Development of Microemulsion Formulations for Topical Delivery of Prodrugs Derived from Methotrexate and 5- Aminolevulinic Acid
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Issue Date
2022-12
Authors
Jadhav, Shreeya Satish '22
Degree
MS in Pharmaceutical Sciences
Advisor
Hass, Martha
Committee Members
Balaz, Stefan
Zheng, HaiAn
Zheng, HaiAn
Journal Title
Journal ISSN
Volume Title
Abstract
Psoriasis is a chronic autoimmune inflammatory disease that is mainly characterized by epidermal plaques on the surface of the skin as a result of increased hyperproliferation of epidermal keratinocytes and inflammation. Systemic and topical treatments are used to treat psoriasis, and the selection of treatment is dependent on the severity of the disease in the patient. Combination therapies and phototherapy are also used to manage the disease. There is no known cure for psoriasis and current treatments focus on suppression of the symptoms to decrease the severity of the disease and extending periods of remission to help limit the impact of the disease on the patient\342\200\231s day-to-day life.
This project focuses on the development and characterization of lipid-based topical formulations for the delivery of 5-aminolevulinic acid (ALA) and methotrexate (MTX), as a potential combination therapy combination for treating psoriasis. MTX supresses inflammation and attenuates uncontrolled growth of keratinocytes and modulates their differentiation by depriving these cells of folic acid through inhibition of an enzyme called dihydrofolate reductase (DHFR). Delivery of MTX directly to the skin by topical administration is hindered by its hydrophilic properties. ALA is a drug precursor used in photodynamic therapy that elevates protoporphyrin IX (PpIX) in the skin. Exposure of skin containing elevated levels of PpIX to blue light irradiation supresses hyperproliferation of keratinocytes. The Hass lab has previously demonstrated that MTX and ALA in combination effectively inhibits DHFR and decreases proliferation of keratinocytes after blue light irradiation. We aimed to further explore this drug combination in an intact skin model by identifying an effective lipid-based vehicle to deliver MTX, its diester prodrug methotrexate dimethyl ester (MTX DME), ALA and its ester prodrug, aminolevulinic acid benzyl ester (ALA BE) to the skin. We further aimed to show that the prodrugs of MTX and ALA hydrolyze in the skin to release the active parent drugs, resulting in an inhibition of DHFR and elevation of PpIX.
Capmul-based microemulsions (MEs) were determined to be suitable formulations for ALA, ALA BE, MTX disodium salt (MTX DSS), and a 1:1 molar combination of ALA + MTX DSS. The prodrugs, ALA BE and MTX DME were formulated in Capmul-based optimized microemulsions (OMEs). These MEs and OMEs were stable and clear, had optimum particle sizes (150 \342\200\223 300 d-nm), polydispersity indices (0.2 \342\200\223 0.5), and zeta potentials (-1 \342\200\223 -12 mV).
Intact porcine skin was used as a model of human skin to assess the penetration of MTX, ALA and their prodrugs, formulated in the Capmul-based MEs and OMEs. Hydrolysis of the ester prodrugs in the porcine skin was also investigated. Porcine skin was treated with ALA and ALA BE formulated as MEs, and PpIX levels after 4h and 8h treatments significantly increased compared to treatment with unloaded ME. Skin treated with the ME of ALA BE caused an elevation in PpIX levels significantly higher than those observed in controls, however the PpIX levels were lower than those observed in porcine skin treated with the ME of ALA. The elevation of PpIX in skin treated with ME of ALA BE suggests ALA BE was delivered to the skin and hydrolyzed to ALA, sufficiently to induce an elevation in PpIX. Treatment of the skin with the ME of MTX DSS did not cause any elevation of PpIX.
Penetration of formulated MTX DME into porcine skin was also investigated. Quantification of MTX DME in the skin using HPLC showed that MTX DME accumulates in the viable epidermis and hydrolyzes to MTX (67%).
Future studies will include assessing the entrapment efficiency of MTX, ALA and their prodrugs in the MEs. Blue-light irradiation studies will also be conducted with the porcine skin model to determine if Capmul-based MEs can deliver enough ALA and ALA BE to the viable skin layers to produce sufficient levels PpIX suppress keratinocyte proliferation. DHFR inhibition assay will be performed in porcine skin model to determine if sufficient MTX DME hydrolyzes to MTX in the skin to inhibit the enzyme.
Our long-term goal is to develop a codrug derived from MTX and ALA. The results of our formulation and penetration experiments in this project, specifically with the ester prodrugs ALA BE and MTX DME, demonstrated that prodrugs of ALA and MTX can be delivered to the skin where they undergo hydrolysis, suggesting that ester codrugs derived from ALA and MTX will also hydrolyze in the skin.
Citation
Jadhav SS. Development of microemulsion formulations for topical delivery of prodrugs derived from methotrexate and 5- aminolevulinic acid [thesis]. Ann Arbor (MI): Proquest LLC; 2022. 112 p.
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