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679 Folic acid conjugated hollow mesoporous silica nanoparticles encapsulating dacarbazine for targeted melanoma drug delivery and therapy
Pharmacology and Drug Development | ABSTRACTS 678
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Biased CXCR3 ligands differentially alter allergic contact hypersensitivity and chemotaxis JS Smith1, L Nicholson1, J Suwanpradid2, R Glenn1, J Gundry1, P Alagesan1, N Knape1, AR Atwater1, MD Gunn1, AS MacLeod2, RJ Lefkowitz1 and S Rajagopal1 1 Duke, Durham, NC and 2 Duke University, Durham, NC Allergic contact dermatitis (ACD) is a disease with few targeted therapies. Chemokines play an important role in ACD through the recruitment of T-cells that express the chemokine receptor (CKR) CXCR3. Chemokines signal through CKRs, a subgroup of the G proteincoupled receptor (GPCR) family, which are targeted in >30% of drugs. However, few drugs target CKRs. Classically, GPCRs were thought to act as simple switches turned on by agonists and off by antagonists. We now appreciate that GPCRs adopt multiple conformations that link to distinct signaling pathways, such as G-proteins and ß-arrestins (ßarrs). These pathways can be selectively activated by a novel class of receptor ligands, termed biased agonists, which signal through some pathways while blocking signaling through others. The purpose of this study was to determine the roles that G-proteins and ßarrs play in ACD by selectively targeting signaling with CXCR3 biased agonists. Mouse and human cell chemotaxis was determined through transwell migration, and the effects of CXCR3 ligands on ACD were assessed in the DNFB allergic contact hypersensitivity (CHS) mouse model. Patient biopsies of patch tested skin were analyzed. Our results show that ßarr signaling through CXCR3 is necessary for full efficacy chemotaxis of both mouse and human T-cells. A topically applied ßarr-biased ligand doubled (ph0.05) the CHS inflammatory response in WT, but not in ßarr2 KO or CXCR3 KO, mice. Flow cytometry of mouse skin demonstrated increased T-cells following ßarr-biased drug treatment. We conclude that CXCR3 ßarr-mediated signaling is critical for effector T-cell recruitment that underlies the inflammatory response in CHS. These findings suggest that biased ligands could be utilized to selectively target CKRs for therapeutic benefit.
Folic acid conjugated hollow mesoporous silica nanoparticles encapsulating dacarbazine for targeted melanoma drug delivery and therapy N Xu1, J Li2, J Zhu3 and J Tao4 1 Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, China, 2 Department of Dermatology, Union Hospital, Tongji Medical College, HUST, Wuhan, Hubei, China, 3 School of Chemistry and Chemical Engineering, HUST, Wuhan, Hubei, China and 4 Department of Dermatology, Union Hospital, Tongji Medical College,HUST, Wuhan, Hubei, China Hollow mesoporous silica nanoparticles (HMSNs) have shown great potential for cancer therapy due to their good biocompatibility, high loading capacity, and easy surface modification. Conjugation with folic acid (FA) on the surfaces of nanoparticles (NPs) to target folic acid receptors (FR), which are over-expressed on the surface of a variety of tumors, advances the therapeutic strategies of a variety of diseases, including melanoma. Here, we present the dacarbazine-loaded HMSNs conjugated with folic acid (DTIC@HMSN-FA) for targeted drug delivery to melanoma. First, flow cytometry analysis revealed that the percentage of FR expression on human melanoma A875 cell line was as high as 70.4%. Compared with unmodified HMSN (DTIC@HMSN), DTIC@HMSN-FA could be taken up more efficiently by A875 cells. Additionally, in vitro studies showed that DTIC@HMSN-FA exhibited prolonged the intracytoplasmic retention period and enhanced cytotoxicity to melanoma cells as compared to DTIC@HMSN. In vivo investigation also identified that DTIC@HMSN-FA could inhibit melanoma growth more efficiently than DTIC in A875 xenografted mouse model. Furthermore, enhanced inhibition effect on melanoma lung metastasis could also be observed in DTIC@HMSN-FA treated groups as compared to that in DTIC treated groups. Finally, DTIC@HMSN-FA did not cause remarkable damage to normal organs, whereas free DTIC resulted in liver damage. In summary, FA conjugated HMSNs provide a promising platform for targeted drug delivery to melanoma with enhanced therapeutic efficacy and good safety profile.
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Melatonin receptors decrease with age in normal human dermal fibroblasts E Pelle1, K Dong2, EC Goyarts2 and N Pernodet3 1 Estee Lauder R&D/ Env. Med., New York University School of Medicine, Melville, NY, 2 Estee Lauder R&D, Melville, NY and 3 Estee Lauder R&D/ MSE, Stony Brook University, Melville, NY Clock gene activity in human skin is modulated by many factors. Melatonin, a ubiquitous small molecular weight molecule with unique pharmacological properties is a critical component in the skin’s ability to regulate clock gene activity. During a normal circadian cycle, melatonin is highest in the evening where it influences per1, a clock gene. Previously, we observed a dose-dependent effect by melatonin on per1 activity in normal human epidermal keratinocytes using a per1 promoter-luciferase reporter assay. In this study, in order to gain further insight into the melatonin activation pathway, we evaluated melatonin receptors, MT1 and MT2, which are G-protein coupled plasma membrane receptors through which endogenous melatonin initially mediates its cellular effects. Additionally, since melatonin levels decrease with age, we evaluated MT1 and MT2 in normal human dermal fibroblasts derived from both young and aged donors. Using immunocytochemistry and confocal microscopy we determined the presence of both receptors in these fibroblasts. Interestingly, we also observed significant decreases in both types of melatonin receptors in fibroblasts derived from aged donors. MT1 knockdown in fibroblasts further revealed a greater sensitivity to UV-induced DNA damage in both young- and aged-derived fibroblasts. These data demonstrate a reduction in melatonin receptors as a function of age and indicate how modulation of melatonin receptors may be used to treat melatonin-associated circadian disorders in elderly populations and to improve overall skin health.
Building an improved humanized mouse model of psoriasis for drug discovery K Lewandowski1, R Edelmayer2, M Guerrero-Zayas1, P Honore3 and AS Paller4 1 Northwestern University Feinberg School of Medicine, Chicago, IL, 2 AbbVie Inc., North Chicago, IL, 3 AbbVie, North Chicago, IL and 4 Northwestern University, Chicago, IL In vivo models of psoriasis are needed for preclinical testing of new therapeutics, but none has adequately reflected the morphology, mRNA, and protein expression of psoriasis. Mouse models incorporating human epidermal, dermal, and immune cells have the potential to more closely reflect human psoriatic tissue. We generated human skin 3D cultures which were grafted onto NSG mice, leading to 80% graft acceptance and 10% contraction compared to 50% graft take and 60% contraction observed in nude mice. Graft acceptance was evaluated by qPCR for human specific markers of differentiation. There was a clear difference in expression patterns between accepted and rejected human grafts (p0.001). Thicker human skin and reduced graft contraction were also noted. The combination of tapestripping and intradermal injections of human IL-17A, IL-22, and Th1 cells was necessary to induce psoriasis in human grafts. Psoriasis in this humanized mouse model was validated based on tissue morphology (acanthosis and patchy parakeratosis), immunohistochemical assessment, RNA Seq, and a qPCR panel of markers of: i) hyperproliferation (increase in hK16, p0.01); ii) epidermal differentiation (decreases in hKRT10 (54%, p0.01), hLOR (49%, p0.01), and hFLG (51%, p0.01) and increases in hIVL (300%, p0.001)); iii) immune activation (increases in hTNF (275%, p0.001), hIL-36a (320%, p<0.001), and hIFNg (300%, p<0.001)); iv) antimicrobial responses (increase in hS100A7 and hDefB4, both p<0.001); and v) cytokine receptors (increases in hIL-17RA (220%, p<0.01) and hIL-22R (275%, p<0.01)). We also observed an increase in hKi67 positive nuclei (p<0.01); further evidence of hyperproliferation in this model. The humanized mouse model of psoriasis closely resembles human disease and can be utilized for psoriasis drug discovery.
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Gas chromatography-mass spectrometry analysis of effects of dietary fish oil on fatty acid composition in mouse skin P Wang1, M Sun2, J Ren3, Z Djuric3, GJ Fisher4, X Wang1 and Y Li5 1 Department of Photomedicine, Shanghai Dermatology Hospital, Shanghai, Shanghai, China, 2 Department of Dermatology, University of Michigan, Ann Arbor, MI, 3 Department of Family Medicine, University of Michigan, Ann Arbor, MI, 4 Department of Dermatology, University of Michigan, Ann Arbor, MI and 5 Department of Dermatology, University of Michigan, Ann Arbor, MI Altering the fatty acid (FA) composition in the skin by dietary fish oil could provide therapeutic benefits. Although it has been shown that fish oil supplementation enhances EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) abundance in the skin, comprehensive skin FA profiling is needed. We established a gas chromatography-mass spectrometry method, which allows precise quantification of FA profile using small (2 for mice and 2 for humans) skin specimens that can be readily obtained from live mice and humans. We determined mouse skin FA composition after 2, 4 and 8 weeks of consuming a control diet or a diet supplemented with fish oil. Fish oil markedly enhanced EPA and DHA in mouse skin within 2 weeks, and this increase plateaued after 4 weeks. The FA composition in mouse skin was different from that of serum, indicating that skin has homeostatic control of FA metabolism. Mice fed the control diet designed to simulate Western human diet displayed similar skin FA composition as that of humans. The present study presents a validated method for FA quantification that is needed to investigate the mechanisms of actions of dietary treatments in both mouse and human skin.
Induced DNA repair in xeroderma pigmentosum group C cells by readthrough of premature termination codons R Ono1, S Khan2, C Kuschal2, D Tamura2, J Chen2, X Luo2, J DiGiovanna2 and K Kraemer2 1 Department of Dermatology, Graduate School of Medicine, Kobe University, Kobe, Japan and 2 National Cancer Institute, Bethesda, MD Xeroderma pigmentosum (XP) is a rare autosomal recessive, cancer-prone DNA repair disease with mutations in nucleotide excision repair genes (XPA through XPG) or DNA polymerase eta. Of 259 XP cell lines examined, 71 had XPC mutations and 14 (20%) of these had primary premature termination codon (PTC) mutations. mRNA with PTC is removed by nonsensemediated decay (NMD) resulting in reduced levels of mRNA. PTC mutations can be readthrough by aminoglycoside antibiotics that disrupt ribosomal fidelity. A small increase in functional message may provide clinical benefit since XP patient XP72TMA had a spice mutation with 3-5 % of normal XPC mRNA and a mild clinical phenotype. Quantitative capillary western analysis showed XPC protein expression of 1-2 % of normal. For a precision medicine directed XP treatment protocol, potential therapeutic compounds were tested on XP-C cell lines. Previously, we observed increased XPC mRNA after treatment with G418, confirming PTC readthrough activity, but not with gentamycin (GM). We now tested 3 other aminoglycosides in XP518BE cells: paromomycin (PM) and neomycin (NM) increased XPC mRNA while kanamycin (KM) did not. Amlexanox is an anti-inflammatory compound with activity as an inhibitor of NMD. We found that amlexanox alone increased XPC mRNA in 2 of 4 XP-C PTC cell lines. The combination of GM and amlexanox further increased XPC mRNA in 1 of 4 XPC cell lines. NM plus amlexanox and KM plus amlexanox increased XPC mRNA compared to NM or KM alone in XP518BE cells. In contrast, PM plus amlexanox did not show increased XPC mRNA compared to PM alone. Pharmacologic XPC PTC bypass with in vitro assessment may provide a precision medical intervention for carefully selected XP patients.
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Biased CXCR3 ligands differentially alter allergic contact hypersensitivity and chemotaxis JS Smith1, L Nicholson1, J Suwanpradid2, R Glenn1, J Gundry1, P Alagesan1, N Knape1, AR Atwater1, MD Gunn1, AS MacLeod2, RJ Lefkowitz1 and S Rajagopal1 1 Duke, Durham, NC and 2 Duke University, Durham, NC Allergic contact dermatitis (ACD) is a disease with few targeted therapies. Chemokines play an important role in ACD through the recruitment of T-cells that express the chemokine receptor (CKR) CXCR3. Chemokines signal through CKRs, a subgroup of the G proteincoupled receptor (GPCR) family, which are targeted in >30% of drugs. However, few drugs target CKRs. Classically, GPCRs were thought to act as simple switches turned on by agonists and off by antagonists. We now appreciate that GPCRs adopt multiple conformations that link to distinct signaling pathways, such as G-proteins and ß-arrestins (ßarrs). These pathways can be selectively activated by a novel class of receptor ligands, termed biased agonists, which signal through some pathways while blocking signaling through others. The purpose of this study was to determine the roles that G-proteins and ßarrs play in ACD by selectively targeting signaling with CXCR3 biased agonists. Mouse and human cell chemotaxis was determined through transwell migration, and the effects of CXCR3 ligands on ACD were assessed in the DNFB allergic contact hypersensitivity (CHS) mouse model. Patient biopsies of patch tested skin were analyzed. Our results show that ßarr signaling through CXCR3 is necessary for full efficacy chemotaxis of both mouse and human T-cells. A topically applied ßarr-biased ligand doubled (ph0.05) the CHS inflammatory response in WT, but not in ßarr2 KO or CXCR3 KO, mice. Flow cytometry of mouse skin demonstrated increased T-cells following ßarr-biased drug treatment. We conclude that CXCR3 ßarr-mediated signaling is critical for effector T-cell recruitment that underlies the inflammatory response in CHS. These findings suggest that biased ligands could be utilized to selectively target CKRs for therapeutic benefit.
Folic acid conjugated hollow mesoporous silica nanoparticles encapsulating dacarbazine for targeted melanoma drug delivery and therapy N Xu1, J Li2, J Zhu3 and J Tao4 1 Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, China, 2 Department of Dermatology, Union Hospital, Tongji Medical College, HUST, Wuhan, Hubei, China, 3 School of Chemistry and Chemical Engineering, HUST, Wuhan, Hubei, China and 4 Department of Dermatology, Union Hospital, Tongji Medical College,HUST, Wuhan, Hubei, China Hollow mesoporous silica nanoparticles (HMSNs) have shown great potential for cancer therapy due to their good biocompatibility, high loading capacity, and easy surface modification. Conjugation with folic acid (FA) on the surfaces of nanoparticles (NPs) to target folic acid receptors (FR), which are over-expressed on the surface of a variety of tumors, advances the therapeutic strategies of a variety of diseases, including melanoma. Here, we present the dacarbazine-loaded HMSNs conjugated with folic acid (DTIC@HMSN-FA) for targeted drug delivery to melanoma. First, flow cytometry analysis revealed that the percentage of FR expression on human melanoma A875 cell line was as high as 70.4%. Compared with unmodified HMSN (DTIC@HMSN), DTIC@HMSN-FA could be taken up more efficiently by A875 cells. Additionally, in vitro studies showed that DTIC@HMSN-FA exhibited prolonged the intracytoplasmic retention period and enhanced cytotoxicity to melanoma cells as compared to DTIC@HMSN. In vivo investigation also identified that DTIC@HMSN-FA could inhibit melanoma growth more efficiently than DTIC in A875 xenografted mouse model. Furthermore, enhanced inhibition effect on melanoma lung metastasis could also be observed in DTIC@HMSN-FA treated groups as compared to that in DTIC treated groups. Finally, DTIC@HMSN-FA did not cause remarkable damage to normal organs, whereas free DTIC resulted in liver damage. In summary, FA conjugated HMSNs provide a promising platform for targeted drug delivery to melanoma with enhanced therapeutic efficacy and good safety profile.
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Melatonin receptors decrease with age in normal human dermal fibroblasts E Pelle1, K Dong2, EC Goyarts2 and N Pernodet3 1 Estee Lauder R&D/ Env. Med., New York University School of Medicine, Melville, NY, 2 Estee Lauder R&D, Melville, NY and 3 Estee Lauder R&D/ MSE, Stony Brook University, Melville, NY Clock gene activity in human skin is modulated by many factors. Melatonin, a ubiquitous small molecular weight molecule with unique pharmacological properties is a critical component in the skin’s ability to regulate clock gene activity. During a normal circadian cycle, melatonin is highest in the evening where it influences per1, a clock gene. Previously, we observed a dose-dependent effect by melatonin on per1 activity in normal human epidermal keratinocytes using a per1 promoter-luciferase reporter assay. In this study, in order to gain further insight into the melatonin activation pathway, we evaluated melatonin receptors, MT1 and MT2, which are G-protein coupled plasma membrane receptors through which endogenous melatonin initially mediates its cellular effects. Additionally, since melatonin levels decrease with age, we evaluated MT1 and MT2 in normal human dermal fibroblasts derived from both young and aged donors. Using immunocytochemistry and confocal microscopy we determined the presence of both receptors in these fibroblasts. Interestingly, we also observed significant decreases in both types of melatonin receptors in fibroblasts derived from aged donors. MT1 knockdown in fibroblasts further revealed a greater sensitivity to UV-induced DNA damage in both young- and aged-derived fibroblasts. These data demonstrate a reduction in melatonin receptors as a function of age and indicate how modulation of melatonin receptors may be used to treat melatonin-associated circadian disorders in elderly populations and to improve overall skin health.
Building an improved humanized mouse model of psoriasis for drug discovery K Lewandowski1, R Edelmayer2, M Guerrero-Zayas1, P Honore3 and AS Paller4 1 Northwestern University Feinberg School of Medicine, Chicago, IL, 2 AbbVie Inc., North Chicago, IL, 3 AbbVie, North Chicago, IL and 4 Northwestern University, Chicago, IL In vivo models of psoriasis are needed for preclinical testing of new therapeutics, but none has adequately reflected the morphology, mRNA, and protein expression of psoriasis. Mouse models incorporating human epidermal, dermal, and immune cells have the potential to more closely reflect human psoriatic tissue. We generated human skin 3D cultures which were grafted onto NSG mice, leading to 80% graft acceptance and 10% contraction compared to 50% graft take and 60% contraction observed in nude mice. Graft acceptance was evaluated by qPCR for human specific markers of differentiation. There was a clear difference in expression patterns between accepted and rejected human grafts (p0.001). Thicker human skin and reduced graft contraction were also noted. The combination of tapestripping and intradermal injections of human IL-17A, IL-22, and Th1 cells was necessary to induce psoriasis in human grafts. Psoriasis in this humanized mouse model was validated based on tissue morphology (acanthosis and patchy parakeratosis), immunohistochemical assessment, RNA Seq, and a qPCR panel of markers of: i) hyperproliferation (increase in hK16, p0.01); ii) epidermal differentiation (decreases in hKRT10 (54%, p0.01), hLOR (49%, p0.01), and hFLG (51%, p0.01) and increases in hIVL (300%, p0.001)); iii) immune activation (increases in hTNF (275%, p0.001), hIL-36a (320%, p<0.001), and hIFNg (300%, p<0.001)); iv) antimicrobial responses (increase in hS100A7 and hDefB4, both p<0.001); and v) cytokine receptors (increases in hIL-17RA (220%, p<0.01) and hIL-22R (275%, p<0.01)). We also observed an increase in hKi67 positive nuclei (p<0.01); further evidence of hyperproliferation in this model. The humanized mouse model of psoriasis closely resembles human disease and can be utilized for psoriasis drug discovery.
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Gas chromatography-mass spectrometry analysis of effects of dietary fish oil on fatty acid composition in mouse skin P Wang1, M Sun2, J Ren3, Z Djuric3, GJ Fisher4, X Wang1 and Y Li5 1 Department of Photomedicine, Shanghai Dermatology Hospital, Shanghai, Shanghai, China, 2 Department of Dermatology, University of Michigan, Ann Arbor, MI, 3 Department of Family Medicine, University of Michigan, Ann Arbor, MI, 4 Department of Dermatology, University of Michigan, Ann Arbor, MI and 5 Department of Dermatology, University of Michigan, Ann Arbor, MI Altering the fatty acid (FA) composition in the skin by dietary fish oil could provide therapeutic benefits. Although it has been shown that fish oil supplementation enhances EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) abundance in the skin, comprehensive skin FA profiling is needed. We established a gas chromatography-mass spectrometry method, which allows precise quantification of FA profile using small (2 for mice and 2 for humans) skin specimens that can be readily obtained from live mice and humans. We determined mouse skin FA composition after 2, 4 and 8 weeks of consuming a control diet or a diet supplemented with fish oil. Fish oil markedly enhanced EPA and DHA in mouse skin within 2 weeks, and this increase plateaued after 4 weeks. The FA composition in mouse skin was different from that of serum, indicating that skin has homeostatic control of FA metabolism. Mice fed the control diet designed to simulate Western human diet displayed similar skin FA composition as that of humans. The present study presents a validated method for FA quantification that is needed to investigate the mechanisms of actions of dietary treatments in both mouse and human skin.
Induced DNA repair in xeroderma pigmentosum group C cells by readthrough of premature termination codons R Ono1, S Khan2, C Kuschal2, D Tamura2, J Chen2, X Luo2, J DiGiovanna2 and K Kraemer2 1 Department of Dermatology, Graduate School of Medicine, Kobe University, Kobe, Japan and 2 National Cancer Institute, Bethesda, MD Xeroderma pigmentosum (XP) is a rare autosomal recessive, cancer-prone DNA repair disease with mutations in nucleotide excision repair genes (XPA through XPG) or DNA polymerase eta. Of 259 XP cell lines examined, 71 had XPC mutations and 14 (20%) of these had primary premature termination codon (PTC) mutations. mRNA with PTC is removed by nonsensemediated decay (NMD) resulting in reduced levels of mRNA. PTC mutations can be readthrough by aminoglycoside antibiotics that disrupt ribosomal fidelity. A small increase in functional message may provide clinical benefit since XP patient XP72TMA had a spice mutation with 3-5 % of normal XPC mRNA and a mild clinical phenotype. Quantitative capillary western analysis showed XPC protein expression of 1-2 % of normal. For a precision medicine directed XP treatment protocol, potential therapeutic compounds were tested on XP-C cell lines. Previously, we observed increased XPC mRNA after treatment with G418, confirming PTC readthrough activity, but not with gentamycin (GM). We now tested 3 other aminoglycosides in XP518BE cells: paromomycin (PM) and neomycin (NM) increased XPC mRNA while kanamycin (KM) did not. Amlexanox is an anti-inflammatory compound with activity as an inhibitor of NMD. We found that amlexanox alone increased XPC mRNA in 2 of 4 XP-C PTC cell lines. The combination of GM and amlexanox further increased XPC mRNA in 1 of 4 XPC cell lines. NM plus amlexanox and KM plus amlexanox increased XPC mRNA compared to NM or KM alone in XP518BE cells. In contrast, PM plus amlexanox did not show increased XPC mRNA compared to PM alone. Pharmacologic XPC PTC bypass with in vitro assessment may provide a precision medical intervention for carefully selected XP patients.
www.jidonline.org S117