Background and Significance. Cryptococcus neoformans and C. gattii are encapsulated yeasts that cause chronic infections in both immunocompetent and immunosuppressed individuals and are responsible for over 625,000 deaths each year. Amphotericin B (AmB) has been the mainstay of treatment for cryptococcal menin- goencephalitis (CME). However, the significant mortality rate (~20%) in these patients treated with available an- tifungal agents indicates the need for better therapies. AmB is a relatively large molecule and does not effec- tively cross the blood brain barrier (BBB). Our goal is to develop formulations of AmB that will cross the BBB in concentrations capable of effectively eliminating fungal organisms from the brain tissue for treating CME. Hypotheses. BBB endothelial cells express transferrin receptor (TfR) at high levels to transport iron from the blood into the brain. A peptide (CRTIGPSVC) has been identified that mimics iron by binding to transferrin and could be used to target drugs to the brain. We have produced a mutant of streptavidin (SAm7) fused to the brain targeting peptide (BTP) CRTIGPSVC. This fusion protein (BTP-SAm7) binds to TfR. In addition, BTP- SAm7 complexed with biotinylated AmB* (BTP-SAm7-[AmB*]) is effective against C. neoformans in vitro, crosses BBB in vitro, and reduces brain CFUs in murine CME. We hypothesize that a fusion protein consisting of a biotin binding protein (e.g., streptavidin) and a BTP will transport AmB* into the brain. We further hypothe- size that brain-targeted AmB will have greater efficacy against CME. Finally, we hypothesize that targeting AmB to the brain will allow use of lower total doses and thus reduce concerns of AmB systemic toxicity.
Specific Aims. 1) to optimize the transport and delivery of AmB across the BBB, 2) to determine the anti- cryptococcal activity of novel AmB analogs, 3) to determine the efficacy of brain-targeted AmB in murine CME, and 4) to examine the toxicity of brain-targeted AmB. Work Proposed.
For Aim 1, Individual components of the transporter BTP-SAm will be optimized in parallel. We will study other peptides as BTP and other monomeric variants of SA that have varying affinity for biotin. We will systematically test these second-generation molecules in vitro on hCMEC/D3 cell monolayers, which has been established as an in vitro model of the BBB, in comparison to CRTIGPSVC-SAm7 and irrelevant peptide controls. We will determine whether BTP-SAm7 can effectively deliver AmB* into the brain parenchyma in mice.
In Aim 2, we will determine whether analogs of AmB and AmB linked to antifungal peptides that are synergistic with AmB have greater activity against C. neoformans and/or less toxicity.
For Aim 3, we will determine efficacy of BTP-SAm-[AmB*] in mice with CME.
For Aim 4, we will evaluate toxicity of BTP-SAm-[AmB*] compared to AmB in primary neuronal cell cultures and uninfected mice.
After HIV, tuberculosis and malaria, Cryptococcus spp. are the fourth most common infectious cause of mortal- ity in the World, causing over 625,000 deaths/year. Cryptococcus spp. is the third most common invasive fun- gal infection in organ transplant patients, occurs in patients with HIV/AIDS, hematologic malignancies, those treated with chemotherapy, and those receiving immunosuppressive drugs for autoimmune or inflammatory conditions. These are all conditions that affect Veterans. Amphotericin B (AmB) is the most effective drug for treating cryptococcal meningoencephalitis (CME), but the drug does not cross the blood brain barrier (BBB) effectively. The significant mortality rate (~20%) in patients with cryptococcal meningoencephalitis treated with currently available antifungal agents indicates the need for better therapies. We propose to develop methods for effectively delivering AmB across the BBB in concentrations capable of eradicating infection.