Dapsone Research Articles

Plasmodium berghei: Efficacy of 5-fluoroorotate in combination with commonly used antimalarial drugs in a mouse model

Resistance to antimalarial antifolates necessitates a search for new antimetabolites targeting other enzymes of the folate metabolic pathway. In this study, 5-fluoroorotate (FOA), reported to be an inhibitor of thymidylate synthase, was assayed against Plasmodium berghei NK 65 in mice, with(out) an oral uridine supplement. FOA (2.5 and 5.0 mg/kg bw.) was tested alone, or in a double and triple combination with a fixed oral dose of 1.25 and 2.5 mg/kg of pyrimethamine (PYR); 1.0 and 2.0 mg/kg of dapsone (DAP); 1.0 and 2.0 mg/kg of artesunate (ART). FOA achieved high suppression which ranged from 95.7% to aparasitaemic, activity that was dose-dependent. At the highest dosages used, FOA–PYR and FOA–DAP–ART combinations were synergistic with 100% cure rate, while FOA–PYR–ART was antagonistic. Drugs in a synergistic combination may exert less resistance selection pressure, thus FOA–PYR and FOA–DAP–ART warrant further evaluation with an ultimate object of possible clinical use against drug-resistant malaria.

Itraconazole Promising for Palmoplantar Pustulosis

Choosing the proper therapy for a palmoplantar pustular (PPP) dermatosis is frequently a clinical challenge. Satisfactory results are seldom realized with any of the available options, including topical and systemic corticosteroids, topical and systemic retinoids, oral dapsone, oral or intramuscular methotrexate, oral colchicine, oral cyclosporine, and topical or systemic psoralen …

Liposome-based immunostrip for the rapid detection of Salmonella

Salmonellae are ubiquitous human pathogens, which pose a danger to the elderly and children. Due to the increased number of outbreaks of human illness associated with the consumption of contaminated products in the USA and many other countries, there is an urgent need to develop rapid assays to detect common food-borne pathogens. This study demonstrates the feasibility of using a detectable label comprising methyl blue (MB), a visible dye, entrapped inside liposomes. Immunoliposomes tagged with anti-Salmonella common structural antigens (CSA) antibody encapsulating MB dye were prepared and used as the signal amplifier for the development of a field-portable colorimetric immunoassay to detect Salmonellae. Tapping mode atomic force microscopy (TMAFM), a scanning probe technique, was utilized to demonstrate the presence of anti-Salmonella antibody at the thus-prepared liposome. A plastic-backed nitrocellulose strip with two immobilized zones formed the basis of a sandwich assay. The first zone was the antigen capture zone (AC zone), used in a sandwich (noncompetitive) assay format; the other was the biotin capture zone (BC zone), used as a quality control index for the strip assay. During the capillary migration of the wicking reagent containing 80μL of immunoliposomes and 40μL of the test sample (heat-killed S. typhimurium), sample pathogens with surface-bound immunoliposomes were captured at the AC zone, while the unbound immunoliposomes continued to migrate and bind to the anti-biotin antibodies coated on the BC zone. The color density of the AC zone was directly proportional to the number of Salmonella typhimurium in the test sample. The detection limit of the current assay with heat-killed Salmonella typhimurium was 1,680 cells. The cross-reactivity of the proposed immunoassay was also investigated, and pathogens including E. coli O157:H7 and Listeria genus specific caused no interference with the detection of Salmonella typhimurium.

Sensitive spectrophotometric methods for the assessment of nitrite in water sample

Two spectrophotometric methods have been developed for the determination of nitrite using dapsone (DAP) with alpha-naphthol and 4-amino-5-hydroxynapthalene-2,7-disulphonic acid monosodium salt (AHNDMS) as chromogenic reagents with maximum absorbance wavelength at 540 and 520 nm respectively. For the method that utilizes dapsone with alpha-naphthol (DAP-alpha-naphthol), the beer's law range is obeyed between 0.05-0.8 microg ml(-1) with molar absorptivity of 5.749 x 104 l mol(-1) cm(-1). The second method that uses dapsone with AHNDMS (DAP-AHNDMS), the beer's law is valid over the range 0.2-1.4 mug ml(-1) and molar absorptivity 2.44 x 104 l mol(-1) cm(-1). The common interfering ions in the analytical procedures have been studied. This proposed methods are reliable, reproducible and have been successfully applied to determine nitrite in various water sources of environmental interest.

Development of Spray Dried Liposomal Dry Powder Inhaler of Dapsone

This investigation was undertaken to evaluate practical feasibility of site specific pulmonary delivery of liposomal encapsulated Dapsone (DS) dry powder inhaler for prolonged drug retention in lungs as an effective alternative in prevention of Pneumocystis carinii pneumonia (PCP) associated with immunocompromised patients. DS encapsulated liposomes were prepared by thin film evaporation technique and resultant liposomal dispersion was passed through high pressure homogenizer. DS nano-liposomes (NLs) were separated by ultra centrifugation and characterized. NLs were dispersed in phosphate buffer saline (PBS) pH 7.4 containing different carriers like lactose, sucrose, and hydrolyzed gelatin, and 15% L-leucine as antiadherent. The resultant dispersion was spray dried and spray dried formulation were characterized to ascertain its performance. In vitro pulmonary deposition was assessed using Andersen Cascade Impactor as per USP. NLs were found to have average size of 137 +/- 15 nm, 95.17 +/- 3.43% drug entrapment, and zeta potential of 0.8314 +/- 0.0827 mV. Hydrolyzed gelatin based formulation was found to have low density, good flowability, particle size of 7.9 +/- 1.1 microm, maximum fine particle fraction (FPF) of 75.6 +/- 1.6%, mean mass aerodynamic diameter (MMAD) 2.2 +/- 0.1 microm, and geometric standard deviation (GSD) 2.3 +/- 0.1. Developed formulations were found to have in vitro prolonged drug release up to 16 h, and obeys Higuchi's Controlled Release model. The investigation provides a practical approach for direct delivery of DS encapsulated in NLs for site specific controlled and prolonged release behavior at the site of action and hence, may play a promising role in prevention of PCP.

Drug-induced agranulocytosis in rheumatology. A retrospective study of 12 cases

the aim of this study is to determine the clinical characteristics of drug-induced agranulocytosis in rheumatology. it is a retrospective monocentric study, including all cases of rheumatologic drug-induced agranulocytosis followed between January 1985 and December 2005. eight female and 4 men, mean age 62.5 years (range: 17-79), were included in the present study. The causative drugs were: anti-inflammatory agents (n=5, 41.6%), methotrexate (n=3, 25%), sulfasalazine (n=2, 16.6%), noramidopyrine (n=1) and dapsone (n=1). Main clinical features included infectious disorders in 9 cases (75%) with 4 cases of septicemia (33.3%) and 2 cases of septic shock (16.6%). The mean neutrophils count was 0.132 x 10(9)/L (range, 0-0.4). Outcome was favorable in 11 patients (91.6%). One patient died of septic complications. in rheumatology, drug-induced agranulocytosis is a rare but life-threatening disorder.

Fixed drug eruption: topical provocation and subsequent phenomena.

To determine the usefulness of topical provocation in detecting the incriminated drug causing fixed eruption. Quasi-experimental study. Department of Dermatology, Shaikh Zayed FPGMI, Lahore, from November 2002 to December 2005. Three hundred and five, clinically diagnosed cases of Fixed Drug Eruption (FDE) of either gender and of any age were subjected to topical provocation with different drugs by using concentration of 1% (n=203), 2% (n=210) and 5% (n=235) in white soft paraffin. Drug ointment of one strength was applied one at a time on normal skin of flexor surface of right or left forearm. The effects of tests on involved and uninvolved skin were observed for 48 hours. The changes in lesions like erythema, hyperpigmentation, itching, burning or appearance of new lesion were considered a positive response. In case of no change, the patients (n=5) were subjected to oral provocation test, by giving half to full therapeutic dose of the suspected drug depending upon the severity of the initial attack. A patient who exhibited see-sawing phenomenon with 5% metamizole TPT was given oral challenge with same drug. Control topical tests were repeated in equal number of normal persons with various drug ointments and in patients of FDE with white soft paraffin on normal and affected skin. One hundred and thirty-seven patients were males and one hundred and sixty-eight patients were females. Maximum number of patients belonged to third decade. With 1% drug preparations 12 out of 316, with 2% drug preparations 28 out of 422 and with 5% drug preparations, 312 out of 523 TPTs were positive. The comparison revealed a highly significant association (Chi-square 448.1 and p < 0.000) among various strengths of preparations and positive response. Sulphamethoxazole was found to be the most commonly incriminated cause of FDE applied in 5% concentration yielded sensitivity rate of 91% compared to 4% with lower concentrations. Positive patch test was also observed with oxytetracycline. Five patients who were given oral provocation with different drugs were found to be positive to tinidazole, dapsone, propylphenazone, belladonna and phenobarbitone. Interesting phenomena like earlier reactivation of lesion situated distal to site of application of preparation, marching, see-sawing (with oral metamizole) and lightening observed, are not yet reported in literature. Topical provocation test with 5% drug concentration applied on normal skin of patient with FDE is a possible first line investigation in finding the incriminated drug.

Crystal settling and crystal growth of olivine in magmatic differentiation − the Murotomisaki Gabbroic Complex, Shikoku, Japan

The Murotomisaki Gabbroic Complex is a sill-like layered intrusion of up to 220 m in thickness and is located at Cape Muroto, Kochi Prefecture, Japan. There are several olivine-rich zones within the intrusion, which may have been formed through accumulation of olivine crystals. However, up to now it has not been clear as to whether all of the olivine-rich zones formed in this way. To clarify this, we reinvestigated the layered structure by collecting a consistent data set of modal composition, crystal size, and crystal number density of olivine from throughout the intrusion. It was found out that nearly all of the olivine crystals, in terms of crystal numbers, occur in the basal olivine-rich zone (within 40 m of the base of the intrusion), and the average value of the crystal number density of olivine throughout the entire intrusion coincides with the crystal number density of olivine in the outermost parts of the lower and upper chilled margins. Hence, we conclude that most primary olivine phenocrysts within the magma settled under the influence of gravity and accumulated to form the basal olivine-rich zone. The crystal number density of olivine within the mid-level zones (40-100 m from the base of the intrusion) is much less than the initial values, as indicated by values recorded in the chilled margins. It is proved that the increase of the olivine mode within this zone is attributed not to the crystal accumulation of olivine but to the increase of the crystal size of olivine, i.e., the crystal growth. In this way, considering the mode, crystal size, and crystal number density of olivine throughout the intrusion, the olivine-rich zones within the intrusion can be classified, in terms of their origin, as either crystal accumulation zone (AC zone) or crystal growth zone (GR zone). The growth of olivine crystals in the GR zone was apparently accompanied by an increase in MgO, FeO, and MnO concentrations to levels well above initial (i.e., the chilled marginal) values. This enrichment suggests that crystal growth occurred within a chemically open system in the sense that the increase in MgO content within the GR zone arose from material transfer between the boundary layer (the GR zone) and the overlying magma.

Magmatic differentiation by means of segregation and diapiric ascent of anorthositic crystal mush - the Murotomisaki Gabbroic Complex, Shikoku, Japan

The Murotomisaki Gabbro is a sill-like layered igneous complex that contains several layers of olivine enrichment. In our previous paper (Hoshide et al., 2006), we have identified two zones of olivine enrichment: ‘the crystal accumulation zone (AC zone)’, formed by gravity settling and accumulation of olivine crystals, and ‘the crystal growth zone (GR zone)’, in which increase of modal olivine was caused by crystal growth of olivine and not by crystal accumulation. Based on whole rock compositional data, we have found that the AC zone rocks define a linear compositional trend (termed as ‘AC trend’) which is consistent with the crystal settling and accumulation hypothesis. However the GR zone data define another linear trend with a slope different from that of the AC trend. Moreover, the compositions of the coarse gabbros and the upper olivine gabbros that occur above the GR zone and an anorthosite vein from the GR zone roughly lie on the same trend, but on the opposite side of the GR zone composition, defining the ‘GR trend’ as a whole. Some anorthositic veins and wavy pegmatitic veins have plume-like structures, suggesting that these veins are remnant of crystal mushes that have been mobilized and ascended diapirically during magmatic differentiation. Considering the observed compositional relationships and the mode of occurrences of the anorthosite and wavy pegmatitic veins, we conclude that the segregation and separation of anorthositic material out of semi-solidified crystallization boundary layers was responsible for the formation of the GR zone and the GR trend. Phase equilibrium calculations reveal that the hypothetical anorthosite material was a mixture of fractionated melt and plagioclase crystals that precipitated from the melt. The GR zone represents a residue from the separation of anorthositic crystal mushes and the coarse gabbros and the upper olivine gabbro parts represent mixtures of the crystal mush and the initial melt.

Magmatic differentiation by means of segregation and diapiric ascent of anorthositic crystal mush - the Murotomisaki Gabbroic Complex, Shikoku, Japan

The Murotomisaki Gabbro is a sill-like layered igneous complex that contains several layers of olivine enrichment. In our previous paper (Hoshide et al., 2006), we have identified two zones of olivine enrichment: ‘the crystal accumulation zone (AC zone)’, formed by gravity settling and accumulation of olivine crystals, and ‘the crystal growth zone (GR zone)’, in which increase of modal olivine was caused by crystal growth of olivine and not by crystal accumulation. Based on whole rock compositional data, we have found that the AC zone rocks define a linear compositional trend (termed as ‘AC trend’) which is consistent with the crystal settling and accumulation hypothesis. However the GR zone data define another linear trend with a slope different from that of the AC trend. Moreover, the compositions of the coarse gabbros and the upper olivine gabbros that occur above the GR zone and an anorthosite vein from the GR zone roughly lie on the same trend, but on the opposite side of the GR zone composition, defining the ‘GR trend’ as a whole. Some anorthositic veins and wavy pegmatitic veins have plume-like structures, suggesting that these veins are remnant of crystal mushes that have been mobilized and ascended diapirically during magmatic differentiation. Considering the observed compositional relationships and the mode of occurrences of the anorthosite and wavy pegmatitic veins, we conclude that the segregation and separation of anorthositic material out of semi-solidified crystallization boundary layers was responsible for the formation of the GR zone and the GR trend. Phase equilibrium calculations reveal that the hypothetical anorthosite material was a mixture of fractionated melt and plagioclase crystals that precipitated from the melt. The GR zone represents a residue from the separation of anorthositic crystal mushes and the coarse gabbros and the upper olivine gabbro parts represent mixtures of the crystal mush and the initial melt.

Pregnancy in relapsing polychondritis: twenty-five pregnancies in eleven patients.

To determine maternal and fetal outcome in pregnant women with relapsing polychondritis (RP). Retrospective review of 11 records selected from among those of 116 female RP (Michet's criteria) patients. In these 11 women, 25 pregnancies occurred after or concomitantly with the onset of RP. The mean (+/- SD) age at RP onset was 25 +/- 5 years (range 15-31). Therapeutic abortion was performed in 1 woman because of ongoing cyclophosphamide treatment. Disease flare occurred in 7 of 24 pregnancies, requiring treatment modification in 2 cases. RP was considered stable in 16 and asymptomatic in 1 of the 24. Treatment of RP was with nonsteroidal antiinflammatory drugs (2 of 24), steroids (13 of 24), dapsone (7 of 24), or plasma exchanges (1 of 24); no treatment was given in 8 of the 24 pregnancies. In 14 pregnancies, the course was uneventful. Ten pregnancies were complicated by ectopic nidation (n = 3), spontaneous abortion (n = 3), premature birth (n = 3), and premature rupture of membranes (n = 1). Eighteen normal children were born. Pregnancy does not seem to modify the course of RP. Successful pregnancies may be achieved in women with RP. No RP was observed in the neonates.

Synthesis of [3H] Dapsone.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Individual and Simultaneous Spectrophotometric Determination of Dapsone and Metoclopramide HCl in Pharmaceutical Dosage Forms and Synthetic Binary Mixtures

A rapid, sensitive and selective spectrophotometric method has been developed for the quantitative determination of dapsone (DAP) and metoclopramide hydrochloride (MCP) in both pure and dosage forms. Individual and simultaneous methods are based on the diazo coupling reaction of these drugs with benzoylacetone (BAC) in alkaline medium. The resulting azo dyes exhibit maximum absorption at 437 and 411 nm with a molar absorptivity of 4.14x10(4) and 2.97x10(4) l mol-1 cm-1 for DAP and MCP, respectively. Simultaneous determination of DAP and MCP was developed utilizing first-order digital derivative spectrophotometry. All variables have been optimized. No interferences were observed from drug excipients and the validity of the methods was tested against reference methods.

Scavenging of photogenerated singlet molecular oxygen and superoxide radical anion by sulpha drugs - Kinetics and mechanism

The ability of the sulfanilic antibiotics (SDs), dapsone (DAP), sulfisoxazole (SFX), sulfadiazine (SFD), and sulfanilic acid (SFNA) to act as scavengers of the visible-light-photogenerated species superoxide radical anion (O2·–) and singlet molecular oxygen (O2(1Δg)) was studied employing the natural pigment riboflavin (Rf) and the artificial dye Rose Bengal as photosensitisers. A complex mechanism, common to all the SDs studied, was elucidated through stationary photolysis, polarographic detection of oxygen uptake, fluorescence spectroscopy, time-resolved phosphorescence detection of O2(1Δg), and laser flash photolysis. Visible-light irradiation of aqueous and aqueous methanolic solutions of Rf (ca. 0.02 mmol/L) plus SD (ca. 0.5 mmol/L) photogenerated excited singlet and triplet Rf (1Rf* and 3Rf*). Under these experimental conditions, only 3Rf* is quenched either by oxygen, giving rise to O2(1Δg) by electronic energy transfer to dissolved ground-state oxygen, or by SD, yielding semireduced Rf through an electron-transfer process. Complementary experiments performed in pure water employing superoxide dismutase and sodium azide inhibition of oxygen uptake, in parallel with laser flash photolysis data, showed that O2·– is also formed, probably due to the reaction of the anion radical from Rf with dissolved oxygen, yielding also neutral, ground-state Rf. Both active oxygen species, namely, O2·– and O2(1Δg), are quenched by the SDs and, as a result, photodegradation of the SDs — each to a different extent — and photodegradation of the sensitiser itself were observed. The SDs that kinetically behave as the better physical quenchers of O2(1Δg), which are in principle good candidates as photoprotectors, namely, DAP and SFD, suffer photooxidation, exhibiting high to moderate oxygen consumption rates due to the O2·– oxidative pathway, whereas for SFNA and SFX, oxidation predominantly occurs through an O2(1Δg)-mediated mechanism. Microbiological results for SFX, taken as a representative SD, indicate that the photodegradation of the drug, upon visible-light Rf-sensitised irradiation, is accompanied by a net loss in bacteriostatic activity.Key words: photooxidation, singlet oxygen, superoxide radical anion, sulpha drugs.

3-Aminophenol as a novel coupling agent for the spectrophotometric determination of sulfonamide derivatives

A rapid, simple and sensitive spectrophotometric method for the determination of some sulfa drugs is described. The method is based on the formation of orange yellow colored azo product by the diazotization of sulfonamides, viz., dapsone (DAP), sulfathiazole (SFT), sulfadiazine (SFD), sulfacetamide (SFA), sulfamethoxazole (SFMx), sulfamerazine (SFMr), sulfaguanidine (SFG) and sulfadimidine (SFDd) followed by a coupling reaction with 3-aminophenol in aqueous medium. Absorbance of the resulting orange yellow product is measured at 460 nm and is stable for 6 days at 27 °C. Beer's law is obeyed in the concentration range of 0.05–8.0 μg/ml at the wavelength of maximum absorption. The method is successfully employed for the determination of sulfonamides in various pharmaceutical preparations and common excipients used as additives in pharmaceuticals do not interfere in the proposed method. Plausible reaction mechanism is proposed for the formation of the azo product.

NOVEL METHODS FOR THE RAPID SPECTROPHOTOMETRIC DETERMINATION OF DAPSONE

ABSTRACT Rapid, sensitive and simple spectrophotometric methods for the determination of dapsone are described. The first method is based on the formation of violet coloured azo product by the diazotisation of dapsone (DAP) followed by a coupling reaction with iminodibenzyl (IDB) in alcohol medium. The second method makes use of N-bromosuccinimide (NBS) and promethazine hydrochloride (PMH) to form a green coloured product with dapsone in hydrochloric acid medium. Absorbances of the resulting violet and green products are measured at 570 nm and 610 nm respectively and are stable for 24 h and 50 min at 27°C respectively. Beer's law is obeyed in the concentration range of 0.1–2.5 µg mL−1 of DAP (for IDB) and 0.5–5.0 µg mL−1 of DAP (for NBS) at 570 nm and 610 nm respectively. The methods are successfully employed for the determination of dapsone in pharmaceutical preparation and common excipients used as additives in pharmaceuticals do not interfere in the proposed methods. The methods offer the advantages of rapidity, sensitivity and simplicity without the need for extraction or heating. Reaction sequences are described for the formation of products in both the methods.

The Role of Sequential Well Testing in Improving Oil Recovery From a Closed Sand Lens in the Bahrain Field

Summary The Ac zone refers to the sandstone facies of the Wara formation, which belongs to the Wasia group of the Middle Cretaceous age.* Owing to variable shale to sand ratios, the Ac zone is realized as different isolated sand lenses scattered all over the field. Owing to the complexity of the geology and the flow mechanism, well testing was used to manage the reservoir. The concept of a sequence of well tests on the same well was found to be essential in improving the oil recovery. Conducted on a specific well situated within a closed sand lens, sequential well testing yielded valuable information that assisted in understanding the drive mechanism and the deteriorating reservoir parameters, permeability, and skin. Such a method was crucial in improving the recovery from the sand lens as well as from the single-well-treatment perspective. This paper presents an example of how such a technique aided in improving the oil recovery from a small sand lens.

A trial of proguanil-dapsone in comparison with sulfadoxine-pyrimethamine for the clearance of Plasmodium falciparum infections in Tanzania

Considerable levels of resistance to sulfadoxine-pyrimethamine (SP) have been reported in Plasmodium falciparum in north-eastern Tanzania, and the identification of a suitable antimalarial to replace SP is now a high priority. We conducted a trial in July 2000 to determine the efficacy of proguanil (PG) plus dapsone (DS), compared with that of SP, for the treatment of asymptomatic falciparum infection. A total of 220 children with parasitaemia ⩾ 2000 per μL completed the study; 112 had received a single dose of SP (dosage calculated for pyrimethamine 1·25 mg/kg and sulfadoxine 25 mg/kg) and 108 had taken PG 10 mg/kgwith DS 2·5 mg/kg each day for 3 days. Clearance of asexual parasites at day 7 was 14·3% with SP, but 93·5% with PG-DS. The remarkably high failure rate with SP was not associated with occurrence of leucine substitution at position 164 of the dhfr gene. Both treatment regimens were well tolerated. Compared with available data on another antifolate combination, chlorproguanil-dapsone (‘Lapdap’), PG-DS was slightly but significantly inferior in achieving parasite clearance (99·5% versus 93·5%). The estimated cost of a 3-day course of PG-DS treatment for a child weighing 18 kg is US $0.15. With the rising incidence of SP-resistant P. falciparum infection, PG-DS could provide an effective, affordable and already available therapeutic alternative for malaria in East Africa at least until chlorproguanil-dapsone is registered.

A spectrophotometric method for the determination of metoclopramide HCl and dapsone.

A rapid, sensitive and selective spectrophotometric method has been developed for the quantitative determination of metoclopramide hydrochloride (MCP) and dapsone (DAP) in both pure and dosage forms. The method is based on the diazo coupling reaction of the drugs with a new coupling agent, dibenzoyl methane in an alkaline medium. The resulting coloured azo dyes exhibit maximum absorption at 440 nm for MCP and at 470 nm for DAP with a molar absorptivity of 2.85x10(4) and 1.71x10(4) l mol(-1) cm(-1) for MCP and DAP, respectively. All variables have been optimized. No interferences were observed from excipients and the validity of the method was tested against reference method.

A novel microassay for the quantitation of the sulfated glycosaminoglycan content of histological sections: its application to determine the effects of Diacerhein on cartilage in an ovine model of osteoarthritis

Objective A new micro-histological method of assessing the sulfated glycosaminoglycan (S-GAG) content in unstained histological sections of articular cartilage was developed and used to study the effects of orally administered Diacerhein (DIA) on joint cartilage in an ovine model of osteoarthritis (OA).Methods Twenty adult, age-matched Merino wethers were subjected to bilateral lateral meniscectomy, while 10 served as non-operated controls (NOC groups). Half of the operated sheep (N=10) remained untreated (MEN groups), while the other 10 animals were given DIA (25mg/kg orally) daily for 3 months, then 50mg/kg daily for a further 6 months (DIA groups). Five animals each of the DIA, MEN and NOC groups, respectively, were sacrificed at 3 months post-operatively, and the remainder 6 months later. For the present study only one knee joint of each animal was used for histological processing. The tissues studied were from the lateral femoral condyles (LFC) and lateral tibial plateaux (LTP). Each of these joint regions was further subdivided into inner (I), middle (M), and outer (O) zones. Unstained histological sections from these AC regions and zones were then analysed for S-GAG content using the following procedure. Images of each section of 6μm thickness were acquired using a flatbed scanner and the area determined with an image analysis software program. The sections were then transferred to wells of a microtiter plate, digested with papain and the S-GAG content quantitated using a modification of the 1,9-dimethylmethylene blue dye binding assay. The data was represented as μg S-GAG/mm3of each tissue section. These data were also compared with toluidine blue stained sections from the same paraffin blocks.Results The results obtained showed that the area of histological sections could be very accurately determined by computer assisted image analysis using a 10mm×10mm calibration grid. Cartilage sections of areas ranging from 1mm2up to 25mm2were analysed for S-GAG content with this simple technique. There was a linear relationship between section thickness (2–10μm) and S-GAG content per unit area (R2=0.993). Sections of 6μm thickness were found to be optimal. S-GAG analyses of serial sections from tibial and femoral articular cartilage (I, M and O zones) revealed an average coefficient of variation of 7.0±2.3% (range 4.9–10.2%) confirming the accuracy and reproducibility of this assay method. A separate experiment showed that no significant losses of S-GAG occurred during the histological sample processing.The different regions and zones of the knee joint AC in the six experimental groups revealed variable levels of S-GAG which did not necessarily correlate with the histochemical distribution of toluidine blue staining. The major S-GAG changes occurred in the middle (lesion zone) and outer zones (hypertrophic zone) of both the LFC and LTP of the MEN groups. In the lesion (M) zone the S-GAG content was reduced while in the O zone levels were increased at both 3 and 6 months post-surgery. In animals receiving Diacerhein S-GAG levels in the M zone were lower than or equivalent to those of non-drug treated OA or non-operated controls for both joint regions at 3 and 6 months. While the hypertrophic response in the outer zone of the LFC, as assessed by S-GAG content, was enhanced by drug treatment, the cartilage of the outer zones of the LTP was not affected by drug treatment.Conclusion The results of this study have demonstrated that the S-GAG (and therefore proteoglycan [PG]) content in different cartilage zones of OA joints can be readily quantitated by direct biochemical analysis of unstained histological sections. By this means subtle changes in PG distribution in different cartilage zones, which were not evident using traditional histochemical staining methods, could be readily detected.