Publisher Summary This chapter provides an insight of the findings of past significant papers with the current knowledge of the recently determined high resolution X-ray structure of serum albumin. The most outstanding property of albumin is its ability to bind reversibly an incredible variety of ligands. The sequences of all albumins are characterized by a unique arrangement of disulfide double loops that repeat as a series of triplets. Albumin belongs to a multigene family of proteins that includes α- fetoprotein (AFP) and vitamin D-binding protein (VDP), also known as G complement (Gc) protein. Although AFP is considered the fetal counterpart of albumin, its binding properties are distinct and it is suggested that AFP may have a higher affinity for some unknown ligands important for fetal development. Domain structure and the arrangement of the disulfides, the surface charge distribution, and the conformational flexibility of the albumin molecule are described. The nature of ligand binding, including small organics, long-chain fatty acids, and metals, to multiple sites on the albumin molecule is clearly depicted. The chapter concludes with the perceptive comments on future directions being taken to explore the structure and function of this fascinating protein.

Structure of serum albumin.

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first broke out in 2019 and subsequently spread worldwide. Chloroquine has been sporadically used in treating SARS-CoV-2 infection. Hydroxychloroquine shares the same mechanism of action as chloroquine, but its more tolerable safety profile makes it the preferred drug to treat malaria and autoimmune conditions. We propose that the immunomodulatory effect of hydroxychloroquine also may be useful in controlling the cytokine storm that occurs late phase in critically ill patients with SARS-CoV-2. Currently, there is no evidence to support the use of hydroxychloroquine in SARS-CoV-2 infection. Methods The pharmacological activity of chloroquine and hydroxychloroquine was tested using SARS-CoV-2-infected Vero cells. Physiologically based pharmacokinetic (PBPK) models were implemented for both drugs separately by integrating their in vitro data. Using the PBPK models, hydroxychloroquine concentrations in lung fluid were simulated under 5 different dosing regimens to explore the most effective regimen while considering the drug's safety profile. Results Hydroxychloroquine (EC50 = 0.72 μM) was found to be more potent than chloroquine (EC50 = 5.47 μM) in vitro. Based on PBPK models results, a loading dose of 400 mg twice daily of hydroxychloroquine sulfate given orally, followed by a maintenance dose of 200 mg given twice daily for 4 days is recommended for SARS-CoV-2 infection, as it reached 3 times the potency of chloroquine phosphate when given 500 mg twice daily 5 days in advance. Conclusions Hydroxychloroquine was found to be more potent than chloroquine to inhibit SARS-CoV-2 in vitro.

In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Microneedles for drug and vaccine delivery.

Macroautophagy (autophagy) is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here we show that pancreatic cancers have a distinct dependence on autophagy. Pancreatic cancer primary tumors and cell lines show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to increased reactive oxygen species, elevated DNA damage, and a metabolic defect leading to decreased mitochondrial oxidative phosphorylation. Together, these ultimately result in significant growth suppression of pancreatic cancer cells in vitro. Most importantly, inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models. These results suggest that, unlike in other cancers where autophagy inhibition may synergize with chemotherapy or targeted agents by preventing the up-regulation of autophagy as a reactive survival mechanism, autophagy is actually required for tumorigenic growth of pancreatic cancers de novo, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on autophagy. As chloroquine and its derivatives are potent inhibitors of autophagy and have been used safely in human patients for decades for a variety of purposes, these results are immediately translatable to the treatment of pancreatic cancer patients, and provide a much needed, novel vantage point of attack.

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Pancreatic cancers require autophagy for tumor growth

BackgroundHydroxychloroquine and chloroquine have been proposed as treatments for coronavirus disease 2019 (Covid-19) on the basis of in vitro activity and data from uncontrolled studies and small, randomized trials.MethodsIn this randomized, controlled, open-label platform trial comparing a range of possible treatments with usual care in patients hospitalized with Covid-19, we randomly assigned 1561 patients to receive hydroxychloroquine and 3155 to receive usual care. The primary outcome was 28-day mortality.ResultsThe enrollment of patients in the hydroxychloroquine group was closed on June 5, 2020, after an interim analysis determined that there was a lack of efficacy. Death within 28 days occurred in 421 patients (27.0%) in the hydroxychloroquine group and in 790 (25.0%) in the usual-care group (rate ratio, 1.09; 95% confidence interval [CI], 0.97 to 1.23; P=0.15). Consistent results were seen in all prespecified subgroups of patients. The results suggest that patients in the hydroxychloroquine group were less likely to be discharged from the hospital alive within 28 days than those in the usual-care group (59.6% vs. 62.9%; rate ratio, 0.90; 95% CI, 0.83 to 0.98). Among the patients who were not undergoing mechanical ventilation at baseline, those in the hydroxychloroquine group had a higher frequency of invasive mechanical ventilation or death (30.7% vs. 26.9%; risk ratio, 1.14; 95% CI, 1.03 to 1.27). There was a small numerical excess of cardiac deaths (0.4 percentage points) but no difference in the incidence of new major cardiac arrhythmia among the patients who received hydroxychloroquine.ConclusionsAmong patients hospitalized with Covid-19, those who received hydroxychloroquine did not have a lower incidence of death at 28 days than those who received usual care. (Funded by UK Research and Innovation and National Institute for Health Research and others; RECOVERY ISRCTN number, ISRCTN50189673. opens in new tab; ClinicalTrials.gov number, NCT04381936. opens in new tab.)

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Effect of Hydroxychloroquine in Hospitalized Patients with Covid-19

1. Five healthy volunteers received, in a randomised crossover design study, a 155 mg oral tablet and an intravenous infusion of 155 mg racemic hydroxychloroquine (200 mg hydroxychloroquine sulphate) to assess the bioavailability of the commercially available tablet (Plaquenil, Winthrop Laboratories, Australia). 2. The terminal elimination half-life of hydroxychloroquine is more than 40 days, thus blood and urine samples were collected for 5 months following each dose to characterise adequately the terminal elimination phase and obtain accurate estimates of the areas under the concentration-time curves. 3. The mean (+/- s.d.) fraction of the oral dose absorbed, estimated from the blood and urine data, was 0.74 (+/- 0.13). A wide range of estimates of the fraction of the oral dose absorbed was calculated from the plasma data (0.41 - 1.53), reflecting the difficulties of accurate measurement of hydroxychloroquine in plasma. 4. A period of 6 months is required to achieve 96% of steady-state levels of hydroxychloroquine with the usual once daily, oral dosage regimen. Pharmacokinetic factors may thus be partly responsible for the delayed action of the drug in rheumatic conditions. 5. Haemodialysis will not aid in the case of oral overdose with hydroxychloroquine. Although the proportionate increase in clearance may be large, the increase in the fraction of the dose excreted will be negligible. The extensive sequestration of the drug by tissues limits effectiveness of haemodialysis.

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Bioavailability of hydroxychloroquine tablets in healthy volunteers

1. The pharmacokinetics of hydroxychloroquine were studied in five healthy volunteers following an intravenous infusion of 155 mg (2.47 +/- 0.25 mg kg-1) racemic hydroxychloroquine. Four of these volunteers also received a further 310 mg (4.92 +/- 0.45 mg kg-1) infusion of hydroxychloroquine and evidence of nonlinearities in the pharmacokinetics of hydroxychloroquine were sought. 2. No nonlinear elimination or distribution processes appeared to be operating at the doses of hydroxychloroquine used in this study, supporting the hypothesis that in the therapeutic dosing range the pharmacokinetics of hydroxychloroquine are linear. 3. Half-life and mean residence time were long (around 40 days) and large volumes of distribution were calculated (5,522 l from blood, 44,257 l from plasma). Sequestration into the tissues is an important feature of the disposition of hydroxychloroquine. The persistence of hydroxychloroquine in the body is due primarily to this extensive tissue distribution, rather than to low clearance (667 ml min-1 based on plasma data, 96 ml min-1 based on blood data). 4. Plasma data were more variable than blood data. Blood to plasma concentration ratios were not constant (mean +/- s.d.: 7.2 +/- 4.2). The data indicate that it is preferable to measure whole blood concentrations of hydroxychloroquine, rather than plasma concentrations, in pharmacokinetic studies. 5. The pharmacokinetics of hydroxychloroquine are similar to those of chloroquine.

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A dose-ranging study of the pharmacokinetics of hydroxy-chloroquine following intravenous administration to healthy volunteers.

To demonstrate rapid (~1 min) lidocaine delivery using 3M’s solid microstructured transdermal system (sMTS) for prolonged, local analgesic action. Polymeric microneedles were fabricated via injection molding and then dip-coated using an aqueous lidocaine formulation. The amount of lidocaine coated onto the microneedles was determined by high performance liquid chromatography (HPLC). To assess drug delivery and dermal pharmacokinetics, lidocaine-coated microneedles were inserted into domestic swine. Skin punch biopsies were collected and analyzed to determine lidocaine concentration in skin using HPLC-mass spectrometry (LC-MS). Commercial lidocaine/prilocaine EMLA (Eutectic Mixture of Local Anesthetic) cream was used as comparative control. Lidocaine dissolves rapidly off the microneedles and into skin such that the 1-min wear time achieves or exceeds lidocaine tissue levels needed to cause analgesia. This therapeutic threshold (100 ng/mg) was estimated by measuring the total amount of lidocaine and prilocaine in skin following a 1 h EMLA application. When co-formulated with 0.03 wt% vasoconstrictor-epinephrine, the concentration of lidocaine in tissue was maintained above 100 ng/mg for approximately 90 min. 3M’s sMTS can be used to provide rapid delivery of lidocaine for local analgesia up to 90 min.

Development of lidocaine-coated microneedle product for rapid, safe, and prolonged local analgesic action.

Purpose
The purpose of this work is to demonstrate rapid intradermal delivery of up to 1.5 mL of formulation using a hollow microneedle delivery device designed for self-application.

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Kenneth F. Brown

Papers

Bioavailability of hydroxychloroquine tablets in healthy volunteers

A dose-ranging study of the pharmacokinetics of hydroxy-chloroquine following intravenous administration to healthy volunteers.

Development of lidocaine-coated microneedle product for rapid, safe, and prolonged local analgesic action.

Rapid intradermal delivery of liquid formulations using a hollow microstructured array.

Displacement of tolbutamide, glibenclamide and chlorpropamide from serum albumin by anionic drugs