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Sixty-nine infants at risk of developing retinopathy of prematurity (ROP) were entered into a prospective study to assess the incidence and natural history of the disease. Seventeen infants developed ROP, and in six eyes the disease progressed to stage IV ROP. The natural regression of a pupillary membrane and physiological vitreous haze was not influenced by the onset of ROP. Progression from stage I to stage III was rapid and the rate was influenced by the zone affected. Congestion and tortuosity of vessels in the posterior pole always signified stage III ROP. Progression from stage III to stage IV ROP was slower; it was characterised by the development of vitreoretinopathy, signified by the sudden onset of a vitreous haze. Iris congestion associated with poor mydriasis may be a grave sign indicating imminent retinal detachment. Cicatricial ROP can be divided into retinal and vitreoretinal cicatricial disease directly related to the stage of active disease reached. ROP is characterised by its self limiting nature, but the stage at which it becomes inactive varies and will influence the final outcome. [ABSTRACT FROM PUBLISHER]

We present a case in which trauma from a broken glass resulted in complete amputation of the upper lid and severe lacerations to the lower lid but with an intact and functioning globe. The avulsed upper lid was repaired as a composite autograft. The possible management of such an unusual case is discussed. [ABSTRACT FROM PUBLISHER]

Background and Aim: The utility of renal replacement therapy (RRT) before liver transplant (LT) in patients without end-stage renal disease (ESRD) or advanced chronic kidney disease (CKD-IV/V) is debatable and lacks data support. We aimed to evaluate the impact of RRT on patients undergoing LT., Methods: We used the National Readmission Database (2016-2019) to identify all index hospitalizations undergoing RRT before LT (cases). A matched comparison cohort of similar hospitalizations without RRT before LT was identified (controls) after 1:1 propensity score matching for age, gender, and available comorbidities., Results: We matched 364 cases (RRT before LT) to 364 controls (LT without prior RRT). There was no statistical difference in all-cause inpatient mortality (4.9% vs 3.6% P  = 0.4). A significantly greater proportion of cases were associated with ICU admission (40.7% vs 17.0%, P  < 0.001) and RRT requirement post LT (100% vs 17%, P  < 0.001). There was no difference in 30- (hazard ratio [HR] 1.1, 0.4-2.6), 60- (HR 0.9, 0.4-1.8), or 90-day (HR 0.8, 0.4-1.6) inpatient mortality between the groups. Also, 180-day survival estimates were comparable ( P  = 0.5). The results were similar in patients with no chronic kidney disease (CKD) and CKD-III., Conclusion: RRT prior to LT, in patients without advanced CKD or ESRD, was associated with greater instances of ICU stay and need for future RRT. Also, 30-, 60-, and 90-day inpatient mortality rates were similar, and 180-day survival estimates were comparable., (© 2024 The Authors. JGH Open published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Mental health services continue to experience rising demand that exceeds capacity. The COVID-19 pandemic exacerbated this crisis, with access to services being reduced. Although video consultations (VCs) are a solution, usage in UK community mental healthcare settings remains limited. This study aims to investigate psychiatrists' and general practitioners' (GPs) perceptions of the benefits and challenges of VC for the diagnosis and follow-up of general adult mental health patients in the community during the COVID-19 pandemic. Semi-structured interviews in NHS community mental healthcare settings were conducted. Psychiatrists ( n = 11) and GPs ( n = 12) were recruited through purposive sampling. An explorative qualitative approach was employed. Data were analysed using thematic analysis. Four key themes were identified: (1) patient access to VC, (2) suitability of VC for mental health consultations, (3) information gathering with VC and (4) clinician satisfaction with VC. This study provides valuable insights into the experiences of psychiatrists and GPs working in the UK during the COVID-19 pandemic. To facilitate a digital-first future for the NHS, greater investment in remote technologies is required, particularly in the context of growing mental healthcare demand. Though face-to-face consultations remain the gold standard, VC provides an efficient way of communicating with patients, particularly those with less severe forms of mental illness.

Here, we explore whether PEGylation of antibodies can modulate their biodistribution to the eye, an organ once thought to be immune privileged but has recently been shown to be accessible to IV-administered large molecules, such as antibodies. We chose to PEGylate an anti-MerTK antibody, a target with known potential for ocular toxicity, to minimize biodistribution to retinal pigment epithelial cells (RPEs) in the eye by increasing the hydrodynamic volume of the antibody. We used site-specific conjugation to an engineered cysteine on anti-MerTK antibody to chemically attach 40-kDa branched or linear PEG polymers. Despite reduced binding to MerTK on cells, site-specifically PEGylated anti-MerTK retained similar potency in inhibiting MerTK-mediated macrophage efferocytosis of apoptotic cells. Importantly, we found that PEGylation of anti-MerTK significantly reduced MerTK receptor occupancy in RPE cells in both naïve mice and MC-38 tumor-bearing mice, with the branched PEG exhibiting a greater effect than linear PEG. Furthermore, similar to unconjugated anti-MerTK, PEGylated anti-MerTK antibody triggered type I IFN response and exhibited antitumor effect in syngeneic mouse tumor studies. Our results demonstrate the potential of PEGylation to control ocular biodistribution of antibodies.

Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients., Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo., Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates., Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor., Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.

Aims: The aim of this study was to evaluate and correlate ultrasound measurement of airway parameters with the Cormack- Lehane (CL) grading observed under direct laryngoscopy for prediction of difficult airway., Methods: This prospective, observational study was conducted in a tertiary care institute. Ninety-six patients were scheduled for elective surgery under general anaesthesia and tracheal intubation. They were categorised as having easy (CL grades 1, 2a, and 2b) or difficult (CL grades 3a, 3b, and 4) laryngoscopy. The sonographically measured airway parameters included anterior neck soft tissue thickness at vocal cord level (ANS-VC), hyomental distance ratio (HMDr), and tongue volume (TV). These parameters were compared and correlated with the CL grading. The statistical analysis was done using SPSS version 21.0., Results: Difficult laryngoscopy was observed in 17.7% patients. Significant difference was noted in ANS-VC 0.28 6 0.09; 0.39 6 0.12, (P < .0001) and HMDr, 1.2 6 0.09; 1.15 6 0.13, (P ¼ .006) for easy and difficult laryngoscopy, respectively. ANS-VC had a sensitivity of 78.9% and specificity of 71.1% (AUC-0.816) followed by HMDr (AUC-0.713) and TV (AUC-0.608). Combined ultrasound parameters had significantly higher AUC value (0.867)., Conclusions: ANS-VC was the most significant parameter with a value of >0.29 cm being a sensitive predictor of difficult intubation. Combined sonographic parameters (ANS-VC, HMDr, and TV) were better predictors of difficult intubation.

Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.

Competing Interests: There are no conflicts of interest.

Background and Aims: The aim of the study was to evaluate and compare three different ultrasonographic calculation methods for tongue volume in a real time 2D ultrasonography and correlate with Modified Cormack-Lehane grading observed under direct laryngoscopy., Methods: This prospective observational study was conducted in a tertiary care institute. Tongue volume was assessed ultrasonically in 50 adult patients using three techniques in all the patients undergoing surgery under general anesthesia and correlated with Modified Cormack-Lehane grading. In METHOD A, the tongue volume was calculated as multiplication of mid sagittal cross-sectional area and width in transverse plane; METHOD B, Cross-sectional area obtained in vertical plane was multiplied with the maximum width of tongue in transverse plane; METHOD C. the volume was calculated by multiplying length, width, and height in vertical, transverse, and mid-sagittal/oblique plane, respectively. The analysis was done using Statistical Package for Social Sciences (SPSS) version 21.0. Receiver operating characteristic (ROC) curve was used to find out cutoff point of different methods for predicting difficult laryngoscopy., Results: The specificity and sensitivity of three different methods were statistically compared and area under the receiver operating characteristic (ROC) curve for method A, B, and C was 0.562, 0.502, and 0.548, respectively., Conclusion: In our study, we found all three methods to calculate tongue volume to be equally good to assess difficult laryngoscopy., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Indian Journal of Anaesthesia.)

Antibodies bearing engineered cysteine residues (termed THIOMAB™ antibodies) enable the site-selective attachment of a drug, label or other payload for specific delivery to certain tissues (e.g., tumors). This Chapter describes detailed methods we have developed and optimized for the conjugation, purification and analysis of THIOMAB™ antibody drug conjugates (TDCs).

Introduction: Constrictive pericarditis is an important cause of diastolic heart failure. The relationship between the presence and degree of pericardial calcification with constrictive pericarditis is variable, however this should be an early warning sign to initiate appropriate investigations., Presentation of Case: A 64-year-old gentleman presented with dyspnoea and dizziness on exertion. Plain posterior-anterior chest X-ray showed mild to moderate pericardial calcification. He had an episode of atrial fibrillation which resolved spontaneously and thought to be the cause of his symptoms. Patient symptoms progressed. Further investigations including CT scan confirmed extensive constrictive pericarditis. He underwent a successful percardiectomy and full recovery., Discussion: Constrictive pericarditis can be the consequence of different conditions such as tuberculosis, radiation, idiopathic or many others. Pericardial calcification is present in less than 25% of all cases of constrictive pericarditis, and patient with it are prone to develop atrial fibrillation. The presence of pericardial calcification on plain chest radiograph is an important indicator for the possibility of pericardial dysfunction. Many cases of pericardial calcification are benign without any clinical significance; however three-dimensional imaging is required to determine the calcium load and aid in reaching appropriate diagnosis., Conclusion: Chest X-ray is valuable detector of pericardial calcification, a surrogate of pericardial constriction, but it is important to remember that pericardial calcification is a three-dimensional condition, and should be further investigated by lateral chest X-ray, CT scan and then appropriate functional imaging., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR ., (©2017 American Association for Cancer Research.)

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC 50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 μg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pK a . We measured the cysteine thiol pK a using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pK a (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pK a and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pK a of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pK a . The influence of cysteine thiol pK a on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

The tubulysins are promising anticancer cytotoxic agents due to the clinical validation of their mechanism of action (microtubule inhibition) and their particular activity against multidrug-resistant tumor cells. Yet their high potency and subsequent systemic toxicity make them prime candidates for targeted therapy, particularly in the form of antibody-drug conjugates (ADCs). Here we report a strategy to prepare stable and bioreversible conjugates of tubulysins to antibodies without loss of activity. A peptide trigger along with a quaternary ammonium salt linker connection to the tertiary amine of tubulysin provided ADCs that were potent in vitro . However, we observed metabolism of a critical acetate ester of the drug in vivo , resulting in diminished conjugate activity. We were able to circumvent this metabolic liability with the judicious choice of a propyl ether replacement. This modified tubulysin ADC was stable and effective against multidrug-resistant lymphoma cell lines and tumors.

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics for the targeted delivery of highly potent cytotoxic drugs to tumor cells to improve bioactivity while minimizing side effects. ADCs are composed of both small and large molecules and therefore have complex molecular structures. In vivo biotransformations may further increase the complexity of ADCs, representing a unique challenge for bioanalytical assays. Quadrupole-time-of-flight mass spectrometry (Q-TOF MS) with electrospray ionization has been widely used for characterization of intact ADCs. However, interpretation of ADC biotransformations with small mass changes, for the intact molecule, remains a limitation due to the insufficient mass resolution and accuracy of Q-TOF MS. Here, we have investigated in vivo biotransformations of multiple site-specific THIOMAB antibody-drug conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass (HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS enabled more comprehensive identification of ADC biotransformations. It was particularly beneficial for characterizing ADC modifications with small mass changes such as partial drug loss and hydrolysis. This strategy has significantly enhanced our capability to elucidate ADC biotransformations and help understand ADC efficacy and safety in vivo.

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Disulfide bonds provide a bioactivatable connection with applications in imaging and therapy. The circulation stability and intracellular release of disulfides are problematically coupled in that increasing stability causes a corresponding decrease in cleavage and payload release. However, an antibody offers the potential for a reversible stabilization. We examined this by attaching a small molecule directly to engineered cysteines in an antibody. At certain sites this unhindered disulfide was stable in circulation yet cellular internalization and antibody catabolism generated a disulfide catabolite that was rapidly reduced. We demonstrated that this stable connection and facile release is applicable to a variety of payloads. The ability to reversibly stabilize a labile functional group with an antibody may offer a way to improve targeted probes and therapeutics.

Background: Postoperative pain is treated incompletely and ineffectively in many circumstances, and chronic postoperative pain causes suffering and diminishes productivity. The objective of this project is to determine whether a recently developed slow-release formulation of bupivacaine was effective in reducing the experimental chronic postoperative pain., Methods: In male Sprague-Dawley rats (250-300 g body weight), bupivacaine-releasing microspheres (MS-Bupi), containing 60 mg of bupivacaine base, were locally injected (MS-Bupi-L) 2 hours preoperatively into the subcutaneous compartment at the locus for experimental thoracotomy. Hypersensitivity to tactile stimulation was assessed by reductions in the threshold force required to induce a response to von Frey filaments (VFH) applied to the hairy back near the incision/retraction site. Pain behavior was assessed using a Qualitative Hyperalgesia Profile. Control groups included rats receiving the same dose of MS-Bupi but at a distant site on the back (MS-Bupi-D, testing for systemic drug actions) and rats receiving the same mass of microspheres with no drug (MS-Placebo) at the wound site. Rats were tested for 3 days before and 28 days (postoperative days [PODs]) after the procedure. Withdrawal threshold differences, which were the primary outcome measure, among all treatment groups were assessed by the Kruskal-Wallis test, after which pairwise comparisons were made by determining Wilcoxon-Mann-Whitney odds (WMWodds), with Bonferroni correction of the confidence intervals., Results: Microsphere bupivacaine released near the incision reduced the chronic tactile allodynia after thoracotomy. The threshold values during PODs 14 to 28 were different among the 3 treatment groups when examined on PODs 14, 16, 18, 23, 25, and 28 but not on POD21 (P = 0.0603). WMWodds showed that threshold of the MS-Bupi-L group differed from those of the MS-Bupi-D and the MS-Placebo groups for all the tested PODs, whereas the thresholds of the MS-Bupi-D group never differed from those of the MS-Placebo group. Area-under-curve analysis for threshold reductions below baseline, using WMWodds, also showed a reduction during the entire 28 PODs that was greater for the MS-Bupi-L group compared with the MS-Placebo or MS-Bupi-D group. The incidence of intense pain scores by the Qualitative Hyperalgesia Profile analysis was observed in 7 of 8 rats in the MS-Placebo group and in 5 of 8 rats in the MS-Bupi-L group., Conclusions: Local slow release of bupivacaine subcutaneously from the MS-Bupi formulation suppresses postoperative mechanical hypersensitivity for ≥4 weeks after experimental thoracotomy. Systemic bupivacaine from this treatment has no effect on this hypersensitivity.

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.

Background: Prolonged local anesthesia, particularly desirable to minimize acute and chronic postoperative pain, has been provided by microspheres that slowly release bupivacaine (MS-Bup). In this study, we report on the systemic drug concentrations and the local dermatopathology that occur after subcutaneous injection of MS-Bup., Methods: Rats (approximately 300 g) were injected under the dorsolumbar skin with MS-Bup containing 40 mg of bupivacaine (base) or with 0.4 mL of 0.5% bupivacaine-HCl (BupHCl; 1.78 mg bupivacaine). Blood was drawn, under sevoflurane anesthesia, at 10 minutes to 144 hours, and the serum analyzed for total bupivacaine by liquid chromatography-tandem mass spectrometry. In different animals, skin punch biopsies (4 mm) were taken at 1, 3, 7, 14, and 30 days after the same drug injections, sectioned at 5 μm, and stained with hematoxylin-eosin. Samples from skin injected with BupHCl, with MS-Bup suspended in carboxymethyl cellulose (MS-Bup.CMC), or in methyl cellulose (MS-Bup.MC) were compared with their respective drug-free controls (placebos)., Results: Serum bupivacaine reached a maximal average value (n = 8) of 194.9 ng/mL at 8 hours after injection of MS-Bup (95% upper prediction limit = 230.2 ng/mL), compared with the maximal average (n = 6) serum level of 374.9 ng/mL (95% prediction limit = 470.6 ng/mL) at 30 minutes after injection of BupHCl. Serum bupivacaine decreased to undetectable levels (<3.23 ng/mL) at 8 hours after BupHCl and was detectable at approximately 20% of the maximal value at 144 hours after MS-Bup injection. BupHCl injection resulted in moderate lymphocytic infiltration of skeletal muscle at 1 and 3 days. MS-Bup.CMC and placebo-CMC caused extensive infiltration of macrophages, lymphocytes, and some neutrophils at 1 to 7 days, whereas MS-Bup.MC and placebo-MC caused only mild inflammation., Conclusions: Subcutaneous administration of microspheres releasing bupivacaine results in lower blood levels lasting for much longer times than those from bupivacaine solution.

Background and Objectives: Postoperative pain alters physiological functions and delays discharge. Perioperative local anesthetics are effective analgesics in the immediate 1- to 2-day postoperative period, but acute pain often lasts longer. The goal of this work was to develop a local anesthetic formulation adhering to an intraoperative implanted device that reduces pain for at least 3 days after surgery., Methods: Six groups, each with 8 rats, were studied. In a control group (group I), one 1.2-cm-long incision of the skin was followed by blunt dissection to separate the skin away from the underlying tissues and closing with 2 sutures. In 3 of the treatment groups, the same surgical procedure was used, with the subcutaneous space formed by the blunt dissection lined with a 1-cm square patch of hernia mesh coated with poly lactide co-glycolic acid microspheres containing approximately 17 mg of bupivacaine (group II), no drug (placebo; group III), or bupivacaine free-base powder (group IV). Uncoated mesh implants (group V) served as a secondary control. A standard bupivacaine solution (0.4 mL, 0.5%; 2-mg dose) was infiltrated subcutaneously 30 minutes before the surgery and served as a standard control (group VI). Mechanosensitivity of the skin was tested by the local subcutaneous muscle responses to cutaneous tactile stimulation by von Frey hairs with forces of 4 g (for allodynia) and 15 g (for hyperalgesia) preoperatively and for 7 postoperative days., Results: Control rats (group I) showed mechanohypersensitivity, indicative of postoperative allodynia and hyperalgesia, for all 7 postoperative days. Mechanohyperalgesia in rats that received mesh coated with bupivacaine-releasing microspheres (group II) was reduced during this period to 13% of control postoperative values (P < 0.001); mesh coated with bupivacaine base (group IV) reduced it by 50% (P = 0.034). The placebo mesh (group III) and uncoated mesh (group V) caused no significant reduction of mechanohypersensitivity, and bupivacaine solution infiltrated before the incision (group VI) reduced hypersensitivity for only approximately 2 hours, an overall insignificant effect., Conclusions: Bupivacaine slowly released for 72 hours from microspheres adsorbed to the hernia mesh significantly suppresses evoked postoperative hypersensitivity for at least 1 week and is more effective than a suspension of these microspheres or preoperative single-shot infiltration of bupivacaine.

Background: Acute postoperative pain causes physiological deficits and slows recovery. Reduction of such pain by local anesthetics that are delivered for several days postoperatively is a desirable clinical objective, which is approached by a new formulation and applied in animal studies reported here., Methods: We subcutaneously injected a new formulation of poly-lactic-co-glycolic acid polymer microspheres, which provides steady drug release for 96+ hours into rats at the dorsal region 2 hours before surgery. A single 1.2-cm-long skin incision was followed by blunt dissection of skin away from the underlying fascia, and closed by 2 sutures, followed by 14 days of testing. Microspheres containing 5, 10, 20, and 40 mg bupivacaine were injected locally 2 hours before surgery; bupivacaine-free microspheres were the vehicle control, and bupivacaine HCl solution (0.5%), the positive control. Mechanical sensitivity was determined by the frequency of local muscle contractions to repeated pokes with nylon monofilaments (von Frey hairs) exerting 4 and 15 g forces, testing, respectively, allodynia and hyperalgesia, and by pinprick., Results: Injection of bupivacaine microspheres (40 mg drug) into intact skin reduced responses to 15 g von Frey hairs for 6 hours and to pinprick for 36 hours. Respective reductions from bupivacaine HCl lasted for 3 and 2 hours. Skin incision and dissection alone caused mechanical allodynia and hyperalgesia for 14 days. Microspheres containing 20 or 40 mg bupivacaine suppressed postoperative hypersensitivity for up to 3 days, reduced integrated allodynia (area under curve of response versus time) over postoperative days 1 to 5 by 51% ± 20% (mean ± SE) and 78% ± 12%, and reduced integrated hyperalgesia by 55% ± 13% and 64% ± 11%, for the respective doses. Five and ten milligrams bupivacaine in microspheres and the 0.5% bupivacaine solution were ineffective in reducing postoperative hypersensitivity, as were 40 mg bupivacaine microspheres injected contralateral to the incision., Conclusions: Significant suppression of postoperative pain by the slow-release bupivacaine preparation outlasts its anesthetic action on intact skin. These findings demonstrate preventive analgesia and indicate the importance of acute processes in the development of chronic postoperative pain.

Antibody-drug conjugates (ADCs) are target-specific anticancer agents consisting of cytotoxic drugs covalently linked to a monoclonal antibody. The number of ADCs in the clinic is growing, and therefore thorough characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance. Cytotoxic drugs such as the tubulin polymerization inhibiting auristatin, monomethyl auristatin E, have been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through internal cysteines. This results in a heterogeneous mixture of antibody species with drug-to-antibody ratios (DAR) ranging from 0 to 8. In order to characterize the assays used to quantitate total MC-vc-PAB-MMAE ADCs (conjugated and unconjugated antibody), we used purified fractions with defined DARs from 6 therapeutic antibodies to evaluate different assay formats and reagents. Our investigations revealed that for quantitation of total antibody, including all unconjugated and conjugated antibody species, sandwich ELISA formats did not always allow for recovery of all purified DAR fractions (DAR 0-8) to within ±20% of the expected values at the reagent concentrations tested. In evaluating alternative approaches, we found that the recovery of DAR fractions with semihomogeneous assay (SHA) formats, in which sample, capture, and detection reagents are preincubated in solution, were less affected by the antibody's MMAE drug load as compared to traditional stepwise sandwich ELISAs. Thus, choosing the optimal assay format and reagents for total antibody assays is valuable for developing accurate quantitative assays.

Background and Objectives: To minimize acute postoperative pain, a new formulation of slowly released bupivacaine was developed., Methods: Bupivacaine was microencapsulated at 60% (wt/wt) in poly-lactide-co-glycolide polymers and characterized for physicochemical properties and bupivacaine release kinetics. This formulation was injected around the rat sciatic nerve to produce an antinociceptive effect to toe pinch. Mechanical hyperalgesia following lateral plantar paw incision in rats was assessed for 7 to 14 days when the bupivacaine slow-release formulation was placed at the ipsilateral sciatic nerve and compared with the hyperalgesia that developed with various controls., Results: Bupivacaine was released in vitro at a relatively constant rate over a period of ≈ 72 to 96 hours. Complete antinociception, shown as no response to toe pinch, lasted for 23 ± 7 hours, with a half-recovery time of 42 ± 8 hours after sciatic nerve injection of 0.4 mL of the microspheres delivering 34 mg of bupivacaine. Solutions of 0.5% (wt/vol) bupivacaine-HCl (0.1 mL) produced complete antinociception for less than 2 hours and recovery half-times of 2 hours. Postincisional mechanical hyperalgesia, shown by increased withdrawal responses to von Frey filaments, was absent for 24 hours and was lower than control for 96 hours, when the sciatic nerve was blocked by bupivacaine microspheres, whereas the 0.5% bupivacaine solution reduced postincisional pain for only 4 hours., Conclusions: Corresponding to its far greater functional blocking time, the microsphere-bupivacaine formulation was able to significantly reduce postoperative pain below control levels for up to 4 days. These findings of several days of postoperative pain relief, for an injectable formulation containing a single active agent, present an improved and potentially promising therapy to prevent acute pain after surgery.

Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.

A series of 3-alkoxy-4-methanesulfonamido acetophenone derivatives were synthesized and evaluated for their anti-inflammatory activity in carrageenan-induced rat paw edema model. The synthesized compounds were also investigated for their gastric ulcerogenic potential. The compounds 4a, 4c and 4d showed comparable anti-inflammatory activity to rofecoxib and indomethacin, the standard drugs taken in both studies and were also non ulcerogenic at the test doses. In silico (docking studies) were done to investigate the hypothetical binding mode of the target compounds to the cyclooxygenase isoenzyme (COX-2). A binding model has been proposed based on the docking studies. Selected physicochemical properties were calculated for theoretical ADME profiling of the compounds and excellent compliance was shown with Lipinski's rules., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Ectopic calcification is a major cause of bioprosthetic heart valve failure. New therapeutic opportunities are offered by the growing understanding that ectopic calcification is an actively regulated process involving several key gene products. One of these products, osteopontin (OPN), is a glycosylated phosphoprotein previously shown to inhibit apatite crystal formation, induce carbonic anhydrase II, and promote mineral resorption. In this study, OPN-deficient mice (OPN-/-) were utilized as an in vivo model to stimulate the ectopic calcification of glutaraldehyde-fixed bovine pericardium (GFBP) tissue and to examine OPN delivery and structure-function relationships with respect to its anti-calcific activity. Significant calcification of GFBP tissue was obtained within 7 days of subcutaneous implantation in OPN-/- mice. Direct rescue of the calcification phenotype was achieved by the administration of exogenous recombinant rat, histidine-fused OPN (rat His-OPN) to the implant site via soluble injection (up to 72% mitigation achieved) or adsorption onto the implant materials (up to 91% mitigation achieved). Effects were specific, since neither fibronectin nor polyhistidine alone could mitigate calcification of GFBP. The maximum anti-calcific effect was achieved only when rat His-OPN was adequately phosphorylated and contained a functional arginine-glycine-aspartate (RGD) cell adhesive domain. Furthermore, CAII levels in host cells surrounding GFBP were greatest when phosphorylated, RGD-containing rat His-OPN was adsorbed. These data suggest that both physical inhibition, mediated by phosphorylation sites in OPN, as well as the induction of CAII and mineral regression, mediated by the RGD domain, contribute to the unique ability of OPN to mitigate ectopic calcification of bioprosthetic valve tissue.

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.

Pathologic calcification is the leading cause of the clinical failure of glutaraldehyde-fixed bovine pericardium used in bioprosthetic valves. A novel surface modification of glutaraldehyde fixed bovine pericardium was carried out with high molecular weight hyaluronic acid (HA). HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional groups onto the HA backbone. Glutaraldehyde-fixed bovine pericardium (GFBP) was modified by grafting this HA to the free aldehyde groups on the tissue via the hydrazide groups. Following a 2-week subcutaneous implantation in osteopontin (OPN)-null mice, the calcification of HA-modified bovine pericardium was drastically reduced (by 84.5%) compared to positive controls (tissue without HA-modification) (p = 0.005). The calcification-mitigating effect of HA surface modification was also confirmed by microscopic analysis of explanted tissue stained with Alizarin Red S for calcium., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 328-334, 2004)

The finding of retinal or vitreous hemorrhage in a child under age 3 years may cause significant controversy with regard to the etiology, because it raises the suspicion of nonaccidental injury. Blood dyscrasias have been documented to cause retinal and vitreous hemorrhages in adults and children, but they have rarely been reported to be the cause of retinal hemorrhages in neonates. We report on a patient with a low plasma fibrinogen level who had bilateral retinal hemorrhage that proceeded to vitreous hemorrhage. This subtle abnormality of the blood clotting cascade caused significant retinal and vitreous hemorrhage in a child without risk factors for abuse.