Harold N. Wright

Chronic toxicity studies of analgesic and antipyretic drugs and congeners

Abstract The chronic toxicity of a group of nonnarcotic antipyretic and analgesic drugs has been studied in mice over periods up to five generations. All the compounds studied significantly shortened survival time and produced a more rapid deterioration of physical condition as compared with the controls. They also greatly reduced both the percentage of mated females having litters and of young born raised to weaning age. No increased incidence of mammary carcinoma or of gross abnormalities was found. Of the three major analgesic drugs acetylsalicylic acid, acetophenetidin, and acetanilid, acetylsalicylic acid was the least toxic on all counts. At the 0.1% dietary level, which yielded doses approximating twice maximal continuous daily intake levels for a human being, acetylsalicylic acid produced a just significant shortening of survival time and a mild depression of physical condition and reduced the raising of young to one-third that of the controls. Acetophenetidin depressed survival more than acetanilid. The percentage of young raised was 15% for acetanilid and 10% for acetophenetidin. The three secondary analgesics salicylamide, paracetamol, and acetoacetanilid were all more toxic on all counts than their primary congeners.

CHEMOTHERAPEUTIC ACTIVITY OF CYANINES AND RELATED COMPOUNDS IN FILARIASIS IN THE COTTON RAT

A group of more than sixty cyanine dyes and related compounds has been studiedla for chemotherapeutic activity against the naturally acquired Litomosoides carinii infestation in the wild cotton rat as part of a coordinated research program on filariasis under the auspices of thc Office of Scientific Research and Development. A similar study with a group of pyrryl, pyrimidyl and related cyanines was carried out simultaneously by Dr. A. D. Welch and his associate^^-^ at Western Reserve University. Care of the animals and administration of drugs was greatly facilitated by the use of special cages, holders, and feed cups designed by one of the authors (J. T. Litchfield, Jr.). The cages were made of a heavy wire mesh, were oblong in shape and of a size suitable for housing the rats individually. One end of the cage was designed as consisting of flanged sheet metal into which fitted a sliding door and to which either the holder or feed cup could be attached (FIGURE 1 ) . The holders were equipped with sliding doors at both ends and had a curved piece of metal extending most of the length inside the top, which was attached by springs to a metal bar outside the holder. With the holder in hand, slight pressure from the palm could be exerted on the bar, thus pressing the abdomen of the rat gently against the wire mesh bottom of the holder, through which intraperitoneal injections were then made. For drug diet administration, feed cups were attached to the metal flanges on the outside of the cages, thus allowing easy removal for daily filling and weighing. . Tests for chemotherapeutic activity were made in uivo against the adult filaria. Administration was usually by intraperitoneal injection every eight hours for eighteen doses with autopsy on the eighth day. The filaria were removed at autopsy, placed in a modified Sim’s solution and examined for motility for 24 hours. The minimum curative dose was taken to be that dose of drug which killed 50 per cent or more of the adult filaria. Therapeutic indices were determined as the ratio of the maximum tolerated dose (M.T.D.) to the minimum curative dose (M.C.D.) based

Relation Between Colloidal Properties and Toxicity of Arsphena-mine and Neoarsphenamine.

Bauer, 1 Klemensiewicz, 2 Sherndal, 3 Raiziss and Gavron, 4 and others, have demonstrated that both arsphenamine and neoarsphenamine belong to the group of emulsoid “semi-colloids”. Hirsch-felder and Wright 5 studied the effects of neoarsphenamine and other semi-colloids on solutions of egg albumin and rabbit plasma and concluded that “the fact that most of these substances react strongly with proteins, producing ultramicroscopic, and in some cases macroscopic, changes in the proteins, lends strong support to the idea that anaphylactoid and febrile reactions following the injection of these substances into the blood stream, are due to definite changes brought about in the hydration and aggregation of the blood proteins”. Raiziss and Gavron determined the degree of colloidality of a few samples of arsphenamine and neoarsphenamine by means of dialysis, using parchment membranes. Such membranes are relatively impermeable, and permitted only a small fraction of the arsenical to pass through. Dialysis experiments using both arsphenamine and neoarsphenamine with viscose and cellophane membranes of varied permeability indicate that both of these arsenicals contain particles of many sizes ranging from the true crystalloid which will pass through membranes of slight permeability to particles which must be large aggregates since they fail to pass through the most permeable membranes we have employed. Arsphenamine HCl may be maintained in aqueous solution under nitrogen indefinitely without evidence of deterioration, since the iodine value remains constant and tests for arsenoxide were always negative. Dialysis through viscose membranes does not produce either oxidation or precipitation. Solutions of sodium arsphenamine under nitrogen are stable in the absence of a membrane.

The acute oral toxicity of commonly used household textile dyes and color removers

Fourteen commonly used household textile dyes and two color removers were tested for their oral toxicity in mice. For this species all fell in the category of “moderately toxic” compounds. Toxic symptoms appeared rapidly and consisted of marked respiratory difficulties and convulsions. Most deaths occurred within 13 to 3 hours. The five most toxic dyes and one color remover were tested for oral toxicity in dogs. In this species all preparations tested rated as “very toxic.” Symptoms resembled those seen in mice. Intravenous administration to dogs showed progressive cardiovascular and respiratory depression. In general the dark colored dyes were more toxic than the pastel shades.

Trypanocidal Activity and Arsenic Content of Rat Blood Following Intravenous Administration of Mapharsen

Fractional dose therapy of rat trypanosomiasis has shown the trypanocidal action of mapharsen in vivo to be of relatively short duration. 1 The brief trypanocidal activity of rabbit serum after administration of the related trivalent arsenical, reduced tryparsamide, is also indicative of this fact. 2 On the contrary, arsenic has been found in the blood and tissues of rats for relatively long periods after mapharsen injection. 3 This study was concerned with an investigation of the apparent lack of parallelism between these two factors. The procedure consisted in determining the minimum trypanocidal concentration (M.T.C.) of the blood arsenic of rats at various intervals after intravenous injection of the maximum tolerated dose (12.5 mg/kg) of mapharsen and comparing this with the M.T.C. of the unijected drug. The blood samples were taken at 1/4, 1/2, 1, 4, 8, 12, 18, 24, 36, 48 or 240 hr after injection, heparin being used as an anticoagulant. The arsenic content was determined by the A.O.A.C. Gutzeit method, 4 and was expressed as μ (γ) of mapharsen per cc of blood. The M.T.C. was determined by a modification of the method of Murgatroyd et al., 2 and Hawking et al., 3 sterile technic being used throughout. A series of dilutions of the blood was made, on the basis of arsenic content, with a medium consisting of 1 part of heparinized rat blood and 2 parts of Lockes solution (0.2% glucose). The tubes were then inoculated with a dilute suspension of Trypanosoma equiperdum from infected rats and incubated for 18 hr at 37°C. After incubation, cultures which showed no motile organisms in microscopic cover-glass preparations were injected intra-peritoneally into young rats, which were observed for septicemia and death for 30 days.

Comparative Distribution and Retention of Crystalloid and Colloid Fraction of Arsphenamine and Neoarsphenamine

Conclusion The separated colloid fraction of either arsphenamine or neoarsphenamine is retained long in the body in comparison with the crystalloid fraction which is rapidly eliminated.

Therapeutic Efficiency of Arsphenamine and Neoarsphenamine Fractions.

Biedermann, Hanssen and Wright 1 have shown that arsphenamine hydrochloride, sodium arsphenamine or neoarsphenamine may be separated by means of dialysis through viscose membranes into 2 fractions, the one consisting of particles which readily pass through the membrane (crystalloid fraction), the other consisting of particles which fail to pass through the membrane after repeated dialysis until no further arsenical passes through the membrane (colloid fraction). The technic and precautions used are similar to those described in the previous paper. In all experiments with sodium arsphenamine and neoarsphenamine it was found necessary to use 1/10,000 sodium formaldehyde sulphoxylate as a stabilizing agent. The previously reported study having shown that the 2 fractions of these semi-colloid arsenicals possessed markedly different properties in regard to toxicity, the present study was undertaken to determine whether or not these separated fractions possessed any differences in therapeutic activity. The experiments on the therapeutic properties of the various arsenicals and their fractions were carried out on albino rats of the Wistar Institute strain, weighing 150-200 gm., and inoculated 24 hours before injection of the arsenical with approximately 300,000 organisms of Trypanosoma equiperdum. † Control animals were included in every experiment and these animals invariably died in 3 to 5 days. Test animals were not recorded as cured unless repeated microscopic examinations of the blood were negative for trypanosomes for 30 days after injection of the arsenical. The results clearly indicate that the crystalloid fraction of neo-arsphenamine has a much higher therapeutic effect than that of the colloid fraction. In general, we have found that the smaller the amount of the crystalloid sample (first 24 hour dialysate) the higher the therapeutic efficiency. Thus in Sample A1 above, the viscose membrane was relatively impermeable and the crystalloid fraction was found to have a very high therapeutic activity, only 3 mg./kg. being required as against 24 mg./kg. for the colloid fraction. In another experiment using the same brand and lot number but a very porous membrane the crystalloid fraction contained 323 mg., the colloid fraction 233 mg., ratio 0.7, and the therapeutic activity of the crystalloid fraction was found to be 11 mg./kg., and 13 mg./kg. for the colloid fraction, giving a ratio of only 1.2 in favor of the crystalloid fraction, a result agreeing well with the poor separation obtained.

Ultramicroscopic Studies Upon Colloidal State of Antiseptics and Arsenicals in Relation to Their Actions.

As Voegtlin 1 and others have shown that the action and toxicity of arsphenamine is closely related to their viscosity and colloidality, and as the action of antiseptics is greatly diminished by the presence of protein and of lipoids (Hirschfelder and Decherd 2 ) we have studied the appearance of solutions of a number of these substances under the Szigmondy slit ultramicroscope. Solutions of arsphenamine and neoarsphenamine show the soap-bubble-like appearance of hydrated lyophillic colloids, the micellae being smallest between pH 7 and 9, and again from 11 to 13. In the presence of proteins they aggregate to form stellate, highly refractive micellae. Rivanol is fluorescent, lyophillic, soap-bubble-like and aggregates with proteins. Triphenyl methane dyes are crystalloidal, but aggregate with proteins. Quinine and the hydrocuprein series (optochin, eucupin and vuzin) are fluorescent, crystalloid and aggregate with proteins. Metaphen is crystalloid and produces little change in the proteins. Mercurochrome and acriflavine show fluorescence, but no visible particles; the micellae formed by mercurochrome and protein appear to be quite small; with acriflavine the protein aggregates are somewhat larger. These colloidal phenomena are probably associated with the toxicity and pharmacological action of the drugs, especially in intravenous injection.

Mucolytic Enzyme Systems. XIV. Effect of Certain Quinolines on Hyalur-onidase and Its Serum Inhibitor.

Summary Two compounds, 6-methoxy-8-(β - diisobutylaminomethyl) aminoquinoline dihydrochloride and 6-methoxy-8-(diisobutyl-aminoisopropyl) aminoquinoline dihydrochloride, which are chemotherapeutically effective in mice infected with schistosoma mansoni, have been shown to be potent inhibitors of hyaluronidase although they inactivate the serum inhibitor of the enzyme. Chemically related rosaniline and plasmochin, that have little chemotherapeutic effect, had no demonstrable direct effect on the hyaluronidase, although both potentiated the serum inhibitor to some degree.

A Parallelism Between Blood Sugar, Blood Calcium and Blood Coagulability in Normal and Jaundiced Dogs.

It has been noted by many early observers, among them Wright, 1 that the coagulation time of blood was decreased after meals. Schreiber 2 and Kehr 3 suggested the preoperative and postoperative use of intravenous glucose in obstructive jaundice, but it was not used extensively until Crile and Walters advocated it 5 years later. Partos and Svec 4 pointed out that a relation existed between the sugar content and coagulability of the blood, namely, that with increasing blood sugar the clotting time is reduced. They demonstrated this effect following the injection of various substances which produce a hyperglycemia, e. g., glucose, epinephrine, theobromine sodium salicylate and morphine. Ravdin 5 presented a series of experiments in which he attempted to justify the use of intravenous glucose in cases having lesions of the biliary apparatus and who were poor operative risks. In determining coagulation time we used 2 methods simultaneously in all cases, (1) that of Lee and White 6 and (2) a modification of the method of Wright 7 (using pieces of glass tubing 40 mm. long and 2 mm. internal diameter). Tests for coagulation were made every half minute. The two methods usually checked within one-half minute; often the results were identical; occasionally a minutes difference was observed, especially in cases where the coagulation time was very long, as in the jaundiced animal. Serum calcium determinations were made by the method of Kramer and Tisdall. 8 , 9 Blood sugars were determined, using the Folin-Wu technique. 10 Serum bilirubin was determined by Thannhauser and Andersons modification of van den Berghs method. All experiments were done on dogs fasted over night. Both normal and jaundiced dogs were used.