Fmoc-L-leucinol

Glioblastoma multiforme (GBM) is the most malignant type of central nervous system tumor that is resistant to all currently used forms of therapy. Thus, more effective GBM treatment strategies are being investigated, including combined therapies with drugs that may cross the blood brain barrier (BBB). Another important issue considers the decrease of deleterious side effects of therapy. It has been shown that nanocarrier conjugates with biotin can penetrate BBB. In this study, biotinylated PAMAM G3 dendrimers substituted with the recognized anticancer agents cyclooxygenase-2 (COX-2) inhibitor celecoxib and peroxisome proliferator-activated receptor γ (PPARγ) agonist Fmoc-L-Leucine (G3-BCL) were tested in vitro on human cell lines with different p53 status: glioblastoma (U-118 MG), normal fibroblasts (BJ) and immortalized keratinocytes (HaCaT). G3-BCL penetrated efficiently into the lysosomal and mitochondrial compartments of U-118 MG cells and induced death of U-118 MG cells via apoptosis and inhibited proliferation and migration at low IC50 = 1.25 µM concentration, considerably lower than either drug applied alone. Comparison of the effects of G3-BCL on expression of COX-2 and PPARγ protein and PGE2 production of three different investigated cell line phenotypes revealed that the anti-glioma effect of the conjugate was realized by other mechanisms other than influencing PPAR-γ expression and regardless of p53 cell status, it was dependent on COX-2 protein level and high PGE2 production. Similar G3-BCL cytotoxicity was seen in normal fibroblasts (IC50 = 1.29 µM) and higher resistance in HaCaT cells (IC50 = 4.49 µM). Thus, G3-BCL might be a good candidate for the targeted, local glioma therapy with limited site effects.

(N-[9-fluorenylmethoxycarbonyl]-)-L-leucine (FMOC-L-leucine) and rosiglitazone, two ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), were evaluated in mature (adult mice) and immature (pups) brain injury models. In adult magnesium-deficient mice, a model responsive to both neuroprotective and anti-seizure compounds, FMOC-L-leucine, but not rosiglitazone, protected against audiogenic seizures. The protection afforded by FMOC-L-leucine was alleviated by the PPARgamma antagonist GW9662 (1-2 mg/kg) and was induced in 50% animals by 4.8+/-1.2 mg/kg. At this dose, FMOC-L-leucine modified audiogenic seizure phase durations in convulsing mice differently than prototype antiepileptic drugs did. FMOC-L-leucine (up to 100 mg/kg) was inactive in the 6 Hz seizure test, an adult animal model largely responsive to anti-seizure drugs. In a model of neonatal brain injury, FMOC-L-leucine (4 microg/kg) was neuroprotective against cerebral ibotenate toxicity. It reduced significantly the size of lesions in grey but not in white matter, while rosiglitazone (10 microg/kg) was inactive. Taken as a whole, the present data support neuroprotective potentialities of FMOC-L-leucine towards both mature and immature brain. The PPAR-based protection of immature brain is more important as it is known that classic adult brain protectants (GABA(A) activators, N-methyl-D-aspartate and sodium channel blockers) may be toxic for immature brain. The PPARgamma agonist FMOC-L-leucine is likely to be devoid of these classic protective mechanisms because of its inactivity in the 6 Hz seizure test, its activity in the audiogenic test being explained by neuroprotective rather than intrinsic anti-seizure mechanisms. Targeting PPARs might be thus a promising way to protect immature brain.

Tumors still remain one of the main causes of mortality due to the lack of effective anti-cancer therapy. Recently it has been shown, that overexpression of inducible cyclooxygenase-2 (COX-2) and decrease of peroxisome proliferator-activated receptor γ (PPARγ) expression accompany many malignances, therefore, it has been proposed, that COX-2 inhibitors and PPARγ agonists are potential candidates for anticancer therapy and their synergistic, antineoplastic action has been described. In the present study a COX-2 inhibitor (celecoxib) and/or PPARγ agonist (Fmoc-l-Leucine) were conjugated with the biotinylated G3 PAMAM dendrimer to form a three different constructs targeted to cells with increased biotin uptake. All conjugates were characterized by the NMR spectroscopy. Investigation of three types of human cells: normal skin fibroblasts (BJ), immortalized keratinocytes (HaCaT) and cancer lines: glioblastoma (U-118 MG) and squamous cell carcinoma (SCC-15) revealed similar biotin labeled ATTO590 accumulation (after 24 h), except for SCC-15 with significantly lower loading. Constitutive expression of COX-2 protein was confirmed in all tested cells with significantly higher levels (2-2.5 times) in both cancer lines. Comparison of cytotoxicity of the new synthetized dendrimers clearly documented the highest cytotoxicity of the G31B16C15L dendrimer conjugated with both drugs (1: 1) as compared with drugs alone and single conjugates. Additive effects of construct with both compounds were shown for fibroblasts and both cancer cell lines in the order BJ > U-118 MG > SCC-15 with IC50 in the range: 0.69, 1.44 and 2.22 μM, respectively and lowest cytotoxicity in HaCaT cells (IC50 = 2.88). Our results showed, that biotinylated G3 PAMAM dendrimers substituted with COX-2 inhibitor, celecoxib, and PPARγ agonist, Fmoc-l-Leucine (1:1) may be a good candidate for local therapy of glioblastoma but not a skin cancer. Since the effect of PPARγ agonists on COX-2 expression vary depending upon the cell type, specificity of used agonist and the presence of other environmental factors, it is necessary to carefully evaluate the response of chosen drugs on the target cells.