Hass, Martha Theses Advised
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Item Assessment of Hydrolysis of Prodrugs and Co-drugs Derived from 5-Aminolevulinic Acid and Mycophenolic Acid(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Strumski, Kaitlyn '23Mycophenolic acid (MPA) and 5-aminolevulenic acid (ALA) have been known to reduce the symptoms associated with autoimmune skin disease, psoriasis. It is hypothesized that the combination of these two drugs, when delivered simultaneously to the skin, will provide synergistic therapeutic benefit in suppressing the symptoms of psoriasis. This research project aims to evaluate the in vitro efficacy of prodrugs and investigational co-drugs derived from ALA and MPA using an immortalized human keratinocyte cell line (HaCaT) as models for drug behavior in human skin. Previously, it was shown that MPA and ALA do not negatively interact, but rather improve treatment when administered together. This project is specifically aimed at using HaCaT cells as a viable “skin” model to determine if the prodrugs and the investigational co-drugs derived from ALA and MPA are sufficiently metabolized to release therapeutically effective doses of the parent compounds that elevate protoporphyrin IX (PpIX) and inhibit inosine-5′-monophosphate dehydrogenase (IMPDH). HaCaT cells were grown, and cultures were treated with ALA-BE and ALA separately to evaluate the increase in PpIX which is hypothesized to correlate with hydrolysis of ALA-BE into the active compound ALA. MPA-ME was synthesized, HaCaT cell lysate was prepared, and the lysates were treated with MPA and MPA-ME. The MPA treated lysates were analyzed using HPLC to determine the extraction efficiency of MPA and the MPA-ME treated lysates were analyzed using HPLC to determine the percent hydrolysis of MPA-ME into the active compound MPA. This research establishes that HaCaT cells contain hydrolytic enzymes capable of promoting hydrolysis of ALA-/MPA- prodrugs and co-drugs that results in release of the parent drugs, ALA and MPA. The activity of the parent compounds correlates with the extent to which the prodrugs and co-drugs hydrolyze in epithelial cells.Item Development of Microemulsion Formulations for Topical Delivery of Prodrugs Derived from Methotrexate and 5- Aminolevulinic Acid(Albany College of Pharmacy and Health Sciences Theses, 2022-12) Jadhav, Shreeya Satish '22Psoriasis 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.Item Assessment of the in vitro efficacy of 5-aminolevulinic acid in combination with methotrexate: A potential new therapy for the treatment of psoriasis(Albany College of Pharmacy and Health Sciences Theses, 2022-08) Quirk, Mackenzie '22Psoriasis is broadly defined as a chronic skin disease marked by raised, itchy scales that can be expressed anywhere but are most found on the knees, elbows, trunk, and scalp. Patients with moderate to severe plaque psoriasis experience physical discomfort in addition to higher levels of anxiety, depression, bullying, and lower employment opportunities than people with healthy skin. There are two main contributors to the development and prolongation of the disease: T-cell activation and recruitment resulting in chronic inflammation, and the hyperproliferation of keratinocytes to form thick, scaly plaques. Current drug therapy targets one or both mechanisms of pathophysiology. This project focused on the potential therapeutic benefits of combining the antiproliferative agent methotrexate (MTX) with the photosensitizing agent 5-aminolevulinic acid (ALA) in an in vitro model for plaque psoriasis using HaCaT cells. To determine the effect of protoporphyrin IX (PpIX) generation, cells were treated with either ALA alone, MTX alone, or a 1:1 molar combination of ALA + MTX at a concentration range of 0.1 \342\200\223 1 mM. Absorbance was measured using fluorescent spectroscopy. To determine the clinical effect of PpIX induced phototoxicity, treated cells were irradiated with blue light and cell viability was assessed using an MTT assay. To assess the antiproliferative effect of drug treatment at a range of 2 \342\200\223 100 nM, a dihydrofolic reductase (DHFR) enzyme inhibition assay was used. The results suggest that MTX does not affect the ability of ALA to cause a dose-dependent increase in PpIX generation. Additionally, blue light irradiation in cells treated with ALA alone or in combination causes significant cytotoxicity compared to cells not exposed to blue light, indicating that MTX does not impede the ability of ALA-induced PpIX production to cause phototoxicity. The DHFR assay demonstrated that ALA does not impair the ability of MTX to inhibit DHFR. In summary, the combination of ALA and MTX provides two mechanisms of action against an in vitro model of psoriasis without either compound interfering with the mechanism of the other, suggesting that the combination of these agents would be feasible and warrants further investigation with in vivo models.Item Anti-proliferative effect of Aminolevulinic acid (ALA) and Mycophenolic acid (MPA) with blue light irradiation(Albany College of Pharmacy and Health Sciences Theses, 2021-12) Capriglione, Noelle Grace '21Combining the photodynamic therapeutic (PDT) effects of aminolaevulinic acid (ALA) coherently with the chemotherapeutic benefits of mycophenolic acid (MPA) for psoriasis treatment, as a codrug has never been pursued. ALA is an endogenous molecule currently used in PDT for psoriasis by limiting the hyperproliferation of keratinocytes which characterizes psoriasis and manifests as scaling layered plaques on the skin. The application of ALA increases the production of photosensitizer protoporphyrin IX-(PpIX) through a PpIX mediated mechanism that reduces cell viability when irradiated. MPA, another reliable treatment for psoriasis, inhibits the function of an enzyme essential for purine synthesis called inosine monophosphate dehydrogenase (IMPDH) which is specific for resident and infiltrating T-cells in psoriatic skin. The goal of this research is to investigate how MPA affects the ability of ALA to induce an elevation of PpIX levels in keratinocytes and subsequently how MPA affects cell viability of ALA-treated HaCaT cells exposed to blue light. In vitro activity of ALA and the effect of MPA on this activity were established by measuring PpIX concentration in HaCaT cells treated with various doses of ALA alone or in combination with MPA, using fluorescence spectroscopy. Cytotoxicity of drug treatments with and without blue-light irradiation was determined using a cell viability assay. PpIX formation in ALA-treated HaCaT cells increased in a dose-response manner as concentration treatments of ALA (in PBS) increased (250, 500, 1000\302\265M). MPA did not significantly hinder ALA functionality for PpIX generation in HaCaT cells when treated in (1:1) combination at 500 and 1000\302\265M. We observed significant cytotoxic effects in HaCaT cells treated with ALA at 500 and 1000\302\265M, after blue-light exposure. This cytotoxicity was observed to the same extent in HaCaT cells treated in an equimolar combination treatment with MPA, at 500\302\265M. The cytotoxicity observed from the phototherapy with ALA treatment alone was enhanced at 1000\302\265M in the (1:1) combination treatment, ALA + MPA. More experiments will need to be done with a wider range of concentration treatments at dosages of ALA where PpIX formation is detectable in HaCaT cells. The combination of ALA + MPA seems to be a suitable co-drug candidate. The possible effects of ALA on MPA functionality, in IMDPH inhibition, must be explored.Item Characterizing Microemulsion Formulations and Their Stability for Topical Delivery of Prodrugs(Albany College of Pharmacy and Health Sciences Theses, 2021-12) Uddin, Zaheen Nafi Sala '21Psoriasis is a chronic and common disease of the skin that is characterized by epidermal hyperplasia, altered keratinocyte differentiation, dilated vasculature in the dermis, and infiltration of leukocytes into both the epidermal and dermal layers of the skin. This autoimmune disease involves the acceleration of the life cycle of skin cells (keratinocytes) resulting in inflammation and plaques. Therapies for treating psoriasis are aimed at suppressing hyperproliferation of keratinocytes and tempering the activation of the immune cells.
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