Application of immunocapture and nanoflow LC-MS/MS to overcome the barriers to the quantitation of oxytocin in plasma of women in third stage labour.

AIMS: Postpartum haemorrhage (PPH) is the leading cause of maternal mortality worldwide. To prevent PPH, the WHO recommends administration of oxytocin (OT) immediately after birth, i.e. during the third stage of labour (TSL). Previous studies demonstrate that methods to quantify OT in biological matrices, e.g. enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) lack the specificity and/or sensitivity to accurately quantify OT in plasma from women administered OT during TSL. This is due to increased metabolic clearance of OT in late-stage pregnancy and at the time of childbirth, resulting in extremely low OT plasma concentrations. This study describes the development of an ultra-sensitive bioanalytical method that overcomes the issues previously reported and enables accurate pharmacokinetic analyses of exogenously administered OT in TSL. METHODS: A selective and sensitive assay to quantify OT in TSL plasma was developed. Immunoprecipitation (IP) was applied to selectively extract OT from the TSL plasma, thereby generating clean extracts compatible with nanoflow LC (nLC). nLC-MS/MS was chosen for its high sensitivity and ability to differentiate between OT and potentially co-captured OT-like immunoreactive products. RESULTS: The presented methodology is accurate and precise, with a good linear fit between 100-10 000 fg mL-1 OT. TSL plasma samples from a clinical phase 1 study (NCT02999100) were analysed successfully, enabling OT quantification down to 100 fg mL-1. CONCLUSIONS: The presented IP-nLC-MS/MS method succeeded in overcoming the sensitivity challenge related to the assay of OT in TSL plasma and thereby revealing the PK profiles of OT in TSL plasma clinical study samples.

Exploring the role of ex vivo metabolism on blood and plasma measurements of oxytocin among women in the third stage of labour: A post hoc study.

AIMS: To examine the role of ex vivo oxytocin metabolism in post-dose peptide measurements. METHODS: The stability of oxytocin (Study 1) and oxytocinase activity (Study 2) in late-stage pregnancy blood was quantified using liquid-chromatography tandem mass-spectrometry (LC-MS/MS) and a fluorogenic assay, respectively. Analyses were conducted using blood from pregnant women (>36 weeks gestation) evaluated in lithium heparin (LH), ethylenediaminetetraacetic acid (EDTA) and BD P100 blood collection tubes with or without protease inhibitors. In addition, plasma oxytocin concentrations following administration of oxytocin 240 IU inhaled, 5 IU intravenous or 10 IU intramuscular in women in third stage of labour (TSL) were analysed using enzyme-linked immunosorbent assay (ELISA) and LC-MS/MS to understand how quantified peptide concentrations differ between these analytical methods (Study 3). RESULTS: Study 1: Oxytocin was stable in blood collected into EDTA tubes with or without protease inhibitors but not in LH tubes. Study 2: Blood collected into all EDTA-containing collection tubes led to near-complete inhibition of oxytocinase (≤100 min). In plasma, a 35% reduction in oxytocinase activity was observed in LH tubes with EDTA added. In plasma from late-stage pregnancy compared to nonpregnant participants, the oxytocinase activity was approximately 11-fold higher. Study 3: Plasma oxytocin concentrations from nonpregnant or women in TSL following exogenous oxytocin administration were ≤33 times higher when analysed using ELISA vs. LC-MS/MS methods. CONCLUSIONS: Collection of blood from late-stage pregnant women into tubes containing EDTA inhibits oxytocinase effectively stabilizing oxytocin, suggesting low concentrations of oxytocin after dose administration reflect rapid in vivo metabolism.

Single-dose pharmacokinetics and lung function of nebulized niclosamide ethanolamine in sheep.

PURPOSE: Niclosamide is approved as an oral anthelminthic, but its low oral bioavailability hinders its medical use requiring high drug exposure outside the gastrointestinal tract. An optimized solution of niclosamide for nebulization and intranasal administration using the ethanolamine salt has been developed and tested in a Phase 1 trial. In this study we investigate the pulmonary exposure of niclosamide following administration via intravenous injection, oral administration or nebulization. METHODS: We characterized the plasma and pulmonary pharmacokinetics of three ascending doses of nebulized niclosamide in sheep, compare it to intravenous niclosamide for compartmental PK modelling, and to the human equivalent approved 2 g oral dose to investigate in the pulmonary exposure of different niclosamide delivery routes. Following a single-dose administration to five sheep, niclosamide concentrations were determined in plasma and epithelial lining fluid (ELF). Non-compartmental and compartmental modeling was used to characterize pharmacokinetic profiles. Lung function tests were performed in all dose groups. RESULTS: Administration of all niclosamide doses were well tolerated with no adverse changes in lung function tests. Plasma pharmacokinetics of nebulized niclosamide behaved dose-linear and was described by a 3-compartmental model estimating an absolute bioavailability of 86%. ELF peak concentration and area under the curve was 578 times and 71 times higher with nebulization of niclosamide relative to administration of oral niclosamide. CONCLUSIONS: Single local pulmonary administration of niclosamide via nebulization was well tolerated in sheep and resulted in substantially higher peak ELF concentration compared to the human equivalent oral 2 g dose.

Polymeric Nanotubes as Drug Delivery Vectors─Comparison of Covalently and Supramolecularly Assembled Constructs.

Rod-shaped nanoparticles have been identified as promising drug delivery candidates. In this report, the in vitro cell uptake and in vivo pharmacokinetic/bio-distribution behavior of molecular bottle-brush (BB) and cyclic peptide self-assembled nanotubes were studied in the size range of 36-41 nm in length. It was found that BB possessed the longest plasma circulation time (t1\2 > 35 h), with the cyclic peptide system displaying an intermediate half-life (14.6 h), although still substantially elevated over a non-assembling linear control (2.7 h). The covalently bound BB underwent substantial distribution into the liver, whereas the cyclic peptide nanotube was able to mostly circumvent organ accumulation, highlighting the advantage of the inherent degradability of the cyclic peptide systems through their reversible aggregation of hydrogen bonding core units.

Biocompatible Cationic Lipoamino Acids as Counterions for Oral Administration of API-Ionic Liquids.

PURPOSE: The use of ionic liquids (ILs) in drug delivery has focused attention on non-toxic IL counterions. Cationic lipids can be used to form ILs with weakly acidic drugs to enhance drug loading in lipid-based formulations (LBFs). However, cationic lipids are typically toxic. Here we explore the use of lipoaminoacids (LAAs) as cationic IL counterions that degrade or digest in vivo to non-toxic components. METHODS: LAAs were synthesised via esterification of amino acids with fatty alcohols to produce potentially digestible cationic LAAs. The LAAs were employed to form ILs with tolfenamic acid (Tol) and the Tol ILs loaded into LBF and examined in vitro and in vivo. RESULTS: Cationic LAAs complexed with Tol to generate lipophilic Tol ILs with high drug loading in LBFs. Assessment of the LAA under simulated digestion conditions revealed that they were susceptible to enzymatic degradation under intestinal conditions, forming biocompatible FAs and amino acids. In vitro dispersion and digestion studies of Tol ILs revealed that formulations containing digestible Tol ILs were able to maintain drug dispersion and solubilisation whilst the LAA were breaking down under digesting conditions. Finally, in vivo oral bioavailability studies demonstrated that oral delivery of a LBF containing a Tol IL comprising a digestible cationic lipid counterion was able to successfully support effective oral delivery of Tol. CONCLUSIONS: Digestible LAA cationic lipids are potential IL counterions for weakly acidic drug molecules and digest in situ to form non-toxic breakdown products.

Digestion of Lipid-Based Formulations Not Only Mediates Changes to Absorption of Poorly Soluble Drugs Due to Differences in Solubilization But Also Reflects Changes to Thermodynamic Activity and Permeability.

The aim of this study was to evaluate the effect of lipid digestion on the permeability and absorption of orally administered saquinavir (SQV), a biopharmaceutics classification system (BCS) class IV drug, in different lipid-based formulations. Three LBFs were prepared: a mixed short- and medium-chain lipid-based formulation (SMCF), a medium-chain lipid-based formulation (MCF), and a long-chain lipid-based formulation (LCF). SQV was loaded into these LBFs at 26.7 mg/g. To evaluate the pharmacokinetics of SQV in vivo, drug-loaded formulations were predispersed in purified water at 3% w/w and orally administered to rats. A low dose (0.8 mg/rat) was employed to limit confounding effects on drug solubilization, and consistent with this design, presolubilization of SQV in the LBFs did not increase in vivo exposure compared to a control suspension formulation. The areas under the plasma concentration-time curve were, however, significantly lower after administration of SQV as MCF and LCF compared to SMCF. To evaluate the key mechanisms underpinning absorption, each LBF containing SQV was digested, and the flux of SQV from the digests across a dialysis membrane was evaluated in in vitro permeation experiments. This study revealed that the absorption profiles were driven by the free concentration of SQV and that this varied due to differences in SQV solubilization in the digestion products generated by LBF digestion. The apparent first-order permeation rate constants of SQV (kapp,total) were estimated by dividing the flux of SQV in the dialysis membrane experiments by the concentration of total SQV on the donor side. kapp,total values strongly correlated with in vivo AUC. The data provide one of the first studies of the effect of digestion products on the free concentration of a drug in the GI fluid and oral absorption. This simple permeation model may be a useful tool for the evaluation of the impact of lipid digestion on apparent drug permeability from lipid-based formulations. These effects should be assessed alongside, and in addition to, the more well-known effects of lipids on enhancing intestinal solubilization of poorly water-soluble drugs.

Quantifying In Vivo Luminal Drug Solubilization -Supersaturation-Precipitation Profiles to Explain the Performance of Lipid Based Formulations.

PURPOSE: To evaluate the role of supersaturation in the in vivo absorption of fenofibrate (FFB), after oral administration in a medium-chain lipid-based formulation (MCLBF). METHODS: FFB was loaded at 90% and 20% w/w of saturated solubility in MCLBF. The two formulations were pre-dispersed in purified water at 5% w/w (ME90% and 20%, respectively) and orally administered to rats to measure in vivo luminal drug concentrations. RESULTS: FFB precipitated in the stomach due to lipid digestion by gastric lipases and loss of solubilization capacity. This was most significant for ME90%. For ME90%, a high degree of supersaturation was also observed in the duodenum, however, precipitated FFB crystals rapidly re-dissolved. The combination of supersaturation and rapid re-dissolution appeared to drive effective absorption in the upper intestine. For ME20%, FFB precipitated in the stomach but not in the crystalline form and rapidly re-dissolved. Supersaturation in the duodenum again appeared to be the major driver of oral absorption. CONCLUSIONS: The data provide one of the first studies of in vivo luminal drug concentration, supersaturation and absorption from lipid based formulations and suggests that for FFB, whilst very high supersaturation may drive in vitro and in vivo precipitation, re-dissolution and drug absorption is rapid and efficient.

Substantial Targeting Advantage Achieved by Pulmonary Administration of Colistin Methanesulfonate in a Large-Animal Model.

Colistin, administered as its inactive prodrug colistin methanesulfonate (CMS), is often used in multidrug-resistant Gram-negative pulmonary infections. The CMS and colistin pharmacokinetics in plasma and epithelial lining fluid (ELF) following intravenous and pulmonary dosing have not been evaluated in a large-animal model with pulmonary architecture similar to that of humans. Six merino sheep (34 to 43 kg body weight) received an intravenous or pulmonary dose of 4 to 8 mg/kg CMS (sodium) or 2 to 3 mg/kg colistin (sulfate) in a 4-way crossover study. Pulmonary dosing was achieved via jet nebulization through an endotracheal tube cuff. CMS and colistin were quantified in plasma and bronchoalveolar lavage fluid (BALF) samples by high-performance liquid chromatography (HPLC). ELF concentrations were calculated via the urea method. CMS and colistin were comodeled in S-ADAPT. Following intravenous CMS or colistin administration, no concentrations were quantifiable in BALF samples. Elimination clearance was 1.97 liters/h (4% interindividual variability) for CMS (other than conversion to colistin) and 1.08 liters/h (25%) for colistin. On average, 18% of a CMS dose was converted to colistin. Following pulmonary delivery, colistin was not quantifiable in plasma and CMS was detected in only one sheep. Average ELF concentrations (standard deviations [SD]) of formed colistin were 400 (243), 384 (187), and 184 (190) mg/liter at 1, 4, and 24 h after pulmonary CMS administration. The population pharmacokinetic model described well CMS and colistin in plasma and ELF following intravenous and pulmonary administration. Pulmonary dosing provided high ELF and low plasma colistin concentrations, representing a substantial targeting advantage over intravenous administration. Predictions from the pharmacokinetic model indicate that sheep are an advantageous model for translational research.

Ionic Liquid Forms of Weakly Acidic Drugs in Oral Lipid Formulations: Preparation, Characterization, in Vitro Digestion, and in Vivo Absorption Studies.

This study aimed to transform weakly acidic poorly water-soluble drugs (PWSD) into ionic liquids (ILs) to promote solubility in, and the utility of, lipid-based formulations. Ionic liquids (ILs) were formed directly from tolfenamic acid (Tolf), meclofenamic acid, diclofenac, and ibuprofen by pairing with lipophilic counterions. The drug-ILs were obtained as liquids or low melting solids and were significantly more soluble (either completely miscible or highly soluble) in lipid based, self-emulsifying drug delivery systems (SEDDS) when compared to the equivalent free acid. In vivo assessment of a SEDDS lipid solution formulation of Tolf didecyldimethylammonium salt and the same formulation of Tolf free acid at low dose (18 mg/kg, where the free acid was soluble in the SEDDS), resulted in similar absorption profiles and overall exposure. At high dose (100 mg/kg), solution SEDDS formulations of the Tolf ILs (didecyldimethylammonium, butyldodecyldimethylammonium or didecylmethylammonium salts) were possible, but the lower lipid solubility of Tolf free acid dictated that administration of the free acid was only possible as a suspension in the SEDDS formulation or as an aqueous suspension. Under these conditions, total drug plasma exposure was similar for the IL formulations and the free acid, but the plasma profiles were markedly different, resulting in flatter, more prolonged exposure profiles and reduced Cmax for the IL formulations. Isolation of a weakly acidic drug as an IL may therefore provide advantage as it allows formulation as a solution SEDDS rather than a lipid suspension, and in some cases may provide a means of slowing or sustaining absorption. The current studies compliment previous studies with weakly basic PWSD and demonstrate that transformation into highly lipophilic ILs is also possible for weakly acidic compounds.

In Vivo Formation of Cubic Phase in Situ after Oral Administration of Cubic Phase Precursor Formulation Provides Long Duration Gastric Retention and Absorption for Poorly Water-Soluble Drugs.

Lipid-based liquid crystalline systems based on the combination of digestible and nondigestible lipids have been proposed as potential sustained release delivery systems for oral delivery of poorly water-soluble drugs. The potential for cubic phase liquid crystal formation to induce dramatically extended gastric retention in vivo has been shown previously to strongly influence the resulting pharmacokinetics of incorporated drug. In vitro studies showing the in situ formation of cubic phase from a disordered precursor comprising a mixture of digestible and nondigestible lipids under enzymatic digestion have also recently been reported. Combining both concepts, here we show the potential for such systems to form in vivo, increasing gastric retention, and providing a sustained release effect for a model poorly water-soluble drug cinnarizine. A mixture of phytantriol and tributyrin at an 85:15 mass ratio, shown previously to form cubic phase under the influence of digestion, induced a similar pharmacokinetic profile to that in the absence of tributyrin, but completely different from tributyrin alone. The gastric retention of the formulation, assessed using micro-X-ray CT imaging, was also consistent with the pharmacokinetic behavior, where phytantriol alone and with 15% tributyrin was greater than that of tributyrin in the absence of phytantriol. Thus, the concept of precursor lipid systems that form cubic phase in situ during digestion in vivo has been demonstrated and opens new opportunities for sustained release of poorly water-soluble drugs.

Addition of 20-kDa PEG to Insulin Lispro Alters Absorption and Decreases Clearance in Animals.

PURPOSE: Determine the pharmacokinetics of insulin peglispro (BIL) in 5/6-nephrectomized rats and study the absorption in lymph duct cannulated (LDC) sheep. METHODS: BIL is insulin lispro modified with 20-kDa linear PEG at lysine B28 increasing the hydrodynamic size to 4-fold larger than insulin lispro. Pharmacokinetics of BIL and insulin lispro after IV administration were compared in 5/6-nephrectomized and sham rats. BIL was administered IV or SC into the interdigital space of the hind leg, and peripheral lymph and/or serum samples were collected from both LDC and non-LDC sheep to determine pharmacokinetics and absorption route of BIL. RESULTS: The clearance of BIL was similar in 5/6-nephrectomized and sham rats, while the clearance of insulin lispro was 3.3-fold slower in 5/6-nephrectomized rats than in the sham rats. In non-LDC sheep, the terminal half-life after SC was about twice as long vs IV suggesting flip-flop pharmacokinetics. In LDC sheep, bioavailability decreased to <2%; most of the dose was absorbed via the lymphatic system, with 88% ± 19% of the dose collected in the lymph after SC administration. CONCLUSION: This work demonstrates that increasing the hydrodynamic size of insulin lispro through PEGylation can impact both absorption and clearance to prolong drug action.

Pluronic-Functionalized Silica-Lipid Hybrid Microparticles: Improving the Oral Delivery of Poorly Water-Soluble Weak Bases.

A Pluronic-functionalized silica-lipid hybrid (Plu-SLH) microparticle system for the oral delivery of poorly water-soluble, weak base drugs is reported for the first time. A highly effective Plu-SLH microparticle system was composed of Labrasol as the lipid phase, Pluronic F127 as the polymeric precipitation inhibitor (PPI), and silica nanoparticles as the solid carrier. For the model drug cinnarizine (CIN), the Plu-SLH delivery system was shown to offer significant biopharmaceutical advantages in comparison with unformulated drug and drug in the silica-lipid hybrid (SLH) system. In vitro two-phase dissolution studies illustrated significantly reduced pH provoked CIN precipitation and an 8- to 14-fold improvement in the extent of dissolution in intestinal conditions. In addition, under simulated intestinal digesting conditions, the Plu-SLH provided approximately three times more drug solubilization than the SLH. Oral administration in rats resulted in superior bioavailability for Plu-SLH microparticles, i.e., 1.6- and 2.1-fold greater than the SLH and the unformulated CIN, respectively. A physical mixture of Pluronic and SLH (Plu&SLH), having the same composition as Plu-SLH, was also evaluated, but showed no significant increase in CIN absorption when compared to unmodified CIN or SLH. This work represents the first study where different methods of incorporating PPI to formulate solid-state lipid-based formulations were compared for the impact on the biopharmaceutical performance. The data suggest that the novel physicochemical properties and structure of the fabricated Plu-SLH microparticle delivery system play an important role in facilitating the synergistic advantage of Labrasol and Pluronic F127 in preventing drug precipitation, and the Plu-SLH provides efficient oral delivery of poorly water-soluble weak bases.

Transformation of poorly water-soluble drugs into lipophilic ionic liquids enhances oral drug exposure from lipid based formulations.

Absorption after oral administration is a requirement for almost all drug products but is a challenge for drugs with intrinsically low water solubility. Here, the weakly basic, poorly water-soluble drugs (PWSDs) itraconazole, cinnarizine, and halofantrine were converted into lipophilic ionic liquids to facilitate incorporation into lipid-based formulations and integration into lipid absorption pathways. Ionic liquids were formed via metathesis reactions of the hydrochloride salt of the PWSDs with a range of lipophilic counterions. The resultant active pharmaceutical ingredient-ionic liquids (API-ILs) were liquids or low melting point solids and either completely miscible or highly soluble in lipid based, self-emulsifying drug delivery systems (SEDDS) comprising mixtures of long or medium chain glycerides, surfactants such as Kolliphor-EL and cosolvents such as ethanol. They also readily incorporated into the colloids formed in intestinal fluids during lipid digestion. Itraconazole docusate or cinnarizine decylsulfate API-ILs were subsequently dissolved in long chain lipid SEDDS at high concentration, administered to rats and in vivo exposure assessed. The data were compared to control formulations based on the same SEDDS formulations containing the same concentrations of drug as the free base, but in this case as a suspension (since the solubility of the free base in the SEDDS was much lower than the API-ILs). For itraconazole, comparison was also made to a physical mixture of itraconazole free base and sodium docusate in the same SEDDS formulation. For both drugs plasma exposure was significantly higher for the API-IL containing formulations (2-fold for cinnarizine and 20-fold for itraconazole), when compared to the suspension formulations (or the physical mixture in the case of itraconazole) at the same dose. The liquid SEDDS formulations, made possible by the use of the API-ILs, also provide advantages in dose uniformity, capsule filling, and stability compared to similar suspension formulations. The data suggest that the formation of lipophilic ionic liquids provides a means of increasing dissolved-drug loading in lipid based formulations and thereby promoting the exposure of poorly water-soluble drugs after oral administration.

Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro.

Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon-ß in relapsing-remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement-dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab-exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25(hi) T-cell population was necessary for the suppressive activity of alemtuzumab-exposed T cells. The mechanism of this suppression was found to be dependent on both cell-cell contact and interleukin-2 consumption. These findings suggest that an alemtuzumab-mediated increase in the proportion of Treg cells may play a role in promoting the long-term efficacy of alemtuzumab in patients with multiple sclerosis.

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization.

BACKGROUND: Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. METHODS: In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. RESULTS: The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. CONCLUSION: Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.

An in vitro digestion test that reflects rat intestinal conditions to probe the importance of formulation digestion vs first pass metabolism in Danazol bioavailability from lipid based formulations.

The impact of gastrointestinal (GI) processing and first pass metabolism on danazol oral bioavailability (BA) was evaluated after administration of self-emulsifying drug delivery systems (SEDDS) in the rat. Danazol absolute BA was determined following oral and intraduodenal (ID) administration of LFCS class IIIA medium chain (MC) formulations at high (SEDDSH-III) and low (SEDDSL-III) drug loading and a lipid free LFCS class IV formulation (SEDDS-IV). Experiments were conducted in the presence and absence of ABT (1-aminobenzotriazole) to evaluate the effect of first pass metabolism. A series of modified in vitro lipolysis tests were developed to better understand the in vivo processing of SEDDS in the rat. Danazol BA was low (<13%) following oral and ID administration of either SEDDS. Increasing drug loading, ID rather than oral administration, and administration of SEDDS-IV rather than SEDDS-III led to higher oral BA. After pretreatment with ABT, however, danazol oral BA significantly increased (e.g., 60% compared to 2% after administration of SEDDSL-III), no effect was observed on increasing drug loading, and differences between SEDDS-III and -IV were minimal. In vitro digestion models based on the lower enzyme activity and lower dilution conditions expected in the rat resulted in significantly reduced danazol precipitation from SEDDS-III or SEDDS-IV on initiation of digestion. At the doses administered here (4-8 mg/kg), the primary limitation to danazol oral BA in the rat was first pass metabolism, and the fraction absorbed was >45% after oral administration of SEDDS-III or SEDDS-IV. In contrast, previous studies in dogs suggest that danazol BA is less dependent on first pass metabolism and more sensitive to changes in formulation processing. In vitro digestion models based on likely rat GI conditions suggest less drug precipitation on formulation digestion when compared to equivalent dog models, consistent with the increases in in vivo exposure (fraction absorbed) seen here in ABT-pretreated rats.

SQ3370, the first clinical click chemistry-activated cancer therapeutic, shows safety in humans and translatability across species.

Background: SQ3370 is the first demonstration of the Click Activated Protodrugs Against Cancer (CAPAC™) platform that uses click chemistry to activate drugs directly at tumor sites, maximizing therapeutic exposure. SQ3370 consists of a tumor-localizing biopolymer (SQL70) and a chemically-attenuated doxorubicin (Dox) protodrug SQP33; the protodrug is activated upon clicking with the biopolymer at tumor sites. Here, we present data from preclinical studies and a Phase 1 dose-escalation clinical trial in adult patients with advanced solid tumors ( NCT04106492 ) demonstrating SQ3370's activation at tumor sites, safety, systemic pharmacokinetics (PK), and immunological activity. Methods: Treatment cycles consisting of an intratumoral or subcutaneous injection of SQL70 biopolymer followed by 5 daily intravenous doses of SQP33 protodrug were evaluated in tumor-bearing mice, healthy dogs, and adult patients with solid tumors. Results: SQL70 effectively activated SQP33 at tumor sites, resulting in high Dox concentrations that were well tolerated and unachievable by conventional treatment. SQ3370 was safely administered at 8.9x the veterinary Dox dose in dogs and 12x the conventional Dox dose in patients, with no dose-limiting toxicity reported to date. SQ3370's safety, toxicology, and PK profiles were highly translatable across species. SQ3370 increased cytotoxic CD3 + and CD8 + T-cells in patient tumors indicating T-cell-dependent immune activation in the tumor microenvironment. Conclusions: SQ3370, the initial demonstration of click chemistry in humans, enhances the safety of Dox at unprecedented doses and has the potential to increase therapeutic index. Consistent safety, toxicology, PK, and immune activation results observed with SQ3370 across species highlight the translatability of the click chemistry approach in drug development. Trial registration: NCT04106492; 7 September 2019.

Click Chemistry Selectively Activates an Auristatin Protodrug with either Intratumoral or Systemic Tumor-Targeting Agents.

The Click Activated Protodrugs Against Cancer (CAPAC) platform enables the activation of powerful cancer drugs at tumors. CAPAC utilizes a click chemistry reaction between tetrazine and trans-cyclooctene. The reaction between activator, linked to a tumor-targeting agent, and protodrug leads to the targeted activation of the drug. Here, tumor targeting is achieved by intratumoral injection of a tetrazine-modified hyaluronate (SQL70) or by infusion of a tetrazine-modified HER2-targeting antigen-binding fragment (SQT01). Monomethyl auristatin E (a cytotoxin hindered in its clinical use by severe toxicity) was modified with a trans-cyclooctene to form the protodrug SQP22, which reduced its cytotoxicity in vitro and in vivo. Treatment of SQP22 paired with SQL70 demonstrated antitumor effects in Karpas 299 and RENCA murine tumor models, establishing the requirement of click chemistry for protodrug activation. SQP22 paired with SQT01 induced antitumor effects in the HER2-positive NCI-N87 xenograft model, showing that tumor-targeted activation could be accomplished via systemic dosing. Observed toxicities were limited, with transient myelosuppression and moderate body weight loss detected. This study highlights the capabilities of the CAPAC platform by demonstrating the activity of SQP22 with two differentiated targeting approaches and underscores the power of click chemistry to precisely control the activation of drugs at tumors.

Incomplete desorption of liquid excipients reduces the in vitro and in vivo performance of self-emulsifying drug delivery systems solidified by adsorption onto an inorganic mesoporous carrier.

The purpose of the current study was to provide a mechanistic basis for in vitro and in vivo performance differences between lipid-based formulations solidified by adsorption onto a high surface area material and their respective liquid (i.e., nonadsorbed) counterparts. Two self-emulsifying formulations (based on either medium-chain or long-chain lipids) of the poorly water-soluble drug danazol were solidified by adsorption onto Neusilin US2. Liquid and adsorbed lipid-based formulations were subjected to in vitro dispersion-digestion tests, and additional in vitro experiments were performed to elucidate the cause of performance differences. The bioavailability of danazol after oral administration to rats was also assessed. The percentage of the dose solubilized in the aqueous phase during in vitro dispersion-digesting was ∼35% lower for the adsorbed formulations when compared to their liquid counterparts. This trend was also reflected in vivo, where the bioavailability of danazol after administration of the adsorbed formulations was ∼50% lower than that obtained after administration of the equivalent liquid formulation. Incomplete desorption of the microemulsion preconcentrate from the carrier on dispersion-digestion was identified as the main contributor to the reduced pharmaceutical performance of the adsorbed formulations. The results of the current study indicate that solidification of lipid-based formulations through adsorption onto a high surface area carrier may limit formulation (and drug) release in vivo and thereby reduce oral bioavailability.

Neuroinflammation in neuronopathic Gaucher disease: Role of microglia and NK cells, biomarkers, and response to substrate reduction therapy.

Background: Neuronopathic Gaucher disease (nGD) is a rare neurodegenerative disorder caused by biallelic mutations in GBA and buildup of glycosphingolipids in lysosomes. Neuronal injury and cell death are prominent pathological features; however, the role of GBA in individual cell types and involvement of microglia, blood-derived macrophages, and immune infiltrates in nGD pathophysiology remains enigmatic. Methods: Here, using single-cell resolution of mouse nGD brains, lipidomics, and newly generated biomarkers, we found induction of neuroinflammation pathways involving microglia, NK cells, astrocytes, and neurons. Results: Targeted rescue of Gba in microglia and neurons, respectively, in Gba-deficient, nGD mice reversed the buildup of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph), concomitant with amelioration of neuroinflammation, reduced serum neurofilament light chain (Nf-L), and improved survival. Serum GlcSph concentration was correlated with serum Nf-L and ApoE in nGD mouse models as well as in GD patients. Gba rescue in microglia/macrophage compartment prolonged survival, which was further enhanced upon treatment with brain-permeant inhibitor of glucosylceramide synthase, effects mediated via improved glycosphingolipid homeostasis, and reversal of neuroinflammation involving activation of microglia, brain macrophages, and NK cells. Conclusions: Together, our study delineates individual cellular effects of Gba deficiency in nGD brains, highlighting the central role of neuroinflammation driven by microglia activation. Brain-permeant small-molecule inhibitor of glucosylceramide synthase reduced the accumulation of bioactive glycosphingolipids, concomitant with amelioration of neuroinflammation involving microglia, NK cells, astrocytes, and neurons. Our findings advance nGD disease biology whilst identifying compelling biomarkers of nGD to improve patient management, enrich clinical trials, and illuminate therapeutic targets. Funding: Research grant from Sanofi; other support includes R01NS110354, Yale Liver Center P30DK034989, pilot project grant.