Low volume resuscitants and methods of making and using the same

Abstract

Method of treating and / or preventing hemorrhagic shock, comprising administering to a subject in need thereof a therapeutically effective amount of a water-soluble polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to the subject thereby functioning as a plasma volume expander.

Description

[0001] LOW VOLUME RESUSCITANTS AND METHODS OF MAKING AND USING THE SAME

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Patent Application No. 63 / 529640, filed July 28, 2023, expressly incorporated herein by reference in its entirety

[0004] STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0005] This invention was made with government support under Grant No. 1R01 HL139007 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Traumatic injuries account for a substantial portion of global morbidity and mortality and is the leading cause of death for persons aged 1-46 in the United States. Among trauma-related fatalities, hemorrhagic shock from blood loss causes up to 40% of civilian deaths. Alarmingly, over half of these hemorrhagic deaths occur within the first minutes to hours after injury, often before patients can reach a hospital. This critical timeframe underscores the urgent need for innovative strategies and therapies that can effectively address hemorrhage and achieve damage control resuscitation, even in the prehospital environment. The extensive blood loss from hemorrhage leads to hemorrhagic shock, a pathophysiologic state in which blood pressure collapses and the hypovolemic circulatory system is unable to adequately perfuse tissues or deliver oxygen. The resulting hypoxia forces tissues into anaerobic respiration, thereby inducing lactic acidosis and the shutdown of cellular sodium pumps. The corresponding influx of sodium ions into cells is accompanied by a large volume of water that flows from the intravascular space and into the interstitium, exacerbating the hypovolemia and hemorrhagic shock. Furthermore, the immune response that follows a traumatic injury generates high levels of inflammatory cytokines and reactive oxygen species. This dysfunctional inflammatory response can cause severe secondary injury by ischemia reperfusion, which can lead to multiple organ failure and death.

[0008] The goal of fluid resuscitation is to restore oxygen delivery to adequate levels to prevent progressive organ damage. However, aggressive fluid resuscitation in the setting of freely-bleeding wounds can disrupt clots and cause rebleeding events that worsen shock. Damage control resuscitation (DCR) is an approach that has been developed to maintain basal perfusion of vital organs without pushing blood pressures high enough to cause rebleeding. In past reports, we detail the development of a polymeric hemostat, PolySTAT, to address the threat of rebleeding during treatment of hemorrhagic shock.

[0009] The current standard of care resuscitants, crystalloid and colloid solutions, operate on the principle of aggressive fluid replacement. These approaches require several liters of treatment that must be warmed to physiological temperature before use, rendering these large volume resuscitants impractical in prehospital settings. The large volumes administered to trauma patients dilute coagulation factors, spike blood pressure, and encourage tissue edema. In austere prehospital environments, such as air evacuations and military settings, it is particularly advantageous for resuscitation fluids to be lower-volume and lighter-weight. Modem resuscitation strategies in accordance with DCR intentionally limit fluid administration and instead rely upon whole blood transfusions, thereby reducing coagulopathy, inflammation, and mortality. However, logistical challenges prevent regular use of blood products in the prehospital setting, particularly as they are of extremely limited supply.

[0010] Alternative approaches in development include synthetic low volume resuscitants (LVRs) having unique and favorable properties. Polyethylene glycol of molecular weight 20kDa (PEG20K) is currently a lead candidate for LVR and operates as a hybrid cell impermeant and oncotic agent. The cell impermeant component of PEG20K allows it to partially extravasate from the vasculature and pull fluid directly out of interstitial cells, while the oncotic component remains in the bloodstream to further draw fluid into the vascular space. These polymers achieve resuscitation by generating oncotic pressure gradients, which relocate fluid from the interstitium into the patient’s vasculature, essentially using the patient’s own interstitial fluid reserve for resuscitation. PEG20K has demonstrated a potency 12-18x greater than crystalloid / colloid solutions, shrinking the volume of therapy needed for resuscitation and comprising a therapy that is exponentially more portable and conducive to prehospital applications.

[0011] Despite promising preclinical results in animal models, PEG20K impairs coagulation, inducing a state of platelet passivation similar to thrombocytopenia. Other concerns arise from the increased prevalence of PEG-induced anaphylaxis and other allergic reactions, which rose correspondingly with PEG-containing CO VID- 19 vaccines. A need exists for improved resuscitants that overcome the disadvantages of associated from present resuscitants and possess advantageous resuscitative profiles, coagulopathic effects, enhanced circulation time, all without disrupting coagulation. The present disclosure seeks to fulfill this need and provides further related advantages.

[0012] SUMMARY

[0013] In one aspect, the disclosure provides, a method of treating and / or preventing hemorrhagic shock, comprising administering to a subject in need thereof a therapeutically effective amount of a water-soluble polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to the subject thereby functioning as a plasma volume expander to treat and / or prevent hemorrhagic shock.

[0014] In another aspect, the disclosure provides a composition comprising two or more different water-soluble synthetic polymers, each having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to a subject thereby functioning as a plasma volume expander, wherein the two or more different water- soluble synthetic polymers are selected from the group consisting of linear polymers, radiant star polymers, and comb polymers.

[0015] DESCRIPTION OF THE DRAWINGS

[0016] The foregoing aspects and many of the attendant advantages of this disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

[0017] FIGS. 1A-1J present characterization of linear LVRs synthesized via RAFT. Molecular structure of L-GmMA (FIG. 1A), L-MPC (FIG. IB), and L-SBMA (FIG. 1C), respectively. Gel Permeation Chromatograms of DP100, 200, and 300 L-GmMA, L-MPC, and L-SBMA LVRs (FIGS. ID- IF), respectively. Each trace is overlaid with PEG20K as a comparison. The Mn and PDI were determined using 100% mass recovery (RI Detector, Wyatt Optilab T-rex) and multiangle light scattering (MALS, Wyatt miniDAWN Treos) gel permeation chromatography (GPC). The running solvent was phosphate buffered saline (PBS) (flow rate: 0.5 mL / min) at room temperature and samples were prepared at 10 mg / mL. Small-angle x-ray scattering (SAXS) radius of gyration measurements as a function of degree of polymerization (FIG. 1G). The dotted line with ± 1 SEM interval band represents the size range of PEG20K measured in literature. The DP200 L-GmMA (MW = 32 kDa), L-MPC (MW = 59 kDa), and L-SBMA (MW = 56 kDa) all have a similar Rgcompared to PEG20K (MW = 20 kDa). All polymers were evaluated at 5 mg / mL and 15 mg / mL in PBS (pH 7.4). Data shown are from monodisperse gaussian coil model fits from scattering data for 15 mg / mL. Fit data confirmed that there is no effect due to concentration between the 5 mg / mL and 15 mg / mL samples. Vapor pressure osmometry measurements of PEG20K and 35K (left), L-GmMA (middle left), L-MPC (middle right), and L-SBMA (right). All polymers were dissolved in normal saline (FIG. 1H), osmolarity of the normal saline for reference. All measurements were done using a Vapro 5600 in triplicate. Error bars are 1 standard deviation from the mean. Vapor pressure osmometry measurements of L-SBMA with no self-assembly (No SA) (FIG. II). All polymers were dissolved in normal saline with additional NaCl to match the number of moles of repeat units, the osmolarity of the normal saline only. All measurements were done using a Vapro 5600 in triplicate. Error bars are 1 standard deviation from the mean. Differential scanning calorimetry measurements of the hydration number (mi) for linear L-GmMA (left), and L- SBMA with (middle) and without (right) self-assembly (No SA) (FIG. 1J), ± 1 SEM interval band represents the nu of PEG20K controls.

[0018] FIGS. 2A-2F compare in vitro (FIGS. 2A-2C) and in vivo (FIGS. 2D-2F) screening of linear LVRs. General schematic of the ROTEM parameters (FIG. 2A). Alpha angle is a measure of clot formation kinetics, while MCF is a measure of the firmness of the clot. Change in alpha angle measured in degrees for human plasma treated with 10% of total blood volume (TBV) of pGmMA, pMPC, and pSBMA (FIG. 2B). Change in MCF measured in mm for human plasma treated with 10% of TBV of pGmMA, pMPC, and pSBMA (FIG. 2C). For all EXTEMs, the data displayed is the difference from a normal saline volume control run at the same time for each donor, ± 1 SEM (standard error of measurement) band is placed at zero to represent the normal saline control. A fit model for a repeated measure, one-way ANOVA with Tukey post-hoc analysis (a = 0.050) with donor as a random source effect was used. All error bars and bands represent ± 1 SEM from the mean. For all the ROTEM data, significance labels were used to indicate if there is a statistically significant difference when comparing the polymers to the normal saline volume control. The following significance labels were used: ns (P > 0.05), * (P < 0.05), ** (P < 0.01), *** (P < 0.001), **** (P < 0.0001). Overview of the severe hemorrhagic shock model in rats (FIG. 2D). Screening study to observe the ability of DP200 and DP300 L-GmMA to rescue and maintain blood pressure in the target MAP range (FIG. 2E). Comparison of the mean arterial pressure (MAP) in mmHg during the infusion and observation time of the study for rats treated with pGmMA, DP200 (20% w / v, n=l, triangles) and DP300 (30% w / v, n=2, circles). Screening study to observe the ability of DP100, DP200, and DP300 L-SBMA to rescue and maintain blood pressure in the target MAP range (FIG. 2F). Comparison of the MAP in mmHg during the infusion and observation time of the study for rats treated with pSBMA of DP100 (10% w / v, n=l, light orange rectangles), DP200 (20% w / v, n=l, triangles) and DP300 (30% w / v, n=l, circles). All rats were given an estimated 10% TB V dose of LVR therapy. PEG20K (10% w / v, n=5, green diamonds) was used as a positive control. The target range for MAP in damage control resuscitation of about 60-80 mmHg is shown. All error bars and bands represent ± 1 SEM from the mean. Due to the low sample size, no statistics were run on the data.

[0019] FIGS. 3A-3I illustrate the synthesis and characterization of radiant star LVRs synthesized via RAFT polymerization. Reaction scheme for copolymerization of either GmMA or SBMA with HEMA-ECT (hECT) to create macro-chain transfer agents (macroCTAs) for the radiant star cores (FIG. 3A). This is followed by chain extension with either GmMA or SBMA to create radiant star LVRs (RS-GmMA and RS-SBMA, respectively). GPC showing successful chain extension of the GmMA macroCTA core to prepare DP400 RS-GmMA (FIG. 3B). GPC trace of DP400 RS-GmMA radiant star (FIG. 3C). GPC showing successful chain extension of SBMA macroCTA core to prepare DP400 RS-SBMA. GPC traces of DP100, 200, and 400 RS-SBMA (FIG. 3D), respectively. For all GPCs PEG20K is overlaid for comparison. The molecular weight (Mn) and polydispersity index (PDI) were determined using 100% mass recovery (RI Detector, Wyatt Optilab T-rex) and multiangle light scattering (MALS, Wyatt miniDAWN Treos) gel permeation chromatography (GPC). The running solvent for both RS-GmMA and its macroCTA was DMF with Ig / L of LiBr flow rate: 0.8 mL / min) at 60°C with samples prepared at 10 mg / mL. The running solvent for the RS-SBMA and their macroCTA was phosphate buffered saline (PBS) (flow rate: 0.5 mL / min) at room temperature with samples prepared at 10 mg / mL (FIG. 3E). Small-angle x-ray scattering (SAXS) radius of gyration (Rg) measurements as a function of DP, with ± 1 SEM interval band represents the size range of PEG20K measured in literature (FIG. 3F). DP400 RS- GmMA (triangle, MW = 70 kDa), DP400 RS-SBMA (circles, MW = 118 kDa), all have larger Rgcompared to PEG20K (MW = 20 kDa). All polymers were evaluated at 5 mg / mL and 15 mg / mL in PBS (pH 7.4). Data shown are from fits to star polymer models from scattering data for 15 mg / mL. Fit data confirmed that there is no effect due to concentration between the 5 mg / mL and 15 mg / mL samples. Vapor pressure osmometry measurements of pGmMA (middle) and pSBMA (right). Linear LVRs (circles) are shown for reference to radiant stars (asterisks) (FIG. 3G). All polymers were dissolved in normal saline, (± 1 standard deviation) shows the osmolarity of the normal saline for reference. All measurements were done using a Vapro 5600 in triplicate. Error bars are ±1 standard deviation from the mean. Vapor pressure osmometry measurements of DP400 RS-SBMA (asterisks) compared to DP300 L-SBMA (circles) with no self-assembly (No SA) (FIG. 3H). AU polymers were dissolved in normal saline with additional NaCl added to match the number of moles of repeat units, the osmolarity of the normal saline for reference. All measurements were done using a Vapro 5600 in triplicate. Error bars are 1 standard deviation from the mean. DSC measurements of the nu for L-GmMA (triangle / left) compared to RS-GmMA (asterisk / 2nd left), and L-SBMA (2nd right) compared to RS- SBMA (asterisk / right) without self-assembly (No SA) (FIG. 31), with ± 1 SEM interval band represents the nu of PEG20K controls. All polymers were dissolved in normal saline. To prevent self-assembly, additional NaCl was added to match the number of moles of repeat units.

[0020] FIGS. 4A-4D compare the characterization of radiant star LVRs synthesized via RAFT polymerization and in vitro characterization of the effect of radiant star LVRs on coagulation in comparison to PEG20K and Hextend. For all clotting assays, Hextend (clinical standard colloid control), RS-GmMA (20% w / v), RS-SBMA (20% w / v), PEG20K (10% w / v), and normal saline were all evaluated at 10% TBV. All treatments were dissolved in veterinarian-grade 0.9% normal saline. General schematic of key ROTEM parameters (FIG. 4A). Clotting time (CT) is the time it takes for clots to begin forming, Alpha Angle is a measure of clot formation kinetics, while A10 and Maximum Clot Firmness (MCF) are measures of clot firmness at ten minutes and the maximum overall during the clotting time period (60 minutes), respectively. ROTEM evaluation of final LVRs in human whole blood (n = 5-7) (FIG. 4B): change in clotting time measured in seconds; change in alpha angle measured in degrees; change in clot firmness time measured in seconds; change in maximum clot firmness (MCF) measured in mm. A fit model for a repeated measure, one-way ANOVA with Tukey post-hoc analysis (a = 0.050) with donor as a random source effect was used. ROTEM evaluation of final LVRs in human plasma (n = 7) (FIG. 4C): change in clotting time measured in seconds; change in alpha angle measured in degrees; change in A10 measured in mm; change in MCF measured in mm. For both whole blood and plasma, the data displayed is the difference from a normal saline volume control run at the same time for each donor. Dotted line with the 95% confidence interval band is placed at zero to represent the normal saline control. A fit model for a repeated measure, one-way ANOVA with Tukey post-hoc analysis (a = 0.050) with donor as a random source effect was used. All diamonds and bands represent the 95% confidence interval. Overview of thrombin generation assay (TGA) metrics measured in human plasma (FIG. 4D). Lag time (tLag) is the time it takes for thrombin to begin being generated, Velocity Index (VI) is the rate of thrombin generation, Time to Peak (tPeak) is how long it takes to reach the maximum thrombin concentration, Peak Height (Peak) is the maximum concentration of thrombin measured, and Area Under the Curve (AUC) is the total amount of thrombin generated during coagulation; n =5 donors were evaluated: change in the area under the TGA curve measured in nM; change in tLag measured in minutes; change in VI or the slope of the initiation phase measured in nM / min; change in tPeak measured in minutes; change in the peak thrombin concentration measured in nM. The data displayed is the difference from a normal saline volume control run at the same time for each donor. A dotted line with the 95% confidence interval band is placed at zero to represent the normal saline control. A fit model for a repeated measure, one-way ANOVA with Tukey post-hoc analysis (a = 0.050) with donor as a random source effect was used. All diamonds and bands represent the 95% confidence interval. For all data, significance labels were used to indicate if there is a statistically significant difference when comparing the polymers to the normal saline volume control. The following significance labels were used: ns (P > 0.05), * (P < 0.05), ** (P < 0.01), *** (P < 0.001), **** (P < 0.0001).

[0021] FIGS. 5A-5E compare in vivo evaluation of radiant star LVRs in severe hemorrhagic shock model in rats (n=5) to PEG20K and Hextend. Overview of the severe hemorrhagic shock model in rats (FIG. 5A). Hextend (rectangles, 6% w / v), PEG20K (diamonds, 10% w / v), RS-GmMA (triangles, 20% w / v), and RS-SBMA (circles, 20% w / v) were all given at 10% TBV. Hextend was given in its standard commercial formulation, while PEG20K and the radiant stars were dissolved in 0.9% veterinarian-grade normal saline. Comparison of the mean arterial pressure (MAP) in mmHg during the infusion period (t=10 minutes) for rats (FIG. 5B). Comparison of the MAP in mmHg during the observation period for rats (FIG. 5C). Hemoglobin (Hb) concentration (g / dL) at various time points during the study (FIG. 5D). Lactate concentration (mM) at various time points during the study (FIG. 5E). For all plots, a dotted line with the ± 1 SEM interval band is placed at zero to represent the baseline levels for the rats. All error bands represent ± 1 SEM. The target range for MAP in damage control resuscitation of 60-80 mmHg is shown by a grey band. A fit model for a repeated measure, one-way ANOVA with Tukey post- hoc analysis (a = 0.050) was used for statistical analysis. For all data, significance labels were used to indicate if there is a statistically significant difference when comparing the LVRs (PEG20K, RS-GmMA, and RS-SBMA) to Hextend at each timepoint. The following significance labels were used: ns (P > 0.05), * (P < 0.05), ** (P < 0.01), *** (P < 0.001), **** (P < 0.0001).

[0022] FIG. 6 is a schematic illustration of the synthesis of representative linear neutral and zwitterionic polymers.

[0023] FIGS. 7A and 7B are schematic illustrations of the synthesis of HEMA-ECT (hECT) (FIG. 7A) and the radiant stars (FIG. 7B), respectively.

[0024] FIG. 8 compares internalization of fluorescently labeled Hextend and RS-GmMA by macrophage cells.

[0025] FIG. 9 compares cell viability of macrophages after a short 15 min incubation with Hextend or RS-GmMA.

[0026] FIG. 10 is a schematic illustration of the synthesis of a representative comb LVR. DETAILED DESCRIPTION

[0027] The present disclosure provides low volume resuscitants engineered for the prehospital care of severe hemorrhagic shock.

[0028] In one aspect, the present disclosure provides a method for treating and / or preventing hemorrhagic shock. In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a watersoluble polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to the subject thereby functioning as a plasma volume expander to treat and / or prevent hemorrhagic shock.

[0029] The method described herein is useful for treating and / or preventing hemorrhagic shock. The method is useful for treating a subject experiencing hemorrhagic shock. The method is also useful for preventing an injured subject from experiencing hemorrhagic shock.

[0030] The method described herein utilizes a water-soluble polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to the subject thereby functioning as a plasma volume expander effective to treat and / or prevent hemorrhagic shock. As used herein the term “polymer” refers to a macromolecule prepared by the polymerization of a monomer to provide the macromolecule having a backbone consisting of repeating units derived from the monomer. For the water-soluble polymers described herein, when the monomer includes a substituent that is not involved in the polymerization reaction, the repeating units of the backbone of the resulting polymer include these substituents as groups that are pendant from the backbone (see, for example, P in formulae (I) and (TI) below).

[0031] By virtue of their pendant groups, the water-soluble polymers useful in the method associate water on administration to the subject and thereby function as a plasma volume expander (i.e., resuscitant). Therefore, the water-soluble polymers described herein are low volume resuscitants (LVRs) in that they are not delivered to the subject in a high- volume state (i.e., not delivered with maximum associated water) and only become high volume resuscitants after administration to the subject (e.g., intravenous injection). In addition to associating water on administration and maintaining associated water in the circulatory system, the water-soluble polymers useful in the method do not negatively impact coagulation.

[0032] It will be appreciated that water-soluble polymer having a polymer backbone and pendant groups covalently coupled thereto useful in the method described herein is not a polypeptide derived from alpha-amino acids (e.g., naturally occurring), a polysaccharide (natural or synthetic), a poly(alkylene glycol) (e.g., PEG or PPG), or a dendrimer.

[0033] The water-soluble polymer useful in the method is a synthetic polymer having a synthetic backbone. It will be appreciated that the polymer backbone is not a polysaccharide or a polypeptide prepared from alpha-amino acids (e.g., naturally-occurring alpha-amino acids). It will also be appreciated that the polymer backbone is not a polyether (i.e., does not include ether groups), such as in the backbones of hyperbranched polyglycerols.

[0034] In certain embodiments, the polymer backbone is a fully carbon polymer backbone having only C-C sigma bonds (see, e.g., formula (I) below). Representative fully carbon polymer backbones useful in the LVRs described herein are prepared by polymerization of suitable acrylate and acrylamide monomers via, for example, RAFT and ATRP synthesis. A representative fully carbon polymer backbone is shown below. The fully carbon polymer backbone having only C-C sigma bonds is depicted within the box:

[0035] In this schematic representation, R1is hydrogen or C1-C3 alkyl, P is the pendant group, n represents the degree of polymerization, and the * represents the terminal groups residual from the polymerization process (e.g., RAFT chain transfer agents, ATRP initiators).

[0036] In other embodiments, the water-soluble polymer useful in the method is a polypeptoid polymer having polymer backbone shown in formula (II) below:

[0037] Representative polypeptoid polymer backbones useful in the LVRs described herein are prepared by polymerization of suitable peptoid monomers via, for example, ring opening polymerization of N-substituted N-carboxyanhydrides. In this schematic representation, P is the pendant group, n represents the degree of polymerization, and the * represents the terminal groups residual from the polymerization process (e.g., carboxylic acids).

[0038] It will be appreciated that the LVRs described herein include terminal groups that are residual from the polymerization process for making them (e.g., RAFT, ATRP, ROP).

[0039] The pendant groups (P in the schematic illustrations above) of the water-soluble polymer useful in the method impart water solubility to the polymer and associate water on administration to the subject and thereby impart to the water-soluble polymer the capability to function as a plasma volume expander. In certain embodiments, the pendant group is an electronically neutral, non-charged group. In other embodiments, the pendant group is a zwitterionic group (electronically neutral, charged group).

[0040] It will be appreciated that the pendant groups are not poly ethers groups (e.g., solely polyether groups), such as poly(oxyethylene) (PEG) or poly(oxypropylene) (PPG) groups. In certain embodiments, the water-soluble polymer has a fully carbon polymer backbone having only C-C sigma bonds. In certain of these embodiments, the water- soluble polymer is an acrylate polymer or acrylamide polymer. Acrylate and acrylamide polymers have fully carbon polymer backbones, as depicted above in formula (I). Representative acrylate polymers include acrylates and methacrylates (e.g., polyacrylate or polymethacylate). Representative acrylamide polymers include acrylamides and methacrylamides (e.g., polyacrylamide or polymethacylamide).

[0041] In other embodiments, the water-soluble polymer has a polypeptoid polymer backbone. In certain of these embodiments, the water-soluble polymer is a polypeptoid polymer. Polypeptoid polymers have polymer backbones depicted as shown above in formula (II).

[0042] The water-soluble polymers useful in the disclosed method have a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to the subject thereby functioning as a plasma volume expander effective to treat and / or prevent hemorrhagic shock.

[0043] In certain embodiments, the pendant group is an electronically neutral, non-charged group. In certain embodiments, the pendant group includes two or more hydroxyl groups (electronically neutral, non-charged neutral). In certain of these embodiments, the pendant group comprises -CH2CH(OH)CH2OH or -CH(CH2OH)2. In other embodiments, the pendant group includes a polyol, such as a sugar. In further embodiments, the pendant group includes a single hydroxyl group and, in a related embodiment, the pendant group includes a single hydroxyl group and one or more ether groups. Representative pendant groups having a single hydroxyl group and one or more ether groups include -CH2- CH(OH)-CH2-OCH3, -CH2-CH(OH)-O(C=O)-(CH2CH2O)n-CH3, where n = 1 or 2.

[0044] Representative water-soluble polymers useful in the method described herein for imparting an electronically neutral, non-charged pendant group into the water-soluble polymer can be prepared from the following monomers: dihydroxypropylacrylate (or methacrylate, methacrylamide, acrylamide), dihydroxy isopropyl acrylate (or methacrylate, methacrylamide, acrylamide), glycerol monomethacrylate, or glycerol acrylate, each of which impart neutral pendant groups having two or more hydroxyl groups.

[0045] Other suitable monomers include 5 -membered or 6-membered cyclic acetal acrylates (or methacrylates, acrylamides, methacrylamides) (e.g., 5-CAA and 6-CAA) and ether-containing monomers (e.g., GM-1 and GM-5) having ether-containing pendant groups. See Pham et al., Various radical polymerizations of glycerol-based monomers, Eur. J. Lipid Sci. Technol. 2013, 115, 28-40. In certain embodiments, the pendant group includes a single hydroxyl group and, in related embodiments, the pendant group includes a single hydroxyl group and one or more ether groups (e.g., -CH2-CH(OH)-CH2-OCH3, - CH2-CH(OH)-O(C=O)-(CH2CH2-O)n-CH3, where n = 1 or 2).

[0046] In other embodiments, the pendant group is a zwitterionic group. In certain of these embodiments, the pendant group is a zwitterionic group selected from a sulfohetaine group, a carboxybetaine group, a phosphobetaine group, a trimethylamine N-oxide group, or a phosphatidylserine group. In certain embodiments, the zwitterionic group is an group that includes a carboxylate (anion) and an ammonium group (cation), for example, - (CH2)nCH(CO2-)(NH3+), where n = 0, 1, 2, or 3.

[0047] In certain embodiments, the water-soluble polymer comprises a repeating unit: wherein

[0048] R1is hydrogen or C1-C3 alkyl (preferably hydrogen or methyl)

[0049] X is O or NH;

[0050] R2is selected from the group consisting of

[0051] (a) a neutral group as described herein (e.g., a neutral group having two or more hydroxyl groups or a single hydroxyl group and one or more ether groups), and

[0052] (b) a zwitterionic group as described herein; n is from about 20 to about 1000 DP; and wherein * represent the remainder of the polymer.

[0053] In certain of the above embodiments, R2is selected from the group consisting of

[0054] (a) -CH2CH(OH)CH2OH (GmMA),

[0055] (b) -CH2CH2N+(CH3)2CH2CH2CH2SO3- (SBMA), and

[0056] (c) -CH2CH2P(=O)(O )OCH2CH2N(CH3)3+ (MFC).

[0057] In certain embodiments, the water-soluble polymer is a methacrylate polymer comprising 50% or more repeating units derived from a hydrophilic monomer selected from the group consisting of (a) glycerol monomethacrylate (GmMA), (b) oligoethylene glycol methacrylate, (c) 2-methacryloyloxyethyl phosphocholine (MPC), and (d) 3-LL2- (methacryloyloxy)ethyl]dimethylammonio]propane-l -sulfonate (DMAPS orSBMA).

[0058] In certain embodiments, the water-soluble polymer is a poly(glycerol monomethacrylate) (pGmMA).

[0059] In other embodiments, the water-soluble polymer is a poly(3-[[2-

[0060] (methacryloyloxy)ethyl]dimethylammonio]propane-l -sulfonate) (pSBMA or pDMAPS).

[0061] In further embodiments, the water-soluble polymer is a poly(2- metbacryloyloxyethyl phosphocholine) (pMPC).

[0062] The polymer architecture of the water-soluble polymer useful as the LVR described herein may vary. In certain embodiments, the water-soluble polymer is a linear polymer. In other embodiments, the water-soluble polymer is a radiant star polymer. In further embodiments, the water-soluble polymer is a comb polymer (also referred to as a bottlebrush polymer).

[0063] The size of the water-soluble polymer useful as the LVR described herein may vary. In certain embodiments, the water-soluble polymer has a degree of polymerization from about 20 to about 1000.

[0064] Table 1 summarizes the minimum (Table 1) and maximum (Table 1 cont’d) values of polymer properties for certain embodiments of representative linear, radiant star, and comb polymers (100) and (20) useful in the methods and compositions described herein: total DP, number of arms, arm DP, backbone DP, and molecular weight for representative GmMA and zwitterionic polymers.

[0065] Table !. Minimum Values. Table 1 cont’d. Maximum Values.

[0066] Table 2 summarizes the minimum (Table 2) and maximum (Table 2 cont’d) values of polymer properties for other embodiments of representative comb polymers (100) and (20) useful in the methods and compositions described herein: total DP, arm density, number of arms, arm DP, backbone DP, and molecular weight for representative GmMA and zwitterionic polymers.

[0067] Table 2. Minimum Values. Table 2 cont’d. Maximum Values.

[0068] Referring to the tables above, N / A is “not applicable” (due to polymer architecture), Total DP is the overall DP of the entire polymer, Backbone DP is the DP of the polymer from which the arms are grown from for the combs, MW GmMA is the molecular weight of the final polymer using GmMA, and MW Zwitterion is the upper limit of molecular weight for a zwitterionic version of the polymer. For the comb polymers, Arm Density is the number of arms per backbone repeating units.

[0069] In certain embodiments, the pendant groups of the water-soluble polymer useful as the LVR described herein are covalently coupled the polymer backbone by a degradable linkage. In certain embodiments, the degradable linkage is a biodegradable linkage. Representative biodegradable linkages include ester linkages, acetal linkages, silyl ether linkages, carbonate linkages, hydrazone linkages, disulfide linkages, and boronic acid linkages.

[0070] In certain embodiments, the water-soluble polymer is administered by intravenous injection.

[0071] In another aspect, the disclosure provides blends of the polymeric LVRs described herein that provide a multi-functional (timing) approach to treating and / or preventing hemorrhagic shock.

[0072] In certain embodiments, the blend comprises two or more different water-soluble synthetic polymers, each having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to a subject thereby functioning as a plasma volume expander, wherein the two or more different water-soluble synthetic polymers are selected from the group consisting of linear polymers, radiant star polymers, and comb polymers.

[0073] In certain of these embodiments, the composition comprises a linear polymer and a radiant star polymer.

[0074] In other of these embodiments, the composition comprises a linear polymer and a comb polymer.

[0075] In further of these embodiments, the composition comprises a linear polymer, a radiant star polymer, and a comb polymer.

[0076] It will be appreciated that the composition may include two of the same type of polymer (e.g., two linear polymers, two radiant star polymers, or two comb polymers) where the two polymers are not the same and therefore each imparts a different LVR property to the composition.

[0077] The compositions described herein are blends of LVRs that may include LVRs with different pharmacokinetic properties to provide resuscitation over different time courses. For example, a blend of a short circulating LVR with a long circulating LVR could provide fast, high resuscitation capacity followed by lower, sustained resuscitation capacity.

[0078] The most desirable profile would be a slow release LVR, where a pressure spike that could lead to bleeding is completely avoided. To accomplish this, the LVR degrades into smaller pieces in the bloodstream. Because the osmotic pressure of a polymer is dictated by the number of moles of polymer in solution, a self-assembled particle or polymer that degrades and results in an increase in the number of moles of polymer in the bloodstream is useful. In such a situation, polymer clearance of the smaller polymers from the body are balanced using blends or self-assembly leads to advantageous resuscitation profiles.

[0079] In a further aspect, the disclosure provides an artificial plasma volume expander, comprising a water-soluble synthetic polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to a subject suffering from or in danger of hemorrhagic shock.

[0080] In a related aspect, the disclosure provides a water-soluble synthetic polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to a subject for use in treating and / or preventing hemorrhagic shock. The following describes the preparation, characterization, and use of low volume resuscitants for treating hemorrhagic shock.

[0081] Synthesis and characterization of linear neutral and zwitterionic hydrophilic polymers for low volume resuscitation

[0082] To begin with, different size ranges of methacrylate-based resuscitants were explored to observe the influence on resuscitation. Theoretical calculations of polymer contour length, average square end-to-end distance, and Rgwere conducted and a methacrylate with a degree of polymerization (DP) of 125 was found to theoretically provide an Rgapproximately half that of PEG20K. A panel of poly(glycerol monomethacrylate) (pGmMA) linear homopolymers with DPs of 100, 200, and 300 were selected. The oncotic potential of these neutral pGmMA LVRs should be closely related to the osmotic pressure (IT) of the polymers in solution which is proportional to their molar concentration, as dictated by van’t Hoff’s equation: where nsoiute = the number of moles of polymer in solution, V = volume, R = molar gas constant, and T = temperature. Interestingly, highly charged macromolecules such as albumin exhibit a larger osmotic pressure than predicted by van’t Hoff’s equation due to the Gibbs-Donan effect. Without being bound by theory, we believed that highly-charged zwitterionic monomers would increase the oncotic potential of our LVRs, enabling the use of a lower polymer dose. Two zwitterionic polymers were selected for evaluation: polymers based on the monomers 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3-sulfopropyl)-N-methacroyloxyethyl-N,N-dimethylammonium betaine (SBMA) to evaluate alongside GmMA. Both monomers exhibit over double the number of coordinated water molecules compared to GmMA, as determined by differential scanning calorimetry (DSC). A panel of linear polymers of DP100, 200, and 300 was synthesized using the three monomers (L-GmMA, L-MPC, and L-SBMA) via RAFT polymerization (FIG. 6) as shown in FIGS. 1 A-1C. LVR polymers were synthesized with molecular weights (number average molecular weight, Mn) of approximately 18 kDa to 90 kDa and with narrow polydispersity indices (PDIs) (1.02-1.25) (FIGS. 1D-1F). The characterization of linear (L) LVRs synthesized is summarized in Table 3. Table 3. Overview of Representative Linear LVRs.

[0083] The polymer panel was characterized using small-angle x-ray scattering (SAXS) to measure radii of gyration (Rg) (FIG. 1G). The DP200 L-GmMA (MW = 32 kDa), L-MPC (MW = 59 kDa), and L-SBMA (MW = 56 kDa) all have similar Rgas PEG20K, as expected from our theoretical calculations. Next, the osmolarity of the linear polymer panel was measured in normal saline (0.9% w / v NaCl, isotonic to blood serum) by vapor pressure osmometry (FIG. 1H). DP200 and DP300 L-GmMA showed similar osmolarity to PEG20K while L-MPC and L-SBMA showed lower osmolarity compared to PEG20K. In the case of L-SBMA, there was a decrease in osmolarity as the molar concentration increased. There are likely two factors contributing to the phenomenon. First, pSBMA can self-assemble as a function of concentration in solutions with low ionic strength, which would create larger macromolecules that decrease the overall number of moles of solute in solution. Second, as polymer concentration is increased, pSBMA may be coordinating salt ions from the normal saline solution. The drop in osmolarity below the 0.9% NaCl solution level (see FIG. 1G) at high polymer concentrations supports this. The apparent drop in osmolarity as polymer concentration is increased is an artifact of the experimental set-up; there are a fixed number of ions in solution during osmometry measurements, whereas in the body there would be large reserves outside the vascular space to compensate for the zwitterions. With this detail in mind, the L-SBMA osmometry measurements (FIG. II) were repeated but included additional NaCl to match the molar concentration of the zwitterionic groups and prevent self-assembly. An approximately 2-6x increase in the osmolarity of pSBMA was observed. With L-MPC, no evidence of self-assembly was observed. Finally, the hydration number (nu) or the number of strongly bound, non- freezing water molecules coordinated per repeat unit was measured using DSC for PEG20K, L-GmMA, and L-SBMA (FIG. 1J and Table 3). In 0.9% NaCl solution, L-GmMA and L-SBMA had similar nu values to PEG20K. However, in agreement with the osmometry measurements for L-SBMA, when additional NaCl was added to coordinate with the zwitterionic groups to prevent self-assembly, an approximately 4x increase was observed in the amount of water coordinated per polymer repeat.

[0084] Linear LVRs underperform in rotational thromboelastometry evaluation and a severe hemorrhagic shock model in rats

[0085] Rotational thromboelastometry (ROTEM) was used to evaluate the effect of the linear LVRs on coagulation using human plasma. ROTEM monitors clot firmness over time, characterizing clot formation and breakdown (FIG. 2A). In the analysis, molar concentrations of polymers were held constant by analyzing 10%, 20%, and 30% w / v solutions of DP100, 200, and 300 polymers, respectively, at 10% total plasma volume in ROTEM. The various linear LVRs were evaluated against a normal saline volume control for their clotting times, alpha angle (FIG. 2B), and maximum clot firmness (MCF) (FIG. 2C).

[0086] L-GmMA minimally affected coagulation, with only the DP300 showing a significant change in alpha angle. In contrast, both L-MPC and L-SBMA induced hypocoagulability in the plasma of human donors, decreasing both the rate of clot formation and the overall clot firmness in ROTEM, significantly impairing coagulation. Since MCF correlates highly with plasma fibrinogen levels, the decrease in clot firmness suggests that the zwitterions are either inhibiting the activation of fibrinogen into fibrin, or that the zwitterions are disrupting fibrin polymerization and changing clot structure, resulting in softer clots.

[0087] L-GmMA and L-SBMA polymers were evaluated in vivo using a lethal and severe rat hemorrhagic shock model (FIG. 2D). Blood pressure as a function of time is shown for DP200 and 300 L-GmMA (FIG. 2E) and DP100, 200, and 300 L-SBMA (FIG. 2F). The target range for mean arterial pressure (MAP) in damage control resuscitation is about 60- 80 mmHg, which balances reperfusion of tissues with hypotension to prevent rebleeding and coagulopathy. From the initial in vivo pilot experiment, DP300 L-GmMA (n=2) temporarily reaches the desired MAP range, but subsequently drops below the range. DP300 L-SBMA of (n=l) maintains MAP within the target range throughout the evaluation. These initial studies indicated that for L-GmMA, it was needed to prevent premature clearance and increase oncotic potency; for L-SBMA, it was needed to prevent coagulopathies. It is believed that that restructuring these linear polymers into a radiant star architecture would increase oncotic potency by increasing the surface area available for interactions with water, leading to a higher density of coordinated water molecules. Similarly, it has been shown that hyper-branched PEG (HPG) has a higher density of coordinated water compared to linear PEG. HPG also exhibited better blood compatibility compared to its linear counterparts of similar molecular weights. Additionally, HPG and cyclic polymers have improved biodistribution compared to their linear counterparts of similar molecular weight due to a decreased ability to reptate through nanopores such as the fenestrated glomerular endothelium in the kidney. We believed that moving to a radiant star architecture would impede this reptation and therefore reduce renal excretion rates.

[0088] Synthesis and characterization of radiant star neutral and zwitterionic hydrophilic polymers for low volume resuscitation

[0089] FIG. 3A below outlines the synthesis of radiant star (RS) polymers using RAFT polymerization adapted from D. Das, S. Srinivasan, F. D. Brown, F. Y. Su, A. L. Burrell, J. M. Kollman, A. Postma, D. M. Ratner, P. S. Stayton, A. J. Convertine, Polym. Chem. 2018, 9, 2134-2146. A schematic illustration of the synthesis is shown in FIGS. 7A and 7B, which depict a reaction scheme for synthesis of HEMA-ECT (hECT) (FIG. 7 A) and a reaction scheme for the synthesis of the corresponding radiant stars (FIG. 7B). Because L- MPC polymers seemed to impact coagulation slightly more compared to L-SBMA in ROTEM experiments, radiant star versions of pGmMA (RS-GmMA) and pSBMA (RS- SBMA) were prepared and evaluated. Table 4 summarizes the representative radiant star LVRs prepared.

[0090] Table 4. Overview of Representative Radiant Star LVRs.

[0091] RS-SBMA of DP 100 and DP200 were synthesized to directly compare to their linear counterparts. It is believed that higher molecular weight radiant star polymers would improve in vivo performance, and therefore DP400 RS-SBMA and RS-GmMA polymers were synthesized. FIGS. 3B-3E show the GPC traces of the final synthesized radiant stars, confirming successful chain extension of the macroCTAs. A range of RS-SBMA and RS- GmMA polymers with a comparable range of PDls (1.02-1.12) and molecular weights (about 35 kDa to 118 kDa) to their linear counterparts (see Table 2) were synthesized. The molecular weights of our macroCTAs (GmMA = 5.7 kDa, SBMA = 7.1 kDa) from GPC were comparable to those reported by Das et al. From the conversion of both the HEMA- ECT and its co-monomer (either GmMA or SBMA) in 'H NMR analysis, the radiant stars were calculated to have an average of about 3.6 arms. This agreed with SAXS analysis, which indicated that approximately 3 arms provided the best fit of the data. From SAXS analysis (FIG. 3F), the DP 100 and DP200 RS-SBMA were found to have similar Rgto their linear counterparts, while DP400 RS-GmMA and RS-SBMA had Rgabout 1 nm larger compared to the DP300 linear polymers. A small in vivo study (n=3 per treatment) was conducted to compare the biodistribution of DP300 L-GmMA to DP400 RS-GmMA during the severe hemorrhagic shock model in rats. The RS-GmMA and L-GmMA showed a similar mass-to- volume plasma concentrations (mg / mL) across all time points despite the L-GmMA being given at a 1.5x higher (30% w / v) infusion compared to the RS-GmMA (20% w / v). This suggests that the L-GmMA is rapidly cleared from the bloodstream, resulting in a lower than expected plasma concentration. The percentage of injected dose in the plasma at T90 for RS-GmMA was significantly higher (p = 0.0005) compared to L- GmMA (about 27% vs. about 15%, respectively). Although not statistically significant, there was a trend towards L-GmMA having a higher percentage of the injected dose at T90 in the kidneys and urine compared to RS-GmMA. Overall, the DP400 RS-GmMA was retained in plasma better than DP300 L-GmMA. Vapor pressure osmometry was conducted for the radiant stars and observed similar osmolarity trends as their linear counterparts, including the decrease in osmolarity with molar concentration in normal saline for RS-SBMA (FIG. 3G). Again, additional NaCl prevented the self-assembly and resulted in a higher osmolarity (FIG. 3H). The DSC measurements of nu again mirrored the trend seen in osmometry (FIG. 31). The DP400 RS-GmMA and RS-SBMA coordinated a similar number of non-freezing water molecules to their linear versions. Using Rgdata from SAXS and nn data from DSC, DP400 RS-SBMA was calculated to have a density of about 2.4 H2O per nm3, whereas the L-SBMA of DP 100, DP200, and DP300 have water densities of about 7.8, 5.1, and 4.6 H2O per nm3, respectively, suggesting that the density of coordinated water is lower for the radiant star architecture compared to linear architecture. DP400 RS-GmMA has a density of about 1 H2O per nm3, whereas L-GmMA of DP100, DP200, and DP300 have water densities of about 1.5, 1.8, and 0.9 H2O per nm3, respectively.

[0092] RS-GmMA is the first resuscitant to ever demonstrate no adverse effects upon coagulation

[0093] Based on preliminary in vivo dosing experiments, a 20% w / v solution of RS- GmMA (DP400) and RS-SBMA (DP400) was selected in order to match concentrations between coagulation assays and in vivo studies. In whole blood, PEG20K and RS-SBMA were each found to increase clotting time compared to normal saline, while Hextend, PEG20K, and RS-SBMA all decrease clotting kinetics and lead to overall softer clots. See FIGS. 4A and 4B. PEG20K and RS-SBMA were not statistically different from each other, and both impacted clotting similarly, leading to a hypocoagulable state with decreased clot firmness. The RS-GmMA did not show a statistical difference from normal saline across the typical ROTEM parameters reported and was therefore the only material that did not adversely affect coagulation.

[0094] The same treatments were evaluated in human plasma to remove platelets and evaluate the treatment effect on fibrinogen (FIG. 4C). Similar to whole blood, it was found that PEG20K increased the clotting time of human plasma, and that Hextend, PEG20K, and RS-SBMA all led to a significant decrease in clot firmness resulting in a hypocoagulable state. To evaluate whether Hextend, PEG20K, and RS-SBMA disrupt thrombin activity, the effect of the treatments on thrombin generation in human plasma was investigated by thrombin generation assay (TGA) (FIG. 4D). PEG20K and RS-SBMA were observed to decrease the peak thrombin concentration (tPeak) and shift the thrombin generation curve down to later time points by increasing the lag phase (tLag) and decreasing the rate of thrombin generation (VI). However, the total amount of thrombin generated, quantified from the AUC, stays the same across treatments. Thrombin concentration affects fibrin fibril structure, suggesting that PEG20K and RS-SBMA are delaying the conversion of prothrombin into thrombin, but not fully disrupting it, leading to softer clots. Knowing that Hextend does not affect thrombin but still demonstrates significant coagulopathy, it was concluded that Hextend is most likely directly acting on fibrin and fibrinogen to impair clot formation.

[0095] The effect of the radiant stars and PEG20K upon platelet activation and aggregation was evaluated to probe any ramification on primary hemostasis with treatment. When compared to the volume negative controls, there was no statistically significant change in platelet activation or aggregation, indicating that PEG20K, RS-GmMA, and RS-SBMA do not cause platelet dysfunction. From ROTEM, the platelet contribution to MCF was estimated by subtracting the MCF in plasma from the MCF measured in whole blood. Once again, a statistically significant effect on platelets was not observed for any of the treatments. In summary, the in vitro coagulation assays indicate that PEG20K, RS-SBMA, and Hextend can induce a hypocoagulable state in human blood, but that RS-GmMA does not adversely affect coagulation.

[0096] Radiant star GmMA and SBMA correct severe hemorrhagic shock in rats to the desired blood pressure range for damage control resuscitation

[0097] Hextend, PEG20K, RS-SBMA (DP400), and RS-GmMA (DP400) were evaluated in a severe hemorrhagic shock rat model. FIG. 5A shows an overview of the model.

[0098] All rats had a similar average percentage of TBV removed during the catheter hemorrhage period, about 58%, about 60%, about 57%, and about 59% for Hextend, PEG20K, RS-SBMA, and RS-GmMA, respectively. After reaching blood lactate > 8 mmol / L (typically 30-45 minutes of shock), the rats were given a single bolus infusion of treatment over ten minutes. All treatments were administered at an estimated 10% of TBV for the rat. FIG. 5B shows the change in blood pressure, heart rate, and the diastolic shock index (DSI) during the infusion and observation periods for each of the treatments. Interestingly, during the infusion the RS-SBMA showed a similar rate of increase in blood pressure to Hextend, whereas the RS-GmMA performed similarly to PEG20K, reflecting the osmolarity and DSC measurements of RS-SBMA. During administration, RS-SBMA is likely self-assembled at its reconstitution concentration of 20% w / v, as it becomes diluted in the bloodstream of the rat, it disassembles, leading to a slower onset of oncotic response.

[0099] All treatments show an initial peak of blood pressure between 15 and 30 minutes after administration, followed by a drop in blood pressure likely due to renal clearance. At 75 min and 90 min, there is no statistical difference between PEG20K, RS-GmMA, and RS-SBMA, whereas Hextend is significantly lower than the other three treatments (FIG. 5C). FIGS. 5D and 5E show blood gas results from the study which indicate how well the metabolic dysfunction of hemorrhagic shock is being corrected. RS-GmMA, RS-SBMA, and PEG20K all similarly decreased lactate levels, while Hextend did not reduce lactate levels (FIG. 5E). The hemoglobin concentration reflects the amount of water drawn into the bloodstream by the resuscitants, as hemoglobin (Hb) is diluted as the vascular space is refilled with interstitial water during resuscitation. Both RS-SBMA and PEG20K were found to have statistically significant lower Hb levels, while RS-GmMA was similar to Hextend (FIG. 5D). The lower osmolarity and nn of RS-GmMA compared to RS-SBMA may have decreased the amount of water drawn in the bloodstream, allowing Hb levels to remain higher. Both PEG20K and RS-GmMA were found to return ion concentrations to near-baseline levels. Recall that hemorrhagic shock shuts down cellular ion pumps, increasing potassium levels while decreasing sodium levels in the blood. Although rats treated with RS-SBMA showed similar blood pressure, hemoglobin, and lactate levels to PEG20K, their potassium and sodium levels were significantly elevated at 60 and 90 min. It is believed that RS-SBMA is coordinating these ions retaining them in the blood. Finally, the kidneys, liver, heart, and lungs of the rats were submitted for histopathologic analysis by a board-certified veterinary pathologist. Histologic lesions were generally minimal to mild when observed, and no significant differences were identified between the treatment groups. In summary, the in vivo blood pressure and hemoglobin levels indicate that PEG20K, RS-GmMA, and RS-SBMA show similar ability to refill the vascular space, while lactate levels support a similar ability to correct the metabolic deficit created by hemorrhagic shock. Even with the high polymer doses no adverse effects were observed in hemodynamics or in histopathology of major organs.

[0100] Polysaccharide-based colloidal resuscitants like Hextend can be retained in tissues for years and may result in renal dysfunction due to rapid internalization in immune and epithelial cells. Radiant star polymers described herein were evaluated for retention in tissues despite no observation of cellular internalization such polymers. A representative radiant star LVR, RS-GmMA, and Hextend were fluorescently labeled and their internalization by macrophage cells was evaluated. See FIG. 8. Minimal cellular association of RS-GmMA was observed, whereas Hextend was rapidly internalized. Hextend accumulates in resident macrophages, generating a foamy phenotype as observed in lysosomal storage disease and in proximal tubular cells inducing cell swelling in the kidney. The viability of macrophages was evaluated after a short 15 min incubation with Hextend or RS-GmMA and a reduction in cell viability was observed for Hextend, but not RS-GmMA, compared to controls. See FIG. 9.

[0101] Comb LVRs and Degradable LVRs

[0102] The present disclosure provides polymeric LVRs with optimized polymer backbone chemistry, polymer architecture, branching arm density, and polymer size. An ideal LVR circulates during the golden hour to facilitate resuscitation but then is excreted after resuscitation. In certain embodiments, biodegradable LVRs are provided that hydrolysable linkages between the polymer backbone and pendant groups. These polymeric LVRs with hydrolysable bonds decrease long-term tissue accumulation of the polymers.

[0103] In addition to linear and radiant star LVRs, the present disclosure also provides comb (or bottlebrush) LVRs. Like radiant star LVR described herein, LVRs with a comb architecture maximize the surface area of polymer interacting with water to provide greater water-recruiting capacity. This is in contrast to linear LVRs that have relatively less polymer surface area interacting with water.

[0104] The branching density of the polymer affects oncotic potency and water-recruiting capacity. For radiant stars, modulating the length of arms extending from the core as well as the number of arms affect these properties. For combs, the density of arms branching off the polymer backbone, the backbone length, and arm length affect these properties. Branching density maximizes oncotic potency by providing high surface area for polymer- water interactions while also reserving enough dimensional space that water molecules are not excluded from coordinating with the polymer.

[0105] FIG. 10 is a schematic illustration of the synthesis of a representative comb LVRs. In certain embodiments, RAFT polymerization is used to synthesize a macroCTA followed by arm extension by RAFT. A representative comb LVR is prepared by synthesizing a pHEMA (or pHEMA analog) homopolymer via RAFT polymerization followed by endcapping. A chain transfer agent (CT A) is grafted to the hydroxyls of this HEMA-type monomer via EDC coupling to create a macroCTA. Chain extending the macroCTA with a GmMA analog via RAFT grows the arms of the combs. By controlling the molar ratio of the CTA to the hydroxyls on the comb backbone during the EDC coupling, the number of arms and arm density is controlled.

[0106] As used herein, the term “about” refers to ±5% of the specified value.

[0107] MATERIALS AND METHODS

[0108] Materials. 2,2'-Azobis(2-methylpropionitrile) (AIBN), 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CCC), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 4-(dimethylamino)pyridine (DMAP), dichloromethane (DCM), N,N-dimethylacetamide (DMAc), and all other reagents were purchased from Sigma- Aldrich (Saint Louis, MO) unless noted otherwise. 4-Cyano-4- (ethylsulfanylthiocarbonyl)sulfanylpentanoic acid (ECT) was purchased from AmBeed (Arlington Hts, IL), 3-[[2-(Methacryloyloxy)ethyl]dimethylammonio]propane-l-sulfonate (SBMA), 2-methacryloyloxyethyl phosphorylcholine (MPC), and 3-sulfopropyl methacrylate potassium salt (SPMK) were purchased from TCI America (Portland, OR). Glycerol monomethacrylate (GmMA) was purchased from Polysciences, Inc. (Warrington, PA). 2,2’-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044) was purchased from FUJIFILM Wako Chemicals USA (Richmond, VA).

[0109] Synthesis of linear LVRs

[0110] Neutral Homopolymers. pGmMA was synthesized via reversible additionfragmentation chain transfer (RAFT) polymerization as described previously (L. W. Chan, X. Wang, H. Wei, L. D. Pozzo, N. J. White, S. H. Pun, Science translational medicine 2015, 7). Briefly, GmMA monomer was combined with CCC and AIBN at the following ratios - 110: 1:0.2 (DP 100), 220: 1:0.2 (DP200), 330: 1:0.2 (DP300) in dimethylacetamide at a monomer concentration of 0.6 M. A small amount of DMF was spiked in as an internal standard for NMR. The mixture was purged with argon for at least 15 min. This mixture reacted for 18 h at 70 °C. pGmMA polymers were precipitated 2x in diethyl ether. Precipitated polymer was collected by centrifugation at 7197 x g. Trithiocarbonate groups were removed via an end-capping reaction with 20x molar excess AIBN at 70 °C for 24 hours. The pGmMA polymers were then dialyzed against DI water for 2 days, with a minimum of six bath changes, followed by lyophilization.

[0111] Zwitterionic Homopolymers. SBMA monomer was combined with CCC and VA- 044 at the following ratios - 100: 1 :0.2 (DP 100), 200: 1 :0.2 (DP200), and 300: 1 :0.2 (DP300) in normal saline (0.9% w / v, NaCl) at a monomer concentration of 0.6 M. A small amount of DMF was spiked in as an internal standard for NMR. The mixture was purged with argon for at least 15 min. This mixture was reacted for 12 h at 44 °C. The solution was then open to atmosphere and diluted to a final polymer concentration of 2 mM. Then, trithiocarbonate groups were removed via an end-capping reaction with 20x molar excess VA-044 at 44 °C for 24 hours. The pSBMA polymers were dialyzed against 0.9% w / v saline (NaCl) for 1 day, with a minimum of three bath changes, and were then dialyzed against DI water for 2 days, with a minimum of six bath changes, which would result in the polymer crashing out of solution. The contents of the dialysis bag were vigorously mixed to suspend the polymer, and then the polymer was precipitated by mixing 5 mL of the suspension, with 40 mL of methanol in a 50 mL conical tube. Methanol was used to remove as many NaCl counterions as possible. Precipitated polymer was collected by centrifugation at 7197 x g, followed by removal of residual methanol overnight under high vac. The same steps were followed for MPC and SPMK.

[0112] Polymer characterization

[0113] Polymers were characterized via gel permeation chromatography (GPC) with multi angle light scattering and refractive index detectors (MiniDawn Treos and OptilabTRex, respectively, both from Wyatt Technology, Santa Barbara, CA) to determine molecular weight from 100% mass recovery and dispersity index (PDI). Polymers were run on a system consisting of a Shimadzu LC-20AD stack running room temperature, pH 7.4 phosphate buffered saline (0.05% w / v NaNa) mobile phase at a flow rate of 0.5 mL min1through a single column setup (Phenomenex PolySep™-GFC-P 4000; separation range: 3kDa- 400KDa). Poly(ethylene glycol) standards (Ready Cal- Kit, PSS Polymer Standards Services GmbH) were used to calibrate the system. The p(GmMA-co-hECT) macroCTA core and chain extension (FIG. 3B) were run on a separate system consisting of an Agilent 1260 HPLC stack running heated LiBr-supplemented (0.1% w / v) DMF mobile phase at a flow rate of 1 mL min1through a semi-prep three-column setup (Tosoh TSKGel alpha- 4000 to Tosoh TSKGel alpha-300 to Phenomenex Phenogel 103A). Poly(methyl methacrylate) standards (PL2020-0100, Agilent Technologies, Inc.) were used to calibrate the system. ' H nuclear magnetic resonance (NMR) spectroscopy on a Bruker GG 500 was utilized to determine conversion of the polymer prior to purification. Vapor pressure osmometry (Vapro 5600) was used to measure osmolarity as a function of molar concentration for the different polymers. Each polymer was dissolved in 0.9% normal saline (NaCl) and was run in triplicate.

[0114] Small-angle X-ray Scattering of radius of gyration

[0115] X-Ray scattering was measured using a Xenocs Xeuss 3.0 (Grenoble, France) SAXS instrument with a copper radiation source. Samples were analyzed in a 1.2 mm diameter quartz capillary and measured at four detector distances (266, 370, 900, and 1800 mm sample-to-detector distances with counting times of 300, 900, 1380, and 1800 seconds, respectively) to cover a q-range of approximately 5xl0-3to 4xl0-1A-1. Polymer samples were run at 15 mg / mL and 5 mg / mL in phosphate buffered saline (PBS). Scattering contribution from PBS and the capillary have been subtracted from polymer solution scattering data. Data reduction and processing were completed using XSACT software (Xenocs, Grenoble, France). Data fitting to monodisperse gaussian coil or star polymer models was completed on 15 mg / mL sample data using SASView 5.0.5. Fits were qualitatively assessed on 5mg / mL sample data and confirmed there was no concentrationdependent structure factor leading to correlations between polymers.

[0116] Differential Scanning Calorimetry (DSC) measurements of bound water

[0117] Polymers were dissolved at a concentration of 10% w / w in 0.9% normal saline (NaCl). Additional samples for SBMA were run with added NaCl that matched the molar concentration of 3-sulfopropyl and dimethyl ammonium groups to prevent self-assembly. 15 uL of polymer solution were added to Tzero hermetic pans (TA Instruments - Waters LLC, New Castle, DE). A Discovery 2500 DSC instrument (TA Instruments - Waters LLC, New Castle, DE) was used to measure the ice-water fusion enthalpy. A ramp from - 60°C to 30°C at a rate of 2°C / min, using an empty pan as a reference. The peak enthalpy analysis tool on the TA TRIOS software (version 5.1.1.46572) was used to quantify the area of the melting peak around 0°C. The following equation was used to calculate the number of bound water molecules per polymer (N): where is the fusion enthalpy of a control, is the fusion enthalpy of free water in the polymer solution, is the mass fraction of polymer in solution, and is the molecular weight was calculated from conversion of the polymer from 'H nuclear magnetic resonance (NMR) spectroscopy. The hydration number, nu, was then calculated by the following equation: where DP is the number of repeats in the polymer based off]H NMR spectroscopy.

[0118] Synthesis of HEMA-ECT (hECT) CTA monomer

[0119] 4-Cyano-4-((ethylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (ECT) (1 g, 3.8 mmol), hydroxyethyl methacrylate (HEMA) (1.2 g, 9.1 mmol), and 4- dimethylaminopyridine (DMAP) (1.5 g, 12.2 mmol) were dissolved in DCM (38 mL, 0.1 M reaction concentration) in a round bottom flask (RBF) with a stir bar. The RBF was capped, and the solution was cooled over ice for 15 minutes, then N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC • HC1) (874 mgs, 4.6 mmol). Pyridine (353 (1L, 4.36 mmol) was added to the mixture. The reaction was stirred over ice and allowed to warm to room temperature while stirring overnight. The mixture was washed 5x with a 1:1 volume with a cold saturated brine solution, followed by a 5x wash with cold 0.1M HC1 solution, a 5 wash with cold saturated sodium bicarbonate solution, and a final wash with cold saturated brine solution. The collected DCM was dried over sodium sulfate. The solvent was rotovapped off to obtain a deep orange oil. From proton NMR there was a mixture of HEMA (54%) and HEMA-ECT (hECT) (46%). The monomer was not further purified and used as a crude mixture. The CT A monomer was stored at -20°C.

[0120] Synthesis of poly(GmMA-co-hECT) and poly(SBMA-co-hECT) Radiant Stars

[0121] The synthesis of radiant stars from macroCTA cores (below) was adapted from D. Das, S. Srinivasan, F. D. Brown, F. Y. Su, A. L. Burrell, J. M. Kollman, A. Postma, D. M. Ratner, P. S. Stayton, A. J. Convertine, Polym. Chem. 2018, 9, 2134-2146.

[0122] Synthesis of p(GmMA-co-hECT) macroCTA core. GmMA monomer was combined with hECT and AIBN at the following molar ratio 2.333: 1:0.1 (GmMA:hECT / HEMA:AIBN) in dimethylacetamide at a monomer concentration of 0.6 M. A small amount of DMF was spiked in as an internal standard for NMR. The mixture was purged with argon for at least 15 min. This mixture reacted for 18 h at 70 °C. p(GmMA-co-hECT) polymers were precipitated 5x in diethyl ether. Precipitated polymer was collected by centrifugation at 7197 x g.

[0123] Chain Extension of p(GmMA-co-hECT) macroCTA core. For a DP400, GmMA monomer was combined with p(GmMA-co-hECT) macroCTA and AIBN at the following molar ratio 600:1:0.00752 (GmMA:MacroCTA:AIBN) in dimethylacetamide at a monomer concentration of 0.6 M. A small amount of DMF was spiked in as an internal standard for NMR. The mixture was purged with argon for at least 15 min. This mixture reacted for 12 h at 70 °C (targeting 67% conversion to hit DP400). p(GmMA-co-hECT) polymers were precipitated 5x in diethyl ether. Precipitated polymer was collected by centrifugation at 7197 x g. Trithiocarbonate groups were removed via an end-capping reaction with 20x molar excess AIBN to the concentration of trithiocarbonate groups at 70 °C for 24 hours. The RS-GmMA polymers were then dialyzed against DI water for 2 days, with a minimum of six bath changes, followed by lyophilization.

[0124] Synthesis of p( SBMA-co-hECT) macroCTA core. SBMA monomer was combined with hECT and AIBN at the following molar ratio 2.333:1:0.1 (GmMA: hECT / HEMA:AIBN) in a mixed solvent of 75% DMSO / 25% 0.9% normal (NaCl) saline at a monomer concentration of 0.6 M. A small amount of DMF was spiked in as an internal standard for NMR. The mixture was purged with argon for at least 15 min. This mixture reacted for 12 h at 70 °C. p(SBMA-co-hECT) polymers were precipitated 5x in 50:50 diethyl ether / acetone. Precipitated polymer was collected by centrifugation at 7197 x g.

[0125] Chain Extension of p(SBMA-co-hECT) macroCTA core. SBMA monomer was combined with p(SBMA-co-hECT) macroCTA and AIBN at the following molar ratios (SBMA:MacroCTA:VA-044) - 100: 1 :0.00752 (DP100), 200: 1 :0.00752 (DP200), and 400: 1:0.00752 (DP400) in normal saline (0.9% w / v, NaCl) at a monomer concentration of 0.6 M. A small amount of DMF was spiked in as an internal standard for NMR. The mixture was purged with argon for at least 15 min. This mixture reacted for 12 h at 44 °C. The solution was then open to atmosphere and diluted to a final polymer concentration of 2 mM. Then, trithiocarbonate groups were removed via an end-capping reaction with 20x molar excess VA-044 to the concentration of trithiocarbonate end groups at 44 °C for 24 hours. The pSBMA polymers were dialyzed against 0.9% w / v saline (NaCl) for 1 day, with a minimum of three bath changes, and were then dialyzed against DI water for 2 days, with a minimum of six bath changes, which would result in the polymer crashing out of solution. The contents of the dialysis bag were vigorously mixed to suspend the polymer, and then the polymer was precipitated by mixing 5 mL of the suspension, with 40 mL of methanol in a 50 mL conical tube. Methanol was used to remove as many NaCl counterions as possible. Precipitated polymer was collected by centrifugation at 7197 x g, followed by removal of residual methanol overnight under high vac.

[0126] Rotational thromboelastography (ROTEM) Evaluation

[0127] Whole human blood was purchased from Bloodworks Northwest (50 mL total blood in 3.2% sodium citrate). Half of the human blood was centrifuged down to make platelet-poor plasma (PPP). Two ROTEM deltas (Werfen, Bedford, MA / Serial #s 3733 and 4515) were used using the standard manufacturer’s protocol for EXTEM (CaCh + recombinant tissue factor + polybrene) for both whole blood and PPP. For each test, 40 pL of treatment was mixed with 360 pL of whole blood or plasma ( 10% TB V) in an Eppendorf tube then immediately evaluated on the ROTEM to prevent any separation of the blood. EXTEM of PPP was used as a surrogate for the standard FIBTEM test to measure the fibrinogen contribution to coagulation. Thrombin Generation Assay (TGA)

[0128] Whole human blood was purchased from Bloodworks Northwest (50 mL total blood in 3.2% sodium citrate). The human blood was centrifuged down to make plateletpoor plasma (PPP) then stored at -80 °C until subsequent testing. An automated Ceveron® alpha instrument with Anorogenic thrombin generation assay (TGA) module (Technoclone, Vienna, Austria; Software Release V 2. 1.2.2 ) was used to evaluate the resuscitants for their impact upon the thrombin generation profile of human plasma samples (n=5). The GmMA and SBMA radiant star LVRs were prepared at 20% w / v, PEG-20k and Hextend were prepared at 10% w / v and 6% w / v HES (commercial formulation), respectively. Saline was evaluated as a negative control. All resuscitants and controls were added to plasma at 10% of total plasma volume. The assay was performed using the Ceveron® TGA RC Low Kit (Technoclone, Vienna, Austria). Within the assay, a CaC12 buffer and a small concentration of recombinant human tissue factor (rhTF) are used to initiate coagulation by activating the platelets and thrombin in the plasma sample. Activated thrombin then cleaves a fluorogenic substrate, Z-Gly-Gly-Arg-AMC (Ceveron® TGA substrate, Technoclone, Vienna, Austria), allowing the quantification of thrombin concentration, which is plotted against time. The results are automatically calculated and displayed in Lag Time (Lag), Time to Peak (tPeak), Peak Thrombin (Peak), Velocity Index (VI) and Area Under the Curve (AUC).

[0129] Platelet Aggregometry

[0130] Healthy donor whole blood (n=4) in 3.2% sodium citrate was incubated with either a saline control or a low volume resuscitant (LVR) for 5 min. LVRs tested were polyethylene glycol (PEG) and synthesized polymers LVR 102 and LVR113. Samples were then activated with either 10 mM adenosine diphosphate or 2 mg / mL collagen (Chrono-Log, Havertown, PA). The platelet aggregation response was then measured by impedance aggregometry using a Chrono-Log Model 700 according to manufacturer specifications. Each condition was run simultaneously. The raw curves were collected along with the metrics reported by the Chrono-Log (Amplitude, Slope, area under the curve (AUC)).

[0131] Platelet Activation

[0132] Aliquots of 450 pL of whole blood were incubated for 5 min at room temperature with 50 uL of treatment (RS-GmMA and RS-SBMA = 20%w / v, PEG20K = 10% w / v, control = PBS), DPBS 2% fetal bovine serum (FBS, Gibco), or adenosine diphosphate (ADP, Bio / Data) for positive activation control. Aliquots of 5 pL were then incubated with antibody solutions of F1TC antihuman PAC-1 (1:10, BD Biosciences), APC antihuman CD62P (1: 10, BD Biosciences), and / or PE antihuman CD61 (1:10, BD Biosciences) for 20 min at room temperature. Cells were fixed in DPBS 1% PFA and analyzed on an Attune NxT (Invitrogen) flow cytometer.

[0133] Rat severe hemorrhagic shock model

[0134] Animal experiments were carried out in accordance with protocols approved hy the University of Washington Institutional Care and Use Committee (Protocol 4053-01: Biomaterials for Biomedical Applications). Model was slightly modified from V. Plant, D. W. Parrish, A. Limkemann, P. Ferrada, M. Aboutanos, M. J. Mangino, J Pharmacol Exp Ther 2017, 361, 334-340. Male Sprague Dawley rats (270g-370g) were anesthetized by isoflurane and continuously given a mix of isoflurane and oxygen via nosecone. During baseline, two femoral artery catheter lines were placed in the hind limbs, one to perform a controlled catheter bleed, and the second to constantly monitor vitals (blood pressure and heart rate) and collect blood samples for blood gas measurements. A venous line is placed in the left hind limb for intravascular administration of the low volume resuscitants. After catheterization, baseline blood gas and metabolites were evaluated to ensure healthy baseline respiration and lactate levels (pCO2 < 55 mmHg, sO2% > 95%, cLac < 1.0 mmol / L) prior to hemorrhage. After the rat is baselined, a single bolus of 500 units / kg of heparin is given to prevent clogging of the catheter lines during hemorrhage. Next, the rat was catheter hemorrhaged down to a mean arterial pressure (MAP) < 25 mmHg over five minutes (typically 7-10 cc of blood). After a MAP < 25 mmHg was reached, a timer was started for the shock period. Blood was withdrawn as needed up to a maximum of 60% total blood volume to maintain MAP < 25 mmHg (shock period) for at least 30 minutes until a lactate > 8 mmol / L is achieved. The total blood volume (TBV) of the rat was estimated as previously described by T. K. Arora, A. K. Malhotra, R. Ivatury, M. J. Mangino, J Trauma Acute Care Surg 2012, 72, 397-402.

[0135] Total Blood Volume (mL) = Weight of Rat (g) * 0.06 + 0.77

[0136] After 30 minutes, blood gas was checked every 15 minutes until the goal lactate was achieved. At the end of the shock period (lac > 8 mmol / L), the low volume resuscitant will be infused intravenously over 10 minutes (10% TBV). Blood gas and MAP will be recorded over 90 minutes, at the end of which the rat will be humanely euthanized via barbiturate overdose. Histopathology

[0137] Hearts, lungs, kidneys, and livers were harvested, fixed in 10% neutral buffered formalin, and paraffin embedded. Tissue sections (5pm) were stained with hematoxylin and eosin and evaluated qualitatively by a board-certified veterinary pathologist who was blinded to treatment group. Images were captured from glass slides using NIS-Elements BR 4.20.01 and plated in Adobe Photoshop 2023. Image white balance, lighting, and contrast were adjusted using auto corrections applied to the entire image. Original magnification is stated, and a scale bar is placed.

[0138] Biodistribution Study

[0139] FITC labeled RS-GmMA (DP400) and L-GmMA (DP300) were made by copolymerizing in 2% of a FITC-methacrylate monomer, AEMA-FL (synthesis of monomer outlined below). For RS-GmMA, AEMA-FL was copolymerized during the chain extension of the macroCTA core. All the same synthesis conditions, characterization, and purification methods were used as outlined previously. For the biodistribution study, animal experiments were carried out in accordance with protocols approved by the University of Washington Institutional Care and Use Committee (Protocol 4053-01: Biomaterials for Biomedical Applications). The same rat severe hemorrhagic shock model was performed as outlined above. Blood samples (1 mL in 10% v / v citrated syringes) were taken at T15, T20, T30, T45, T60, and T90. Samples were immediately spun down to collect plasma, then snap frozen, and stored at -80°C until analyzed. The rats were given an overdose of ketamine / xylazine, and heavy anesthesia was confirmed via toe pinch. The rats were then perfused with heparinized saline (10 units / mL). The urine was collected from the bladder, then the heart, lungs, both kidneys, spleen, and two liver lobes were collected, rinsed, weighed, snap frozen, then stored in a -80°C until analyzed. Organs were non-enzymatically homogenized and analyzed against standard curves generated from the organs of a control rat following the protocol by D. M. DiPema, A. V. Prakapenka, E. P. Chung, R. W. Sirianni, in Targeted Drug Delivery: Methods and Protocols (Eds.: R.W. Sirianni, B. Behkam), Springer US, New York, NY, 2018, pp. 191-199. FITC fluorescence was measured in both plasma and urine samples, and analyzed against standard curves generated from a control rat.

[0140] Estimation of the percentage of injected dose at T90

[0141] For the kidneys, heart, lungs, and spleen, the volume of each organ was calculated from its mass (density was assumed to be 1 g / cm3). For the liver, since only two lobes were taken, the theoretical total mass of the liver was assumed to be 3.756% of total body weight. For plasma, the total blood volume at T90 was estimated using a similar method to Hedin and Hahn, Clinical Science 2005, 108, 217-224. The volume of withdrawn blood, hemoglobin concentrations measured at the end of shock and T90, and a hematocrit of 48.3% were used in the following equations:

[0142] Where the BVT9O is the estimated total blood volume at 90 minutes, TBVbase is the total blood volume estimated at baseline, CATHHEM is the volume of blood removed, Hbesis the hemoglobin concentration measured at the end of shock, Hb go is the hemoglobin concentration measured at T90, LVRT9O is the measured concentration of LVR in the plasma by fluorescence, HCT is the hematocrit percentage for the rat (assumed to be 48.3%).

[0143] Synthesis of 2-Propenoic acid, 2-methyl-, 2-[[[(3',6,-dihvdroxy-3-oxospiro iisobenzofuran-l(3H),9'-i9Hlxanthenl-5-yl)aminolthioxomethyl1aminolethyl _ ester

[0144] (AEMA-FL): Fluorescein 5(6)-isothiocyanate (1 g / 2.58 mmol) and 1.1 equivalents of 2- aminoethyl methacrylate hydrochloride (470 mgs / 2.84 mmol) were added to a round bottom flask with a stir bar. DMSO (25.8 mL / 0.1 M) was added. Once the constituents were dissolved (pale yellow solution), 2.6 equivalents of DIPEA (1.29 m ) were added dropwise (turned to deep orange color). The mixture was stirred at RT overnight. The solution was precipitated one time in cold 6% HC1 solution (lOx volume of reaction) constantly swirled over ice. The orange precipitate was filtered off with a glass frit. The precipitate was collected into a glass vial then lyophilized overnight. The solid, orange monomer was stored at -20 °C. Yield was > 90%. The monomer was pure by 1H nuclear magnetic resonance (NMR) spectroscopy on a Broker AV 300 and TLC (not shown).

[0145] Theoretical Calculations

[0146] From literature, PEG with molecular weights of 20-40 kDa were active as LVRs. Leveraging RAFT, different size ranges of methacrylates was explored. However, compared to PEG, methacrylates are very different chemically which leads to a different size in solution. PEG has a very low molecular weight repeat unit at 44 g / mol, whereas the methacrylate with the smallest side chain polymethacrylic acid (PMMA), has a repeat unit of 88 g / mol. Additionally, PEG has a backbone of alternating C-0 and C-C o-bonds, while methacrylates instead have a purely carbon-carbon backbone. Therefore, 20-40 kDa methacrylates were not directly synthesized, there was a need to explore different molecular weight ranges compared to PEG. Additionally, one benefit of methacrylates is the chemical flexibility and tailorability of the sidechain. Due to the different sidechains, degree of polymerization (DP) was fixed across different side chains, instead of fixing molecular weight.

[0147] The contour length of polymers only considers the bond length of the repeat unit and the DP, and assumes the polymer is fully stretched out:

[0148] Contour Length (L) = nl where L is the contour length, n is the number of repeats, and 1 is the bond length. However, polymer chains are coiled up in solution, so to consider chain rigidity the average squared end-to-end distance of the polymer < R2> is used:

[0149] < R2> = COTnl2where (Coo) is the characteristic ratio, or a dimensionless factor for chain rigidity, n is the number of repeats, and 1 is the bond length. The end-to-end distance is a descriptor that is easy to calculate and only describes the distance between the two ends of the polymer coil. To get a real sense of the size of a polymer, radius of gyration (Rg) is the average squared distance of a point on the polymer coil from its center of mass (moment of inertia) is a better descriptor that can be measured using small-angle x-ray scattering. For a freely jointed chain the mean average of Rg2can be estimated by dividing the mean end-to-end distance squared by 6:

[0150] Table 5 below shows repeat unit bond lengths and characteristic ratios (C«>) from literature. Table 5. Comparison of bond length for each monomer repeat unit and characteristic ratio.

[0151] As can be seen in Table 5, although PEG has a longer bond length, it has a much more flexible backbone (lower C / ) compared to methacrylates. It is well documented that PEG graft density on the surfaces dictates whether the polymer forms a mushroom-shape (bending back on itself) or an extended (more rigid) conformation. Interestingly for methacrylates, the side chain can be used as a tool to tailor the rigidity of the backbone. As the side chain of the methacrylate increases in length, C<» increases as a result, and the backbone of the methacrylate is more rigid and extended in solution. Given the values from Table 5, the following estimates for the size of PEG compared to poly glycerol monomethacrylate (pGmMA) of 20 kDa are given in Table 6 below. Since there is not a reported C, for pGmMA, the value for polyhexyl methacrylate was used.

[0152] Table 6. Theoretical contour length, root mean squared end to end distance, and the radius of gyration for PEG 20k and pGmMA 20k.

[0153] Contour _ !

[0154] Polymer °.PLength <1>< >

[0155] (kDa) (umts)(nm) (nm) (nm)

[0156] PEG 20k 20 454 199 78 32 pGmMA 20k 20 125 38 38 16

[0157] There is a large difference in the contour length, root mean square of the end-to- end-distance, and the radius of gyration for PEG, indicating that the polymer bends back on itself, and is more compact in solution due to the flexibility of its backbone. Methacrylates on the other hand can achieve a relatively high radius of gyration at a lower degree of polymerization of PEG. Following these calculations, we decided to synthesize a panel of DP100, 200, and 300 pGmMA polymers. Although methacrylates, from a theoretical standpoint, could match the range of Rgfor PEG, we were concerned about the larger molecular weight of the higher DP pGmMAs (DP200 = 32 kDa and DP300 = 48 kDa). The osmotic pressure (fl) of a polymer is proportional to the number of moles of solute (nsoiute), where V = volume, R = molar gas constant, and T = temperature.

[0158] J-J _nsoluteRT

[0159] Since the oncotic potential of these LVRs is dictated by their osmotic pressure, the molar concentration in the blood is critical. Therefore, due to the 2-3x higher molecular weight of the methacrylate-based LVRs, a greater mass concentration of methacrylate- based LVR is needed for the same molar concentration of PEG-based LVRs. There was a concern that there would be issues reaching these high concentrations in the blood, and if this would impact performance. Therefore, additional methacrylate side chains were sought that may be able to increase the water-carrying capacity of the methacrylate compared to pGmMA. The hydration number (nn) for a polymer is the number of water molecules coordinated per repeat unit, and this is typically understood to be the amount of non-freezing water coordinated (strongly bound) with the polymer measured by dynamic scanning calorimetry. In general, it is known for methacrylates that the following trend is seen for water-coordinating strength of the oxygens on their sidechains Ohydiuxyi > CLarbouyi > O alkoxy- The hydroxyl group can hydrogen-bond via both the oxygen and the hydrogen, while the carbonyl is highly electronegative, and a water molecule is bridged between two monomers, and the alkoxy group typically presents more steric hinderance. Unfortunately, pGmMA has not been measured in literature so we estimated it from data for pHEMA and assumed that the extra hydroxyl group would coordinate one additional water (very conservative estimate). Moving from pHEMA to pGmMA resulted in a large increase in water-solubility for PolySTAT. Table 7 shows estimates for PEG and various methacrylates from literature. Table 7. Hydration numbers from literature measured by DSC for various polymers. Based on literature, it was decided to move forward with the commercial zwitterionic monomers 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3- sulfopropyl)-N-methacroyloxyethyl-N,N-dimethylammonium betaine (SBMA), which showed ~2x the water-carrying capacity compared to pGmMA. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of treating and / or preventing hemorrhagic shock, comprising administering to a subject in need thereof a therapeutically effective amount of a water- soluble polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to the subject thereby functioning as a plasma volume expander to treat and / or prevent hemorrhagic shock.

2. The method of Claim 1, wherein the water-soluble polymer has a fully carbon polymer backbone having only C-C sigma bonds.

3. The method of Claim 1, wherein the water-soluble polymer is an acrylate polymer or acrylamide polymer.

4. The method of Claim 1, wherein the water-soluble polymer has a polypeptoid polymer backbone.

5. The method of Claim 1, wherein the water-soluble polymer is a polypeptoid polymer.

6. The method of any one of Claims 1-5, wherein the pendant group is a neutral group or a zwitterionic group.

7. The method of any one of Claims 1-5, wherein the pendant group is a neutral group having two or more hydroxyl groups.

8. The method of any one of Claims 1-5, wherein the pendant group comprises -CH2CH(OH)CH2OH or -CH(CH2OH)2.

9. The method of any one of Claims 1-5, wherein the pendant group is a zwitterionic group.

10. The method of any one of Claims 1-5, wherein the pendant group is a zwitterionic group selected from a sulfobetaine group, a carboxybetaine group, a phosphobetaine group, a trimethylamine N-oxide group, or a phosphatidylserine group.

11. The method of Claim 1, wherein the water-soluble polymer comprises a repeating unit:whereinR1is hydrogen or C1-C3 alkyl;X is O or NH;R2is selected from the group consisting of(a) a neutral group having two or more hydroxyl groups, and(b) a zwitterionic group; n is from about 20 to about 1000 DP; and wherein * represent the remainder of the polymer.

12. The method of Claim 11, wherein R2is selected from the group consisting of(a) -CH2CH(OH)CH2OH (GmMA),(b) -CH2CH2N+(CH3)2CH2CH2CH2SO3- (SBMA), and(c) -CH2CH2P(=O)(O )OCH2CH2N(CH3)3+ (MPC).

13. The method of Claim 1, wherein the water-soluble polymer is a methacrylate polymer comprising 50% or more repeating units derived from a hydrophilic monomer selected from the group consisting of (a) glycerol monomethacrylate (GmMA), (b) oligoethylene glycol methacrylate, (c) 2-methacryloyloxyethyl phosphocholine (MPC), and (d) 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-l-sulfonate (DMAPS or SBMA).

14. The method of Claim 1, wherein the water-soluble polymer is a poly(glycerol monomethacrylate) (pGmMA).

15. The method of Claim 1, wherein the water-soluble polymer is a poly(3-[[2- (methacryloyloxy)ethyl]dimethylammonio]propane-l -sulfonate) (pSBMA).

16. The method of Claim 1, wherein the water-soluble polymer is a poly(2- methacryloyloxyethyl phosphocholine) (pMPCi.

17. The method of any one of Claims 1-16, wherein the water-soluble polymer is a linear polymer.

18. The method of any one of Claims 1-16, wherein the water-soluble polymer is a radiant star polymer.

19. The method of any one of Claims 1-16, wherein the water-soluble polymer is a comb polymer (also referred to as a bottlebrush polymer).

20. The method of any one of Claims 1-16, wherein the pendant groups are covalently coupled the polymer backbone by a biodegradable linkage.

21. The method of Claim 20, wherein the biodegradable linkage is an ester linkage, an acetal linkage, a silyl ether linkage, a carbonate linkage, a hydrazone linkage, a disulfide linkage, or a boronic acid linkage.

22. The method of any one of Claims 1-21, wherein the water-soluble polymer has a degree of polymerization from about 20 to about 1000.

23. The method of any one of Claims 1-22, wherein the water-soluble polymer is administered by intravenous injection.

24. A composition comprising two or more different water-soluble synthetic polymers, each having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to a subject thereby functioning as a plasma volume expander, wherein the two or more different water-soluble synthetic polymers are selected from the group consisting of linear polymers, radiant star polymers, and comb polymers.

25. The composition of Claim 24, wherein the composition comprises a linear polymer and a radiant star polymer.

26. The composition of Claim 24, wherein the composition comprises a linear polymer and a comb polymer.

27. The composition of Claim 24, wherein the composition comprises a linear polymer, a radiant star polymer, and a comb polymer.

28. An artificial plasma volume expander, comprising a water-soluble synthetic polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to a subject suffering from or in danger of hemorrhagic shock.

29. A water-soluble synthetic polymer having a polymer backbone and pendant groups covalently coupled thereto that associate water on administration to a subject for use in treating and / or preventing hemorrhagic shock.

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