Transdermal composition for the local introduction of therapeutically and/or prophylactically active agent, tape or bandages for transdermal delivery

 

(57) Abstract:

The invention relates to the field of medicine and relates to transdermal compositions and tapes or dressings for transdermal delivery. The invention consists in that the transdermal composition for the local introduction of therapeutically and /or prophylactically active agent contains a fatty acid with a long chain molecular structure or an ester of a lower alkyl fatty acid with a long chain molecular structure, where this long chain is a chain of C13-C21and preferably contains at least one CIS-olefinic bond. Preferred components of long-chain are oleic acid and etiloleat, and the preferred fatty acid is a low molecular weight is propionic acid. Tape or bandage for transdermal delivery provides impermeable back layer, the adhesive means can hold the tape or bandage on the epidermis, local therapy or prophylactic composition for PP.1 - 13 and the permeable membrane. The proposal provides sustained relief oral administration with a reduced frequency of administration. 2 C. and 12 C.p. f-crystals, 5 tab., 7 want to make their penetration through the skin, which are especially valuable for delivery into the body of acetylcholinesterase inhibitors urethane basic type, and more specifically the type of physostigmine.

Carbamates mestinon and physostigmine are noncompetitive inhibitors of acetylcholinesterase, but tend to dissociate of this enzyme so that in a relatively short period of time, for example about 15 min, achieved a significant recovery of the activity of the esterase. This effect is the complete opposite of that, which is observed in organophosphate substances nerve, such as sarin and Tabun, which modify the chemically active steps esterase, permanently destroying the activity of esterase and thereby related cholinergic system.

It was found that the specific dose for prevention of such carbamates can be used to protect a partial amount of acetylcholinesterase subject from interaction with organophosphates, because of their ability to block the site or the active site of this enzyme. Although treated the subject may lose consciousness or pichetto esterase, preserved in the protection of physostigmine may be sufficient to prevent a fatal outcome, as and when it is released from a locked state by means of carbamate dissociate from the enzyme.

Obviously, to ensure long-term protivoglistotnoe protection or other methods of processing and long-term acting reversible inhibitor would require the drug that brings sustainable relief. Bringing sustainable relief medication oral administration could reduce the frequency of injecting it while ensuring a convenient way of dispensing, which does not require special equipment. Still to achieve this modest goal does not appear satisfactory preparation.

As a preventive measure to protect against nerve agents currently used carbamate in the form of tablets mestinon. Pyridostigmine does not pass the blood-brain barrier and therefore does not provide protection of the Central nervous. The same chemical property that does not allow mestinon to enter the brain, namely its positive charge, predotvrashchala for transdermal delivery into the body. But physostigmine has a short half-life from plasma and has a low therapeutic index that determines the use of the composition, bringing sustainable relief.

Although the known method of injection of physostigmine by injection, an attractive alternative way of introducing a protective doses seems transdermal (i.e. through the skin) using a patch bearing a suitable composition of the transdermal delivery. This known composition delivery of physostigmine is disclosed in UK patent N GB 2163347-A; it contains a mixture of physostigmine with propionic acid in a ratio of 50 wt.% to the volume. In recent work on the treatment of Alzheimer's disease (a type of presenilny dementia), authors (Christie et al, Br. J. Psychiatry, 138 46-50 (1981)) have identified such transdermal pharmaceutical compositions which deliver into the body through the skin of physostigmine in therapeutically useful doses, and in a further recent study (Pardo et al, J. Pharm. Sci. 79 p. 573-578 (1990)) have shown that thermodynamic activity of physostigmine, which directs the diffusion of drugs into the skin, it appears higher in the case of a binary media: isopropyl myristate and from servicestech nervous system disorders, but with the introduction of dosages required in cases of poisoning organophosphates, it became clear that it was necessary to impose inconvenient larger patches of plaster. Moreover, large quantities of physostigmine, which should be available on such flaps could be lethal in case the patient has a skin injury, such as penetrating wound, in the area of the overlapping flap therapeutic patch.

The invention provides improved compositions to deliver through the skin increased the number of fixed connections per connection in the bandage. Very beneficial data composition is used for delivery of basic compounds, non-ionic form, i.e., those that are not subject to ionization by, for example, stereoselectivity nitrogen groups. Thus, proposed by the present invention compositions can reduce the number of connections that you want to put in a bandage, achieving the advantage that the size of the bandage may be reduced.

Note that the composition of the present invention can be used for transdermal delivery of basic (protonarratives) compounds frequent in the x treatment processes. So, possible treatment, among others, Alzheimer's disease (senile sclerosis of the brain or the kind presenilny dementia), diseases of Parkinsona (shaking palsy), syndrome of Peak (Predtechenskaya limited atrophy of the brain), syndrome of Gerstman-Straussler, myasthenia-gravis spinal paralysis) and glaucoma is carried out using such compositions. The UK patent N GB 2163347-And makes the further assumption regarding cholinergic dysfunction, where you could apply such transdermal delivery, and they will also represent a potentially valuable area for application of the compositions of the present invention.

Specific preferred compositions according to this invention not only achieves the required dosages, but also provide stable suppression of enzyme activity.

In its broadest form the present invention provides compositions containing a fatty acid of low molecular weight, characterized in that they further contain a fatty acid with a long chain molecular structure or an ester of a lower alkyl fatty acid with a long chain molecular structure, where this long chain, not only at the more the present invention provides the use of these compositions as carriers of transdermal delivery.

The preferred chain length for the specified long chain fatty acid or a complex fatty acid ester, excluding carbon hydroxyl group, will be C15-C19and most preferably it is C17. Preferably, the long chain was straight, unbranched (n) chain, and that she had preferably only one CIS-olefinic (C=C) bond. When the acid is esterified with lower alkyl, as a group, the lower alkyl is preferably take alkyl, C1-C4more preferably alkyl, C1-C2. Thus, miristinovaya acid is usable saturated acid, but more preferred are unsaturated acids with longer chains of the molecular structure, as, for example, oleic acid. Component fatty acids of low molecular weight preferably is a fatty acid C2-C4and even more preferably propionic acid or butyric acid. Particularly preferred component fatty acids of low molecular weight acelerou patterns fatty acids can cause more probable rupture of tissue lipid layers, while decreasing chain length, as can be expected, reduces the penetration ability of such compositions, and therefore leads to the decrease in the amount to be delivered through the skin of the target compounds (i.e., drug); thus, these factors, among others, cannot be overlooked when selecting a track to select a specific active ingredient. It should also be noted that when used herein, the term "active ingredient" refers primarily preventive or therapeutic activity, and not the increased activity associated with the transition process of the skin.

Preferential treatment of fatty acids with long chain molecular structure or of ester to fatty acid of low molecular weight are in the range from 40:60 to 60:40 (V/V), more preferably in the range from 40:55 to 55:45 (V/V) and most preferably this ratio is approximately 50:50 (V/V), while the preferred concentration of active ingredient in the mixture has a value of from 10 to 50 wt.% the volume of the mixture. More preferably it is in the range from 20 to 30 wt.% the volume of the mixture and most preferably a value of 25% wt/abgenix main anticholinesterase inhibiting compounds, urethane type, in particular physostigmine and its analogues. Of further consideration is clear that this composition suitable for transdermal delivery and many other types of major, especially non-ionic primary connections.

Preferred components of fatty acids with long chain - oleic acid and etiloleat are toxicologically acceptable for injection of drugs, but was never used in the compositions of physostigmine for transdermal application. Either oleic acid or etiloleat not affect the stability of physostigmine, while conducted by the inventors of the research of some long-chain fatty acids and esters, such as isopropylmyristate showed the slow deterioration of the quality of physostigmine. The use of the acid component, and this case fatty acids with low molecular weight, for example propionic acid, is required in order to solutionat and neutralize primary active agent, for example, the free base of physostigmine.

As mentioned above, the prevention of the carbamate poisoning caused by substance nerve depends on the share of acetylcholinesterase, carbamate-inhibited (for example, fittest, when all unbound acetylcholinesterase is fosfaurilirovanna organophosphate substance nerve. After poisoning carbamylcholine share acetylcholinesterase available for spontaneous circulation in a functional enzyme that restores the body to its normal physiological functions. Thus, the subject is either fully protected or at least saved from airborne hazards of toxic substances nerve. The researchers found that the implementation of this prevention must be protected (to accumulate and store in the body) as specified approximately 30% of the available acetylcholinesterase.

The inventors have studied the absorption of physostigmine through human skin to evaluate the feasibility of its transdermal delivery into the body as a pre-administration of medication under influence of toxic substances nerve. The penetration of radioactively labeled physostigmine through human epidermis was measured in vitro using a glass diffusion cell, where optimization of absorption of physostigmine was achieved by applying comb the body by transdermal is an area of increased interest and offers the advantage when it comes to long-term and stable input medicines in the blood. However, the excellent properties of the skin as a barrier, impose a serious limitation on the remedy in relation to the desired dosage. Therefore, before attempting to develop a system transdermal delivery, it is necessary to determine whether the medication to be injected into the body through human skin in the desired therapeutic amount.

Clinical studies show that the rate of intravenous infusion of 400 mg/h creates cholinesterase inhibition in the blood by about 30%. This inhibition does not cause obvious signs of poisoning by cholinesterase and does not affect its clinical effect. When delivering medication transdermal way, if the permeability of the skin is becoming a limiting factor, the bioavailability can be increased by using a greater surface area. Practical considerations limit the size bandages (or flap patch) applied to the human body, the size of about 20 cm, which determines the rate of permeability of at least 20 µg/cm2/H. practicable for transdermal delivery of physostigmine. Higher speed values of permeability can reduce prepared to wetston, thereby minimizing the risk of intoxication by physostigmine after being wounded at the site specified headbands.

The suitability of the compositions of the present invention for the above transdermal delivery into the body of the active ingredients will now be illustrated by specific examples of such compositions are described to facilitate understanding of the advantages of the last experts in this field.

Examples are given to illustrate the invention but do not restrict it.

Fig. 1 is a graph of the absorbed active compounds (cumulative amount of absorbed substances µg/time in hours), illustrating the penetration of physostigmine through human epidermis from the following substances:

(o) - saturated propylene glycol;

(+) - isopropylmyristate;

() - propylene glycol/oleic acid in the ratio 10:90.

Points on the graphs represent the average values of the results 3-5 identical experiments.

Fig. 2 is a graph of the absorbed active compounds (cumulative amount of absorbed substances µg/time in hours), illustrating the penetration of physostigmine through human epidermis from the following substances:

(+) the tat (in the ratio 50:50./vol.);

() - 25% of physostigmine in a mixture of propionic acid/oleic acid (in the ratio 50:50./vol.).

Points on the graphs represent the average results of 3 to 5 identical experiments.

Fig. 3 is a graph of the absorbed active compounds (cumulative amount of absorbed substances µg/time in hours), illustrating the penetration of physostigmine through human epidermis from a mixture of propionic acid/oleic acid (mixed in a ratio of 50:50 volume units) with the following additives of physostigmine:

() - 1% (weight/volume);

(+) - 25% (weight/volume);

(e) - 10% (weight/volume).

Fig. 4 is a graph of the absorbed active compounds (cumulative amount of absorbed substances µg/time in hours), illustrating the penetration of physostigmine through human epidermis from 10%-aqueous solution of propionic acid/oleic acid (mixed in a ratio of 10:90 volume units).

Fig. 5 is a schematic section transdermal applicator for local application of physostigmine on the skin of Guinea pigs; this applicator is glued to the skin with korotkostrizhenih hair on the back of the chest, about 2 cm down from the ears.

Fig. 6 is a graph of inhibition imagenum at time zero timestamp and removed after 72 hours (arrow). Dashed lines indicate the values of cholinesterase activity in plasma, continuous lines are the same, in erythrocytes, and triangles indicate data for animals of the control group not treated with the drug. Points on the graphs provide average values +/- standard deviation of 5 animals. The area of application is 1 cm2.

Fig. 7 is a graph of inhibition of cholinesterase in the blood by 25% (weight/volume) of physostigmine in a mixture of propionic/oleic acid (in the ratio 50: 50. units ). The applicator set at the zero time point and was removed after 72 hours after the time of withdrawal indicated by arrow). Dashed lines indicate the values of cholinesterase activity in plasma, continuous lines are the same, in erythrocytes, and triangles indicate data for animals of the control group not treated with the drug. Points on the graphs represent mean values +/- standard deviation of 5 animals. The area of application is 0.5 cm2.

Materials and methods Chemical reagents

The name of the reagent supplier

[3H] water and [3H]physostigmine degree of radiochemical purity: more than 90%. - Amersham International plc (Amersham, UK)
Isopropylmyristate (98%), N - hydroxyethylated (70% aqueous solution) and physostigmine (free base). Sigma Chemical Company Ltd (Poole, UK)

Scintillation fluid and Salwen-350. - United Techologies, Packard (Illinois, USA)

Oleic acid (purity > 99%). - BDH chemicals Ltd (Eastleigh, UK)

Onomatology broadcast dipropyleneglycol. - Dow Chemicals (Rotterdam)

Physostigmine Foundation in the amount of 10 - 20 mg was weighed and placed in a volumetric flask (1 ml divisions). There also added the original volume of 25 ml of a selected medium, and the mixture was subjected to ultrasonic stirring for 30 minutes. If at the end of a specified period of time of dissolution had not occurred, to the flask was added an additional 10 - 50 ál - aliquot quantity of the medium and the process repeated until then, had not yet achieved complete dissolution.

2% solution of physostigmine was prepared in the media to be evaluated. Two milliliters of this solution was placed in a vial, adding a known amount of radioactive physostigmine (A0 counts per minute). This solution was subjected to shaking and 50 µl-aliquot was taken for counting in 5 ml of scintillation fluid (A1 counts per minute). Weighted piece of human epidermis (g) domesticado at a temperature of 30oC, thus exposing it from time to time shaken; then took 50 µl-aliquot and counted in 5 ml scintillation fluid (A2 counts per minute).

2 ml of 2% aqueous solution of physostigmine in each medium, the candidate was placed in separate test tubes and each tube was added 10 μl of the stock of radioactive physostigmine. An additional amount of 10 μl was added to 5 ml scintillation fluid and counted radioactivity with the receipt of doses of total added radioactivity (A0 counts per minute). Then made counting the radioactivity of the aliquot samples of each solution candidate with the receipt of doses of the total radioactivity in the solution after quenching (A1 counts per minute). Amendment damping for each solution is given by the expression:

A0/A1

A known sample of the epidermis (g) was then added to each solution with incubation for 48 hours at a temperature of 30oC and periodic shaking. Then he measured level of radioactivity remaining in each dvuhkilometrovom volume of media (A2 counts per minute), and the concentration of radioactive substances in solution (CS) was calculated according to the following formula:

CS= (A0/A1)(A2/2) decays and 1 ml of Toluene-350, counting the radioactivity in 10 ml of scintillation fluid (S1 counts per minute). Twenty-five microlitres standard physostigmine solution with a known level of radioactivity (S0 counts per minute) was added to the vial, after which it was conducted a recount of radioactivity (S2 counts per minute). Taking the density of the skin is equal to 1 g/ml, was calculated concentration of radioactivity in tissues (Ct) according to the following formula:

Ct= S1S0/W(S2-S1) disintegrations per minute/ml (2)

Were also determined values of the background account with the introduction of appropriate amendments to the fabric, toluene and scintillation fluid (in the formed systems). The partition coefficient (Km) was then calculated as follows:

Km= concentration in tissue/concentration in solution = Ct/Cs(3)

Saturated solutions of physostigmine in isopropylmyristate, propylene glycol and propylene glycol/oleic acid (50:50 vol.ed.) were prepared by adding excess amount of physostigmine to 1 ml of media. These solutions were subjected to rapid stirring with a vortex mixer and then allowed to stand. After holding five sessions ochered the s'", was collected using a Pasteur pipette, and then "drew" it by 5 to 10 ál of [3H]-of physostigmine before you can use it for studies of percutaneous absorption (i.e. absorption of the drug into the skin).

All other compositions (weight/volume) were prepared as follows. Physostigmine was weighed and transferred into a 10 ml flask with a round bottom. The media was injected into the flask by pipette and the sample obtained was concentrated using 5 - 10 ál of [3H]-of physostigmine. The resulting mixture was subjected to turbulent mixing in an ultrasonic bath until then, until dissolution occurred. All developed formulations (compositions) were protected from light and stored at a temperature of 4oC Poste restante.

The skin of the abdomen of the deceased man was taken by placing it in airtight plastic bags and keep them stored at a temperature of minus 20oC. Upon consumption, the tissue was allowed to thaw at room temperature and remove subcutaneous fat. Epidermal film (i.e., the skin or outer layer of the epidermis) was separated from the full thickness of the skin by immersing it in water at a temperature of 60oC for 45 sec, followed by slow and careful OTDELENIE epider the glass diffusion cells. Epidermal membranes were mounted in diffusion cells Franz with the cross-sectional area of 2.54 cm2. The cell was placed in a water bath, which maintained the temperature of the 30oC, the upper surface of the membrane was subjected to the donor solution. The bottom surface of this membrane was washed continuously stir the receptor fluid (in the amount of 4 - 5 ml).

Before applying the composition containing physostigmine, have assessed the integrity of each cooked skin-membrane element by determining the permeability of the latter for water, tritium-labeled. Experiments on the permeability to water flowed over a 6-hour period that ensures the achievement of steady state conditions. As the receptor fluid was used physiological saline, and 1 ml of saline solution containing tritium-labeled water (0.2 millicurie/cm3) were added to the donor chamber. Then with an interval of one hour produced probatory (25 ál) of the receptor of the cell. At the end of the cycle driving on tritium-labeled water was removed as the donor and receptor solutions, and using distilled water, made 2-3 leaching surfaces to which mpany, depriving them of residual activity.

During the examination the next day, any membrane with a permeability of more than 1.5 103cm/h were considered invalid and were excluded from subsequent measurements of permeability. The receptor chamber was filled with 50% aqueous solution of ethanol, and in the donor chamber was brought to 200 μl of labeled physostigmine (1 mccoury). The degree of transmission of physostigmine through the membrane was observed by probatorul receptor solution (50 μl) over a period of 3 days.

Counting the radioactivity of receptor samples were produced in 5 ml volume of scintillation fluid in a liquid scintillation counter model 1215 of Rackbeta - II.

The results of experiments on skin absorption of drugs is presented in the form of graphs, as the cumulative amount of absorbed substances depending on time. Speed stable penetration or stream J (MGSM-2h-1) was calculated by the method of linear regression analysis based on linear plots graphs. The coefficient of permeability Kp(SMC-1) was obtained using the following mathematical expression:

J = KmDc/S = Kpwith (4)

where

c - concentration of pronikayushie between the carrier and the skin;

D is the diffusion coefficient of penetrating substances in the epidermis;

S - thickness of the membrane.

The intersection point on the time axis is called latency and is related to the diffusion coefficient D as follows:

< / BR>
The flow rate can also be expressed on the basis of the activity of the drug in the carrier (a)

< / BR>
where

f is the activity coefficient of the drug in the skin barrier, it is generally assumed f to be constant, so that changes to the penetration factor associated with changes in the activity of drugs in the media. By definition, a solid substance is thermodynamic activity equal to one, and therefore a saturated solution that is in equilibrium with a given solid, should also have this activity equal to one. Therefore, under ideal conditions, the maximum rate of penetration will be achieved with saturated solutions of the drug. Perform measurement activity is a difficult task, and usually determine the ratio of the concentration of dissolved substances to the value of its solubility (percent saturation) to get an indication (or characteristic) of this activity, or "the tendency of care" penetrating substances in the media. In practice, the number of drugs is esta in the donor, the effect on the rate of penetration is negligible. If the content of the drug in the donor solution is reduced by less than 10% during the experiment, it can be assumed that the donor solution is an inexhaustible source and the experiment takes place in the presence of inorganic dose.

The coefficients violations permeability was determined for evaluation of destroying the membrane potential of various compositions of physostigmine. This is due to the measurement of the permeability coefficient of water, labeled with tritium, through the membrane before and after the application of the test composition on the epidermal surface within a specified period of time. Then the coefficient of damage (DR) is defined by the following ratio:

DR = Kpwater after the introduction of the carrier/Kpwater prior to the introduction of the carrier (7)

Propylene glycol, isopropylmyristate and a mixture of propylene glycol/oleic acid (in the ratio 50:50.ed.) were selected as comparative compositions to evaluate them as carriers for transdermal delivery of physostigmine. Composition of physostigmine in two-component systems consisting of mixtures of propionic acid/isopropylmyristate (in the ratio 50:50.ed.) and poeteray 50% of physostigmine in propionic acid, described in the patent N GB 2163347A. Also explored the impact of changes in concentrations of physostigmine and proportions in the media propionic acid/oleic acid.

Studies of the distribution and solubility factor was used to estimate olejowego alcohol, ethyloleate, squalene, nanometrology ether dipropyleneglycol and N-hydroxyethylated as alternative carriers for transdermal delivery into the body of physostigmine.

In Fig. 1 - 4 presents the temporal characteristics of the process of penetration of physostigmine through human epidermis from the tested mixtures. Relevant data on permeability are given in table.1. With the exception of saturated isopropylmyristate, and 1%- and 10%-aqueous composition of propionic acid/oleic acid, the penetration index was below 10% of the applied dose that satisfies the conditions of "limitless" drug dose (i.e., inexhaustible donor source), ensuring the achievement and maintenance of regime stability condition. Note that from isopropylmyristate (see Fig.1) the number of physostigmine is rapidly decreased. Only transient, a steady state was observed when 50 hours was the period of measurement of diffusion. Physostigmine is decomposed into rupestris and other products of molecular degradation by hydrolysis and subsequent oxidation, which contributes to the effect of light and heat. Rupestris has a bright red color, and of the compounds studied in this work, only propylene glycol, propylene glycol/oleic acid and N-hydroxyethylated purchased the pink/red color (after 10 to 24 hours). The exact degree of disintegration of physostigmine is not defined. Called "rich" solutions isopropylmyristate, propylene glycol and a mixture of propylene glycol/oleic acid used to obtain the flow rates listed in tab.1, were obtained by vortex mixing, as indicated above. Subsequent analysis method ultraviolet spectroscopy and comparison with solutions prepared by the method of homogenization ultrasound confirmed that they have values of degree of saturation listed in table 1. It is likely that the discrepancy between the results occurred due to improper mixing of the solutions of the vortex mixer as opposed to that of using an ultrasonic homogenizer. The values of the rate of diffusion is too low, to be able to use them with bandages etc saturation. The difference in the values of the rate of diffusion between the media and those containing propionic acid, is too great to be overcome by increasing the concentration at saturation.

The use of propionic acid as co-solvent with isopropylmyristate and oleic acid increases the solubility and stability of physostigmine (Fig. 2). The compositions of physostigmine on the basis of two solvents, namely 25% in a mixture of propionic acid/isopropylmyristate and 25% in a mixture of propionic acid/oleic acid, provided higher values transdermal fluxes (17 µg cm-2h-1and 24 µg cm-2h-1respectively) than 50% composition propionic acid (13 µg cm-2h-1) (table. 1). The use of saturated solutions of physostigmine may increase flows in two-component systems and it can be concluded that both two-component system capable of delivering therapeutic amounts of physostigmine through the skin from dressing with a contact area specified in table 2.

Increased values of flows observed in the cases of two-component systems, there are, apparently, due to the combination effects of the distribution of/R is BL.3), contributing to the implementation of physostigmine in the skin. Was made without evidence that oleic acid and isopropylmyristate stimulate penetration, creating an obstacle to the lipid layers of the stratum corneum and opening the passages for diffusion (Albery et al, J. Pharm. Pharmacol.31 140-147 (1979)). This mechanism causes the diffusion of the stimulator in the stratum corneum before will show a synergistic effect than can be explained by the increased value of time delay from these carriers.

The decrease in the concentration of physostigmine in the media, "propionic acid/oleic acid resulted in large increases in the permeability of the skin (Fig. 3). However, these increases were associated with higher values of the coefficient of water damage (DR), as shown in table 4, which indicates damage to the membrane as the responsible for the increased permeability at 1% and 10% different compositions compared to oleic acid (DR = 1,5). The value of DR, 2.4, caused 25% composition, is a little damage to the barrier layer. Although propionic acid and is an irritant tool, it is likely that physostigmine, being in propionic acid, forms propionate, which reduces or even eliminates irritating action on the part dostatochno the amount of free acid, to cause membrane damage.

In order to obtain not injure the composition containing 10% of physostigmine, a volume ratio in a mixture of propionic acid/oleic acid was reduced from 50 : 50 to 10 : 90 (Fig.4). This media was saturated with physostigmine and, as expected, would not be annoying tool. Apparently, oleic acid still interacted with skin lipid, which is indicated by a large value of the time delay, but the transdermal flux of physostigmine was very small. This pattern may be due to a less favorable ratio Department of physostigmine from oleic acid than from the mixture (in the ratio 50 : 50.ed) propionic acid/oleic acid. Therefore, increased oleic acid may lower branch of physostigmine (from the material of the carrier) and incorporate it into the epidermis.

Concentration and separation are critical determinants transdermal flux, as is evident from the dependence of flow steady state (see equation (4)). This implies that the increase in Kmand c will lead to increased values of the flow rate. However, these two parameters are interdependent in the sense that the wear is mine to separate and to penetrate into the skin, and Vice-versa. As predicted, squalene, isopropylmyristate and etiloleat all have a relatively low degree of solubility for physostigmine, but high values of Km(PL. 3). Conversely, alerby alcohol, onomatology broadcast dipropyleneglycol, N-hydroxyethylated and oleic acid have a higher degree of solubility for physostigmine, but lower values of Km. It is obvious that there must be a compromise between the Kmand c when optimizing the composition of the media, what was done and achieved in two-component systems tested to date. Of the five preferred alternative media in the evaluation process, apparently, etiloleat in combination with propionic acid is the best carrier for transdermal delivery of physostigmine. He has more favorable Kmthan oleic acid, and contains a CIS-double bond, which, as I believe, is the cause of enhancing/stimulating action of this compound.

While isopropylmyristate increases percutaneous absorption by increasing the separation and introduction of physostigmine in the skin, oleic acid and etiloleat increase as the separation and diffusion of physostigmine, moreover, the application of the compositions of the present invention containing isopropylmyristate, leads to a very slow appearance of pink color, indicating the decay products. The inclusion of oleic acid and ethyloleate in transdermal formulations allows the use of smaller transdermal bandage for the correct dose to a person (for example, the bandage with the area of contact with skin, about 16 - 20 cm2), containing a smaller number of physostigmine for the demand in the dosage, resulting in decreased risk of poisoning from mechanical damage headbands and below her skin.

Note to the table. 3. Values of solubility was determined by ultrasonic homogenization of mixtures, as described in "Materials and methods". All the proportions of components in mixtures are given in volumetric units (for example, 10:90).

Note to the table. 4. The destruction factor is the ratio of constant permeability for water, labeled with tritium, before and after exposure of the membrane effects of the drug compound, as described in section "Materials and methods".

For further research on the effectiveness of the compositions of the present invention was the rate of physostigmine in the body of the Guinea pig. These experiments show that the compounds of preparations containing oleic acid, have no irritant effect after 48 hours after overlay applicator.

Materials

Physostigmine (supplier - firm Sigma chemical company, Poole, Dorset);

Propionic acid (supplier - firm Aldrich, Gillingham, Dorset);

Oleic acid (supplier - British Drag Houses, Poole, Dorset).

All these substances had a degree of purity higher than 99% and were used in the experiment immediately upon delivery.

Animals - Guinea pigs (Dunkin - Hartley) were purchased from the company Interfauna Ltd, Animals were kept in the experimental nursery during all experiments, allowing them free access to food and water. In the experiments for the measurement of cholinesterase activity were used in female Guinea pigs (weighing 700 - 1200 grams), and in experiments on the study of irritant drugs were used males of these animals (weighing 300 to 500 grams).

Compositions with physostigmine.

Physostigmine in propionic acid was prepared by dissolving the free base of physostigmine in propionic acid in equal proportions (weight/volume) rim base of physostigmine in a mixture of propionic acid/oleic acid", made in the ratio of 50 : 50./about.

Prepared composition (1) was applied to the cut area of the skin (2) rubber cap (3), removed from disposable piston of the syringe; the resulting device will be called the "applicator". The dead space of the internal cavity of the applicator filled silicone rubber (4), leaving the place with a volume of about 100 μl, which was filled with a composition candidate. The cap is glued with an adhesive means (5) to the site pre-cut skin mesothorax region of the animal, about 2 cm down from the ears, with the help of cyanoacrylate glue. The experimenters found that the "Superglue Extra (RTM), which are produced in the form of a gel is the most effective glue, as it fills the area around the hair bristles, providing a more reliable seal than liquid adhesives.

Applicators containing physostigmine, was established after taking blood samples on the first day, and was shot after taking blood samples on the third day of the experiment. Animals were slightly anestesiologi using Halothane to facilitate removal of applicators, which can be torn off by hand, and place applications have purified water concrete is in propionic acid and 0.5 cm2for physostigmine in a mixture of propionic and oleic acids.

Sampling of blood produced by the puncture of the ear vein, every day, for five days, and again on the seventh day, with a selection directly into the sample tube containing ethylendiaminetetraacetic acid as an anticoagulant. Taking blood samples were kept on ice until analysis. The erythrocytes were separated by centrifugation for 30 seconds at 14,000 rpm in a centrifuge company Eppendorf model 5415. The plasma is reserved, and the erythrocytes were washed in cold (ice) physiological solution. The washed cells were re-suspended to the original volume in the cold, ice, salt (physiological) solution. Hematocrit initial blood and resuspending cells was measured using microhematocrit system Hokki (Hawksley microhaematocrit system).

The acetylcholinesterase activity was quantified by the method of Ellman and others (Ellman et al; Biochem. Pharmacol. 7 88 (1961)). Mixture for analysis contained either erythrocytes or plasma (diluted in a ratio of 1 : 600 in the final mixture for analysis), 0.01 M 5,5'-dithiobis-2-nitrobenzoic acid and 1 mm acetylthiocholine in 0.1 M phosphate buffetaamiaisessa monitoring using a spectrophotometer manufactured by Pai-Unicam, England, model SP 8-100. The reaction was monitored on a wave of 412 nm within 10 minutes (neighbourly process) or for 20 minutes (inhibited), and the initial reaction rate was calculated from the graphs of absorbance (or optical density) depending on time. The analysis was subjected to daily calibration using the standard (m) solutions of cysteine. The reaction was started by adding substrate.

Forms used in the spectrophotometric reference cells that contained all the components of the reaction except for the substrate. The values of the rate of spontaneous hydrolysis was measured separately and subtracted from the values of the rate of catalysis of the enzyme. Amendments were introduced using hematocrit values, so that values of enzyme activity was expressed as the number of product min-1ml-1the initial volume of blood.

Enzyme activity on the first day was used as the base values for each individual, and the result was expressed as the relative inhibition (inhibition%) of this value. The control group of animals were also samples of blood each day during the weeks the CLASS="ptx2">

Male Guinea pigs (weighing 300 - 500 grams each) were subjected to percutaneous impact of the drug on the basis of oleic acid (25 ml) for 48 hours, and the site of application was previously vestigal from wool and closed. Place of application of the applicator washed with a water-alcohol solution, to eliminate the excess of the preparation and assessment of places applications were made on a scoring system of Draza (Draize Scoring system). Number of points were averaged in accordance with the published methodology of this system of counting (EEC Guideline N L/257; Off. J. Eur. Community 26 19 (1983)).

Physostigmine in propionic acid (50 : 50 weight/vol.) given the increasing inhibition of cholinesterase in plasma and erythrocytes before removing the transdermal applicator after 72 hours (Fig. 6). Growth inhibition continued for the next 24 hours. After 72 hours after removal of the applicator began to recover activity, however, the cholinesterase in plasma and erythrocytes were still suppressed by more than 50% at this time. Within 24 hours of the drug marked the death of one animal in the test group, and all other animals showed no signs of poisoning by cholinesterase (i.e. left "asymptomatic").

As it turned out, 25% of the contents (weight/volume) of physostigmine in a mixture of propionic and oleic acid (in the ratio 50 : 50./about. units) no irritants (i.e., is not a drug-stimulus) in a modified test Drazu. Of the six treated animals three animals showed signs of mild forms eritem 24 hours after application of the drug and two XB - 48 hours later. After 6 to 7 days after treatment 5 out of 6 animals showed slight scattered flaking area of application (table 5). The average point count of the manifestations of erythema, edema and desquamation at any time does not exceed two units.

In this study, physostigmine was delivered into the body transdermalniho, the inclusion of oleic acid in the formulation of the drug reduces the concentration of physostigmine, and the area of application required to create the desired degree of inhibition of cholinesterase in the blood (approximately 80% of plasma and 60% in erythrocytes).

It was impossible to reproduce the results presented in document N GB 2163347A, where it achieved a 30% inhibition of cholinesterase in whole blood of the Guinea pig from physostigmine in composition with propionic acid, and this value of inhibition was constant over the period of time from 5 hours to 3 days and the resulting area applications preparation of 1 cm2; proposed by the present invention the formulation of the mixture has not been received constant level of inhibition.

Physostigmine labile blood of Guinea pigs, and the elimination half-life from plasma is approximately 40 - 50 minutes (Lukey et al, J. Pharm. Sci, 79 796-798 (1990)). Because of this rapid Samovodene active physostigmine, a continuation of the inhibitory effect on cholinesterase blood within 3 days after stopping treatment applicator indicates the ongoing delivery (and preserved receipt) of physostigmine in blood from some depot. This "depot effect" may be dosrochnoe applique bandage reduces the risk of irritation or sensitization from repeated multiple treatments medication or treatment sessions. Conversely, the presence of the loaded drug depot will support dosing after you remove the bandage, which may be undesirable if the patient has been the victim of injury, accident, etc) and requires treatment with medication.

The location of this depot in the body is a matter of conjecture, but since the "depot effect" is not observed when intravenous physostigmine, it is likely that this depot is located in the skin. The stratum corneum is the most likely location of the depot. The drug, which is separated from the carrier and into this layer will slowly diffuse into the blood. However, based on currently available data cannot exclude the possibility that the depot is located elsewhere, for example in the upper layers of the skin.

The degree of inhibition of cholinesterase in the blood, obtained in this study is higher than that required for protection in case of poisoning of the body by substances nerve (30%). Observed deaths at an early stage of the treatment period were related to the fact that the experimental animals were able to scrape off the drug from the applicator. It is likely that these animals swallowed Bo is the area of application should be commensurate with the specific recipient with the in order to achieve the required degree of inhibition, as it will be obvious to specialists in this field.

The inclusion of the drug oleic acid, ethyloleate or isopropylmyristate let reduce by half as the number of physostigmine in the composition and size of the dressing. This agrees well with the data of experiments conducted in vitro, namely, that oleic acid has doubled the speed of penetration of the drug through the skin.

To be clinically applicable, transdermal drug physostigmine should not have irritating effect. Although propionic acid, as such, is an irritant to skin, being mixed with physostigmine (in the ratio 50 : 50) it is not an irritant (according to GB patent N 2163347). According to the results of the present work, a 48-hour skin contact with the product containing oleic acid, gave a very faint erythema and scaling with the average count of less than two points, according to the guidelines of the European economic community EEC Guideline L/257" does not allow to classify this drug as an irritant.

Adding oleic acid to the mixture was allowed to halve the area of application of the esterase in the blood, however, reaching a constant value of inhibition before the expiration of 24 hours from the beginning of the application. The effectiveness of these formulations for delivery of physostigmine in the body transdermal method, in particular compositions on the basis of oleic acid and ethyloleate, indicates the possibility of further reducing the size of the bandage when the task is to get only 30% inhibition of cholinesterase. Both composition give the degree of inhibition of cholinesterase in the blood that is stored for 72 hours after removal of physostigmine from the skin (i.e., after removal of the applicator). The inclusion of oleic acid in the composition does not create an irritable response in Guinea pigs after application of the drug within 48 hours.

Table 5 presents the primary manifestations of irritation from skin exposure of physostigmine (25% weight/volume) in propionic and oleic acids (a mixture in the ratio of 50 : 50 volume units). The average score indicator on Drazu specified for groups of six animals depending on the time that has elapsed since the first application. It should be noted that for the classification of compounds as the stimulus, the average must be at least two points.

Example of a filled liquid is malinow bandage, saturated liquid containing the composition according to example 2, was collected using (sticky) tape and permeable membranes, supplied by the firm 3M-Medica. The diffusion of physostigmine through capillary membranes containing different concentrations of vinyl acetate, was measured using diffusion cells Franz. Through the membrane, containing 19% vinyl acetate, (cat. N MSP 987192) physostigmine diffuse faster than the separated epidermis. This membrane was used for the manufacture of fluid-filled bandages with impermeable back side (material company "Scotchpak" cat. N 1006-RTM), which was taped to the skin using a pressure-sensitive adhesive means on the basis of silicone (material company "Dow-Corning, cat. N 282A).

1. Transdermal composition for the local introduction of therapeutically and/or prophylactically active agent containing 10 to 50 wt.%/about. therapeutically and/or prophylactically active agent and a fatty acid of low molecular weight, wherein the composition further comprises a fatty acid with a long chain or an ester of a lower alkyl fatty acids, and long chain fatty acids, excluding the carboxyl carbon is the flail is Inna chain of from 40 : 60 to 60 : 40.

2. The composition according to p. 1, characterized in that the preventive and/or therapeutic compound includes a compound urethane type, inhibiting the acetylcholinesterase.

3. The composition according to p. 2, characterized in that said prophylactic or therapeutic agent is physostigmine or equivalent.

4. Composition according to any one of p. 1 - 3, characterized in that the long chain fatty acids with long chain molecular structure, or a complex ester of fatty acids with long chain molecular structure contains at least one CIS-olefinic bond.

5. Composition according to any one of p. 1 - 4, characterized in that the specified fatty acid of low molecular weight is C2-C4fatty acid.

6. The composition according to p. 5, characterized in that the specified C2-C4fatty acid is propionic acid.

7. Composition according to any one of p. 1 - 6, characterized in that the long chain fatty acids with long chain molecular structure, or a complex ester is a chain of C15-C19.

8. Composition according to any one of p. 1 - 7, characterized in that the long chain fatty acids with long CE is>9. Composition according to any one of p. 1 - 8, characterized in that it includes a specified ester of lower alkyl fatty acid with a long chain molecular structure and a complex ester of the lower alkyl is an ester of alkyl (C1-C4.

10. Composition according to any one of p. 1 - 9, characterized in that the specified fatty acid with a long chain molecular structure, or a complex ester are oleic acid or etiloleat.

11. Composition according to any one of p. 1 - 10, characterized in that said complex ester of fatty acids with long chain molecular structure is isopropylmyristate.

12. Composition according to any one of p. 1 - 11, characterized in that the ratio of the volume of fatty acids with long chain or a complex ester of fatty acid with a long chain to the amount of fatty acids of low molecular weight is approximately 50:50./about.

13. Composition according to any one of p. 1 - 12, characterized in that it comprises 25% (wt./about.) preventive and/or therapeutic agent.

14. Tape or bandage for transdermal delivery, containing impermeable back layer, the adhesive means can hold the tape or bandage on the epidermis, local therapeutic or prophylact lakticheskoj composition it contains a composition according to any one of p. 1 - 13.

 

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