CYCLODEXTRINES AS FUNCTIONAL AGENTS FOR DECONTAMINATION OF THE SKIN CONTAMINATED BY NERVE AGENTS

Cyclodextrines [CDs] are cyclic oligosacharides formed by a number of glucose units linked with alpha-(1–4) glycosidic bonds. There are three known basic structures that differ only in the number of units. The alpha-cyclodextrine [α-CD], of six units, beta-cyclodextrine [β-CD] of seven units and the gama-cyclodextrine [χ-CD]. All of them have as a common feature the existence of a hydrophobic central cavity surrounded by a hydrophilic ring formed by hydroxyl groups (7). These seminatural artificial receptors can bind a variety of organic, inorganic and biological guest molecules inside their apolar cavities in aqueous solution to form host-guest complexes or supramolecular systems (17). Therefore, this fascinating property enables them to be successfully used as drug carriers (10,16), separation reagents (6,25), enzyme mimics (2,18) and catalysts of the chemical reactions (20). Relatively little of this work has been concerned with the hydrolysis of the organophosphorus compounds (1,3). Organophosphorus compounds used at the present time as pesticides [paraoxon] or as chemical warfare agents [sarin, soman, cyclosarin, tabun] belong among irreversible inhibitors of the enzyme acetylcholinesterase [AChE; EC 3.1.1.7] (13,15). AChE plays a key role in the physiological function of the cholinergic nervous system and, therefore, its inhibition is life-endangering (14). The relatively unsatisfactory antidotal treatment available for acute poisonings with organophosphates has prompted the study of prophylaxis possibilities that allow survival of organisms exposed to organophosphates (4). There is considerable interest in methods for the decontamination of the skin after contamination with these agents (8,11). Several methods of nerve agents decontamination are based on the sorption into the porous materials [currently used decontamination mean in the Czech Army, called DESPRACH] (21), on the micelar catalysis (5,12) or on the cyclodextrine host-guest interactions (3,22), as mentioned above. In this study, we have prepared decontamination solutions of beta-cyclodextrines and tested their ability to decontamine rat skin contamined with nerve agent soman. Decontamination efficacy of the tested cyclodextrine solutions was compared with the same decontamination means but without the cyclodextrines.


Introduction
Cyclodextrines [CDs] are cyclic oligosacharides formed by a number of glucose units linked with alpha-(1-4) glycosidic bonds. There are three known basic structures that differ only in the number of units. The alpha-cyclodextrine [α-CD], of six units, beta-cyclodextrine [β-CD] of seven units and the gama-cyclodextrine [χ-CD]. All of them have as a common feature the existence of a hydrophobic central cavity surrounded by a hydrophilic ring formed by hydroxyl groups (7).
These seminatural artificial receptors can bind a variety of organic, inorganic and biological guest molecules inside their apolar cavities in aqueous solution to form host-guest complexes or supramolecular systems (17).
Therefore, this fascinating property enables them to be successfully used as drug carriers (10,16), separation reagents (6,25), enzyme mimics (2,18) and catalysts of the chemical reactions (20). Relatively little of this work has been concerned with the hydrolysis of the organophosphorus compounds (1,3 (13,15). AChE plays a key role in the physiological function of the cholinergic nervous system and, therefore, its inhibition is life-endangering (14).
The relatively unsatisfactory antidotal treatment available for acute poisonings with organophosphates has prompted the study of prophylaxis possibilities that allow survival of organisms exposed to organophosphates (4).
There is considerable interest in methods for the decontamination of the skin after contamination with these agents (8,11). Several methods of nerve agents decontamination are based on the sorption into the porous materials [currently used decontamination mean in the Czech Army, called DESPRACH] (21), on the micelar catalysis (5,12) or on the cyclodextrine host-guest interactions (3,22), as mentioned above.
In this study, we have prepared decontamination solutions of beta-cyclodextrines and tested their ability to decontamine rat skin contamined with nerve agent soman. Decontamination efficacy of the tested cyclodextrine solutions was compared with the same decontamination means but without the cyclodextrines. All other chemicals used in this work were commercial products of Merck or Sigma-Aldrich.

Animals
Male white albino Wistar rats weighing 180-200 g were purchased from Velaz Praha [Czech Republic]. They were kept in an air-conditioned room [22 ± 1 °C and 50 ± 10 % relative humidity] with lights from 07.00 to 19.00 and were allowed free access to standard food and tap water ad libitum. The rats were divided into groups of five animals each [n = 5]. Before the experiment they were shaved on their dorsal part [3 x 5 cm]. Experiments were performed under the supervision of the Ethics Committee of Medical Faculty of Charles University and Purkyně Military Medical Academy in Hradec Králové, Czech Republic.

Decontamination solutions
There were prepared six kinds of decontamination solutions -tetraborate buffer pH =9, tetraborate buffer with the addition of the β-CD, tetraborate buffer with acetone, tetraborate buffer with acetone and with the addition of the β-CD, water solution of 2-aminoethanol and finally water solution of 2-aminoethanol with the addition of the β-CD. Composition of all tested decontamination solutions is described in the Table 1.

In vivo experiments
The efficacy of the cyclodextrine solutions to decontaminate soman contaminated rat skin was determined using modified in vivo decontamination test (4). To determine the decontaminating efficacy of the tested solutions, the rats were poisoned percutaneously [p.c.] with the appropriate dose of organophosphate soman and then they were decontaminated using CDs solutions. The control group were rats contaminated with soman, but decontamination was not performed. The decontamination with CD solutions was started 2 min following p.c. poisoning. The decontamination solution was used at a dose of 1.6 ml per animal and it was spread on the contaminated skin area using defined tampons [weight 327 + 23.5 mg] within 30 s.

Data analysis
The LD 50 values and their 95% confidence limits were calculated by probit analysis of deaths occurring within 24 hours after p.c. administration of soman at five different doses with five animals per dose (23). Efficacies of decontamination solutions were compared using ID 50 values [Protective ratio]. Index ID 50 was calculated from the values of LD 50 measured with the decontamination, and LD 50 without the decontamination. ID 50 = LD 50 p.c. decontaminated / LD 50 p.c. non-decontaminated Just as the index ID 50 is high, the decontamination solution is potent.

Results
Contamination of the skin of experimental animals with soman was performed as described above. The decontamination action with one of different six decontamination prescriptions tested in our study, was started  water solution of 30 ml 2-aminoethanol 2-aminoethanol with 70 ml distilled water the addition of the 1.70 g β-CD β-CD *composition for 100 ml of the final solution after application of toxic organophosphorus compound. The efficacy of tested decontaminants was evaluated by the assessment of the ID 50 values using Weil's test (23). Results for all decontamination receptures are presented in Table 2.
All the decontaminants tested show the value of ID 50 higher than 1. Whence it follows that all the prescriptions for decontamination solutions effect as decontaminants of the skin contaminated with soman. When solutions 1 and 2 are compared, it is clear that the decontamination efficacy is decreased by CD addition. On the contrary, in case of tetraborate buffer with acetone [3,4], the addition of CD resulted in ID 50 increase. Acetone addition into the decontamination solution only slightly increases the ID 50 value. Nevertheless the substitution of tetraborate buffers [solutions 1-4] for 2-aminoethanol solution [solutions 5, 6] significantly enhances the decontamination efficacy of the appropriate solutions. Even in case of solution 6, the value of decontamination efficacy ID 50 achieves 16.7. From results presented in Table 2 it can be concluded that the most effective decontaminant is the prescription consisting of aqueous solution of 2-aminoethanol and β-CD [6].

Discussion
Well-timed skin decontamination is the crucial step after percutaneous poisoning with chemical warfare agents (4). Several modalities of skin decontamination can be recently applied as described above (5,12,21,22). Cyclodextrines can serve as suitable decontaminants after skin contamination with nerve agents due to their ability to form host-guest complexes (17).
Our in vivo results show that addition of β-CD into the solution of tetraborate buffer and tetraborate buffer with acetone does not cause significant increase in decontamination efficacy. Only in case of aqueous solution of 2-aminoethanol, the addition of β-CD resulted in significant increase [32%] in decontamination efficacy. Ineffectiveness of tetraborate decontaminants has two reasons. At first, neat soman applied on the skin has considerably higher affinity to mixture of lipophile and hydrophile medium represented by skin than to aqueous medium of decontaminating solution (24). In consequence soman does not reach the CD molecules in short time and sufficient amount and thus the process of decontamination does not run at desirable rate. Slow diffusion from the solid surface into the solution is the direct action of the whole process, instead of the rapid chemical reaction. Secondly, during our experiment it was found that the animal skin is within 1 to 2 minutes after decontamination action completely dry and thus the decontaminating reaction is stopped, because the suitable medium for reaction running is not available and soman with respect to its low volatility proceeds further with penetrating into the skin (24).
The addition of acetone into the decotaminant solution should avoid problems with soman insolubility but it resulted only in slight increase in ID50 value. Rapid evaporation of solvents from the surface, even potentiated by acetone adding, probably caused lower efficacy than we expected. Therefore acetone was replaced by 2-aminoethanol with much lower volatility in comparison with acetone (9). In addition with regard to 2-aminoethanol, structure there is no presumption of competition with soman for CD cavity occupation.
In conclusion, decontamination prescriptions with CD [2,4] do not show significantly better decontamination efficacies in comparison with prescriptions without CD. In case of decontaminants with 2-aminoethanol [6], the satisfactory decontamination efficacies were achieved.