Melanin targeting for intracellular drug delivery: Quantification of bound and free drug in retinal pigment epithelial cells

Melanin binding affects drug distribution and retention in pigmented ocular tissues, thereby affecting drug response, duration of activity and toxicity. Therefore, it is a promising possibility for drug targeting and controlled release in the pigmented cells and tissues. Intracellular unbound drug concentrations determine pharmacological and toxicological actions, but analyses of unbound vs. total drug concentrations in pigmented cells are lacking. We studied intracellular binding and cellular drug uptake in pigmented retinal pigment epithelial cells and in non-pigmented ARPE-19 cells with five model drugs (chloroquine, propranolol, timolol, diclofenac, methotrexate). The unbound drug fractions in pigmented cells were 0.00016–0.73 and in non-pigmented cells 0.017–1.0. Cellular uptake (i.e. distribution ratio Kp), ranged from 1.3 to 6300 in pigmented cells and from 1.0 to 25 in non-pigmented cells. Values for intracellular bioavailability, Fic, were similar in both cells types (although larger variation in pigmented cells). In vitro melanin binding parameters were used to predict intracellular unbound drug fraction and cell uptake. Comparison of predictions with experimental data indicates that other factors (e.g. ion-trapping, lipophilicity-related binding to other cell components) also play a role. Melanin binding is a major factor that leads to cellular uptake and unbound drug fractions of a range of 3–4 orders of magnitude indicating that large reservoirs of melanin bound drug can be generated in the cells. Understanding melanin binding has important implications on retinal drug targeting, efficacy and toxicity.Peer reviewe

Binding of Small Molecule Drugs to Porcine Vitreous Humor

Pharmacokinetics in the posterior eye segment has therapeutic implications due to the importance of retinal diseases in ophthalmology. In principle, drug binding to the components of the vitreous, such as proteins, collagen, or glycosaminoglycans, could prolong ocular drug retention and modify levels of pharmacologically active free drug in the posterior eye segment. Since drug binding in the vitreous has been investigated only sparsely, we studied vitreal drug binding of 35 clinical small molecule drugs. Isolated homogenized porcine vitreous and the drugs were placed in a two compartment dialysis system that was used to separate the bound and unbound drug. Free drug concentrations and binding percentages were quantitated using LC-MS/MS. Drug binding levels varied between 21 and 74% in the fresh vitreous and 0 and 64% in the frozen vitreous. The vitreal binding percentages did not correlate with those in plasma. Our data-based pharmacokinetic simulations suggest that vitreal binding of small molecule drugs has only a modest influence on the AUC of free drug or drug half-life in the vitreous. Therefore, it is likely that vitreal binding is not a major reason for interindividual variability in ocular drug responses or drug-drug interactions.Peer reviewe

The expression and prognostic value of toll-like receptors (TLRs) in pancreatic cancer patients treated with neoadjuvant therapy

Publisher Copyright: Copyright: © 2022 Nurmi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Objectives Toll-like receptors (TLRs) play a pivotal role in the immune system and carcinogenesis. There is no research on TLR expression and association with survival among preoperatively treated pancreatic cancer patients. We studied the expression intensity and prognostic value of TLRs in pancreatic cancer patients treated with neoadjuvant therapy (NAT) and compared the results to patients undergoing upfront surgery (US). Method Between 2000 and 2015, 71 borderline resectable patients were treated with NAT and surgery and 145 resectable patients underwent upfront surgery at Helsinki University Hospital, Finland. We immunostained TLRs 1–5, 7, and 9 on sections of tissue-microarray. We classified TLR expression as 0 (negative), 1 (mild), 2 (moderate), or 3 (strong) and divided into high (2–3) and low (0–1) expression for statistical purposes. Results Among TLRs 1, 3, and 9 (TLR1 81% vs 70%, p = 0.008; TLR3 92% vs 68%, p = 0.001; TLR9 cytoplasmic 83% vs 42%, p<0.001; TLR9 membranous 53% vs 25%, p = 0.002) NAT patients exhibited a higher immunopositivity score more frequently than patients undergoing upfront surgery. Among NAT patients, a high expression of TLR1 [Hazards ratio (HR) 0.48, p<0.05] associated with a longer postoperative survival, whereas among US patients, high expression of TLR5 (HR 0.64, p<0.05), TLR7 (HR 0.59, p<0.01, and both TLR7 and TLR9 (HR 0.5, p<0.01) predicted a favorable postoperative outcome in separate analysis adjusted for background variables. Conclusions We found higher immunopositive intensities among TLRs 1, 3, and 9 in NAT patients. A high TLR1 expression associated with a longer survival among NAT patients, however, among US patients, high expression intensity of TLR5 and TLR7 predicted a favorable postoperative outcome in the adjusted analysis.Peer reviewe

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology

Impact of histological response after neoadjuvant therapy on podocalyxin as a prognostic marker in pancreatic cancer

Podocalyxin overexpression associates with poor survival in pancreatic cancer (PDAC). We investigated whether podocalyxin expression correlates with treatment response or survival in neoadjuvant-treated PDAC. Through immunohistochemistry, we evaluated podocalyxin expression in 88 neoadjuvant and 143 upfront surgery patients using two antibodies. We developed a six-tier grading scheme for neoadjuvant responses evaluating the remaining tumor cells in surgical specimens. Strong podocalyxin immunopositivity associated with poor survival in the patients responding poorly to the neoadjuvant treatment (HR 4.16, 95% CI 1.56-11.01, p=0.004), although neoadjuvant patients exhibited generally low podocalyxin expression (p=0.017). Strong podocalyxin expression associated with perineural invasion (p=0.003) and lack of radiation (p=0.036). Two patients exhibited a complete neoadjuvant response, while a strong neoadjuvant response (Peer reviewe

Основные проблемы агротуризма в Республике Беларусь на современном этапе

Материалы VI Междунар. науч. конф. студентов, аспирантов и молодых ученых, Гомель, 26 апр. 2013 г

Increased MIB-1 expression in salivary gland pleomorphic adenoma that recurs and undergoes malignant transformation

Correction: Volume12, Issue1 Article Number10828 DOI: 10.1038/s41598-022-15267-7The objective of this retrospective study was to explore possible changes in histopathological features and expression of cyclin D1 and MIB-1 in salivary gland pleomorphic adenoma (PA) that recur or undergo malignant transformation. Knowledge of these characteristics might help to guide the management of these rare tumors. The histopathology and immunohistochemical staining characteristics of such tumors were analyzed in a cohort of 65 patients constituting three different groups of tumors: PA, recurrent pleomorphic adenoma (RPA) and carcinoma ex PA (CxPA). The RPAs were divided into two subgroups: primary PA that were known to recur later (PA-prim) and recurrent tumors appearing after a primary tumor (PA-rec). RPAs and CxPAs were compared with PAs without recurrence, which served as a control group. In our study, CxPA and PA-rec, but not PA-prim, showed increased MIB-1 expression compared with the control group. Neither cyclin D1 expression nor any histopathological features showed any association in statistical analyses. CxPA showed increased mitotic activity, squamous metaplasia, and nuclear atypia. Tumor multifocality was more frequent in PA-rec and CxPA. The different MIB-1 expression in CxPA and PA-rec in comparison to PA-prim suggests that the changes in expression could develop after the primary tumor.Peer reviewe

The expression and prognostic value of toll-like receptors (TLRs) in pancreatic cancer patients treated with neoadjuvant therapy

ObjectivesToll-like receptors (TLRs) play a pivotal role in the immune system and carcinogenesis. There is no research on TLR expression and association with survival among preoperatively treated pancreatic cancer patients. We studied the expression intensity and prognostic value of TLRs in pancreatic cancer patients treated with neoadjuvant therapy (NAT) and compared the results to patients undergoing upfront surgery (US).MethodBetween 2000 and 2015, 71 borderline resectable patients were treated with NAT and surgery and 145 resectable patients underwent upfront surgery at Helsinki University Hospital, Finland. We immunostained TLRs 1–5, 7, and 9 on sections of tissue-microarray. We classified TLR expression as 0 (negative), 1 (mild), 2 (moderate), or 3 (strong) and divided into high (2–3) and low (0–1) expression for statistical purposes.ResultsAmong TLRs 1, 3, and 9 (TLR1 81% vs 70%, p = 0.008; TLR3 92% vs 68%, p = 0.001; TLR9 cytoplasmic 83% vs 42%, pConclusionsWe found higher immunopositive intensities among TLRs 1, 3, and 9 in NAT patients. A high TLR1 expression associated with a longer survival among NAT patients, h</p

Prognostic Role of Porphyromonas gingivalis Gingipain Rgp and Matrix Metalloproteinase 9 in Oropharyngeal Squamous Cell Carcinoma

Background/Aim: The oral bacteria involved in the development of periodontitis alter the tissue conditions and modify immune responses in a way that may also influence tumor development. We investigated the prevalence of R gingipain (Rgp), a key virulence factor of the oral pathobiont Porphyromonas gingivalis, and the tissue-destructive enzymes matrix metalloproteinase 8 (MMP-8) and 9 (MMP-9) in 202 unselected consecutive oropharyngeal squamous cell carcinomaPeer reviewe