Modifying amphotericin B for the treatment of cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a neglected tropical disease. It presents as disfiguring and deforming skin lesions. There is an urgent need for a simple, low-cost and non-toxic treatment for use in resource poor countries. As amphotericin B (AmB) has transformed visceral leishmaniasis treatment, I have developed and optimised a poly (methacrylic acid)-AmB drug (AmB-PMA). This involved the use of a commercially available, non-toxic, well-defined narrow molecular weight distribution polymer, poly (methacrylic acid sodium salt) (PMA) associated with AmB to create AmB-PMA. After chemical synthesis optimisation, AmB-PMA was shown to be less toxic than clinical grade AmB, and it was effective against Leishmania spp. promastigotes and amastigotes in vitro. To determine in vivo efficacy, studies were performed using BALB/c mice. They develop progressive non-healing skin lesions and are unable to control parasite growth. Mice were infected subcutaneously with L. major (LV39) promastigotes and treated locally with AmB-PMA. Studies were then conducted to evaluate the optimum route of administration, treatment frequency and dosing schedules. The results showed a reduction in both parasite burden and lesion size of the infected treated footpad with no adverse toxicity. Cytokine production was determined by quantitative real-time PCR for mRNA at the lesion site and the draining lymph node. I found that the AmB-PMA could effectively heal an established lesion and cure a CL infection. This was associated with: - (i) increased IFN-Y and TNF-α production, (ii) reduced IL-10 and MIP-1β production, when compared to infected, untreated lesions. The success of this treatment approach was also evaluated by histological studies. In addition, treated mice with resolved primary cutaneous lesions mounted a delayed type hypersensitivity response 24 h after challenge of the contra-lateral footpad with L. major. My thesis demonstrates the new opportunity for a cost-effective AmB based polymer drug that can be used locally to cure CL

Local Increase of Arginase Activity in Lesions of Patients with Cutaneous Leishmaniasis in Ethiopia

The leishmaniases are a complex of diseases caused by Leishmania parasites. Currently, the diseases affect an estimated 12 million people in 88 countries, and approximately 350 million more people are at risk. The leishmaniases belong to the most neglected tropical diseases, affecting the poorest populations, for whom access to diagnosis and effective treatment are often not available. Leishmania parasites infect cells of the immune system called macrophages, which have the capacity to eliminate the intracellular parasites when they receive the appropriate signals from other cells of the immune system. In nonhealing persistent leishmaniasis, lymphocytes are unable to transmit the signals to macrophages required to kill the intracellular parasites. The local upregulation of the enzyme arginase has been shown to impair lymphocyte effector functions at the site of pathology. In this study, we tested the activity of this enzyme in skin lesions of patients presenting with localized cutaneous leishmaniasis. Our results show that arginase is highly upregulated in these lesions. This increase in arginase activity coincides with lower expression of a signalling molecule in lymphocytes, which is essential for efficient activation of these cells. These results suggest that increased arginase expression in the localized cutaneous lesions might contribute to persistent disease in patients presenting with cutaneous leishmaniasis

Modifying amphotericin B for the treatment of cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a neglected tropical disease. It presents as disfiguring and deforming skin lesions. There is an urgent need for a simple, low-cost and non-toxic treatment for use in resource poor countries. As amphotericin B (AmB) has transformed visceral leishmaniasis treatment, I have developed and optimised a poly (methacrylic acid)-AmB drug (AmB-PMA). This involved the use of a commercially available, non-toxic, well-defined narrow molecular weight distribution polymer, poly (methacrylic acid sodium salt) (PMA) associated with AmB to create AmB-PMA. After chemical synthesis optimisation, AmB-PMA was shown to be less toxic than clinical grade AmB, and it was effective against Leishmania spp. promastigotes and amastigotes in vitro. To determine in vivo efficacy, studies were performed using BALB/c mice. They develop progressive non-healing skin lesions and are unable to control parasite growth. Mice were infected subcutaneously with L. major (LV39) promastigotes and treated locally with AmB-PMA. Studies were then conducted to evaluate the optimum route of administration, treatment frequency and dosing schedules. The results showed a reduction in both parasite burden and lesion size of the infected treated footpad with no adverse toxicity. Cytokine production was determined by quantitative real-time PCR for mRNA at the lesion site and the draining lymph node. I found that the AmB-PMA could effectively heal an established lesion and cure a CL infection. This was associated with: - (i) increased IFN-Y and TNF-α production, (ii) reduced IL-10 and MIP-1β production, when compared to infected, untreated lesions. The success of this treatment approach was also evaluated by histological studies. In addition, treated mice with resolved primary cutaneous lesions mounted a delayed type hypersensitivity response 24 h after challenge of the contra-lateral footpad with L. major. My thesis demonstrates the new opportunity for a cost-effective AmB based polymer drug that can be used locally to cure CL.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Leishmania infantum proteophosphoglycans regurgitated by the bite of its natural sand fly vector, Lutzomyia longipalpis, promote parasite establishment in mouse skin and skin-distant tissues

We demonstrate that a proteophosphoglycan-rich gel secreted by Leishmania infantum inside the midgut of Lutzomyia longipalpis sand flies (promastigote secretory gel) is regurgitated along with an average dose of 500 L. infantum metacyclic promastigotes per infected bite. Using both low (10³) and high (10⁵) doses of parasites in the ears of BALB/c mice we show that the infections benefit from the presence of vector saliva and parasite gel in the skin. However, chronic infection of the spleen was only enhanced in high dose co-infections with gel. These results provide the framework for a more natural experimental model of visceral leishmaniasis

Accelerated healing of cutaneous leishmaniasis in non-healing BALB/c mice using water soluble amphotericin B-polymethacrylic acid.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that causes prominent skin scaring. No water soluble, non-toxic, short course and low cost treatment exists. We developed a new water soluble amphotericin B-polymethacrylic acid (AmB-PMA) using established and scalable chemistries. AmB-PMA was stable for 9 months during storage. In vitro, it was effective against Leishmania spp. promastigotes and amastigote infected macrophages. It was also less toxic and more effective than deoxycholate-AmB, and similar to liposomal AmB. Its in vivo activity was determined in both early and established CL lesion models of Leishmania major infection in genetically susceptible non-healing BALB/c mice. Intradermal AmB-PMA at a total dose of 18 mg of AmB/kg body weight led to rapid parasite killing and lesion healing. No toxicity was seen. No parasite relapse occurred after 80 days follow-up. Histological studies confirmed rapid parasite clearance from macrophages followed by accelerated fibroblast mediated tissue repair, regeneration and cure of the infection. Quantitative mRNA studies of the CL lesions showed that accelerated healing was associated with increased Tumour Necrosis Factor-α and Interferon-γ, and reduced Interleukin-10. These results suggest that a cost-effective AmB-PMA could be used to pharmacologically treat and immuno-therapeutically accelerate the healing of CL lesions

Frequency of CD4⁺ and CD8⁺ T cells in PBMCs and biopsies of LCL patients.

<p>The frequencies of CD4⁺ and CD8⁺ T cells was determined in cells isolated from the blood and from the biopsies of LCL patients (n = 15) by flow cytometry. (A) % of CD4⁺ T cells; (B) % of CD8⁺ T cells; (C) ratio of CD4/CD8. Values represent median with interquartile range. Statistical significance was determined by a two-tailed Mann-Whitney test, ns = not significant.</p

Ratio of CD4/CD8⁺ T cells in PBMCs isolated from controls (n = 10) and LCL patients (n = 12).

<p>Values represent median ± sem.</p

L-arginine levels and CD3ζ expression in CD4⁺ and CD8⁺ T cells.

<p>Plasma was isolated by centrifugation of the blood of controls (n = 11), VL patients (n = 11) and treated VL patients (n = 11) and (A) the levels of L-arginine were measured by HPLC. PBMCs were isolated by density gradient from the blood of controls (n = 10), VL patients (n = 10) and treated VL patients (n = 10) and the mean fluorescence intensity of CD3ζ was determined in CD4⁺ T cells (B) and CD8⁺ T cells (C). Box = interquartile range and median; whiskers = range. Statistical significance was determined by a two-tailed Mann-Whitney test. NS = not significant. Patients = VL patients before treatment; treated patients = VL patients after 3–4 weeks treatment.</p

Frequency of neutrophils and monocytes in PBMCs.

<p>PBMCs were isolated by density gradient from the blood of controls (n = 10) and LCL patients (n = 15) and the frequencies of CD15⁺ (A), CD14⁺ (B) cells and the ratio of CD15/CD14 (C) were determined by flow cytometry. Values represent median with interquartile range. Statistical significance was determined by a two-tailed Mann-Whitney test, ns = not significant.</p