Supplementary Figure S5. DNA damage signaling activated by low concentrations of DP68. U251 and T... more Supplementary Figure S5. DNA damage signaling activated by low concentrations of DP68. U251 and T98G cells were exposed to DP68 for 24 hrs and whole cell and nuclear extracts were processed for Western blotting. Representative blots from three independent experiments are shown.
Supplementary Figure S4. DP86 and DP68 activity is independent of MMR expression. (A) Western blo... more Supplementary Figure S4. DP86 and DP68 activity is independent of MMR expression. (A) Western blot confirmed knockdown in T98G cells infected with empty vector, sheGFP, and two shRNAs targeting MLH1. (B) Infected cells were treated with TMZ, DP86, or DP68 and cell survival was analyzed via CyQuant assay. Mean IC50 {plus minus} SEM from three independent experiments are graphed. * (p<0.05)
Supplementary Figure S2. Summary of prodrug activation. Summary of prodrug activation and drug me... more Supplementary Figure S2. Summary of prodrug activation. Summary of prodrug activation and drug mechanisms of action for TMZ, DP68 and DP86 highlighting the roles of diazonium and aziridinium ion intermediates. (A-B) TMZ hydrolysis, generation of methyldiazonium ions and reaction with DNA; (C) DP86 monoalkylation; (D) DP68 crosslink formation.
The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indometha... more The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indomethacin in healthy children. The participants (n = 31, aged 4–144 months) received indomethacin (0.35 mg/kg) as a 10‐minute intravenous infusion prior to surgery under spinal anaesthesia. A single CSF and plasma sample from each individual was collected 14 to 225 minutes after the infusion. Indomethacin concentrations were determined from the CSF, plasma, and protein‐free plasma. Total plasma, protein‐free plasma, and CSF concentrations of indomethacin ranged between 90 and 2200 ng/mL (median, 780 ng/mL), 0.3 and 0.8 ng/mL (median, 0.5 ng/mL), and 0.2 and 5.0 ng/mL (median, 1.4 ng/mL), respectively. The CSF to plasma concentration ratio remained less than 0.01. There was no correlation between the administration time and CSF concentrations. Eleven children developed 12 nonserious adverse effects, from which 5 were central nervous system (CNS) effects (agitation). In conclusion, indomethacin permeated into the CSF of children, which enables both desired and adverse CNS effects of indomethacin.
European Journal of Pharmaceutical Sciences, Feb 1, 2023
Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly solub... more Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly soluble yet readily absorbable parent drug amprenavir. In this study, a novel cell-free in vitro setup with quasi-continuous monitoring of the dynamic dissolution/bio-conversion/permeation of fosamprenavir was designed and tested. It consists of side-by-side diffusion cells, where the donor and acceptor compartments are separated by the biomimetic barrier PermeaPad®, and sampling from the donor compartment is accomplished via a microdialysis probe. Externally added bovine alkaline phosphatase induced bioconversion in the donor compartment. Microdialysis sampling allowed to follow the enzymatic conversion of fosamprenavir to amprenavir by the bovine alkaline phosphatase in an (almost) real-time manner eliminating the need to remove or inactivate the enzyme. Biomimetic conversion rates in the setup were established by adding appropriate amounts of the alkaline phosphatase. A substantial (6.5-fold) and persistent supersaturation of amprenavir was observed due to bioconversion at lower (500 µM) concentrations, resulting in a substantially increased flux across the biomimetic barrier, nicely reflecting the situation in vivo. At conditions with an almost 10-fold higher dose than the usual human dose, some replicates showed premature precipitation and collapse of supersaturation, while others did not. In conclusion, the proposed novel tool appears very promising in gaining an in-depth mechanistic understanding of the bioconversion/permeation interplay, including transient supersaturation of phosphate-ester prodrugs like fosamprenavir.
Various novel morpholinyl-(3a,b) and methylpiperazinylacyloxyalkyl (3c-f) esters of 2-(6methoxy-2... more Various novel morpholinyl-(3a,b) and methylpiperazinylacyloxyalkyl (3c-f) esters of 2-(6methoxy-2-naphthyl)propionic acid were synthesized and evaluated in vitro for topical drug delivery as potential prodrugs of naproxen (1). Compounds 3a-f were prepared by coupling the corresponding naproxen hydroxyalkyl ester with the morpholinyl-or (4-methyl-1-piperazinyl)acyl acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) and quantitatively hydrolyzed (t 1/2) 1-26 min) to naproxen in human serum. Compounds 3c-f showed higher aqueous solubility and similar lipophilicity, determined by their octanol-buffer partition coefficients (log P app), at pH 5.0 when compared to naproxen. At pH 7.4 they were significantly more lipophilic than naproxen. The best prodrug 3c led to a 4and 1.5-fold enhancement of skin permeation when compared to naproxen at pH 7.4 and 5.0, respectively. The present study indicates using a methylpiperazinyl group yields prodrugs that are partially un-ionized under neutral and slightly acidic conditions, and thus, a desirable combination is achieved in terms of aqueous solubility and lipophilicity. Moreover, the resulting combination of biphasic solubility and fast enzymatic hydrolysis of the methylpiperazinylacyloxyalkyl derivatives gave improved topical delivery of naproxen.
Clozapine (CZP) is the only effective drug in schizophrenia resistant to typical antipsychotics. ... more Clozapine (CZP) is the only effective drug in schizophrenia resistant to typical antipsychotics. However, existing dosage forms (oral or orodispersible tablets, suspensions or intramuscular injection) show challenging limitations. After oral administration, CZP has low bioavailability due to a large first-pass effect, while the i.m. route is often painful, with low patient compliance and requiring specialised personnel. Moreover, CZP has a very low aqueous solubility. This study proposes the intranasal route as an alternative route of administration for CZP, through its encapsulation in polymeric nanoparticles (NPs) based on Eudragit® RS100 and RL100 copolymers. Slow-release polymeric NPs with dimensions around 400–500 nm were formulated to reside and release CZP in the nasal cavity, where it can be absorbed through the nasal mucosa and reach the systemic circulation. CZP-EUD-NPs showed a controlled release of CZP for up to 8 h. Furthermore, to reduce mucociliary clearance and incre...
l-type amino acid transporter 1 (LAT1) is an amino acid transporter that is overexpressed in seve... more l-type amino acid transporter 1 (LAT1) is an amino acid transporter that is overexpressed in several types of cancer and, thus, it can be a potential target for chemotherapy. The objectives of this study were to (a) synthesize LAT1-targeted chlorambucil derivatives and (b) evaluate their LAT1-mediated cellular uptake as well as antiproliferative activity in vitro in the human breast cancer MCF-7 cell line. Chlorambucil was conjugated to l-tyrosine—an endogenous LAT1 substrate—via either ester or amide linkage (compounds 1 and 2, respectively). While chlorambucil itself did not bind to LAT1, its derivatives 1 and 2 bound to LAT1 with a similar affinity as with l-tyrosine and their respective cellular uptake was significantly higher than that of chlorambucil in MCF-7. The results of our cellular uptake study are indicative of antiproliferative activity, as a higher intracellular uptake of chlorambucil derivatives resulted in greater cytotoxicity than chlorambucil by itself. LAT1 thus ...
Bioorganic & Medicinal Chemistry Letters, 2018
To achieve the sustained release of dopamine in the brain for the symptomatic treatment of Parkin... more To achieve the sustained release of dopamine in the brain for the symptomatic treatment of Parkinson's disease, dopamine was conjugated to L-tyrosine, an L-type amino acid transporter 1 (LAT1)-targeting vector, using a secondary carbamate linker. The resulting prodrug, dopa-CBT, inhibited the uptake of the LAT1 substrate [ 14 C]-L-leucine in LAT1-expressing MCF-7 cells with an IC 50 value of 28 µM, which was 3.5-times lower than that of the gold standard for dopamine replacement therapy, L-dopa (IC 50 ca. 100 µM). Despite its high affinity for LAT1, dopa-CBT was transported via LAT1 into MCF-7 cells 850-times more slowly (V max < 3 pmol/min/mg) than Ldopa (V max 2.6 nmol/min/mg), most likely due to its large size compared to L-dopa. However, dopa-CBT was significantly more stable in 10% rat liver homogenate than L-dopa, releasing dopamine and L-tyrosine, an endogenous dopamine precursor, slowly, which indicates that it may serve as a dual carrier of dopamine across the blood-brain barrier selectively expressing LAT1.
Supplementary Figure S5. DNA damage signaling activated by low concentrations of DP68. U251 and T... more Supplementary Figure S5. DNA damage signaling activated by low concentrations of DP68. U251 and T98G cells were exposed to DP68 for 24 hrs and whole cell and nuclear extracts were processed for Western blotting. Representative blots from three independent experiments are shown.
Supplementary Figure S4. DP86 and DP68 activity is independent of MMR expression. (A) Western blo... more Supplementary Figure S4. DP86 and DP68 activity is independent of MMR expression. (A) Western blot confirmed knockdown in T98G cells infected with empty vector, sheGFP, and two shRNAs targeting MLH1. (B) Infected cells were treated with TMZ, DP86, or DP68 and cell survival was analyzed via CyQuant assay. Mean IC50 {plus minus} SEM from three independent experiments are graphed. * (p&lt;0.05)
Supplementary Figure S2. Summary of prodrug activation. Summary of prodrug activation and drug me... more Supplementary Figure S2. Summary of prodrug activation. Summary of prodrug activation and drug mechanisms of action for TMZ, DP68 and DP86 highlighting the roles of diazonium and aziridinium ion intermediates. (A-B) TMZ hydrolysis, generation of methyldiazonium ions and reaction with DNA; (C) DP86 monoalkylation; (D) DP68 crosslink formation.
The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indometha... more The objective of this study was to evaluate the cerebrospinal fluid (CSF) permeation of indomethacin in healthy children. The participants (n = 31, aged 4–144 months) received indomethacin (0.35 mg/kg) as a 10‐minute intravenous infusion prior to surgery under spinal anaesthesia. A single CSF and plasma sample from each individual was collected 14 to 225 minutes after the infusion. Indomethacin concentrations were determined from the CSF, plasma, and protein‐free plasma. Total plasma, protein‐free plasma, and CSF concentrations of indomethacin ranged between 90 and 2200 ng/mL (median, 780 ng/mL), 0.3 and 0.8 ng/mL (median, 0.5 ng/mL), and 0.2 and 5.0 ng/mL (median, 1.4 ng/mL), respectively. The CSF to plasma concentration ratio remained less than 0.01. There was no correlation between the administration time and CSF concentrations. Eleven children developed 12 nonserious adverse effects, from which 5 were central nervous system (CNS) effects (agitation). In conclusion, indomethacin permeated into the CSF of children, which enables both desired and adverse CNS effects of indomethacin.
European Journal of Pharmaceutical Sciences, Feb 1, 2023
Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly solub... more Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly soluble yet readily absorbable parent drug amprenavir. In this study, a novel cell-free in vitro setup with quasi-continuous monitoring of the dynamic dissolution/bio-conversion/permeation of fosamprenavir was designed and tested. It consists of side-by-side diffusion cells, where the donor and acceptor compartments are separated by the biomimetic barrier PermeaPad®, and sampling from the donor compartment is accomplished via a microdialysis probe. Externally added bovine alkaline phosphatase induced bioconversion in the donor compartment. Microdialysis sampling allowed to follow the enzymatic conversion of fosamprenavir to amprenavir by the bovine alkaline phosphatase in an (almost) real-time manner eliminating the need to remove or inactivate the enzyme. Biomimetic conversion rates in the setup were established by adding appropriate amounts of the alkaline phosphatase. A substantial (6.5-fold) and persistent supersaturation of amprenavir was observed due to bioconversion at lower (500 µM) concentrations, resulting in a substantially increased flux across the biomimetic barrier, nicely reflecting the situation in vivo. At conditions with an almost 10-fold higher dose than the usual human dose, some replicates showed premature precipitation and collapse of supersaturation, while others did not. In conclusion, the proposed novel tool appears very promising in gaining an in-depth mechanistic understanding of the bioconversion/permeation interplay, including transient supersaturation of phosphate-ester prodrugs like fosamprenavir.
Various novel morpholinyl-(3a,b) and methylpiperazinylacyloxyalkyl (3c-f) esters of 2-(6methoxy-2... more Various novel morpholinyl-(3a,b) and methylpiperazinylacyloxyalkyl (3c-f) esters of 2-(6methoxy-2-naphthyl)propionic acid were synthesized and evaluated in vitro for topical drug delivery as potential prodrugs of naproxen (1). Compounds 3a-f were prepared by coupling the corresponding naproxen hydroxyalkyl ester with the morpholinyl-or (4-methyl-1-piperazinyl)acyl acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) and quantitatively hydrolyzed (t 1/2) 1-26 min) to naproxen in human serum. Compounds 3c-f showed higher aqueous solubility and similar lipophilicity, determined by their octanol-buffer partition coefficients (log P app), at pH 5.0 when compared to naproxen. At pH 7.4 they were significantly more lipophilic than naproxen. The best prodrug 3c led to a 4and 1.5-fold enhancement of skin permeation when compared to naproxen at pH 7.4 and 5.0, respectively. The present study indicates using a methylpiperazinyl group yields prodrugs that are partially un-ionized under neutral and slightly acidic conditions, and thus, a desirable combination is achieved in terms of aqueous solubility and lipophilicity. Moreover, the resulting combination of biphasic solubility and fast enzymatic hydrolysis of the methylpiperazinylacyloxyalkyl derivatives gave improved topical delivery of naproxen.
Clozapine (CZP) is the only effective drug in schizophrenia resistant to typical antipsychotics. ... more Clozapine (CZP) is the only effective drug in schizophrenia resistant to typical antipsychotics. However, existing dosage forms (oral or orodispersible tablets, suspensions or intramuscular injection) show challenging limitations. After oral administration, CZP has low bioavailability due to a large first-pass effect, while the i.m. route is often painful, with low patient compliance and requiring specialised personnel. Moreover, CZP has a very low aqueous solubility. This study proposes the intranasal route as an alternative route of administration for CZP, through its encapsulation in polymeric nanoparticles (NPs) based on Eudragit® RS100 and RL100 copolymers. Slow-release polymeric NPs with dimensions around 400–500 nm were formulated to reside and release CZP in the nasal cavity, where it can be absorbed through the nasal mucosa and reach the systemic circulation. CZP-EUD-NPs showed a controlled release of CZP for up to 8 h. Furthermore, to reduce mucociliary clearance and incre...
l-type amino acid transporter 1 (LAT1) is an amino acid transporter that is overexpressed in seve... more l-type amino acid transporter 1 (LAT1) is an amino acid transporter that is overexpressed in several types of cancer and, thus, it can be a potential target for chemotherapy. The objectives of this study were to (a) synthesize LAT1-targeted chlorambucil derivatives and (b) evaluate their LAT1-mediated cellular uptake as well as antiproliferative activity in vitro in the human breast cancer MCF-7 cell line. Chlorambucil was conjugated to l-tyrosine—an endogenous LAT1 substrate—via either ester or amide linkage (compounds 1 and 2, respectively). While chlorambucil itself did not bind to LAT1, its derivatives 1 and 2 bound to LAT1 with a similar affinity as with l-tyrosine and their respective cellular uptake was significantly higher than that of chlorambucil in MCF-7. The results of our cellular uptake study are indicative of antiproliferative activity, as a higher intracellular uptake of chlorambucil derivatives resulted in greater cytotoxicity than chlorambucil by itself. LAT1 thus ...
Bioorganic & Medicinal Chemistry Letters, 2018
To achieve the sustained release of dopamine in the brain for the symptomatic treatment of Parkin... more To achieve the sustained release of dopamine in the brain for the symptomatic treatment of Parkinson's disease, dopamine was conjugated to L-tyrosine, an L-type amino acid transporter 1 (LAT1)-targeting vector, using a secondary carbamate linker. The resulting prodrug, dopa-CBT, inhibited the uptake of the LAT1 substrate [ 14 C]-L-leucine in LAT1-expressing MCF-7 cells with an IC 50 value of 28 µM, which was 3.5-times lower than that of the gold standard for dopamine replacement therapy, L-dopa (IC 50 ca. 100 µM). Despite its high affinity for LAT1, dopa-CBT was transported via LAT1 into MCF-7 cells 850-times more slowly (V max < 3 pmol/min/mg) than Ldopa (V max 2.6 nmol/min/mg), most likely due to its large size compared to L-dopa. However, dopa-CBT was significantly more stable in 10% rat liver homogenate than L-dopa, releasing dopamine and L-tyrosine, an endogenous dopamine precursor, slowly, which indicates that it may serve as a dual carrier of dopamine across the blood-brain barrier selectively expressing LAT1.
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