pH-sensitive nanoparticles for improved oral delivery of dapsone: risk assessment, design, optimization and characterization.

AIM: To optimize the production of pH-sensitive dapsone (DAP) nanoparticles based on Eugradit L100 (NPs-EL100-DAP) for oral delivery. MATERIALS & METHODS: NPs-EL100-DAP were optimized using a Plackett-Burman design and a Box-Behnken design. The physicochemical properties of the obtained nanoparticles were monitored by microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, differential scanning calorimetry, in vitro release assays, and examined for cytotoxicity and permeation across intestinal barrier. RESULTS: The in vitro release assay of NPs-EL100-DAP confirmed the nanoparticles' pH sensitivity and the ability to deliver DAP at intestinal environment. NPs-EL100-DAP demonstrated enhanced intestinal interactions in comparison to free DAP, across Caco-2 monolayers. CONCLUSION: These studies demonstrate the potential of NPs-EL100-DAP as a therapeutic platform for oral treatment of leprosy.

Use of morphostructural reaction of blood serum for toxicological evaluation of drugs.

We proposed a simple and economic method for determination of general toxic effects of drugs consisting in evaluation of serum morphology by polarization light microscopy.

Tissue concentration, systemic distribution and toxicity of clofazimine--an autopsy study.

There are very few autopsy studies available on systemic distribution of clofazimine, a drug with anti-mycobacterial activity, used in multidrug therapy (MDT) regimen of leprosy and in erythema nodosum leprosum (ENL). An autopsy study was done on a 45 year old female of lepromatous leprosy (LL) on MDT and long term high dosage of clofazimine. Patient succumbed to intractable abdominal pain, diarrhoea, hypokalemia following clofazimine treatment. Autopsy study revealed yellowish brown discoloration of skin, viscera and body fluids. Chemical extraction of the drug revealed the highest concentration of the drug in jejunum (1.5mg/gm),followed by spleen (1.2mg/gm), pancreas (0.4mg/gm), adrenal (0.25mg/gm), liver (0.21mg/gm), and less than 0.2mg/gm in lung, fat, large intestine and stomach. It can be inferred from the present study that the drug is absorbed from the jejunum and gets deposited in fat, reticulo-endothelial cells (R-E cells) and hepatocytes. The drug is best demonstrated in cryostat sections and is lost partly during tissue processing and staining. The drug toxicity can be fatal as seen in the present case.

Effects of H(2)-receptor antagonists on dapsone-induced methaemoglobinaemia in rats.

Dapsone (DDS) (4,4'diaminodiphenylsulfone), the drug of choice for the treatment of leprosy, frequently induces haemolytic anaemia and methaemoglobinaemia. N-hydroxylation, one of the major pathways of biotransformation, has been constantly related to the methaemoglobinaemia observed with the use of the drug. In order to determine the reversible inhibition of this toxicologic bioactivation pathway without changing the detoxification pathways of the drug or cytosolic acetylation, cimetidine (CIM), ranitidine and famotidine were administered in combination with DDS to male Wistar rats weighing 200-220 g. The animals were divided into nine groups of eight: group 1 received a single dose of 40 mg kg (-1) DDS in dimethylsulfoxide (DMSO) and groups 2-4 received the same treatment as group 1 but after the administration of a single dose of 100, 150 and 200 mg kg (-1) CIM, respectively, injected 2 h prior DDS administration. Groups 5-9 received the same treatment as group 2 but after the treatment of ranitidine (50 and 100 mg kg (-1) intraperitoneally (i.p.) in 200 microl DMSO) and famotidine (10, 50 and 100 mg kg (-1) i.p. in 200 microl DMSO), respectively. The animals were then anaesthetized with ether and blood was collected from the aorta for the determination of plasma DDS and monoacetyldapsone concentrations by HPLC and later for the determination of methaemoglobinaemia by spectrophotometry. CIM showed a higher affinity for cytochrome P-450 than famotidine and ranitidine. The results obtained showed the potentiality of the pharmacological effects of DDS with a low risk of adverse reactions, especially methaemoglobinaemia, which is dose dependent.

Thalidomide: 40 years on.

Thalidomide was marketed in the late-1950s as a sedative and tranquilliser of exceptionally low general toxicity, but in 1961 it was implicated separately by Lenz and MacBride as the cause of the epidemic of congenital malformations that had been puzzling the world for some years. It is a very potent teratogen in humans, but in few other mammalian species; damage to the embryo is produced at specific stages of gestation, but the mechanism of embryopathic action is still not understood. Following the withdrawal of the drug worldwide, it was consigned to the history of medical tragedies. In 1965, however, Sheskin discovered that it was effective in treating erythema nodosum leprosum, a distressing complication of leprosy. As the drug is neither an antibiotic nor an analgesic, its action was assumed to be immunosuppressive. In Brazil the drug was used widely with few regulatory controls, since when more than 100 cases of congenital malformation have appeared. Sheskin's discovery led to the experimental use of thalidomide in many other indications thought to possess some immunological component. In some cases, e.g. Behçet's syndrome, graft-versus-host disease and aphthous ulceration in HIV-positive patients, the drug has been shown to possess some efficacy. And there is some evidence that it inhibits the replication of one of the immunodeficiency viruses. The AIDS community in the US has exerted much pressure on the FDA to allow the drug on to the market, although the use of a potent immunosuppressive drug of unknown mechanism in an immunodeficiency condition raises further questions. Thalidomide is not always beneficial; its use is associated with an increased mortality in epidermal necrolysis. In 1991, D'Amato confirmed it possessed antiangiogenic properties and this led to further trials in malignant conditions. Results were mixed, but those in multiple myeloma gave some grounds for optimism. In 1998, the FDA announced its extraordinary decision to grant marketing approval for thalidomide.

Lack of peripheral neuropathy in Beagle dogs after 53 weeks oral administration of thalidomide capsules.

Thalidomide (Thalomid) is approved for use in the US to treat complications from leprosy. Peripheral neuropathy is a dose-limiting adverse event in humans. As part of a nonrodent regulatory toxicology study, Beagle dogs were fed orally via encapsulation for 53 weeks. A component of this study was to determine if the dogs developed peripheral neuropathy. Twenty-eight male and 28 female Beagle dogs approximately 8-10 months of age were used. They were dosed at 43, 200 or 1000 mg/kg for 53 weeks followed by a 4-week treatment-free recovery period. Nerve function was assessed by electrophysiological measurements of the tibial nerve prior to dosing and at weeks 13, 27, 38 and 51. Representative dogs from each group were sacrificed at 26, 53 and 58 weeks and histologic and ultrastructural evaluations were performed on the sural nerve. Thalidomide had no effect on sensory nerve conduction velocity, duration or amplitude of the action potential. At 27 weeks, mean sensory nerve action potential amplitude for females at 43 mg/kg was significantly greater than control but was not evident at 39 weeks. Mean duration of sensory nerve action potential seemed to increase with similar magnitude over time in all dose groups including controls. Histological and ultrastructural evaluation of sections of sural nerve did not identify treatment-induced differences between control and thalidomide-dosed animals after 26 and 53 weeks of treatment. Additionally, no differences were observed following a 5-week treatment-free period at week 58. In contrast to humans, Beagle dogs did not develop thalidomide-induced peripheral neuropathy under conditions of the study.

Hemolytic drugs aniline and dapsone induce iron release in erythrocytes and increase the free iron pool in spleen and liver.

Incubation of rat erythrocytes with the hydroxylated metabolites of aniline and dapsone (4-4'-diaminodiphenylsulfone), phenylhydroxylamine and dapsone hydroxylamine, respectively, induced marked release of iron and methemoglobin formation. On the contrary, no release of iron nor methemoglobin formation was seen when the erythrocytes were incubated with the parent compounds (aniline and dapsone). The acute intoxication of rats with aniline or dapsone induced a marked increase in the erythrocyte content of free iron and methemoglobin, indicating that the xenobiotics are effective only after biotransformation to toxic metabolites in vivo. Prolonged administration of aniline or dapsone to rats produced continuous release of iron from erythrocytes. Marked iron overload was seen in the spleen and in the liver Kupffer cells, as detected histochemically. The spleen weight in these subchronically treated animals was significantly increased. The free iron pool was markedly increased in the spleen and to a lower extent in the liver. The possible relationships between iron release in erythrocytes, oxidative damage seen in senescent cells, hemolysis, overwhelmed capacity of spleen and liver to keep iron in storage forms and subsequent increase in low molecular weight, catalitically active iron is discussed.

Tissue distribution and deposition of clofazimine in rat following subchronic treatment with or without rifampicin.

Tissue distribution and deposition characteristics of clofazimine (CAS 2030-63-9), an antileprotic drug in rats have been investigated following controlled sub-chronic administration (p.o.) for a period of 1-2 months. The drug was administered alone at a dose of 20 mg/kg body weight and in combination with rifampicin (CAS 13292-46-1) (20 mg/kg p.o.). Various tissues (liver, lung, spleen, small intestine, brain, heart, kidney, skin, stomach and subcutaneous fat) were analyzed for clofazimine in all the treated groups. High levels (range 0.9-3.6 mg/g of wet tissue) were observed in tissues having reticuloendothelial components. In other tissues the levels were relatively lower (range 3-114 micrograms/g of wet tissue). Histopathological studies revealed that clofazimine is deposited in many tissues in the form of reddish-orange crystals. Concomitant treatment with rifampicin did not significantly alter tissue distribution or deposition profile of clofazimine nor did it influence the histopathology.

[Immunocorrective properties of new antileprotic preparations]. / Immunokorrigiruiushchie svoistva novykh protivoleproznykh preparatov.

Antimycobacterial and immunotropic characteristics of new antileprosy drugs, made in this country, have been studied and compared with those of dapsone. Animal experiments have demonstrated a high immunostimulating activity of the new drugs; this calls for clinical trials of these agents, for they may improve the therapy efficacy, help prevent the disease recurrences, and cut down the length of treatment for leprosy.

Mutagenic activity of antileprosy drugs and their derivatives.

We tested the mutagenic activity of antileprosy drugs (clofazimine, ethionamide, prothionamide, prothionamide-S-oxide, rifampin, and dapsone and many of its derivatives) using the Ames Salmonella/microsome assay system. None of these, including N-acetylated and N-hydroxylated derivatives of dapsone, were found to be positive with or without metabolic activation of this test. However, the sulfoxide and sulfide analogs of dapsone were found to be mutagenic with metabolic activation. These two analogs could not be detected in pharmaceutical preparations of dapsone (less than 0.01%), nor could they be found (in either unconjugated or conjugated form) in urine from volunteers taking a single oral dose of 50 mg of dapsone or from patients receiving daily oral doses of 100 mg of dapsone. Also, urine concentrates from volunteers taking 50 mg of dapsone did not exhibit mutagenic activity in the Ames screen. These results indicate that patients receiving antileprosy therapy with clofazimine, dapsone, ethionamide, prothionamide, and/or rifampin are not being exposed to mutagenic (and thereby possible carcinogenic) drugs.

Thiosemicarbazone in the treatment of leprosy

(I) The previous work with thiosemicarbazone in the treatment of leprosy is summarised. (2) Siocarbazone brand of thiosemicarbazone has been used in 52 cases of leprosy, 9 untraeded neural cases, 27 untreated lepromatous cases and 16 previously treated lepromatous cases, 15 with sulphones, and I with hydnocarpus oil. The duration of treatment has from 2 months to 16 months with an average of I0 months. (3) The patients have stood the drug well and there has been satisfactory clinical and bacteriological response. In addition to usual therapeutic affects, special features with thiosemicarbazone treatment have been the complete or partial restoration of sensation in the anaesthetic patches and in the limbs with polyneuritic type of anaesthesia, replacement of deseased nails by new ones, and growth of new hair in depilated areas. Considering the short period of treatment those features are rather remarkable. (4) In the toxicity of thiosemicarbazone seems to be less than that of the sulphones specially in relation to its effect on the haemopoietic system. However, the thiosemicarbazone is not as free from toxicity as described by some workers eho have used it in leprosy. A special kind of toxicity which indicates specific intolerance has been noticed and this has been manifested by sudden rise of temperature even after small doses of the drug

Toxicity of certain proposed antileprosy dyes: fluorescein, eosin, erythrosin, and others

Fluorescein, eosin, erythrosin and methylene green were found to be lethal at 300, 350, 200 and 150 mgm. per kilogram, respectively when administered intravenously to rabbits, and at 600, 500, 300 and 125 mgm. per kilogram intraperitoneally in rats. Methylene green is lethal for anesthetized cats in doses of 50 to 75 mgm. per kilogram. Orally in rats these dyes are tolerated in doses of 1.0 gm. per kilogram with the exception of methylene green, which killed 2 of 5 rats at 500 mgm. per kilogram. Data are presented on the chronic toxicity of trypan blue, gentian violet, brillant green and mercurochrome. Three of 6 rabbits dying under repeated intravenous administrations of trypan blue had received a total cumulative dose, approximately equivalent to but one acute lethal dose, i.e., 120 to 150 mgm. per kilogram. The dangers of repeatedly using high doses in human lepers, the superiority of oral administration over intravenous, and the danger of certain synergizing agents, including photodynamic effects, are discussed.

A preliminary report on the action of certain dyes in leprosy

1- A number of indications and analogies suggest the trial of coal-tar dyes and their derivatives in leprosy. 2- Intravenous injections of a large number of dyes have been given to 85 patients. 3- A number of these dyes show marked selective concentration in the leprotic lesions, a phenomenon not seen in certain non-leprotic lesions. 4- With trypan blue, brilliant green, fluorescein and perhaps eosin a definite diminution of the external manifestations of leprosy has been observed, accompanied by other signs of clinical improvement. 5- With the other dyes administered no signs of clinical improvement were observed. 6- This report is a study of the immediate reaction of the lesions; we have as yet no knowledge of the later effects of the injection of these dyes. We consider from this preliminary report that further trial and study of these dyes is definitely indicated in the hope that it may lead to further possibilities in the chemotherapy of this disease.

The toxicity of sodium hydnocarpate

1-High concentrations and large doses of commercial preparations of sodium hydnocarpate administered to rabbits and dogs intravenously and subcutaneouly produce albuminuria, hematuria, and emaciation. 2- There is loss os weight when either the sodium salts or the ethyl esters are given subcutaneously in amounts more than 50 milligrams per kilogram of body weight. A certain degree of tolerance can be developed with suitable dosage. 3-Pronounced albuminuria is observed in rabbits after subcutaneous injection of 12.5 milligrams of the salts. With larger or smaller doses it is not so pronunced. In dogs, a 4 per cent solution of esters in olive oil is apparently most rapidly absorbed and gives the earliest traces of albumen. $- The less readily absorbed and less reactive concentrations also produce albuminuria, but only after an interval of several days...