Salvarsan

SALVARSAN, Salvarsan (Alt-Salvarsan Ehrlich-Hata 606), 4,4'-dihydroxy-3,3'-diaminoarsenobenzene dihydrochloride. Molecular weight 475. Released in 1910 by the firm Meister, Lucius & Brüning in Germany. In other countries, Salvarsan is manufactured under the following names: in France—Arsenobenzol, Arsenobillon, Arsenobenzene; in England—Kharsivan; in Italy—Jacol; in Japan—Arsaminol, Tanvarsan, Arsemin; in the USA—Arsphenamine; in 1915 it was released into circulation in Russia under the name Arsol (production discontinued); synthesized by Ostromyslensky. Salvarsan is included in the Russian Pharmacopoeia. Physicochemical properties. Salvarsan is a pale yellow powder; a colloid. It has no melting point. It dissolves easily in methyl alcohol, ethylene glycol, glycerin, and water; it is more difficult to dissolve in ethyl alcohol, and is insoluble in ether, acetone, and benzene. Aqueous solutions have an acidic reaction to litmus and a weakly violet reaction to Congo red. Upon adding 2 moles of caustic alkali to an aqueous solution of Salvarsan, the Salvarsan base precipitates, which, upon further addition of alkali, returns to solution, forming a phenolate. In a soda solution, Salvarsan dissolves poorly and is completely insoluble in bicarbonate. Consequently, phenolate solutions become cloudy when standing in air, absorbing carbon dioxide. Salvarsan is precipitated from concentrated aqueous solutions by the addition of concentrated hydrochloric acid. Salvarsan, unlike neosalvarsan, does not decolorize indigo carmine. Paradimethylaminoazobenzene in dilute HCl gives an orange solution with Salvarsan, from which an orange precipitate then falls out. Upon adding benzaldehyde and mercuric chloride to the solution, the reaction becomes significantly sharper and can be used to detect Salvarsan in body tissues. Hydrogen sulfide in an acidic medium does not produce a precipitate. Bettendorff's reagent releases a yellow amorphous precipitate. With ferric chloride, a red coloration of the solutions is obtained. Silver nitrate gives a dark red coloration of the solution, and upon the addition of nitric acid, a brown precipitate falls out. The amino group in an acidic medium is diazotized with sodium nitrite, and upon the addition of an alkaline solution of resorcinol, an intensely red coloration is obtained. This reaction is used for the determination of Salvarsan in urine. Salvarsan solutions decompose easily during storage, even without access to air, with alkaline solutions being less stable than acidic ones. Moist Salvarsan decomposes when heated to 65°, while dry Salvarsan is stable up to 175°. Salvarsan in dry form is easily oxidized by atmospheric oxygen, turning into amino-oxyphenylarsine oxide, which is 20 times more toxic. Salvarsan, prepared by reduction with hydrosulfite (blankit) from nitro-oxyphenylarsinic acid, always contains a certain amount of Salvarsan with sulfino- and sulfo-groups connected to the benzene ring. These sulfo-compounds are approximately 2 times more toxic than Salvarsan itself and have only half the therapeutic effect. The sulfur content is determined by the Carius method and ranges from 1% to 3%. Methods of production of Salvarsan. The starting product for the production of Salvarsan is 3-nitro-4-hydroxyphenylarsinic acid. 1) Reduction in one phase with hydrosulfite proceeds according to the following scheme:

+20NaHSO₃. As=As. Into an enameled vessel with a capacity of 30 liters equipped with a stirrer, 13 liters of water, 513 g of magnesium chloride, and 2,950 g of hydrosulfite are loaded, and a solution of 197 g of nitrooxyphenylarsinic acid in 4.5 liters of water and 135 cm³ of a 10n solution of sodium hydroxide is added. The mixture is heated on a water bath at 55-60° until the complete precipitation of the Salvarsan base, which takes about 2 hours. The resulting precipitate is suction-filtered and washed with water. The crude product is dissolved in 1,700 cm³ of methyl alcohol with the addition of methyl-alcoholic hydrochloric acid. The solution is filtered and Salvarsan is precipitated by pouring the solution into a 10-fold volume of ether. The precipitate is filtered off, washed with ether, and dried in a vacuum over sulfuric acid. All operations are conducted in an atmosphere of carbon dioxide or nitrogen. 2) A purer product can be obtained by conducting the reduction gradually: a) from nitrooxyphenylarsinic acid, 3-amino-4-oxyphenylarsinic acid is obtained; b) 3-amino-4-oxyphenylarsinic acid is reduced with sulfur dioxide in the presence of potassium iodide and sulfuric acid to aminooxyphenylarsine oxide; c) the resulting oxide is converted into Salvarsan by hydrosulfite or sodium amalgam. 3) Excluding the production of the oxide as an intermediate product, aminooxyphenylarsinic acid can be reduced to Salvarsan with hydrosulfite or, better, electrolytically in a sulfuric acid medium on lead electrodes in the presence of iodine. Upon cooling the solutions reduced by current, the sulfate of diamidodioxyarsenobenzene precipitates. The sulfate is dissolved in dilute hydrochloric acid, and its hydrochloride, i.e., Salvarsan, is precipitated with concentrated hydrochloric acid; it is then suction-filtered, washed with 20% hydrochloric acid, alcohol, and ether, and dried in a vacuum desiccator. Analysis of Salvarsan: 1) Determination of the impurity aminooxyphenylarsine oxide: 1 g of Salvarsan is dissolved in a 100 cm³ volumetric flask in 10 cm³ of methyl alcohol, and 25 cm³ of water is added. The Salvarsan base is precipitated by the gradual addition of 1.5 g of pure chalk. Water is added to make 100 cm³, and the mixture is filtered through a dry filter into a dry flask. 50 cm³ of the filtrate is taken, and 75 cm³ of water and 5 cm³ of n hydrochloric acid are added to it. It is titrated with an n/20 iodine solution in the presence of starch. Good preparations require no more than 0.5-0.8 cm³ of iodine solution, which corresponds to an oxide content of 0.5-0.8%. 2) Salvarsan is titrated with iodine in a hydrochloric acid medium, oxidizing into aminooxyphenylarsinic acid: OHNH₂C₆H₃As=AsC₆H₃OHNH₂ + 8J + 6H₂O = 2OHNH₂C₆H₃AsO₃H₂ + 8HJ. The Gehe method for the volumetric determination of Salvarsan is based on this reaction. The reaction is reversible to a certain extent, and therefore a corresponding correction is introduced. Experience has shown that for 1 mole of Salvarsan, not 8, but only 7.509 atoms of iodine are consumed, so that 1 cm³ of n/10 iodine corresponds to 0.006323 g of Salvarsan. In addition, the content of arsenic and nitrogen is determined (for methods, see below—Neosalvarsan). Arsenic is usually contained in an amount a few tenths less than the theoretical value, i.e., 31.6%, and nitrogen at 5.8%. Salvarsan is weighed into ampoules of 0.1, 0.2, 0.3, 0.4 g. The ampoules are evacuated and sealed. The preparation is stored in the dark and in a cool place. Testing of Salvarsan on animals according to Kolle-Zieler: 0.5 cm³ of a 1:150 Salvarsan solution, brought to dissolution by the addition of alkali, is injected into 6 mice. The solution is injected slowly to eliminate the possibility of shock. If 75% survive, the preparation is considered "hyperideal"; if 60% survive, the preparation is considered "ideal"; if less than 60% survive, the preparation is rejected. The preparation is used in the treatment of syphilis, relapsing fever, and other diseases caused by spirochetes, as well as malaria. According to Kolle-Zieler, the German preparation acts as follows: for rabbit syphilis, doses per 1 kg of weight: MLD (minimum lethal dose) - 1.25; MTD (maximum tolerated dose); MCD (maximum curative dose); for relapsing fever in mice, doses per 20 g of weight: MLD - 1/10, MTD - 1/20, MCD - 1/600, MTD = 0.1. Before being released for sale, after testing on mice, the preparation undergoes clinical examination. At the present time, Alt-Salvarsan ("606") is almost never used in medical practice, as the preparation of the solution requires the addition of alkali, and due to its high toxicity, it is possible to use only weak, therapeutically less active solutions; Alt-Salvarsan has therefore been replaced by easily soluble preparations of the Neosalvarsan type. Neosalvarsan, 914, Neosalvarsan (novarsenol, neoyacol, etc., are names for Salvarsan in different countries with the addition of the prefixes "neo" or "novo"); the preparation is accepted by the Pharmacopoeia VII. To replace Alt-Salvarsan, "Salvarsan-sodium" was first released, which eliminates alkalinization when preparing solutions, but it did not find wide application. Most widely used is Neosalvarsan, released in 1912 by the firm Meister, Lucius & Brüning, which is a compound of Salvarsan with Rongalite (formaldehyde-sulfoxylate). According to German data, the preparation has the following formula: OH OH H₂N-C₆H₃-As=As-C₆H₃-NH-CH₂OSONa, molecular weight 466, but precise analyses show that there is an admixture of a disubstituted product: OH OH NaOSO-CH₂-NH-C₆H₃-As=As-C₆H₃-NH-CH₂OSONa. Disubstituted Salvarsan is less toxic than monosubstituted, but possesses a lower therapeutic effect. The commercial preparation is not an individual substance, which is shown by the arsenic content in the preparation in the amount of 18-20%, whereas theoretically, monosubstituted Salvarsan should contain 32.2%. From this, it follows that the active substance in commercial Neosalvarsan is contained in the amount of 52-60%. The 40-48% of impurities consist not only of the disubstituted product but also of Rongalite, sulfate, sulfite, sodium chloride, and others. Neosalvarsan is a yellow or orange-yellow powder, easily soluble in water. The purer the preparation, the more easily it dissolves. It is easily soluble in glycerin, insoluble in methyl alcohol, ethyl alcohol, ether, and benzene. The reaction for the unsubstituted amino group is the same as for Salvarsan. An aqueous solution of Neosalvarsan decolorizes indigo carmine—this is a difference from Salvarsan. With ferric chloride, solutions of Neosalvarsan and Salvarsan are initially colored green; upon further addition of ferric chloride, the Salvarsan solution turns red, and the Neosalvarsan solution turns violet. When heated with bisulfite, a Salvarsan solution gives a yellow precipitate, whereas Neosalvarsan gives no precipitate. The preparation is obtained by the method of P. F. Ryumpin: the filtered and washed base, after the reduction of nitrooxyphenylarsinic acid with hydrosulfite, is dissolved in methyl alcohol with the addition of HCl and filtered. To the resulting solution, a calculated amount of Rongalite in aqueous solution is added and heated to 30°. Neosalvarsan acid precipitates in the form of a clumping precipitate. The alcohol is decanted. Fresh alcohol is added, and the precipitate is ground into a powder. To convert the Neosalvarsan acid into a salt, alkali is added to its suspension in alcohol. The alcohol is decanted from the Neosalvarsan that has separated in the form of a gum, it is dissolved in water, table salt is added, filtered, and poured into 96% alcohol. The precipitated Neosalvarsan is suction-filtered, washed with alcohol and ether, and dried in a vacuum, first at ordinary temperature, and then at an elevated one. All reactions are conducted in an atmosphere of carbon dioxide or in a vacuum. The preparation is stored in evacuated, sealed ampoules (it is unstable in air!), in the dark and in a cool place. Analysis of Neosalvarsan (chemical control). 1) Arsenic content. 0.2 g of Neosalvarsan and 1 g of powdered potassium permanganate are placed into a dry Erlenmeyer flask with a ground-glass stopper. 5 cm³ of 10% sulfuric acid is added and shaken for several minutes. 5-10 cm³ of concentrated sulfuric acid is added dropwise over the course of 5-10 minutes. Hydrogen peroxide is added until the solution is decolorized, then 25 cm³ of water is added, and the mixture is boiled for 10 minutes. The mixture is diluted with 50 cm³ of water, and the residue of hydrogen peroxide is destroyed by dripping n/10 potassium permanganate solution until a faint pink color appears. By adding n/10 oxalic acid, the solution is decolorized and boiled for 15 minutes. Upon cooling, 2.5 g of chemically pure potassium iodide is added, the flask is closed with a stopper, and left for 1 hour in the dark. The solution is diluted with 150 cm³ of water and the liberated iodine is titrated with n/10 thiosulfate. 1 cm³ of n/10 thiosulfate corresponds to 0.00375 g of arsenic. 2) Determination of nitrogen. 0.5 g of Neosalvarsan is heated with 20 cm³ of concentrated sulfuric acid and a small amount of copper sulfate until the liquid is decolorized. Upon cooling, the flask is connected to a condenser, 80-100 cm³ of 33% sodium hydroxide is added from a dropping funnel inserted into the stopper, and the ammonia is distilled into a flask with 20-30 cm³ of n/10 sulfuric acid, cooled with ice. The excess acid is titrated with n/10 alkali. 3) The impurity of sodium chloride is determined as follows: 0.5 g of Neosalvarsan is dissolved in 15 cm³ of water, and 5 cm³ of 10% nitric acid is added. The flask is placed on ice for 10 minutes. The yellow precipitate of Neosalvarsan acid is filtered off and washed with water.

To the filtrate, add 10 cm³ of 10% sulfuric acid, heat to 70-80°, oxidize with ⁿ/₁₀ potassium permanganate until a constant color is achieved, destroy the color with a few drops of 3% hydrogen peroxide, and precipitate the chlorine with 10 cm³ of 5% silver nitrate. Leave overnight in a dark place. In the morning, filter the silver chloride through a Gooch crucible and wash the precipitate with water. Dry first at 100°, then at 130° for an hour, and weigh. 4) Determination of the iodine number. This number is understood as the quantity of cubic centimeters of ⁿ/₁₀ iodine required for the oxidation of 0.1 g of neosalvarsan. This number provides a comparative assessment of the content of reducing impurities in neosalvarsan (rongalite, sulfite). Dissolve 0.1 g of neosalvarsan in 100 cm³ of water, add 20 cm³ of ⁿ/₁₀ iodine, and titrate the unconsumed iodine back with ⁿ/₁₀ thiosulfate until decolorization. 5) The determination of sulfur in neosalvarsan is of very great importance, as it allows one to judge not only the impurities and their composition but also what part of the product is in the form of mono- and what part is in the form of disubstituted S. Sulfur in neosalvarsan is found in the form of: 1) S. linked to amino groups as its formaldehyde sulfoxylate compound; 2) S. linked to the benzene ring in the form of sulfinic and sulfonic groups, if the neosalvarsan was prepared from S. obtained with hydrosulfite; 3) in the form of organic compounds not linked to S. (excess rongalite), and in the form of inorganic compounds (sulfate, sulfite). Determination of total sulfur (A): the substance is heated in a sealed tube with nitric acid according to Carius. The resulting sulfuric acid is precipitated as barium sulfate and weighed. Only with such strong oxidation is the sulfur linked to the benzene ring split off. Determination of sulfur linked to amino groups, together with sulfur in impurities (B): dissolve 0.2 g of neosalvarsan in water. Boil the solution for 8 hours, maintaining a dark yellow color the entire time by the gradual addition of ⁿ/₁₀ iodine; replace evaporated water with fresh water. Filter the liquid and wash the filter with water. To the filtrate, add 3 cm³ of 10% hydrochloric acid, heat to boiling, and precipitate the resulting sulfuric acid with barium chloride as barium sulfate, which is filtered, dried, and weighed. Determination of sulfur contained in impurities (C): dissolve 0.5 g of neosalvarsan in a small amount of water. Add 5 cm³ of 10% hydrochloric acid to the solution and place on ice. Filter the precipitated neoacid and wash with water. Oxidize the filtrate with iodine as before and isolate the sulfuric acid from it as barium sulfate. Filter the barium sulfate on a Gooch crucible, dry, calcine to constant weight, and weigh. Calculation of results: A-B = quantity of sulfur linked to the benzene ring. B-C = quantity of sulfur linked to amino groups. C = quantity of sulfur in impurities (organic and inorganic). In addition to determining the chemical composition, the physical properties of the preparation are also established, and the concentration of hydrogen ions in its aqueous solutions is determined. The color of the preparation must be yellow. Solubility: pour 0.2 g of the preparation in small portions, waiting for dissolution each time, into 5 cm³ of distilled water poured into a small beaker 40 mm in diameter. The total duration of the dissolution process must not exceed 5 minutes. The solution must not become cloudy upon the addition of a few drops of sodium bicarbonate solution (impurity C). Determination of pH in a neosalvarsan solution: dissolve 0.6 g of neosalvarsan in 15 cm³ of distilled water that has been well-boiled and cooled without access to air. Prepare buffer mixtures from solutions of 0.2 mole of disodium phosphate in 1 liter and 0.1 mole of citric acid in 1 liter according to Kolthoff. Add 1 drop of 0.04% bromothymol blue solution as an indicator to all test tubes and compare the colors in a comparator: the pH of neosalvarsan ranges from 6 to 7.8; according to data from the State Venereological Institute, the pH ranges from 6.1 to 7.82 (L. Smorodintseva); the best, least toxic preparations have a pH of 7-7.5. Batches of the preparation with an acidic pH yield a significant percentage of toxicity; high alkalinity can also cause toxicity; a neutral pH (about 7.0) guarantees to a significant degree both the non-toxicity of the batches and their stability (data from the State Venereological Institute). Biological testing of neosalvarsan. The necessity of biological testing of salvarsan preparations was firmly established by Ehrlich when he became convinced through numerous experiments on both animals and humans that a specific chemical composition of the preparation does not guarantee a strictly defined degree of toxicity and its therapeutic effect. An explanation for this discrepancy between chemical composition and biological influence on a living organism was given by Bauer, who proved the colloidal nature of salvarsan preparations and, consequently, the importance of their physical-chemical state (dispersion) in addition to their chemical structure for their physiological and toxic influence on the organism. The biological influence of salvarsan and its derivatives was studied by Ehrlich and his students on various animals (rabbits, rats, mice). There was no need for a specific instruction for broad guidance on biological testing at that time, as the manufacture of salvarsan preparations was carried out according to a patent by only one chemical plant (Hoechst-Farbwerke), where the entire production process—up to the biological testing—took place under the direct control of Ehrlich himself. In other states, similar biological control was performed on the same animals, but according to standards not regulated by the state. In recent years, instructions of an official nature have been developed in a number of states (America, England, France, Italy, Poland). In the USSR, for a number of years, the testing of Soviet neosalvarsan was performed according to an instruction developed by the State Commission for the Testing of Salvarsan Preparations and approved by the Scientific Council of the People's Commissariat of Health. In 1933, in connection with the large amount of both Soviet and foreign material accumulated on biological testing, a special commission reviewed this issue and came to the following conclusion: existing literary data on the question of methods for the biological testing of salvarsan are highly contradictory—while German researchers (the Kolle school) prefer the white mouse as the object, French authors (Launoy and Nicolle) consider the white mouse completely unsuitable for these experiments and recommend rabbits; American researchers (Kolmer, Raiziss) conduct experiments in parallel on mice and rats. The experiments of Soviet researchers also diverge significantly: I. Krichevsky and Petrov saw satisfactory results in experiments on mice; Bets, Savelyev, and Yu. Finkelstein worked on rabbits. Stepun, although he worked on mice, nevertheless emphasized their increased sensitivity to salvarsan. In general, in experiments on any animal, researchers do not consider it possible to predict with absolute certainty the course of salvarsan reactions in humans based on the reaction in an animal. As for the so-called nitritoid crises observed in humans, these, in the opinion of Jadassohn, are not observed at all in animals. The above-mentioned literary discrepancies compel the State Commission for the Testing of Salvarsan Preparations for the present moment to adopt as the basis for the biological testing of Soviet novarsenol the German method on mice and rats as the most standard (in the form of the German instruction) and approved by the Sanitary-Hygienic Section of the League of Nations, and which bears to a certain extent an international character. Biological testing of neosalvarsan breaks down into the determination of toxicity and the determination of therapeutic effect in an experiment on animals. Determination of toxicity. A batch is tested by three different researchers. Two ampoules of 0.3 are taken, each of which serves for an experiment on 5 mice with an average weight of 15 g. In addition to this, another researcher determines the toxicity on 5 rats with an average weight of 150 g from one ampoule. Thus, the preparation is administered to 30 mice and 5 rats. The testing is conducted by intravenous administration to mice and rats of the limit doses indicated below, from which a portion of the animals may perish (the borderline tolerated dose). Doses are established for mice per 20 g, and for rats per 1 kg of weight (well-fed, but fasted for 4 hours). The duration of observation is 3 days for mice and 6 days for rats. 0.3 of a batch is poured into a Jena glass flask with a capacity of 10 cm³, filled with 6 cm³ of double-distilled water, so that the powder rests on the surface of the water. In the case of the formation of a precipitate, the latter is dissolved by light shaking of the flask or with the help of a pipette. From this 5% stock solution, the necessary dilutions are prepared with the help of 0.6% physiological saline. In the first series of experiments, 5 mice receive intravenously 1 cm³ of a 1:135 dilution each, in the second—1 cm³ of a 1:120 dilution each per 20 g of body weight. The solution is administered heated to 30°. In addition to this, 5 rats (average weight 150 g) are injected into the tail vein with 4.5 cm³ of a 5% solution per 1000 g of weight (0.225 g of neosalvarsan per 1 kg).

The injection of the solution must be performed slowly to prevent shock—approximately in half a minute. The evaluation of the flawlessness of a manufactured batch in terms of toxicity is performed by comparing individual series of experiments with each other on the basis of the generalized result of all tests. A manufactured batch is considered suitable if 60% of mice that received a dilution of 1:135 survive, and 50% of mice that received a concentration of 1:120 survive, and 60% of rats. Determination of therapeutic effect. The therapeutic effect is tested by experiments on infected mice, by comparison with standard neosalvarsan. 24 mice are infected with freshly obtained serum from the blood of a mouse infected with trypanosomes. The serum is diluted with 0.85% physiological solution in such a way that in each field of view at a magnification of about 400, there should be 8-10 trypanosomes. Each mouse is injected subcutaneously with 0.5 cm³ of this serum. The next day, about 4-9 trypanosomes should be found in 40 fields of view in these mice. With such an intensity of infection, the animals are used for testing the therapeutic effect. 9 mice are injected intravenously with standard neosalvarsan, the same number of mice are injected with the test batch, and in both series of experiments, every 3 mice receive the same doses. The remaining 6 mice remain untreated as a control for the outcome of the infection. The table below shows the average doses used when testing the therapeutic effect: of the blood taken on a cover glass from the tail, likewise at a magnification of about 400 with recording of the counting results. The blood examination data are recorded as follows: Standard neosalvarsan Test neosalvarsan 3 mice - 1:12,000 3 mice - 1:8,000 3 mice - 1:5,000 1:12,000 1 cm³ per 20 g 1:8,000 1 cm³ per 20 g 1:5,000 1 cm³ per 20 g. In all animals, during the 10-day observation, a blood examination is performed daily, with at least 40 fields of view of the blood preparation being examined ⁺ \ +++ 1-3 parasites in 40 fields of view 1-9

» » each field of view ......9 and more parasites in each field of view. The table below gives the average assessment of the course of infection produced by the strain of trypanosomes used in treatment with standard neosalvarsan. Standard salvarsan (mice infected the day before and showing +). Day 1:12,000 1:8,000 1:5,000 + + + + + 1 - +-m- ++ - + - + j - - +++ - ++ - 1 - - +++ j ~ 7 8 | + + ++ ++!-{- Myosalvarsan (Myosalvarsan-France), sulfosalvarsan (Sulfosalvarsan-Germany), sulfarsphenamine (Sulfarsphenamin-USA). Disodium salt of 3,3'-diamino-4,4'-dihydroxyarsenobenzene dimethane-disulfonic acid: OH OH NaO₃SOCH₂NH-C₆H₃-As=As-C₆H₃-NHCH₂OSO₃Na. These preparations were not included in the Russian Pharmacopoeia. Myosalvarsan is manufactured in France by the firm R. Pluchon, Paris, and was introduced into use in 1919. It is a light yellow powder (French is purely yellow, German has an orange tint), extremely easily soluble in water. Aqueous solutions are significantly more stable than solutions of neosalvarsan. It is insoluble in alcohol, ether, and acetone. Upon heating, the preparation decomposes without melting, with the release of sulfurous acid. Indigo carmine decolorizes only if the myosalvarsan solution is treated with zinc dust and acetic acid and indigo carmine is added to the filtrate (a distinction from neosalvarsan). Free myoacid can be isolated from aqueous solutions with acetic acid. Upon its analysis (see above, neosalvarsan and salvarsan), ratios of sulfur, nitrogen, and arsenic atoms of 1:1:1 are obtained, which indicates a disubstituted product. Theoretically, the arsenic content in the preparation should be 25.06%, but the commercial product contains only 18.5-19.5%, which indicates foreign impurities (as in neosalvarsan). For the preparation of myosalvarsan, one starts from the base C., which is weighed in 3 parts of water, 0.3 parts of 40% formaldehyde and 1 part of 40% sodium bisulfite are added. Heat until completely dissolved. The myoacid is precipitated with hydrochloric acid, which is suction-filtered, washed with water, dissolved in water, neutralized with soda, and precipitated with alcohol or acetone. The product, suction-filtered and washed with absolute alcohol, is dried in a vacuum. It is also possible to start from S. (hydrochloride salt), dissolving it in 10% alcohol and treating it with formaldehyde and bisulfite. The precipitate that falls out initially dissolves again. When poured into strong alcohol, myosalvarsan precipitates. Analysis is conducted as for neosalvarsan. The preparation is used in the same cases as neosalvarsan, but, in contrast to the latter, it can be administered intramuscularly. The trypanocidal properties of myosalvarsan are somewhat weaker than those of neosalvarsan. According to Raiziss, per 1 kg of rats upon intravenous administration: MLD-320-480 mg, MCD-15.9-31.5 mg.

Silbersalvarsan (Silbersalvarsan). The preparation is not included in the Russian Pharmacopoeia. ONa ONa As=C₆H₃-NH₂-Ag-NH₂-C₆H₃=As (OH) This formula (Binz) is controversial, as Ehrlich and Karrer, and Binz and Bauer give others. Silbersalvarsan was first obtained by Ehrlich and Karrer. It was biologically and clinically studied by Kolle and introduced by him into medical practice in 1918. The preparation is a dark brown powder, easily soluble in water, with an alkaline reaction. It is insoluble in dilute mineral acids and bicarbonate. Silver ions are not detected by ordinary reagents. In ampoules without access to air, it keeps well; in air, it easily oxidizes and then gives cloudy solutions in water. Method of preparation: to a solution of 1 mole of S. in methyl alcohol, a methyl alcoholic solution of 6 moles of caustic soda is added. To the resulting solution, 1 mole of silver nitrate in methyl alcohol is added. From the solution, silbersalvarsan is precipitated with absolute ether. The precipitate is suction-filtered, washed with ether, and dried in a vacuum. Analysis: arsenic is determined as for S., it contains about 22%. Silver is determined as follows: 0.3 g of the preparation is mixed with 2 g of permanganate, 10 cm³ of 10% and then 10 cm³ of concentrated sulfuric acid are added. Allow to cool, add 50 cm³ of water, and titrate by the Volhard method with ammonium thiocyanate. 1 cm³ of 1/100 ammonium thiocyanate corresponds to 0.00108 g of silver. Usually, it contains 13-14% silver. The preparation has the best index of all known arsenic preparations. For rabbits per 1 kg of weight, the toxic dose is 0.125, the tolerated dose is 0.1, the therapeutic dose is 0.004, index 1:25. For mice per 20 g of weight, the toxic dose is 1/225, the tolerated dose is 1/300, the therapeutic dose is 1/1000, index 1:3.5. Despite a good index, it is tolerated worse than neosalvarsan, because side effects are observed more often with it. Neosilbersalvarsan (Neosilbersalvarsan). The preparation is included in the Russian Pharmacopoeia. Its structure has not been established. It is a brown powder, easily soluble in water, with a neutral reaction; the solutions are slightly fluorescent. It is stable in air, in contrast to S. and neosalvarsan. It does not precipitate from aqueous solutions with carbonic acid (a distinction from silbersalvarsan). Method of production, developed by Aniltrust: 100 g of neosalvarsan are dissolved in 3 liters of water, a solution of 240 g of silver nitrate in 480 cm³ of water is added. The solution is poured into a mixture of alcohol and ether 1:1. The precipitated sediment is suction-filtered, washed with ether, and dried in a vacuum. Yield 960 g. The preparation contains 20% arsenic and 8% silver. It is used in the same cases as neosalvarsan. For rabbits per 1 kg of weight, MLD-0.15; MTD-0.13; MCD-0.01; index-1:13. For mice per 20 g of weight, MLD-1/150; MTD-1/180; MCD-1/500; index-1:3.

Osarsol (Osarsol-USSR), Stovarsol (Stovarsol-France), Spirozid (Spirozid-Germany), Fourneau 190 (Fourneau 190). The preparation is not included in the FUP. 4-hydroxy-3-acetylaminophenylarsinic acid, OH NH-COCH₃, AsO₃H₂, molecular weight 275. Arsenic content-27.27%. Colorless needles. Without melting, it decomposes at 208-210°. Very slightly soluble (about 1%) in water, alcohol, and 80% acetic acid. Readily soluble in soda and alkalis. Upon heating with alkalis and acids, it splits off the acetyl group. It was first released for sale in France in 1922 by the firm Poulenc freres. The starting product for osarsol is 4-nitro-3-hydroxyphenylarsinic acid, which is reduced to the corresponding amino acid. The amino acid is acetylated OH OH

3-amino-4-hydroxyphenylarsinic acid (Fourneau 189) is an important intermediate product for the production of not only Osarsol, but also Salvarsan; it consists of pale pink crystals, which are difficult to dissolve in water and alcohol. Its salts, both with mineral acids (compounds with an amino group) and with alkalis (with the arsenic acid group), are easily soluble in water. Alkaline solutions turn brown in air due to oxidation, while acidic ones are stable. Methods for producing amido-hydroxyphenylarsinic acid are based on the reduction of nitro-hydroxyphenylarsinic acid 1) with iron in a hydrochloric acid medium or 2) electrolytically in a sulfuric acid medium. In both cases, the resulting amino acid is isolated in the form of a hydrochloride that is difficult to dissolve in a saturated solution of table salt. Other production methods are poorly suited for factory installations. 1) 263 g (1 mole) of nitro-hydroxyphenylarsinic acid is added to 1,750 cm³ of 5N hydrochloric acid with good stirring, maintaining the temperature at 40-60°, and 170 g of cast iron shavings are gradually introduced. The resulting solution is filtered and the hydrochloride is salted out with 250 g of table salt. Upon cooling, the hydrochloride is suction-filtered. 2) 263 g of "nitro-oxy" is weighed into 2 liters of 4N sulfuric acid. It is loaded into an electrolyzer with a diaphragm and a copper cathode. 40 g of ferrous sulfate is added and a current is passed (about 170 A/hours until vigorous evolution of hydrogen). The solution is filtered and 500 g of table salt is added. Upon cooling, the precipitated hydrochloride of amido-hydroxyphenylarsinic acid is filtered off. The crude hydrochloride is purified by crystallizing from 4N hydrochloric acid. Production of Osarsol. The hydrochloride of amido-hydroxyphenylarsinic acid, obtained from 1 mole of nitro-hydroxyphenylarsinic acid, is dissolved in 1 liter of water with slight heating. Hydrosulfite (5 g) and charcoal are added, and the mixture is filtered. First, a concentrated solution of sodium acetate is added until turbidity appears, then, simultaneously with good stirring and as quickly as possible, 200 cm³ of acetic anhydride and 140 cm³ of a 33% solution of sodium acetate are added. The solution is acidified with hydrochloric acid until it turns blue to Congo red, and after some time, the precipitated Osarsol is suction-filtered. For purification, Osarsol is dissolved in soda, filtered, and table salt is added. The sodium salt of Osarsol precipitates; it is suction-filtered, washed with a concentrated solution of table salt, redissolved in water, and pure Osarsol is precipitated by acidifying with hydrochloric acid. The product is suction-filtered, washed with water, alcohol, and ether, and dried in air. Osarsol is tested for arsenic content (like Salvarsan). The commercial product contains 27.2-27.4% arsenic (theory requires 27.27%). Solutions of the preparation in dilute soda should be almost colorless, which indicates that the product does not contain free, non-acetylated amido-hydroxyphenylarsinic acid, which oxidizes easily and yields toxic compounds.

G. Kirchhoff. Clinical use of Salvarsan. Ehrlich, who introduced Salvarsan (Alt-Salvarsan) into the therapy of syphilis, strove with the help of this agent to achieve therapia magna sterilisans, i.e., the cure of the disease with the help of a single administration of the medication (Weise). However, Ehrlich himself had to admit, and the entire future history of Salvarsan preparations confirmed, the impossibility of curing syphilis with the help of a single injection of Salvarsan. Most of the Salvarsan preparations listed above are currently administered almost exclusively intravenously. For intramuscular injections, the following compounds have been proposed: 1) Myosalvarsan; 2) Sulfarsenol and Sulfotreparsenan; these preparations are used particularly successfully in pediatric practice, and according to the reports of French authors, the latter preparation is distinguished by extremely easy local and general tolerance and convenience of use; 3) Acetylarsan, which, despite high therapeutic activity, is still inferior to preparations of the Neosalvarsan type; 4) very recently, in Germany, I. G. Farbenindustrie released a new preparation '4686' (Solusalvarsan); it was released for use in a 10% solution and is currently being clinically tested in Germany. In addition to the preparations indicated above, complex organic compounds of pentavalent arsenic, proposed by Levaditi and Navarro-Martin in 1922 in a preparation released under the name Stovarsol (No. 190), have become widely used. These preparations were soon manufactured in Germany under the name Spirocid, and in the USSR under the names Stovarsolan, Stovarsol, and later Osarsol. The main advantage of preparations of the Osarsol type lies in their oral administration, which significantly simplifies the methodology of syphilis treatment. However, having been tested on a vast amount of material in France, Germany, and the USSR, these preparations proved to be somewhat inferior to Neosalvarsan in therapeutic effect. They find particularly wide application in pediatric practice (M. Lesser, Mettenheim, Smelov). Sezary and Barbé proposed injecting the sodium salt of Stovarsol intravenously in the treatment of general paralysis. Indications for the use of Salvarsan preparations serve primarily all periods of syphilis, especially its infectious manifestations, malaria, and relapsing fever. Stovarsol, in addition to syphilis, is also used for certain helminthic infestations (Trichocephalus dispar), amoebic dysentery, and lambliasis. In the treatment of syphilis, all the preparations indicated above are usually used in combination with bismuth, mercury, and iodine, as well as various types of nonspecific therapy (malaria therapy, spa treatment of syphilis). Absolute contraindications for the use of all Salvarsan preparations are: severe diseases of the kidneys, liver, febrile conditions of non-syphilitic origin, acute widespread dermatitis, acute gastrointestinal diseases, hemorrhagic diathesis, round ulcer of the stomach and duodenum, and sharply expressed degenerative changes in the central nervous system. Special caution and individualization, both in terms of dosages and intervals between infusions, are required in the following cases: during pregnancy, heart and vascular disease, age over 50 years in men and 45 years in women, chronic intoxications, severe exhaustion, severe anemia, epilepsy, in the first 3 days of the menstrual period, in chronic diseases of the gastrointestinal tract, kidney diseases not accompanied by reduced diuresis and edema, the presence in the body of purulent and putrefactive foci, disease of the auditory and optic nerves, Addison's disease, Basedow's disease, diabetes, myxedema, and status thymicolymphaticus, widespread dermatoses, the presence in the anamnesis of a large number of acute infectious diseases, as well as liver and kidney diseases, diseases of the central nervous system not accompanied by sharp degenerative changes, lesions of the larynx with difficulty breathing, and poor tolerance of arsenic preparations in the past. When preparing solutions of Neosalvarsan, it is necessary to use freshly distilled water, boiled before the infusion, the temperature of which should be about 20°. The solution is prepared for each patient immediately before the infusion; the latter is recommended to be done 2 hours after a light breakfast so that the subsequent intake of food is no earlier than 2-3 hours after the infusion. The ampoule must be carefully inspected; in the presence of cracks in the glass, as well as the slightest changes in the physical properties of the preparation (in terms of color, consistency, flowability of the powder), the latter can in no way be allowed for use. Neosalvarsan solutions must be prepared at a rate of 1.0-2.0 of water per 0.1-0.2 cm³ of the preparation and be completely transparent; infusions should be made only by doctors well-versed in the technique of intravenous infusions, since the entry of even insignificant amounts of Neo solutions into the paravenous tissue leads to very painful and very persistent infiltrates, often hindering further treatment, especially in subjects with poorly developed veins. Infusions should be performed slowly (1-2 minutes). During treatment, especially on the eve of and on the days of Neo infusions, patients should avoid physical exertion and the consumption of alcoholic beverages. Salvarsan (Alt-Salvarsan) is used in single doses of 0.1-0.2-0.3-0.4, and per course 2.5-3.0-4.5; Myosalvarsan - 0.15-0.3-0.45-0.6, per course 4.0-5.0-6.0; Neosilbersalvarsan - 0.1-0.3-0.4-0.45, per course 3.5-4.5-5.0; preparations of the Neosalvarsan type (Russian Novarsenol) are recommended to be used in the following doses: for the first infusion 0.15 for women, 0.15-0.3 for men; for subsequent infusions, the dosage is increased by 0.15 for each infusion, with the highest single dose for women being 0.45, and for men 0.6. The course dose for adults should not exceed 4.0-6.0 of the preparation, depending on the general condition of the organism and the period of syphilis. For infants, the single dose for the first infusion should not be more than 0.01 per 1 kg of weight; for subsequent ones, it can be increased to 0.02-0.03 of the preparation per 1 kg of weight; for children from 1 year to 5 years, the single dose should not be higher than 0.01-0.015 of the preparation per 1 kg of the child's weight, however, in general, not higher than 0.15-0.2 of Novarsenol. For children from 5 years and older, 0.01 cm³ of the preparation per 1 kg of weight is used, but calculated so that the single dose is not higher than 0.15-0.25 for children from 5 to 10 years and 0.25-0.45 for children from 10 to 16 years. Intervals between infusions for adults after a dose of 0.15 are two to three days, after 0.3 three to four days, after 0.45 five to six days, after 0.6 six to seven days; for children, the intervals should be at least 5 days. When using Osarsol, we have approximately the same absolute and relative contraindications (see above) as when using Neosalvarsan. During treatment here, careful attention is paid to the state of the gastrointestinal tract. The preparation is used in the form of tablets or powder. Each tablet contains 0.25 of Stovarsol. The daily dose is divided into 2 intakes: the first in the morning on an empty stomach, the second in the evening on an empty stomach (3 hours after eating), washing it down with a small amount of water. In adults, the preparation is used in doses starting from 0.25 to 1.0 per day, with the preparation being given for 5 days in a row, after which a 3-5 day break is taken, and then again 5 days of taking the preparation, and so on until a total course dose of 30.0-40.0 g is reached. For children, the following dosages are recommended, used at the State Venereological Institute in Moscow: Age, Single dose, Daily dose, Course dose: 1-6 months, 0.06, 0.06 x 2 = 0.12, 4.0; 6 months-1 year, 0.03, 0.06 x 4 = 0.24, 6.0; 1-2 years, 0.1, 0.1 x 3 = 0.3 / 0.1 x 4 = 0.4, 8.0; 2-5 years, 0.25, 0.1 x 4 = 0.4 / 0.25 x 2 = 0.5, 10.0; 5-8 years, 0.25, 0.25 x 2 = 0.5, 15.0; 8-12 years, 0.25, 0.25 x 2 = 0.5 / 0.25 x 3 = 0.75, 20.0; 12-16 years, 0.25, 0.25 x 3 = 0.75, 25.0. Osarsol is used in children according to the following scheme: on the 1st day, one single dose is given, on the 2nd day 2 single doses (morning and evening), on the 3rd day 3 single doses (2 in the morning and 1 in the evening), on the 4th day 4 single doses (2 in the morning and 2 in the evening). After this, it is necessary to take a 5-day break in treatment. Then, for 5 days, the full daily dose is given (1/2 in the morning and 1/2 in the evening), after which there is again a break of 5 days; thus, treatment is carried out until the full course dose is obtained. Osarsol is used according to a somewhat different scheme in the treatment of trichocephalosis (see Whipworm). Osarsol in the treatment of syphilis should be used in combination with Bi or Hg. Side effects of Salvarsan. When using all Salvarsan preparations, side effects can be observed. Their quantity and quality are extremely different according to different authors. According to Kaplun and Schoenfeld, out of 63,324 infusions of various Salvarsan preparations, side effects were noted in 376 cases, with 165 being mild, 41 nitritoid crises, 84 exanthemas, 81 cases of jaundice, 4 serous apoplexies, and 1 Salvarsan shock; there was a fatal outcome in 3 cases (2 after the use of Neosalvarsan and 1 after Novarsenol).

According to American authors, out of 1212 patients treated with various Salvarsan preparations, 252 experienced one complication or another, with a fatal outcome noted in 12 cases; the Cologne Commission of 1918 established 12 deaths per 225,730 injections, however, when accounting for injections of Neosalvarsan in doses no higher than 0.6, according to the data of this commission, 1 death occurs per 162,792 injections. The pathogenesis of side effects is extremely complex and diverse. While in some cases the cause of the complication is: 1) insufficiently sterile water, 2) a defective ampoule of the preparation, 3) a toxic batch of Salvarsan, 4) overly careless single or total dosages, 5) short intervals between individual injections, 6) insufficient caution in using the preparation with relative contraindications—in other cases, in view of the absence of the indicated causes, various theories are put forward to explain the side effects: E. Hoffmann considers functional liver damage to be the cause of Salvarsan dermatitis; I. L. Krichevsky sees the cause of side effects in a change in the physical-chemical properties of blood colloids and cells; according to Stuhmer, Salvarsan complications are phenomena of anaphylaxis; P. Tachau believes that in a number of cases of side effects after the use of Salvarsan, we are dealing with idiosyncrasy, just as is observed with the use of mercury, iodoform, antipyrine, quinine, etc., as pointed out by Jadassohn, and along with rare cases of congenital idiosyncrasy, cases of acquired idiosyncrasy are more often observed, namely when side effects occur after repeated injections of Salvarsan, often even only during repeated courses of treatment. This hypersensitivity in some cases proves to be extremely persistent and forces one to abandon further use of the preparation; in other cases, habituation to the preparation occurs, and treatment with it is well tolerated by patients. The clinical picture of side effects is diverse. In the vast majority of cases, a complex toxic symptom complex develops here, in the picture of which phenomena from the skin, liver, or central nervous system, etc., come to the fore. Completely isolated lesions of any organ are observed less frequently. Thus, lesions of the skin or internal organs are often combined with each other, and can also be simultaneously accompanied by meningeal phenomena and a rise in temperature of toxic origin. By the time of onset,

side effects can be divided into 3 groups: 1) those arising during the injection or shortly after it; 2) those developing in a period from several days to 1–2 weeks after the last injection; 3) those appearing after longer intervals. Early complications include: 1) the smell of ether or garlic, nausea and vomiting arising during the injection, 2) skin itching and rapidly disappearing urticarial rashes, 3) temperature reactions, which must be distinguished from the Jarisch-Herxheimer reaction, 4) nitritoid crisis (Milian), which received this name in view of the fact that approximately the same phenomena are observed with it as with the use of amyl nitrite. The latter complication, described by the majority of German authors under the name of angioneurotic symptom complex, consists of either redness or pallor of the skin of the face and neck, the appearance of edema in these places, as well as on the lips and tongue; all these external signs of poisoning can be accompanied by a decline in cardiac activity, and sometimes by complications from the gastrointestinal tract in the form of nausea, vomiting, and diarrhea. Complications of the 2nd group include lesions of the skin (see Dermatitis), liver (see Jaundice, Acute yellow atrophy of the liver), kidneys (see Nephrosis—clinical forms), blood (see Leukemia), joints, and the nervous system (see Neurorecurrence). Among the most formidable side effects from the central nervous system is hemorrhagic encephalitis, purpura cerebri or serous apoplexy (Milian). Sicard, Sézary, Sobolev, and others have described cases of Salvarsan peripheral neuritis. The clinical picture of the disease in this case boils down to various paresthesias, and sometimes to the disappearance of Achilles reflexes; such cases can simulate true tabes dorsalis, which is why French authors introduced the term “arsenical pseudotabes” (pseudo-tabes arsenobenzenique) for these forms of peripheral neuritis. Rare side effects include lesions of the oral mucosa (stomatitis), first described in most detail by Cl. Simon. Differential diagnosis of side effects often presents great difficulties, especially in cases where the complication developed after a more or less long period after the end of treatment. In such cases, one often has to think about so-called monorecurrences, i.e., an isolated syphilitic lesion of one organ or another (more often when it comes to neurorecurrences or monohepatorecurrences), not accompanied by external signs of syphilis, as well as positive seroreactions. Careful clinical observation, and sometimes cautiously undertaken iodine, bismuth, or mercury therapy, allow one, for the most part, to resolve the question of whether one is dealing with a toxic or syphilitic lesion of an organ. Side effects in their course can turn out to be extremely insidious: a mild onset does not guarantee against a further severe course and, conversely, a stormy onset often ends in rapid recovery. It is also necessary to note the ability of some side effects (dermatitis) to spontaneously recur. To prevent severe side effects, it is recommended to adhere to the following rules: 1) it is necessary to instruct patients to consult a doctor regarding any malaise occurring after a Salvarsan injection; 2) upon the onset of side effects, all specific treatment must be stopped and subsequent injections made upon complete recovery, however, not earlier than 7–8 days, and in a reduced dose; 3) all severe side effects require a long cessation of Salvarsan treatment (not less than 3–4 months after the disappearance of all signs of the complication) and the observance of special caution in dosages in the future. Treatment of side effects is usually reduced to symptomatic therapy. In addition to this, upon the onset of a nitritoid crisis or hemorrhagic encephalitis, subcutaneous injection of adrenaline (1 cm³ of 1‰ Sol. Adrenalini hydrochlorici) is recommended; for all side effects, intravenous injections of 10–20% Sol. Natr. hyposulfurosi, 10% Sol. Calcii chlorati are recommended; Mühlpfordt recommends prescribing the so-called “Mixtura acida” according to the following prescription: Acid. hydrochlorici dil. 20.0, Sir. Rubi Idaei 60.0, Aq. destillat. ad. 200.0. MDS 1 teaspoon 3 times a day. In cases of poor tolerance of Salvarsan, in order to prevent side effects, it is recommended to dissolve the preparation in a 10% solution of calcium chloride, 50% glucose solution, and to inject 0.5–1.0 cm³ of 1‰ adrenaline solution under the skin shortly before the Salvarsan injection; Colle, Schlosberger, and Leupold inject 0.05–0.1 of this preparation to particularly sensitive subjects 24 hours before the injection of a normal dose of Salvarsan. When using Osarsol, the same relatively mild side effects are observed as with Salvarsan, however, in the vast majority of cases, according to the unanimous opinion of most Russian and foreign authors, they are encountered significantly less frequently and their course is milder. Upon their onset, treatment with Osarsol is stopped until the complete disappearance of the complication. Resumption of treatment is permitted no earlier than 5–7 days after the regression of side effects. In cases of severe side effects, as well as when they develop repeatedly, it is recommended to stop treatment with Osarsol completely. The therapy for Osarsol complications is the same as for Salvarsan side effects. N. Smelov. Detection in forensic cases. Salvarsan as such cannot be detected in parts of a corpse due to decomposition, and its detection boils down to the detection of arsenic (see). In urine, Salvarsan (and its derivatives) can be detected in the first hours after introduction into the organism by conversion into an azo dye. The urine is acidified with hydrochloric acid, cooled to 0°, and 0.5% sodium nitrate solution is added dropwise (until a piece of paper moistened with potassium iodide solution and starch paste turns blue from a drop of urine). The resulting liquid is poured as a layer onto a 1% resorcinol solution alkalized with caustic soda: a red ring appears at the boundary of the two layers. Instead of a resorcinol solution, alpha-naphthylamine can be used.