Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 36, Num. 4, 2004, pp. 234-237

Indian Journal of Pharmacology, Vol. 36, No. 4, August, 2004, pp. 234-237

Research Paper

Cardio-respiratory effects of DRDE-07, a new prophylactic agent for sulphur mustard toxicity, in anesthetized rats

Malviya V, Singh R, Kumar D, Pathak U, Kumar P, Jaiswal DK, Mathur R, Vijayaraghavan R

Defence Research and Development Establishment. Jhansi Road, Gwalior - 474 002
Correspondence Address:Defence Research and Development Establishment. Jhansi Road, Gwalior - 474 002 jai_vijay@hotmail.com

Code Number: ph04077

ABSTRACT

OBJECTIVE: To study the cardio-respiratory effects of DRDE-07, a new prophylactic agent for sulphur mustard. MATERIAL AND METHODS: The effect of DRDE-07 was studied in anesthetized male rats (Wistar), by administering it either by intraperitoneal or oral routes. Different doses (0.25, 0.5, 1.0 and 2.0 LD₅₀) were used and various cardio-respiratory parameters viz., mean arterial blood pressure, heart rate, respiratory rate and tidal volume were monitored on an oscillograph. RESULTS: Intraperitoneal administration of 0.5 and 1.0 LD₅₀ and oral administration of 0.25 LD₅₀ of DRDE-07 did not produce any effect on the cardio-respiratory variables. Oral administration of 0.5 and 1.0 LD₅₀ showed a significant decrease in mean arterial blood pressure after 60 min. None of the animals died in the two-hour monitoring period. Two (2.0) LD₅₀ administration of DRDE-07 either by intraperitoneal or oral routes induced a sudden decrease in the mean arterial blood pressure and the animals died within one hour. CONCLUSION: DRDE-07 did not show any significant effect on the cardio-respiratory variables at a dose of 0.25 LD₅₀ by oral route and 1.0 LD₅₀ by intraperitoneal route. The death after intraperitoneal or oral administration (2.0 LD₅₀) of DRDE-07 is due to the sudden fall in the mean arterial blood pressure.

Keywords: Alkylating agent, bis (2-chloroethyl) sulphide, chemical weapon, toxicity

INTRODUCTION

The Chemical Weapons Convention (CWC) came into force from 29 April 1997, prohibiting the production, storage, transport and use of chemicals on enemy forces.[1] Countries which had already stockpiled the chemical weapons were given a time of 10 years to destroy them. One such chemical is sulphur mustard (SM), commonly known as mustard gas. Reports are available of its use in several instances.[2],[3],[4] Huge quantities of the stockpiled SM are being destroyed by several countries. In spite of the CWC, SM can be used clandestinely during war or by terrorists due to its simple method of preparation.

SM, chemically bis [2-chloroethyl] sulphide, is an alkylating agent that causes serious blisters upon contact with human skin. In animal models it is extremely lethal. Just one microliter applied on the skin of a mouse or a rat may be lethal in a weeks time. SM forms sulphonium ion in the body and alkylates DNA leading to DNA strand breaks and cell death.[5],[6] Due to the high electrophilic property of the sulphonium ion, SM binds to a variety of cellular macromolecules and fatality may occur due to multi-organ failure.[7],[8] The eyes, skin and the respiratory tract are the principal target organs of SM toxicity.[5], [9], [10]

SM is highly lipophilic and is absorbed very quickly after contact with the skin. Though several antidotes have been reported for the systemic toxicity of SM in experimental animals,[8],[11],[12],[13],[14] none of them have been approved so far and decontamination of SM immediately after contact is recommended as the best protection.[7] Hence, the decontamination has to be achieved quickly to limit the absorption. The most commonly used decontaminant is Fuller′s earth (a native form of aluminum silicate) that removes SM by adsorption, thereby reducing the toxicity.[15] A few chemical decontaminants for human use have also shown very good efficacy.[16],[17]

An effective prophylactic agent against SM toxicity is the requirement of the day, especially for personnel engaged in the destruction of SM and during inspection by the Organization for Prohibition of Chemical Weapons. From a series of aminothiols, two compounds amifostine [organophos-phorothioate] and DRDE-07 [S-(w-aminoalkylamino) alkylaryl sulphide] gave very good protection as prophylactic agents against SM.[18],[19],[20] When given orally DRDE-07 was more efficacious than amifostine as a prophylactic agent against SM induced percutaneous toxicity.[19],[21] The acute toxicity studies (LD₅₀) of DRDE-07 have also been reported.[21]

As DRDE-07 is an investigational drug, to introduce it as a prophylactic agent to the armed forces, various pharmacological and toxicological studies are needed. We initiated several of them and here we report its effect on cardio-respiratory variables.

MATERIAL AND METHODS

Chemicals: DRDE-07 was synthesized in the chemistry laboratory. The compound was characterized by elemental analysis, IR, 1H NMR and mass spectral analysis. The purity was checked by thin-layer chromatography. All other chemicals used were of analytical grade.

DRDE-07: NH₂ - CH₂ - CH₂ - NH - CH₂ - CH₂ - S - C₆H₅

Animals : Wistar male rats (225-300 g, body weight), randomly bred and maintained in the animal house were used for the study. Thirty-six rats were used in the present study and they were divided into nine groups of four animals each. The animals were housed in polypropylene cages on dust-free rice husk as the bedding material. The animals were provided with pellet diet (Amrut Ltd., India) and water ad libitum. The care and maintenance of the animals were as per the approved guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, India. This study has the approval of the Establishment′s Ethics Committee.

Cardio-respiratory parameters in anesthetized rats: The mean arterial blood pressure, heart rate, respiratory rate and tidal volume were recorded using a six-channel polygraph (Graphtec Linerecorder, Germany). The rats were anesthetized with urethane (1.6 g.kg^-1, i.p.) and the neck region was dissected and exposed. The trachea was cannulated and connected to a pneumotachograph. The right jugular vein was cannulated and a syringe was connected for intravenous administrations. The left carotid artery was cannulated with a thin polypropylene tube connected to a pressure transducer (Statham P23Dc), filled with heparinised normal saline. The pressure transducer was connected to a preamplifier (low-level DC, Grass Instruments, USA) and arterial blood pressure was recorded on the polygraph. Mean arterial pressure was calculated from the recorded blood pressure. The signals from the DC Driver amplifier recording the blood pressure were taken from J6 output and fed into the external triggering (High) input of EKG Tachograph preamplifier (Grass Instruments, USA) for recording the heart rate. The pneumotachograph was connected to a differential pressure transducer (SWEMA, Germany) and connected to a pre-amplifier (Low level DC, Grass Instruments, USA), and inspiration was recorded as an upward deflection and expiration as a downward deflection.[22] A polythene tube was inserted through the esophagus to the stomach for feeding DRDE-07. After the surgical procedures, the rat was allowed to stabilize for about 1 h. Three doses of DRDE-07 viz., 0.5, 1.0 and 2.0 LD₅₀ (in distilled water) were administered intraperitoneally (LD₅₀ = 141 mg/kg).[21] Four doses of DRDE-07 viz., 0.25, 0.5, 1.0 and 2.0 LD₅₀ (in distilled water) were administered orally (LD₅₀ = 1599 mg/kg).[21] The control group was administered distilled water.

Statistical analysis
All values were converted as percent of control and represented as mean±SEM. The data were analyzed by one-way ANOVA followed by Student-Newmann-Keuls multiple comparison procedure. P values <0.05 were considered statistically significant. SigmaStat (Jandel Sci., USA) was used for the statistical calculations.

RESULTS

The control values (mean±SE, n=4) of the mean arterial blood pressure, heart rate, respiratory rate and tidal volume in the anesthetized rats were 110±4 mmHg, 338±11 per minute, 37.0±3.3 per minute and 0.66±0.03 ml/min respectively. During the two-hour monitoring period the control animals showed a steady response.

Intraperitoneal administration of DRDE-07 did not show any effect on the blood pressure, heart rate, respiratory rate and tidal volume at 0.5 and 1.0 LD₅₀ doses. At 2.0 LD₅₀ dose the animals died within 60 minutes and a significant decrease in blood pressure and tidal volume was observed [Table - 1]. Oral administration of 0.25 LD₅₀ of DRDE-07 did not show any significant change on the cardio-respiratory variables. 0.5 and 1.0 LD₅₀ of DRDE-07 administered orally showed a significant decrease in the mean arterial blood pressure after 60 min. There was no change in the heart rate, respiratory rate and tidal volume. None of the animals died in the two-hour monitoring period following 1.0 LD₅₀ dose. Oral administration of 2.0 LD₅₀ of DRDE-07 induced a sudden decrease in the mean arterial blood pressure and the animals died within 60 min [Table - 2].

DISCUSSION

Antidotes to SM can act by four different mechanisms: (a) prevention of SM from entering the system (personal decontamination at the site of contact), (b) prevention of SM from alkylating critical target molecules mainly DNA, (c) retrieval of SM alkylated DNA and (d) prevention and reversal of the cascade of secondary biochemical reactions of alkylation.[5] SM is known to react with a variety of macromolecules viz., DNA, proteins and several thiol-containing molecules including GSH.[7] A decrease in the GSH content was reported following percutaneous administration of SM.[6] If thiol-containing compounds are administered as prophylactic agents they can spare GSH by neutralizing SM. Amifostine is one such thiol-containing compound recommended as an adjunct to anticancer agents viz., cisplatin and cyclophosphamide which selectively protects normal tissues without reducing the effect of the anticancer agents on the cancer cells.[23],[24],[25],[26],[27] Analogues of amifostine with better lipophilicity were synthesized that were expected to have better efficacy. Among the several compounds synthesized, DRDE-07 gave better protection when it was administered through the oral route, against percutaneously administered SM.[19],[21]

Amifostine and DRDE-07 did not protect when SM was administered by oral, subcutaneous and inhalation routes. When SM was administered through percutaneous route amifostine and DRDE-07 were effective, showing that the mechanism of the toxic effect of SM varies with different routes. Interestingly, percutaneously administered SM was more toxic in the rodent species viz., mice and rats.[28]

The cardio-respiratory effects of DRDE-07 were studied at various doses for two reasons: (i) to evaluate the safety of DRDE-07 and (ii) to ascertain whether DRDE-07 causes a profound fall in blood pressure that reduces the absorption of SM when the latter is administered through the percutaneous route. The LD₅₀ of DRDE-07 by intraperitoneal and oral routes in male rats were 141 and 1599 mg/kg respectively. The doses that were employed for the prophylactic effect were 0.05, 0.1 and 0.2 LD₅₀ doses, orally.[19],[21] In the present study up to 0.5 and 1 LD₅₀ of DRDE-07 by the intraperitoneal route did not produce any change in the cardio-respiratory parameters. Oral administration of 0.25 LD₅₀ did not show any effect on the blood pressure. So the possibility of the reduced absorption of SM when it is administered through the percutaneous route is ruled out at lower doses of DRDE-07.

In conclusion, DRDE-07 did not show any significant effect on the cardio-respiratory variables at a dose of 0.25 LD₅₀ by the oral route and 1.0 LD₅₀ by the intraperitoneal route. The death after intraperitoneal or oral administration (2.0 LD₅₀) of DRDE-07 is due to a sudden fall in the mean arterial blood pressure.

ACKNOWLEDGEMENT

REFERENCES

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