer necessary for removal of a lysine residue on the surface of the material to facilitate its interaction with plasminogen. Upon contact with blood, the modified material to selectively react with plasminogen, concentrating the protein on the surface. Approximation of protein molecules promoted self-activation of plasminogen, resulting in the material surface layer arose most powerful fibrinolytic enzyme, blood — plasmin. Active way of plasmin is not much inferior to the activity of plasmin from human plasma production company «Sigma» (USA) — 350 — 400 mmol of tyrosine / mg protein * min. It does not show any immune reactions or other complications associated with the use of alien fibrinolytic agents. With regard to the service life of the material, they were quite long — the loss of capacity for plasminogen did not exceed 1% per month.
Thus considered design principles hemocompatible polymers, which are based on a controlled concentration on the surface of the polymer ALS present in a living organism, and even spontaneous activation of BAS, will synthesize a wide and varied range of modified materials, largely to meet the needs cardio-vascular and plastic surgery, and other areas of medicine. They were also helpful in solving the problems associated with storing, blood transfusion, her isolation from the individual components, etc.
This will greatly reduce the toxicity of drugs and to extend their range of medical applications. Therefore, the most promising will provide polymer systems including cardiovascular and anti-tumor agents, drugs acting on the central nervous system, hormones, prostaglandins, vitamins, etc.