Polyoxazoline polymers with methyl (PMOZ), ethyl (PEOZ), and propyl (PPOZ) side chains were prepared by the living cationic polymerization method and purified by ion-exchange chromatography. The ...following properties of polyoxazoline (POZ) were measured: apparent hydrodynamic radius by aqueous size-exclusion chromatography, relative lipophilicity by reverse-phase chromatography, and viscosity by cone–plate viscometry. The PEOZ polymers of different molecular weights were first functionalized and then conjugated to model biomolecules such as bovine serum albumin, catalase, ribonuclease, uricase, and insulin. The conjugates of catalase, uricase, and ribonuclease were tested for in vitro activity using substrate-specific reaction methods. The conjugates of insulin were tested for glucose lowering activity by injection to naïve Sprague–Dawley rats. The conjugates of BSA were injected into New Zealand white rabbits and serum samples were collected periodically and tested for antibodies to BSA. The safety of POZ was also determined by acute and chronic dosing to rats. The results showed that linear polymers of POZ with molecular weights of 1 to 40 kDa can easily be made with polydispersity values below 1.10. Chromatography results showed that PMOZ and PEOZ have a hydrodynamic volume slightly lower than PEG; PEOZ is more lipophilic than PMOZ and PEG; and PEOZ is significantly less viscous than PEG especially at the higher molecular weights. When PEOZ was attached to the enzymes catalase, ribonuclease, and uricase, the in vitro activity of the resultant bioconjugates depended on the extent of protein modification. POZ conjugates of insulin lowered blood glucose levels for a period of 8 h when compared to 2 h for insulin alone. PEOZ, like PEG, was also able to successfully attenuate the immunogenic properties of BSA. The POZ polymers (10 and 20 kDa) are safe when administered intravenously to rats, and the maximum tolerated dose (MTD) was greater than 2 g/kg. Blood counts, serum chemistry, organ weights, and the histopathology of key organs were normal. These results conclude that POZ has the desired drug delivery properties for a new biopolymer.
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The potential of poly (2-oxazoline) or POZ to be a versatile and broad based platform for drug delivery with wide utility in multiple therapeutic areas has long been recognized by ...experts in the field. This feature article provides a case study which describes the chemistry and preclinical studies underlying the Investigational New Drug Application for SER-214, a POZ conjugate of rotigotine, for the treatment of Parkinson’s disease. We report the chemistry, preclinical safety and pharmacology, and the early clinical safety, tolerability and pharmacokinetic data from the Phase I study in patients. SER-214 utilizes a POZ polymer and proprietary custom linker technology to deliver a sustained dose of rotigotine over a period of seven days following a single subcutaneous administration – a result not observed by any other polymer approach that we are aware of. As such, this candidate drug has the promise to be a major advancement in the treatment of Parkinson’s disease. Furthermore, this feature article also highlights the versatility of the POZ polymer platform (POZ™) to deliver cancer drugs by actively targeting cancer cells. Preclinical data reported in this feature showcase the polymer’s attributes in facilitating targeted delivery with folic acid and antibody targeting agents (ADCs). The ability of POZ to reliably delivery large payloads of anticancer drugs is of particular importance when pursuing low-receptor-density targets on the cancer cell. The data presented in this feature, much of it for the first time, establish the broad utility of the POZ polymer platform in drug development. Together with its ease of manufacture, ability to attach drugs to the polymer, and ability to administer an appropriate dose to patients, the results underscore the need to further explore and expand the untapped potential of POZ for the development of new therapeutics for unmet medical needs.
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe ...cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2), we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, as well as after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally oriented regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and the possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates and broadly support the investigation of agents with adequate penetration into relevant regions of the CNS.
To examine the anatomy of Schlemm's canal (SC) and collector channels (CCs) in normal human and primary open-angle glaucoma (POAG) eyes under low and high perfusion pressure.
In normal (n = 3) and ...POAG (n = 3) eye pairs, one eye was perfused at 10 mm Hg while the fellow eye was perfused at 20 mm Hg for 2 hours. Eyes were perfusion fixed at like pressures, dissected into quadrants, embedded in Epon Araldite, and scanned by three-dimensional micro-computed tomography (3D micro-CT). Schlemm's canal volume, CC orifice area, diameter, and number were measured using ANALYZE software.
Normal eyes showed a larger SC volume (3.3-fold) and CC orifice area (9962.8 vs. 8825.2 μm(2)) and a similar CC diameter (34.3 ± 17.8 vs. 32.7 ± 13.0 μm) at 10 mm Hg compared to 20 mm Hg. In POAG eyes, SC volume (2.0-fold), CC orifice area (8049.2 μm(2)-6468.4 μm(2)), and CC diameter (36.2 ± 19.1 vs. 29.0 ± 13.8 μm) were increased in 10 mm Hg compared to 20 mm Hg perfusion pressures. Partial and total CC occlusions were present in normal and POAG eyes, with a 3.7-fold increase in total occlusions in POAG eyes compared to normal eyes at 20 mm Hg. Visualization of CCs increased by 24% in normal and by 21% in POAG eyes at 20 mm Hg compared to 10 mm Hg. Schlemm's canal volume, CC area, and CC diameter were decreased in POAG eyes compared to normal eyes at like pressures.
Compensatory mechanisms for transient and short periods of increased pressure appear to be diminished in POAG eyes. Variable response to pressure change in SC and CCs may be a contributing factor to outflow facility change in POAG eyes.
Claudin-low breast cancer is an aggressive subtype that confers poor prognosis and is found largely within the clinical triple-negative group of breast cancer patients. Here, we have shown that ...intrinsic and immune cell gene signatures distinguish the claudin-low subtype clinically as well as in mouse models of other breast cancer subtypes. Despite adaptive immune cell infiltration in claudin-low tumors, treatment with immune checkpoint inhibitory antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1) were ineffective in controlling tumor growth. CD4+FoxP3+ Tregs represented a large proportion of the tumor-infiltrating lymphocytes (TILs) in claudin-low tumors, and Tregs isolated from tumor-bearing mice were able to suppress effector T cell responses. Tregs in the tumor microenvironment highly expressed PD-1 and were recruited partly through tumor generation of the chemokine CXCL12. Antitumor efficacy required stringent Treg depletion combined with checkpoint inhibition; delays in tumor growth were not observed using therapies that modestly diminished the number of Tregs in the tumor microenvironment. This study provides evidence that the recruitment of Tregs to the tumor microenvironment inhibits an effective antitumor immune response and highlights early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint blockade antibodies in specific subtypes of cancer that are heavily infiltrated with adaptive immune cells.
The site of outflow resistance leading to elevated intraocular pressure in primary open-angle glaucoma is believed to be located in the region of Schlemm’s canal inner wall endothelium, its basement ...membrane and the adjacent juxtacanalicular tissue. Evidence also suggests collector channels and intrascleral vessels may have a role in intraocular pressure in both normal and glaucoma eyes. Traditional imaging modalities limit the ability to view both proximal and distal portions of the trabecular outflow pathway as a single unit. In this study, we examined the effectiveness of three-dimensional micro-computed tomography (3D micro-CT) as a potential method to view the trabecular outflow pathway. Two normal human eyes were used: one immersion fixed in 4% paraformaldehyde and one with anterior chamber perfusion at 10 mmHg followed by perfusion fixation in 4% paraformaldehyde/2% glutaraldehyde. Both eyes were postfixed in 1% osmium tetroxide and scanned with 3D micro-CT at 2 μm or 5 μm voxel resolution. In the immersion fixed eye, 24 collector channels were identified with an average orifice size of 27.5 ± 5 μm. In comparison, the perfusion fixed eye had 29 collector channels with a mean orifice size of 40.5 ± 13 μm. Collector channels were not evenly dispersed around the circumference of the eye. There was no significant difference in the length of Schlemm’s canal in the immersed versus the perfused eye (33.2 versus 35.1 mm). Structures, locations and size measurements identified by 3D micro-CT were confirmed by correlative light microscopy. These findings confirm 3D micro-CT can be used effectively for the non-invasive examination of the trabecular meshwork, Schlemm’s canal, collector channels and intrascleral vasculature that comprise the distal outflow pathway. This imaging modality will be useful for non-invasive study of the role of the trabecular outflow pathway as a whole unit.
► 3D micro-CT is an effective, non-invasive imaging method to study the trabecular outflow pathway. ► 3D micro-CT enables outflow pathways to be studied in the context of the whole eye. ► 3D micro-CT enables identification of collector channel number, size, and location around the eye. ► 3D micro-CT will enable study of regional differences in normal and diseased eyes.
To examine the anatomical variation of normal human collector channel orifices and their relationship with Schlemm's canal.
Ten human anterior segments fixed by immersion or perfusion were dissected ...radially and further divided by fine dissection into corresponding inner and outer wall segments. The tissues were dehydrated, critical-point dried, sputter coated, and examined by scanning electron microscopy. Images were obtained at magnifications from ×200 to ×10,000. Selected radial collector channel regions were processed for plastic embedding.
Two classes of collector channel orifices were identified. Simple oval orifices (54.7 ± 4.6-μm diameter) were lined with endothelial cells and most often occurred on a planar region of Schlemm's canal outer wall. Complex orifices (62.7 ± 3.4-μm diameter) were often found associated with septal columns and bridges, and typically covered with flap-like structures (10-40 μm) that extended between the inner and outer wall and over the collector channel orifices. Both simple and complex orifices had complete or partial lip-like rims. In orifices with partial rims, a trough-like groove was often visible on the outer wall surface opposite the lip. Transected septa and inner and outer wall adhesion sites were often found in association with complex collector channel orifices.
Collector channel orifice structure varied from simple ovals to complex tethered flaps and bridges. Collector channel orifices with complex flaps connect the inner and outer walls of Schlemm's canal, and may serve to enhance and regulate aqueous outflow in these regions.
The distal outflow pathway of the human eye consists of the outer wall of Schlemm's canal, collector channels, and the deep-scleral, mid-scleral and episcleral vessels. It is the last region of ...transit for aqueous humor before returning to the venous system. While the trabecular meshwork, scleral spur, and inner wall of Schlemm's canal have been extensively analyzed to define their contributions to aqueous outflow, the role of the distal outflow pathway is not completely understood. Collector channels, emanating from Schlemm's canal were previously thought to be passive conduits for aqueous humor. However, recent studies have shown many collector channels contain flap-like appendages which move with changes in pressure. These findings, along with studies demonstrating innervation of episcleral vessels, have led to questions regarding whether other structures in the distal outflow pathway are under neural regulation and how this may influence aqueous humor outflow. This study evaluates the innervation of the outer wall of Schlemm's canal and collector channels, along with the deep-scleral, mid-scleral and episcleral vasculature with microcomputed tomography and 3-dimensional reconstruction, correlative light microscopy, immunohistochemistry, and transmission electron microscopy. Peripheral, autonomic, and sensory nerve fibers were found to be present adjacent to Schlemm's canal outer wall endothelium, collector channel endothelium, and in the different regions of the distal outflow vasculature. Nerves were more commonly identified in regions that contained collector channels when compared to regions without collector channels. These findings regarding the neural anatomy suggest an active neural regulation of aqueous humor outflow throughout the proximal and distal regions of the conventional outflow pathway.
•Peripheral, autonomic and sensory nerve were found throughout the distal outflow pathway in the human eye.•Nerve is more prominent near collector channels and their innervation is associated with smooth muscle and endothelial cells.•Nerve on the inner and outer wall of Schlemm's canal suggest a possible pathway of communication to regulate aqueous outflow.
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Poly(2-oxazoline)-drug conjugates with drugs attached via releasable linkages are being developed for drug delivery. Such conjugates with pendent ester linkages that covalently bind ...drugs to the polymer backbone exhibit significantly slower hydrolytic release rates in plasma than the corresponding PEG- and dextran-drug conjugates. The slow drug release rates in-vitro of these POZ-drug conjugates contribute to extended in-vivo pharmacokinetic profiles. In some instances, the release kinetics may be relatively sustained and ideal for once-a-week subcutaneous injection, whereas the native drug by itself may only have an in-vivo half-life of a few hours. The origin of this unusual kinetic and pharmacokinetic behavior is proposed here to involve folding of the POZ conjugate such that the relatively hydrophobic drug forms a central core, and the relatively hydrophilic polymer wraps around the core and slows enzymatic attack on the drug-polymer chemical linkage. Here we present evidence supporting this hypothesis and demonstrate how the hypothesis can be used to tune hydrolytic release rates and pharmacokinetics. Evidence for the folding hypothesis is taken from hydrolysis kinetics of a range of drugs in plasma, pharmacokinetics of a range of drugs following subcutaneous injection in laboratory animals, and nuclear magnetic resonance (NMR) studies showing folding of the POZ-rotigotine molecule. The drugs included in this study to test the hypothesis are: rotigotine, buprenorphine, dexanabinol, cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC) and cannabigerol (CBG).
Alternative payment models have been proposed to deliver high-quality, cost-effective care. Under these models, payments may be shared between the hospital and the post-acute care services. ...Post-acute care services may account for one-third of the episode costs for total hip or knee arthroplasty (THA/TKA). Because hospitals or episode initiators bear notable financial risks in these payment models with minimal risk adjustment for complexity, it has been suggested these models may lead to prospective selection of healthier and younger patients. Studies evaluating the effect of patient demographics, medical complexity, and surgical characteristics on the cost of index hospitalization have been limited. We aimed to (1) quantify the impact of patient demographics, medical complexity, and surgical characteristics (type of anesthesia and operating time) on variation in direct cost of index hospitalization and (2) examine the association of these characteristics with discharge with home health services or to rehabilitation facility.
Retrospective study of 3,542 patients admitted to our hospital for elective THA/TKA between 2012 and 2017. Multivariable generalized estimating equations were used for analysis.
Patient demographics and medical complexity accounted for 6.2% (THA) and 5.6% (TKA) of variation in direct cost of index hospitalization. Surgical characteristics accounted for 37.1% (THA) and 35.3% (TKA) of the cost variation. One thousand one hundred eighty-three (53.4%) patients were discharged with home health services, and 1,237 (29.4%) were discharged to rehabilitation facility. Patient demographics and higher medical complexity were markedly associated with discharge with home health services or to rehabilitation facility after THA/TKA.
Patient demographics and medical complexity had minimal impact on variation in direct cost of index hospitalization for elective THA/TKA compared with surgical characteristics but were markedly associated with discharge with home health services or to rehabilitation facility. Having additional risk adjustment in these payment models could mitigate concerns about access to care for higher risk, higher cost patients.
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