Publications by authors named "Jarkko Rautio"

68 Publications

Molecular characteristics supporting l-Type amino acid transporter 1 (LAT1)-mediated translocation.

Bioorg Chem 2021 Jul 22;112:104921. Epub 2021 Apr 22.

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland. Electronic address:

l-Type amino acid transporter 1 (LAT1) is an interesting protein due to its peculiar expression profile. It can be utilized not only as a carrier for improved or targeted drug delivery, e.g., into the brain but also as a target protein by which amino acid supply can be restricted, e.g., from the cancer cells. The recognition and binding processes of LAT1-ligands, such as amino acids and clinically used small molecules, including l-dopa, gabapentin, and melphalan, are today well-known. Binding to LAT1 is crucial, particularly when designing the LAT1-inhibitors. However, it will not guarantee effective translocation across the cell membrane via LAT1, which is a definite requirement for LAT1-substrates, such as drugs that elicit their pharmacological effects inside the cells. Therefore, in the present study, the accumulation of known LAT1-utilizing compounds into the selected LAT1-expressing cancer cells (MCF-7) was explored experimentally over a time period. The differences found among the transport efficiency and affinity of the studied compounds for LAT1 were subsequently explained by docking the ligands into the human LAT1 model (based on the recent cryo-electron microscopy structure). Thus, the findings of this study clarify the favorable structural requirements of the size, shape, and polarity of the ligands that support the translocation and effective transport across the cell membrane via LAT1. This knowledge can be applied in future drug design to attain improved or targeted drug delivery and hence, successful LAT1-utilizing drugs with increased therapeutic effects.
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http://dx.doi.org/10.1016/j.bioorg.2021.104921DOI Listing
July 2021

Species differences in the intra-brain distribution of an L-type amino acid transporter 1 (LAT1) -utilizing compound between mice and rats.

Int J Pharm 2021 Mar 1;596:120300. Epub 2021 Feb 1.

School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.

The prodrug approach targeting influx transporters has been extensively studied as a means of central nervous system drug delivery. Transporter and enzyme expression, localization and activity may contribute to significant species differences in preclinical studies. However, data about the possible species differences in the intra-brain distribution of transporter utilizing compounds is scarce. Here, we investigated the species differences in the intra-brain distribution of an L-type amino acid transporter 1 (LAT1)-utilizing L-lysine analogue of ketoprofen (KPF) (compound 1) and KPF itself by the whole tissue and brain microdialysis methods in mice, and compared the results to those previously reported in rats. Their pharmacodynamic responses in both species were assessed by measuring the brain prostaglandin E (PGE) levels by LC-MS/MS. The intracellular delivery of compound 1 was much lower in mice than in rats. Higher target site concentrations of compound 1 and released KPF were reflected on a more pronounced effect on PGE levels in the rat brain. In conclusion, these results highlight the need for cross-species characterization of prodrug pharmacokinetics and pharmacodynamics in preclinical studies.
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http://dx.doi.org/10.1016/j.ijpharm.2021.120300DOI Listing
March 2021

Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and Evaluation of the Entire Positional Isomer Library of -Carboranylmethyl-Bearing Glucoconjugates.

Mol Pharm 2021 01 1;18(1):285-304. Epub 2020 Dec 1.

Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014 Helsinki, Finland.

Boron neutron capture therapy (BNCT) is a noninvasive binary therapeutic modality applicable to the treatment of cancers. While BNCT offers a tumor-targeting selectivity that is difficult to match by other means, the last obstacles preventing the full harness of this potential come in the form of the suboptimal boron delivery strategies presently used in the clinics. To address these challenges, we have developed delivery agents that target the glucose transporter GLUT1. Here, we present the chemical synthesis of a number of -carboranylmethyl-substituted glucoconjugates and the biological assessment of all positional isomers. Altogether, the study provides protocols for the synthesis and structural characterization of such glucoconjugates and insights into their essential properties, for example, cytotoxicity, GLUT1-affinity, metabolism, and boron delivery capacity. In addition to solidifying the biochemical foundations of a successful GLUT1-targeting approach to BNCT, we identify the most promising modification sites in d-glucose, which are critical in order to further develop this strategy toward clinical use.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00917DOI Listing
January 2021

Intranasal Nanoemulsions for Direct Nose-to-Brain Delivery of Actives for CNS Disorders.

Pharmaceutics 2020 Dec 18;12(12). Epub 2020 Dec 18.

Faculty of Pharmacy, Uttar Pradesh University of Medical Sciences, Saifai, Etawah 206130, India.

The treatment of various central nervous system (CNS) diseases has been challenging, despite the rapid development of several novel treatment approaches. The blood-brain barrier (BBB) is one of the major issues in the treatment of CNS diseases, having major role in the protection of the brain but simultaneously constituting the main limiting hurdle for drugs targeting the brain. Nasal drug delivery has gained significant interest for brain targeting over the past decades, wherein the drug is directly delivered to the brain by the trigeminal and olfactory pathway. Various novel and promising formulation approaches have been explored for drug targeting to the brain by nasal administration. Nanoemulsions have the potential to avoid problems, including low solubility, poor bioavailability, slow onset of action, and enzymatic degradation. The present review highlights research scenarios of nanoemulsions for nose-to-brain delivery for the management of CNS ailments classified on the basis of brain disorders and further identifies the areas that remain unexplored. The significance of the total dose delivered to the target region, biodistribution studies, and long-term toxicity studies have been identified as the key areas of future research.
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http://dx.doi.org/10.3390/pharmaceutics12121230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767046PMC
December 2020

Addressing the Biochemical Foundations of a Glucose-Based "Trojan Horse"-Strategy to Boron Neutron Capture Therapy: From Chemical Synthesis to Assessment.

Mol Pharm 2020 10 2;17(10):3885-3899. Epub 2020 Sep 2.

Department of Chemistry, University of Helsinki, Finland, P.O. Box 55, FI-00014 Helsinki, Finland.

Boron neutron capture therapy (BNCT) for cancer is on the rise worldwide due to recent developments of in-hospital neutron accelerators which are expected to revolutionize patient treatments. There is an urgent need for improved boron delivery agents, and herein we have focused on studying the biochemical foundations upon which a successful GLUT1-targeting strategy to BNCT could be based. By combining synthesis and molecular modeling with affinity and cytotoxicity studies, we unravel the mechanisms behind the considerable potential of appropriately designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing the biochemical premises of the approach in detail, we report on a hit glucoconjugate which displays good cytocompatibility, aqueous solubility, high transporter affinity, and, crucially, an exceptional boron delivery capacity in the assessment thereby pointing toward the significant potential embedded in this approach.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539299PMC
October 2020

Anti-bacterial activity of inorganic nanomaterials and their antimicrobial peptide conjugates against resistant and non-resistant pathogens.

Int J Pharm 2020 Aug 12;586:119531. Epub 2020 Jun 12.

Pharmaceutical Sciences Laboratory, Faculty of Science & Engineering, Åbo Akademi University, 20500 Turku, Finland. Electronic address:

This review details the antimicrobial applications of inorganic nanomaterials of mostly metallic form, and the augmentation of activity by surface conjugation of peptide ligands. The review is subdivided into three main sections, of which the first describes the antimicrobial activity of inorganic nanomaterials against gram-positive, gram-negative and multidrug-resistant bacterial strains. The second section highlights the range of antimicrobial peptides and the drug resistance strategies employed by bacterial species to counter lethality. The final part discusses the role of antimicrobial peptide-decorated inorganic nanomaterials in the fight against bacterial strains that show resistance. General strategies for the preparation of antimicrobial peptides and their conjugation to nanomaterials are discussed, emphasizing the use of elemental and metallic oxide nanomaterials. Importantly, the permeation of antimicrobial peptides through the bacterial membrane is shown to aid the delivery of nanomaterials into bacterial cells. By judicious use of targeting ligands, the nanomaterial becomes able to differentiate between bacterial and mammalian cells and, thus, reduce side effects. Moreover, peptide conjugation to the surface of a nanomaterial will alter surface chemistry in ways that lead to reduction in toxicity and improvements in biocompatibility.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119531DOI Listing
August 2020

Prodrug Approach for Posterior Eye Drug Delivery: Synthesis of Novel Ganciclovir Prodrugs and in Vitro Screening with Cassette Dosing.

Mol Pharm 2020 06 5;17(6):1945-1953. Epub 2020 May 5.

School of Pharmacy, University of Eastern Finland, Yliopistonranta 1, Kuopio 70211, Finland.

Because of poor ocular drug bioavailability, intravitreal injections have become the gold standard for drug delivery to the posterior eye. The prodrug approach can be used for optimizing the biopharmaceutical properties of intravitreal drugs. The preclinical screening of prodrugs' properties, such as hydrolysis and bioconversion, should be conducted in a resource-efficient way for an extensive set of synthesized compounds with validated methods. Our objective was to explore cassette dosing in in vitro prodrug hydrolysis and bioconversion studies in buffer, vitreous, and retinal pigment epithelium (RPE) homogenate for rapid medium-throughput screening. Moreover, our aim was to correlate the prodrug structure with hydrolytic behavior. We synthesized 18 novel ganciclovir prodrugs and first studied their hydrolysis in aqueous buffer and porcine vitreous in vitro with cassette dosing for 35 h. A method for vitreous homogenate pH equilibration to a physiological level by using buffer and incubation under 5% carbon dioxide was validated. The hydrolysis of the prodrugs was evaluated in porcine RPE homogenate in vitro with cassette dosing, and five prodrugs were assayed individually to examine their bioconversion into ganciclovir in RPE after 2 h. Lastly, the prodrugs' binding to melanin was studied in vitro. The prodrugs showed a wide spectrum of hydrolysis rates, ranging from a few percentages to 100% in the vitreous and RPE; in general, hydrolysis in RPE was faster than in vitreous. Prodrugs with long carbon chains and disubstitution showed lability in the tissue homogenates, whereas prodrugs with branched carbon chains and aromatic groups were stable. All five prodrugs chosen for the bioconversion study in RPE were hydrolyzed into ganciclovir, and their hydrolytic behavior matched results from the cassette mix experiment, supporting the cassette mix approach for hydrolysis and bioconversion studies. None of the prodrugs bound highly to melanin (<50% bound). In conclusion, cassette dosing proved useful for the rapid screening of prodrug hydrolysis and bioconversion properties. Analyzing several compounds simultaneously can complicate the analytics, and thus, choosing the compounds of the cassette mix should be done carefully to avoid mutual interference of the compounds with the results. The methodology and results of the work are applicable in ocular drug research and prodrug design.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00037DOI Listing
June 2020

Tyrosine-Chlorambucil Conjugates Facilitate Cellular Uptake through L-Type Amino Acid Transporter 1 (LAT1) in Human Breast Cancer Cell Line MCF-7.

Int J Mol Sci 2020 Mar 20;21(6). Epub 2020 Mar 20.

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.

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 and , respectively). While chlorambucil itself did not bind to LAT1, its derivatives and 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 contributes to intracellular uptake of chlorambucil derivatives and, therefore, increases antiproliferative activity. The understanding gained from our research can be used in the development of LAT1-targeted anticancer drugs and prodrugs for site-selective and enhanced chemotherapeutic activity.
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http://dx.doi.org/10.3390/ijms21062132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139360PMC
March 2020

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-6.

Molecules 2019 Dec 28;25(1). Epub 2019 Dec 28.

Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências, Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials that is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal [...].
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http://dx.doi.org/10.3390/molecules25010119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983133PMC
December 2019

Role of L-Type Amino Acid Transporter 1 (LAT1) for the Selective Cytotoxicity of Sesamol in Human Melanoma Cells.

Molecules 2019 Oct 27;24(21). Epub 2019 Oct 27.

School of Pharmacy, University of Eastern Finland, Kuopio 70211, Finland.

Sesamol is effective against melanoma cells with less damage to normal cells. The underlying selective cytotoxicity of sesamol in melanoma vs. non-cancerous cells is undefined. Melanoma cells differ from normal cells by over-expression of the L-type amino acid transporter 1 (LAT1). We sought to clarify the transport mechanism on selective cytotoxicity of sesamol in melanoma cells. A human melanoma cell line (SK-MEL-2) and African monkey epithelial cell line (Vero) were used to study the cellular uptake and cytotoxicity of sesamol. The intracellular concentration of sesamol was quantified by UV-HPLC. The cytotoxicity was determined by neutral red uptake assay. Sesamol showed a higher distribution volume and uptake clearance in SK-MEL-2 than Vero cells. Sesamol was distributed by both carrier-mediated and passive transport by having greater carrier-mediated transport into SK-MEL-2 cells than Vero cells. Higher mRNA expression and function of LAT1 over LAT2 were evident in SK-MEL-2 cells compared to Vero cells. Sesamol uptake and sesamol cytotoxicity were inhibited by the LAT1 inhibitor, suggesting LAT1 had a role in sesamol transport and its bioactivity in melanoma. The LAT1-mediated transport of sesamol is indicative of how it engages cytotoxicity in melanoma cells with promising therapeutic benefits.
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http://dx.doi.org/10.3390/molecules24213869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6865181PMC
October 2019

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-5.

Molecules 2019 Jun 30;24(13). Epub 2019 Jun 30.

Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona, Av. Joan XXIII, 27-31, E-08028 Barcelona, Spain.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials which is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal [...].
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http://dx.doi.org/10.3390/molecules24132415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650823PMC
June 2019

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes⁻4.

Molecules 2018 Dec 31;24(1). Epub 2018 Dec 31.

Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona, Av. Joan XXIII, 27-31, Barcelona E-08028, Spain.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials, which is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal . [...].
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http://dx.doi.org/10.3390/molecules24010130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337331PMC
December 2018

Discovery of polar spirocyclic orally bioavailable urea inhibitors of soluble epoxide hydrolase.

Bioorg Chem 2018 10 17;80:655-667. Epub 2018 Jul 17.

Saint Petersburg State University, Saint Petersburg 199034, Russian Federation. Electronic address:

Spirocyclic 1-oxa-9-azaspiro[5.5]undecan-4-amine scaffold was explored as a basis for the design of potential inhibitors of soluble epoxide hydrolase (sEH). Synthesis and testing of the initial SAR-probing library followed by biochemical testing against sEH allowed nominating a racemic lead compound (±)-22. The latter showed remarkable (> 0.5 mM) solubility in aqueous phosphate buffer solution, unusually low (for sEH inhibitors) lipophilicity as confirmed by experimentally determined logD of 0.99, and an excellent oral bioavailability in mice (as well as other pharmacokinetic characteristics). Individual enantiomer profiling revealed that the inhibitory potency primarily resided with the dextrorotatory eutomer (+)-22 (IC 4.99 ± 0.18 nM). For the latter, a crystal structure of its complex with a C-terminal domain of sEH was obtained and resolved. These data fully validate (+)-22 as a new non-racemic advanced lead compound for further development as a potential therapeutic agent for use in such areas as cardiovascular disease, inflammation and pain.
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http://dx.doi.org/10.1016/j.bioorg.2018.07.014DOI Listing
October 2018

Secondary carbamate linker can facilitate the sustained release of dopamine from brain-targeted prodrug.

Bioorg Med Chem Lett 2018 09 18;28(17):2856-2860. Epub 2018 Jul 18.

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland. Electronic address:

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 [C]-l-leucine in LAT1-expressing MCF-7 cells with an IC value of 28 µM, which was 3.5-times lower than that of the gold standard for dopamine replacement therapy, l-dopa (IC ca. 100 µM). Despite its high affinity for LAT1, dopa-CBT was transported via LAT1 into MCF-7 cells 850-times more slowly (V < 3 pmol/min/mg) than l-dopa (V 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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http://dx.doi.org/10.1016/j.bmcl.2018.07.030DOI Listing
September 2018

The expanding role of prodrugs in contemporary drug design and development.

Nat Rev Drug Discov 2018 08 27;17(8):559-587. Epub 2018 Apr 27.

School of Pharmacy, University of Kansas, Lawrence, KS, USA.

Prodrugs are molecules with little or no pharmacological activity that are converted to the active parent drug in vivo by enzymatic or chemical reactions or by a combination of the two. Prodrugs have evolved from being serendipitously discovered or used as a salvage effort to being intentionally designed. Such efforts can avoid drug development challenges that limit formulation options or result in unacceptable biopharmaceutical or pharmacokinetic performance, or poor targeting. In the past 10 years, the US Food and Drug Administration has approved at least 30 prodrugs, which accounts for more than 12% of all approved small-molecule new chemical entities. In this Review, we highlight prodrug design strategies for improved formulation and pharmacokinetic and targeting properties, with a focus on the most recently marketed prodrugs. We also discuss preclinical and clinical challenges and considerations in prodrug design and development.
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http://dx.doi.org/10.1038/nrd.2018.46DOI Listing
August 2018

Structural properties for selective and efficient l-type amino acid transporter 1 (LAT1) mediated cellular uptake.

Int J Pharm 2018 Jun 15;544(1):91-99. Epub 2018 Apr 15.

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.

l-Type amino acid transporter 1 (LAT1) is a sodium-independent exchanger transporting large neural amino acids and several amino-acid mimicking drugs across the cell membranes. LAT1 is highly expressed at the blood brain barrier (BBB) and in numerous cancer cells and is therefore a potential drug target. However, structural features affecting the ability to bind to LAT1 and the cellular translocation by LAT1 are unclear. In the present study we determined the binding to and transport through human LAT1 of several compounds into the human breast adenocarcinoma cells (MCF-7). We show that the meta-conjugation of l-phenylalanine increases binding to human LAT1 compared to para-conjugation or aliphatic amino acid moiety. Furthermore, large, rigid and aromatic meta-substituted l-phenylalanine derivative enabled selective and efficient LAT1-mediated cellular uptake. Our results also demonstrates that in addition to binding studies, it is of utmost importance to determine the cellular accumulation of compounds. It provides crucial information on transport efficiency and selectivity of transport mechanisms that the compounds are able to utilize. Overall, these structural findings and the methodology used herein are exploitable to design LAT1-utilizing compounds, such as markers for cancer imaging and drug molecules, enabling more effective and safer treatments for cancer in the future.
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http://dx.doi.org/10.1016/j.ijpharm.2018.04.025DOI Listing
June 2018

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-2.

Molecules 2017 Dec 28;23(1). Epub 2017 Dec 28.

ICOA UMR CNRS 6005, Universite d'Orleans, Rue de Chartres, 45067 Orleans CEDEX 2, France.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials, which are published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules [...].
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http://dx.doi.org/10.3390/molecules23010065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017245PMC
December 2017

Prodrugs - Recent approvals and a glimpse of the pipeline.

Eur J Pharm Sci 2017 Nov 4;109:146-161. Epub 2017 Aug 4.

Department of Medicinal Chemistry, College of Pharmacy, University of Florida, United States.

Prodrugs offer a versatile strategy to overcome flaws of viable drug candidates or clinically approved drugs. However, the strategic importance of prodrugs in the pharmaceutical industry has often been challenged, and prodrugs are often considered as the last option after lead optimization and when the selected drug candidate has faced significant pharmaceutical and pharmacokinetic limitations. Based on recent success in marketed drugs, prodrug strategy should clearly be considered already in early stages of lead optimization. During the past five years or so, prodrugs have accounted for about 10% of all small molecular weight drugs that have come to the market. In 2015 alone, the FDA approved seven prodrugs, which gives a prodrug prevalence of over 20% among the small molecules or over 15% among the total amount of the FDA approved drugs that year. A great number of various prodrugs are also undergoing late stage clinical trials. The pharmaceutical industry will therefore continue to depend on prodrugs for the foreseeable future. In this review, we will present the state of the art in the design of the prodrugs launched by the FDA since 2015. We will also provide an overview of some interesting late stage clinical prodrug candidates. We hope this review will demonstrate potential of prodrug strategies and facilitates the use of prodrugs in drug discovery projects.
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http://dx.doi.org/10.1016/j.ejps.2017.08.002DOI Listing
November 2017

Prodrugs in medicinal chemistry and enzyme prodrug therapies.

Adv Drug Deliv Rev 2017 09 1;118:65-77. Epub 2017 Jul 1.

Department of Chemistry, Aarhus University, Denmark; iNano Interdisciplinary Nanoscience Centre, Aarhus University, Denmark. Electronic address:

Prodrugs are cunning derivatives of therapeutic agents designed to improve the pharmacokinetics profile of the drug. Within a prodrug, pharmacological activity of the drug is masked and is recovered within the human body upon bioconversion of the prodrug, a process that is typically mediated by enzymes. This concept is highly successful and a significant fraction of marketed therapeutic formulations is based on prodrugs. An advanced subset of prodrugs can be engineered such as to achieve site-specific bioconversion of the prodrug - to comprise the highly advantageous "enzyme prodrug therapy", EPT. Design of prodrugs for EPT is similar to the prodrugs in general medicinal use in that the pharmacological activity of the drug is masked, but differs significantly in that site-specific bioconversion is a prime consideration, and the enzymes typically used for EPT are non-mammalian and/or with low systemic abundance in the human body. This review focuses on the design of prodrugs for EPT in terms of the choice of an enzyme and the corresponding prodrug for bioconversion. We also discuss the recent success of "self immolative linkers" which significantly empower and diversify the prodrug design, and present methodologies for the design of prodrugs with extended blood residence time. The review aims to be of specific interest for medicinal chemists, biomedical engineers, and pharmaceutical scientists.
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http://dx.doi.org/10.1016/j.addr.2017.06.013DOI Listing
September 2017

Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835.

Bioorg Med Chem 2016 11 3;24(21):5481-5494. Epub 2016 Sep 3.

Molecular Therapeutics, School of Pharmacy, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, UK.

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly's drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC=55nM) was reached. Four lead compounds (EC range 55-410nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F=10.3%) and plasma levels achieved on oral administration.
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http://dx.doi.org/10.1016/j.bmc.2016.09.004DOI Listing
November 2016

Amino Acid Promoieties Alter Valproic Acid Pharmacokinetics and Enable Extended Brain Exposure.

Neurochem Res 2016 Oct 14;41(10):2797-2809. Epub 2016 Jul 14.

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, P.O. Box 1627, 70211, Kuopio, Finland.

Valproic acid (VPA) has been used to treat epileptic seizures for decades, but it may also possess therapeutic potential in other nervous system diseases. However, VPA is extensively bound to plasma proteins, asymmetrically transported across the blood-brain barrier and metabolized to toxic species in the liver, which all contribute to its severe off-target adverse effects and possible drug-drug interactions. In this study, we evaluated seven amino acid prodrugs of VPA that were targeted to utilize L-type amino acid transporter 1 (LAT1), if they could alter the brain uptake mechanism and systemic pharmacokinetics of VPA. All prodrugs had affinity for LAT1 studied as competitive inhibition of [C]-L-leucine in human breast cancer (MCF-7) cell line. However, since the ester prodrugs were unstable they were not studied further, instead the corresponding amide prodrugs were used to evaluate their systemic pharmacokinetics in rats and the uptake mechanism via LAT1 into the rat brain. All amide prodrugs were bound to a lesser extent to plasma proteins than VPA and this being independent of the prodrug concentration. Amide prodrugs were also delivered into the brain after intravenous bolus injection. One of the prodrug showed greater brain uptake and high selectivity for LAT1 and it was able to release VPA slowly within the brain. Therefore, it was concluded that the VPA brain concentrations can be stabilized as well as the problematic pharmacokinetic profile can be altered by a LAT1-selective prodrug.
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http://dx.doi.org/10.1007/s11064-016-1996-8DOI Listing
October 2016

Evaluation of novel imidazotetrazine analogues designed to overcome temozolomide resistance and glioblastoma regrowth.

Mol Cancer Ther 2015 Jan 28;14(1):111-9. Epub 2014 Oct 28.

Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota.

The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multiforme (GBM) cell lines and primary culture models are reported. Dose-response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. On the basis of a serial neurosphere assay, DP68 inhibits repopulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR functions. DP68-induced interstrand DNA cross-links were demonstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell-cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biologic response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced antiglioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collectively, these data demonstrate that DP68 is a novel and potent antiglioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to cross-link strands of DNA.
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http://dx.doi.org/10.1158/1535-7163.MCT-14-0113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297195PMC
January 2015

Brain pharmacokinetics of ganciclovir in rats with orthotopic BT4C glioma.

Drug Metab Dispos 2015 Jan 27;43(1):140-6. Epub 2014 Oct 27.

Faculty of Health Sciences, School of Pharmacy (M.G., T.M.K., J.J.H., A.J.J., A.P., M.L., L.P., J.R., M.M.F.) and A. I. Virtanen Institute for Molecular Sciences (J.K., H.S., S.Y.-H.), University of Eastern Finland, Kuopio, Finland.

Ganciclovir (GCV) is an essential part of the Herpes simplex virus thymidine kinase (HSV-tk) gene therapy of malignant gliomas. The purpose of this study was to investigate the brain pharmacokinetics and tumor uptake of GCV in the BT4C rat glioma model. GCV's brain and tumor uptakes were investigated by in vivo microdialysis in rats with orthotopic BT4C glioma. In addition, the ability of GCV to cross the blood-brain barrier and tumor vasculature was assessed with in situ rat brain perfusion. Finally, the extent to which GCV could permeate across the BT4C glioma cell membrane was assessed in vitro. The areas under the concentration curve of unbound GCV in blood, brain extracellular fluid (ECF), and tumor ECF were 6157, 1658, and 4834 μM⋅min, respectively. The apparent maximum unbound concentrations achieved within 60 minutes were 46.9, 11.8, and 25.8 μM in blood, brain, and tumor, respectively. The unbound GCV concentrations in brain and tumor after in situ rat brain perfusion were 0.41 and 1.39 nmol/g, respectively. The highly polar GCV likely crosses the fenestrated tumor vasculature by paracellular diffusion. Thus, GCV is able to reach the extracellular space around the tumor at higher concentrations than that in healthy brain. However, GCV uptake into BT4C cells at 100 μM was only 2.1 pmol/mg of protein, and no active transporter-mediated disposition of GCV could be detected in vitro. In conclusion, the limited efficacy of HSV-tk/GCV gene therapy may be due to the poor cellular uptake and rapid elimination of GCV.
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http://dx.doi.org/10.1124/dmd.114.059840DOI Listing
January 2015

Amino acid ester prodrugs conjugated to the α-carboxylic acid group do not display affinity for the L-type amino acid transporter 1 (LAT1).

Eur J Pharm Sci 2015 Jan 8;66:36-40. Epub 2014 Oct 8.

School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.

L-type amino acid transporter (LAT1) is an intriguing target for carrier-mediated transport of drugs as it is highly expressed in the blood-brain barrier and also in various types of cancer. Several studies have proposed that in order for compounds to act as LAT1 substrates they should possess both negatively charged α-carboxyl and positively charged α-amino groups. However, in some reports, such as in two recent publications describing an isoleucine-quinidine ester prodrug (1), compounds having no free α-carboxyl group have been reported to exhibit high affinity for LAT1 in vitro. In the present study, 1 was synthesized and its affinity for LAT1 was evaluated both with an in situ rat brain perfusion technique and in the human breast cancer cell line MCF-7 in vitro. 1 showed no affinity for LAT1 in either model nor did it show any affinity for LAT2 in an in vitro study. Our results confirm the earlier reported requirements for LAT1 substrates. Thus drugs or prodrugs with substituted α-carboxyl group cannot bind to LAT with high affinity.
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http://dx.doi.org/10.1016/j.ejps.2014.09.025DOI Listing
January 2015

Glutathione-S-transferase selective release of metformin from its sulfonamide prodrug.

Bioorg Med Chem Lett 2014 Nov 16;24(21):5034-6. Epub 2014 Sep 16.

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland. Electronic address:

In this study, three sulfonamide prodrugs of metformin were designed and synthesized. The bioconversion of the sulfonamide prodrugs by glutathione-S-transferase (GST) was evaluated in rat and human liver S9 fractions as well as with recombinant human GST forms. One of the prodrugs (3) was bioactivated by GST and released metformin in a quantitative manner, whereas the two others were enzymatically stable. Prodrug 3 had a much higher logD value relative to metformin and it was reasonably stable in both acidic buffer and rat small intestine homogenate, which indicates that this prodrug has the potential to increase the oral absorption of metformin.
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http://dx.doi.org/10.1016/j.bmcl.2014.09.019DOI Listing
November 2014

Quantitative insight into the design of compounds recognized by the L-type amino acid transporter 1 (LAT1).

ChemMedChem 2014 Dec 9;9(12):2699-707. Epub 2014 Sep 9.

School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio (Finland).

L-Type amino acid transporter 1 (LAT1) is a transmembrane protein expressed abundantly at the blood-brain barrier (BBB), where it ensures the transport of hydrophobic acids from the blood to the brain. Due to its unique substrate specificity and high expression at the BBB, LAT1 is an intriguing target for carrier-mediated transport of drugs into the brain. In this study, a comparative molecular field analysis (CoMFA) model with considerable statistical quality (Q(2) =0.53, R(2) =0.75, Q(2) SE=0.77, R(2) SE=0.57) and good external predictivity (CCC=0.91) was generated. The model was used to guide the synthesis of eight new prodrugs whose affinity for LAT1 was tested by using an in situ rat brain perfusion technique. This resulted in the creation of a novel LAT1 prodrug with L-tryptophan as the promoiety; it also provided a better understanding of the molecular features of LAT1-targeted high-affinity prodrugs, as well as their promoiety and parent drug. The results obtained will be beneficial in the rational design of novel LAT1-binding prodrugs and other compounds that bind to LAT1.
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http://dx.doi.org/10.1002/cmdc.201402281DOI Listing
December 2014

Design, synthesis and brain uptake of LAT1-targeted amino acid prodrugs of dopamine.

Pharm Res 2013 Oct;30(10):2523-37

School of Pharmacy, University of Eastern Finland, Kuopio, Finland.

Purpose: Drug delivery to the brain is impeded by the blood-brain barrier (BBB). Here, we attempted to enhance the brain uptake of cationic dopamine by utilizing the large amino acid transporter 1 (LAT1) at the BBB by prodrug approach.

Methods: Three amino acid prodrugs of dopamine were synthesized and their prodrug properties were examined in vitro. Their LAT1-binding and BBB-permeation were studied using the in situ rat brain perfusion technique. The brain uptake after intravenous administration and the dopamine-releasing ability in the rat striatum after intraperitoneal administration were also determined for the most promising prodrug.

Results: All prodrugs underwent adequate cleavage in rat tissue homogenates. The prodrug with phenylalanine derivative as the promoiety had both higher affinity for LAT1 and better brain uptake properties than those with an alkyl amino acid - mimicking promoiety. The phenylalanine prodrug was taken up into the brain after intravenous injection but after intraperitoneal injection the prodrug did not elevate striatal dopamine concentrations above those achieved by corresponding L-dopa treatment.

Conclusions: These results indicate that attachment of phenylalanine to a cationic drug via an amide bond from the meta-position of its aromatic ring could be highly applicable in prodrug design for LAT1-mediated CNS-delivery of not only anionic but also cationic polar drugs.
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http://dx.doi.org/10.1007/s11095-012-0966-3DOI Listing
October 2013