COMPARISON OF THE EFFECTS OF ORAL AND TRANSDERMAL ADMINISTRATION OF CHLOROQUINES ON SELECTED HEMATOLOGICAL PARAMETERS AND INFLAMMATORY CYTOKINES IN P.BERGHEI- INFECTED MALE SPRAGUE-DAWLEY RATS. Finally, I would like to thank the National Research Foundation (NRF) and the College of Health Sciences for providing the funds to continue my studies.
R ed cell distribution width , M ean Platelet volume and M ean corpuscular haemoglobin 22
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Oral CHQ decreased proinflammatory cytokine concentration in infected rats on the day (day 8 of the experiment) of the treatment period compared to pretreatment (baseline) measurements. In non-infected rats, oral CHQ treatment showed adverse morphological effects on the architecture of these organs, whereas no changes were observed after transdermal CHQ delivery.
INTRODUCTION 1.0 Background
Literature review
The schizonts produced from the erythrocytic stage of the life cycle either infect more red blood cells or differentiate into sexual forms, macro- or microgametocytes. The erythrocytic phase of the Plasmodium life cycle is responsible for most malaria-associated pathologies, including fever and anemia (Haldar and Mohandas, 2009).
HUMAN STAGE
- Anaemia
- Hepcidin
- Cytokines
- TNF-α
- Effects of CHQ on the immune system
- Management of malaria
- Chloroquine
- Inflammation and tissue damage
- C-reactive protein
- Transdermal delivery
- Problem statement and significance
- Aims/ Objectives of the study
Research shows that elevated levels of pro-inflammatory cytokines play a role in the pathogenesis of malaria (Jacobs et al., 1996). In addition, current literature shows substantial evidence for the role of this cytokine in the development of anemia (Clark et al., 2006).
Terminal studies: Blood, Heart, liver, kidney, spleen and skin were collected for biochemical and histological analysis
Physiological and morphological effects after oral and transdermal administration of CHQ in different tissue organs were evaluated in separate groups of uninfected and P.berghei-infected male Sprague-Dawley rats (n=6 in each group) for a period 21 days. . The animals were divided into separate groups malaria-infected mice and non-infected mice (body weight 90-120 g).
This manuscript will be submitted to Malaria Journal. The manuscript has been written according to the Journal guidelines
Oral and transdermal administration effects of chloroquine on selected haematological parameters & pro-inflammatory cytokine concentration in malaria infected rats
Gumede, NM., and Mabandla, MV
- Drugs and chemicals
- Animals
- Experimental design
- Terminal studies
- Measurement of blood parameters
- Data analysis
- Effects of CHQ treatment on Red blood cells, haematocrit and haemoglobin
The effects of oral CHQ (30 mg/kg) and a single topical application of pectin-CHQ patch (56 mg/kg) treatment on uninfected and P. berghei -infected rats were assessed for factors that may be responsible for development of anemia. Topical application of the pectin-CHQ patch had no effect on the above parameters in non-infected animals (NIP) when compared with pre-treated control animals (NIC). Table 1: Comparison of the effect of twice daily oral CHQ treatment (30 mg) or once topical application of pectin-CHQ matrix patch (56 mg), on red blood cells (RBC), hematocrit (HCT) and hemoglobin ( HGB) in uninfected and P.berghei-infected animals.
The effects of CHQ treatment on RDW, MCH and MPV were measured on samples collected from uninfected and P.berghei -infected rats treated with either oral CHQ or the pectin-CHQ matrix patch. A single topical application of the pectin-CHQ patch had no effect on RDW, MCH and MPV in the non-infected group (NIP) compared to the pre-treatment control values (NIC). Topical application of the pectin-CHQ matrix patch had no effect on the above parameters in the non-infected animals compared to non-infected control animals.
The effect of CHQ on TNF-α, IL-1β and IL-6 was evaluated in uninfected and P.berghei-infected male Sprague-Dawleys rats treated with either oral CHQ or a topical skin patch application.
Time (days)[TNF- pg\ml]
Infected control animals had a higher concentration of IL-1 β when compared to non-infected α animals (NIO vs. day 8 IC, p<0.05, Figure 2).
Time (days )
This removal of unparasitized RBCs is believed to be one of the mechanisms that results in persistent anemia (Eric et al., 2013). Furthermore, literature suggests that the weak base of CHQ accumulates in acidic organelles of the cell, where it blocks acidification (Moss et al., 1992). Our results indicate that treatment with oral CHQ may lead to bleeding problems (Vinik et al., 2001).
Pro-inflammatory cytokines, such as TNF-α and IL-10, are believed to play a role in the development of anemia (Kurtzhals et al., 1998). These results confirm findings that have shown an inhibitory effect of CHQ on pro-inflammatory cytokine concentration (Van den Born et al., 1997). CHQ is a potent autophagic drug that can lead to cellular breakdown of hepatocytes in the liver (Abraham et al., 1968).
However, a small part of it accumulates in renal epithelial cells (Gustafsson et al., 1983).
This manuscript will be submitted to Malaria journal. The manuscript has been written according to the Journal guidelines
Identifying morphological changes in visceral organs following oral or transdermal administration of chloroquine in P.berghei-infected rats
Experimental design
- Effects of oral and transdermal CHQ treatments on various visceral organs
The effects of oral CHQ treatment (30 mg/kg) and single application of the pectin-CHQ matrix patch (56 mg/kg) on the heart, kidney, liver and spleen were studied in uninfected and infected rats. The studies were conducted over a period of 3 weeks divided into pre-treatment periods (days 0-7), treatment periods (days 8-12) and post-treatment periods (days 13-21). Animals were housed individually in Makrolon polycarbonate metabolic cages (Techniplasts, Labotec, South Africa) at the Biomedical Resource Unit, University of KwaZulu-Natal.
During the treatment period, the orally treated group was given CHQ diphosphate solution CHQ (30mg/kg), twice a day, 8 hours apart by means of a ball-tipped, 18-gauge probe needle (Kyron Laboratories (Pty) LTD, Benrose, South Africa ) attached to a 1 ml syringe, for 5 consecutive days. For transdermal administration of CHQ, the animals were treated with pectin CHQ matrix patches containing CHQ (56mg/kg). Before treatment, the rats were shaved on the dorsal neck region in preparation for the application of the pectin CHQ matrix patch.
On the first day of the treatment period, a pectin CHQ matrix patch was applied at 9:00 am.
Methods
- Preparation of the pectin-CHQ matrix patches
- Induction of malaria
Sacrifice and removal of skin
- Haematoxylin and Eosin analysis
Briefly, standards and samples were added to wells coated with an antibody specific for CPR in rats. A biotinylated detection antibody specific for rat CPR and AVIDIN-HRP conjugate was added to each microtiter plate well before incubation for 1 hour at 37°C. The enzyme-substrate reaction was stopped by adding a sulfuric acid solution, after which the color turned yellow.
The concentration of rat CPR in the samples was measured by extrapolating the OD of the samples from the standard curve. After incubation, the reaction volume was emptied by inverting the plate and tapping gently against a clean absorbent paper. Substrate solution (90µL) was added to each well after which the plate was covered with a new plate sealer and tin foil (to protect from light), incubated at 37°C for 15 minutes or until the color change reaction was complete.
The reaction was stopped by adding stop solution (50μL) as soon as the color change became visible.
Data analysis
After the final wash, plates were inverted onto absorbent paper to remove excess liquid.
Liver histology
Compared with the untreated uninfected control rats (Figure 3A), P.berghei-infected rats (Figure 3B) showed thickened glomerular basement membrane (TGBM) and thickened basement membrane in Bowman's capsule (BC). Photomicrograph 4 represents the kidney from uninfected rats treated with either oral CHQ or transdermal patch. Photomicrograph 6 represents the heart of uninfected rats treated with either oral CHQ or transdermal patches.
Orally treated uninfected rats Figure 6: Figure 6 shows the heart of uninfected treated rats. Orally treated uninfected rats (Figure 6A) showed moderate cardiomyocyte hypertrophy compared to rats treated with uninfected CHQ pectin patches (Figure 6B). Photomicrograph 8 represents the spleen of uninfected rats treated with oral CHQ or transdermal patch.
The heart mass of non-infected control animals (NIC) did not change significantly throughout the experimental period (Table 1).
Effect of CHQ treatment on C-reactive protein concentration in plasma
Time (days)
We speculate that these changes could be attributed to increased CHQ deposition in the liver after oral treatment. This accumulation of CHQ in the liver after oral administration has been reported to cause hepatocellular injury (McChesney et al., 1976). This is supported by the fact that no morphological changes in liver histology were observed in uninfected animals given CHQ transdermally.
These changes are thought to be attributed to the increased deposition of CHQ in the heart and kidney after treatment (Dondo and Mubagwa, 1990). However, a small fraction of CHQ accumulates in the epithelial cells of the kidneys (Gustafsson et al., 1983). This deposition of CHQ in the kidney is reported to alter renal function (McChesney et al., 1976).
Furthermore, part of the administered CHQ is deposited in the heart (Baguet and Fabre, 1999), where CHQ exerts antiarrhythmic actions to increase heart rate (Dondo and Mubagwa, 1990).
Conclusion
Synopsis
The main aim of the present study was to investigate and compare the effects of using oral chloroquine (CHQ) treatment or a transdermal CHQ patch in the management of malaria infection. The results of the present study showed that both malaria infection and oral CHQ delivery resulted in a reduction in the concentration of red blood cell (RBC), hematocrit (HCT), hemoglobin (HGB), red cell distribution width (RDW) and mean corpuscular hemoglobin (MCH). Interestingly, topical application of pectin CHQ matrix patch did not alter RBC indices of non-infected animals, suggesting the ability of the patch to circumvent the toxic effects of oral CHQ delivery.
The present study measured plasma cytokines to elucidate the mechanism of the observed changes in RBC indices. Our results showed an increase in the concentration of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in mice infected with P.berghei. This suggests better handling of the drug by the body in this mode of treatment.
Topical application of a pectin CHQ matrix patch did not alter the erythrocyte indices of uninfected animals, indicating the ability of the patch to avoid the toxic effects of oral CHQ administration.
Recommendations
The ability of the pectin-CHQ matrix patch to provide slow, sustained releases of CHQ into the circulation avoids drug dumping in various tissue organs and thus circumvents the adverse effects associated with oral administration of CHQ. Serum levels of the pro-inflammatory cytokines Interleukin-1 Beta (IL-1), IL-6, IL-8, IL-10, Tumor Necrosis Factor Alpha, and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and corresponding Uncomplicated malaria or healthy controls. Investigations of the use of bioavailability data to adjust occupational exposure limits for active pharmaceutical ingredients.
Comparative Study of Hematology and Serum Biochemistry of Male Wistar Rats Treated with Chloroquine and Artesunate. Chloroquine-induced inhibition of the production of TNF, but of IL-6, is affected by disruption of iron metabolism. Evaluation of the efficacy of transdermal delivery of chloroquine on Plasmodium berghei-infected male sprague-dawley rats: Effects on blood glucose and renal electrolyte handling.MMeD thesis, University of KwaZulu Natal.
Evaluation of the efficacy of Masilic acid on malaria parasites in plasmodium berghei-infected rats: effects on blood glucose and renal fluid electrolyte handling.
RITH TOWER BUILDING
