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Investigation of new neuromuscular blocking agents was started 30 years ago in Richter Ltd. This paper presents the results obtained by Richter's scientists. 2 compounds out of 100 bisquaternary ammonio steroid having androstane skeleton were selected for further pharmacological study. One of these agents, pipecuronium bromide (Arduan) elicited long-lasting block of neuromuscular transmission without cardiovascular side effects in both animal experiments and clinical studies. Arduan is a powerful competitive antagonist of acetylcholine, since it can bind pre- and postsynaptic (N1) receptors of the transmitters. It has no remarkable cumulative effect. Neostigmine rapidly and completely antagonized the neuromuscular blockade caused by pipecuronium. Arduan was introduced into clinical practice. The second compound, RGH-4201 (Duador) evoked a neuromuscular block of short duration. It showed slight atropin-like cardio-vagolytic effect in animal experiments. In the clinical studies, however, the cardiovascular side effects were found to be too strong. Therefore, it was not introduced in clinical practice.

The following methods are described for the analytical investigation of pipecuronium bromide. 1. HPLC method. Of the several systems tried for the separation and quantification of impurities and degradation products the best results were obtained using silica as the stationary phase and 43:43:14 mixture of methanol, acetonitrile and concentrated aqueous ammonia containing 0.1 mole/l each of ammonium chloride and ammonium carbonate as the eluent. The validation of this method is presented. The above described aggressive eluent can be successfully replaced by an ion-pairing system using silica as the stationary phase and 96:4 mixture of acetonitrile and water containing 0.1 mole/l sodium perchlorate as the eluent. 2. Thin-layer chromatography. TLC systems are described for the separation and densitometric quantification of the impurities and degradation products of pipecuronium bromide. 3. Spectrophotometry. Two methods are described. The ester groups of the molecule can be determined by the iron(III)-hydroxamate method while for the ion-pair extraction of the quaternary ammonium steroid picric acid or bromthymol blue are used as the reagents. 4. Titrimetry. In addition to the titration with acetous perchloric acid for the assay of the bulk material a microtitration method is described for the determination of pipecuronium bromide in individual lyophylized ampoules (potentiometric titration with 0.1 M silver nitrate).

The mass spectral behaviour of the bis-quaternary salt pipecuronium bromide and some related Cn+ A-n type ammonium salts (n = 1, 2, 3) has been studied and compared. Under EI-MS conditions the evaporation of the samples preceded by in situ dealkylation led to formation of alkyl bromide and amine bases. It has been demonstrated that this technique, completed with quantitation of the relative amount of the decomposition products is applicable to get structure information about the basis part as well as the number and positions of the quaternary centres and the substituents at these centres. The FAB mass spectra of these mono-, bis- and this quaternary ammonium salts were found to be very interesting and provide direct structural information about the salt. They exhibit primary Cn+ intact cations and (Cn+ A-n + 1)+ single charged cluster ions and also fragments characteristic of partial structures at the quaternary centres.

The paper presents clinicopharmacological investigations of Arduan carried out in Hungary and in other countries. The aim of the research work is to find an ideal muscle relaxant without side-effects, which are mainly cardiovascular. Pipecuronium bromide (Arduan) is a compound with steroid structure; it has a long duration of action and, most importantly, it does not affect circulation. The properties of pipecuronium bromide will be discussed and compared to those of pancuronium and vecuronium bromide.

As a consequence of the presence of two ester groups in its molecule the main decomposition route of pipecuronium bromide (active ingredient of Arduan injection) is hydrolysis. In addition to this, autoxidation can also take place. In order to obtain a stable dosage form liophylized ampoules containing mannitol were prepared. Mannitol is not only a filling material but its role is also to decrease the dielectric constant of the solvent water thus increasing the stability of the drug substance before and during the liophylization procedure and after the dissolution of the content of the ampoules for administration. In order to protect the drug molecule against oxidation inert gas atmosphere was used during the production of the ampoules. The preparation is compatible with basic infusion solutions.

A series of bisquaternary ammonio steroids having androstane skeleton have been prepared some of which possessed high neuromuscular blocking activity. One of the series 3 alpha, 17 beta-diacetoxy-2 beta, 16 beta-bis (4,4-dimethyl-1-piperazinyl)-5 alpha-androstane dibromide (19, pipecuronium bromide, ARDUAN) has proved to be a clinically useful agent of long duration of action without side effects. The preparation of pipecuronium bromide and its metabolites and the impurities are also described. The structure of 19 and related compounds was elucidated by spectrometric methods IR, NMR and MS.

The synthesis of Pipecuronium bromide together with the investigation of its impurity profile required the solution of a number of analytical and structural problems. NMR spectroscopical methods received a major role in the structure elucidation of the synthesized compounds. The quantitative assessment of contaminations was also realized by 1H NMR spectroscopy. Out of the large amount of undertaken investigations, here we provide the structural characterization of the contaminating regiosimers of an important intermediate (5 alpha-androst-2-en-17-on) formed in the course of the synthesis, together with the NMR signal assignments of the end-product (Pipecuronium bromide) as measured in D2O and a mixture of CDCl3/DMSO-d6. The structure of a minor contamination of the end-product is also given.

The following methods are described for the analytical investigation of the intermediates of the synthesis of pipecuronium bromide (Arduan) (for the numbering of the intermediates and their impurities see Figure 1.). 1. Gas chromatographic methods (capillary GC using fused silica capillaries Ultra-2 and Silar 10C WCOT) for the impurity profiling of intermediates I, II, IV and V including the identification and spectroscopic characterization of their impurities; 2. TLC methods for the similar characterisation of the further intermediates (III, VI, VII and VIII); 3. Gas chromatographic assay methods for IV and V using fused silica capillary technique and internal standards; 4. Potentiometric titration methods for the determination of VII and VIII using 0.1 M hydrochloric acid as the titrant.

Pipecuronium bromide molecule contains two slightly basic nitrogen atoms in its two piperazine rings. Their pK values are quite similar to each other in consequence of their almost equivalent surroundings. Although the potentiometric titration used mostly for determination of pK values of organic compounds can be performed in this case too, but the polarimetric titration applied by us is more advantageous. Change of pH considerably influences the values of optical rotation; consequently, the curve obtained in this way is more utilizable than that of potentiometric titration. Protonation of two nitrogen atoms one after the other changes the optical rotation of the molecule in opposite direction. Consequently, a minimum can be seen on the curve of optical rotation versus pH value. The exact pK values were determined on the basis of the estimated parameters of a curve established corresponding to an equation and fitted to the measuring points. The sequence of deprotonation of nitrogen atoms could only be ascertained on the basis of the curves of polarimetric titration of compounds containing one piperazine ring; on the basis of the pK values and of the changing direction of optical rotation.

The pharmacokinetics of pipecuronium bromide (Arduan) studied in animals (rat, dog, cat) and in humans can be described by a two-compartment open model. The elimination half-life in animals was found to be around 40 min, in humans between 44-137 min. The main route of elimination is the excretion of the unchanged molecule in urine. In kidney patients the elimination becomes longer. The metabolism in the elimination is less important, in rats 3-hydroxy-, 17-hydroxy and 3,17-dihydroxy metabolites have been identified.

The experimental results in animals suggest that pipecuronium bromide offers the possibility of a neuromuscular blocking agent without side effects for surgical procedures of long duration. Its mechanism of action is twofold: 1. antagonism of acetylcholine effect at neuromuscular junction (postsynaptic nicotine receptors), 2. inhibition of acetylcholine release (presynaptic nicotine receptors). Its neuromuscular blocking potency is somewhat greater (2.0-3.0) than that of pancuronium in all species studied, and the duration of action is twice of that. It has no remarkable cumulative effect. Neostigmine rapidly and completely antagonises the neuromuscular blockade caused by pipecuronium. Certain structural properties (e.g. pipecuronium has no acetylcholine-like fragments in contrast with pancuronium and the interonium distance is also considerably larger than in pancuronium) may predict advantages. This has been proved by low vagal blocking--and ganglion--blocking potencies. On the basis of these a wide margin of safety can be expected in humans as well in preventing cardiovascular side effects. Pipecuronium is also characterized by interactions--only slight interactions--with other drugs used mainly in perioperative period.

The authors elaborated a method for the determination of Arduan and its desacetyl metabolites in biological fluids. The method is based on the use of labelled Arduan and on the determination of radioactivity of the parent drug and metabolites separated by ion-pair TLC in the development system of chloroform-dichloromethane-methanol 6 : 2 : 2 (v/v) 3% NaI (w/v).