Beta-amino-ester and beta-thiol-ester containing polymers for drug delivery and pharmaceutical uses

Abstract

Disclosed herein are beta-amino-ester and beta­thiol-ester containing polymers. In certain embodiments, the polymers comprise: monomers with a hydrophobic linker disubstituted with an alkane polyol; monomers of a branched thiol alkanoate; and monomers of an alkyl diamine. In certain embodiment, the polymer further comprises monomers of an amine alkyl acetal. In certain embodiment, the polymer further comprises monomers of an aminoalkyl carbamimidothioate. In certain embodiments, the polymers are used in methods of delivering a pharmaceutical agent to the lungs.

Description

[0001] BETA-AMINO-ESTER AND BETA-THIOL-ESTER CONTAINING POLYMERS FOR DRUG DELIVERY AND PHARMACEUTICAL USES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 712,358 filed October 25, 2024. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under HR0011-19-2-0008 awarded by Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

[0006] BACKGROUND

[0007] The lungs are vital organs and subject to a number of human diseases and conditions. Pulmonary administration of drugs is sometimes ineffective particularly for large sized molecules such as DNA and RNA based vaccines, antibodies and peptides. Thus, there is a need to identify improved therapeutic delivery methods to the lungs.

[0008] Zugates et al. report synthesis of poly beta-amino esters with thiol-reactive side chains for DNA delivery. JACS, 2006, 128, 12726-12734. See also US Patent No. 8,562,966.

[0009] Lakes et al. report highly thiolated poly (beta-amino ester) nanoparticles for acute redox applications. Gels, 2018, 4(4): 80.

[0010] Vanover et al. report nebulized mRNA-encoded antibodies protect hamsters from SARS-CoV-2 Infection. Adv Sci, 2022, 9, 220277.1

[0011] Rotolo et al. report species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung. Nat Mater, 2023, 22(3):369-379. See also WO 2023 / 108111.

[0012] Kavanagh et al. report ligand-free biodegradable poly(beta-amino ester) nanoparticles for targeted systemic delivery of mRNAto the lungs. Biomaterials, 2024, 313, 122753.

[0013] References reported herein are not an admission of prior art. SUMMARY

[0014] Disclosed herein are improved beta-amino-ester and beta-thiol-ester containing polymers. In certain embodiments, the polymers comprise monomers with a hydrophobic linker disubstituted with an alkane polyol; monomers of a branched or tri-branched thiol alkanoate; and monomers of an alkyl diamine. In certain embodiment, the polymer further comprises monomers of an amine alkyl acetal (two ethers attached to a central carbon atom). In certain embodiment, the polymer further comprises monomers of an aminoalkyl carbamimidothioate.

[0015] In certain embodiments, the hydrophobic linker is propane-2, 2-diyldibenzene. In certain embodiments, the alkane polyol is propane-polyol (1,2, 3 -triol). In certain embodiments, the branched or tri-branched thiol alkanoate is tri-branched 2-ethyl-2-(((3-thiolpropanoyl)oxy)methyl)propane- 1,3 -diyl bis(3-thiolpropanoate). In certain embodiments, the alkyl diamine is N1-methylpropane- 1,3-diamine. In certain embodiments, the amine alkyl acetal is 4,4-diethoxybutan-l -amine. In certain embodiments, the aminoalkyl carbamimidothioate is 2-aminoethyl carbamimidothioate.

[0016] In certain embodiments, the beta-amino-thiol-ester polymers are the reaction products of a multi -aery lated polyol comprising a hydrophobic linker, a branched compound having two or three or more terminal thiol groups, a compound comprising a primary amine and acetal or a primary amine and a morpholine group. In certain embodiments, the compounds are further reacted with a compound comprising two amine groups, e.g., a primary amine and a secondary amine group. In certain embodiments, the previous components are further reacted with a compound comprising a primary amine and a carbamimidothioate group or a compound comprising a first primary amine and a second primary amine group.

[0017] In certain embodiments, the beta-amino-thiol-ester polymers are the reaction products of ((propane-2,2-diylbis(4,l-phenylene))bis(oxy))bis(2-hydroxypropane-3,l-diyl) diacrylate; 2-ethyl-2-(((3-thiolpropanoyl)oxy)methyl)propane-l,3-diyl bis(3-thiolpropanoate); 4,4-diethoxybutan-l -amine; Is -m ethylpropane- 1,3 -diamine; and 2-aminoethyl carbamimidothioate (P81).

[0018] In certain embodiments, the beta-amino-thiol-ester polymers are made by the process of contacting: a first compound comprising a first acrylate polyol group and a second acrylate polyol group; a second compound comprising a first thiol group and a second thiol group; a third compound comprising a primary amine and a second group, wherein the second group is an acetal group or a morpholine; a fourth compound comprising two amine groups, e.g., a primary amine and a secondary amine group; and a fifth end-capping compound comprising a primary amine and a second chemical group, wherein the second chemical group is a carbamimidothioate group or the second chemical group is a second amine group; providing a beta-amino-thiol-ester polymer. In certain embodiments, the first through fourth compounds are heated prior to contacting with the fifth end-capping compound.

[0019] In certain embodiments, the first compound comprises a hydrophobic group linking the first acrylate polyol group, and the second acrylate polyol group. In certain embodiments, the hydrophobic group is a propane-2, 2-diyldibenzene group. In certain embodiments, the first compound is ((propane-2, 2-diylbis(4, l-phenylene))bis(oxy))bis(2-hydroxypropane-3, 1-diyl) di acrylate.

[0020] In certain embodiments, the second compound is ethane- 1,2-di thiol. In certain embodiments, the second compound comprises a third thiol group. In certain embodiments, the second compound is 2-ethyl-2-(((3-mercaptopropanoyl)oxy)methyl)propane-l,3-diyl bis(3-m ercaptopropanoate) .

[0021] In certain embodiments, the third compound comprises a primary amine and a second group, wherein the second group is a glycol, cyclic glycol, or morpholino group. In certain embodiments, the third compound is 2-morpholinoethane-l -amine. In certain embodiments, the third compound comprises a primary amine and an acetal group. In certain embodiments, the acetal is a diethyl acetal. In certain embodiments, the third compound is 4,4-diethoxybutan-l -amine.

[0022] In certain embodiments, the fourth compound comprises a primary amine and a secondary amine. In certain embodiments, the fourth compound is N’-methylpropane-l^-diamine.

[0023] In certain embodiments, the fifth compound comprises a primary amine and a carbamimidothioate group. In certain embodiments, the fifth compound is 2-aminoethyl carb amimi dothi oate .

[0024] In certain embodiments, the fifth compound comprises a first primary amine and a secondary primary amine group. In certain embodiments, the fifth compound is 2-methylpentane-1,5-diamine.

[0025] In certain embodiments, the contacting is mixing 0.5 to 1.5 equivalents of the first compound, 0.05 to 0.20 equivalents of the second compound, 0.3 to 0.5 equivalents of the third compound, 0.1 to 0.3 equivalents of the fourth compound, and 1.5 to 5 or 1.5 to 10 or more equivalents of the fifth compound.

[0026] In certain embodiments, the polymer further comprises a therapeutic agent such as nucleobase polymer, peptide, an antibody, or small molecule drug. In certain embodiments, the nucleobase polymer is single or double stranded RNA, or DNA or. In certain embodiments, the DNA or RNA encode a therapeutic protein, e.g., mRNA. In certain embodiments, the RNA is an aptamer or antisense oligonucleotide.

[0027] In certain embodiments, this disclosure relates to methods of treating a subject for a disease or condition by administering or implanting a polymer reported herein comprising a nucleobase polymer, peptide, or small molecule drug a to a subject in need thereof. In certain embodiments, the polymer is administered through the mouth or nose to the lungs. In certain embodiments, the disease or conditions is a pulmonary or vascular disease, pathogenic, bacterial, viral infection, cancer, or hematological cancer.

[0028] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) Figure 1 A illustrates the preparation of P77 and P78 of this disclosure.

[0029] Figure IB illustrates the preparation of P79 and P80 of this disclosure.

[0030] Figure 1C illustrates the preparation of P81.

[0031] Figure ID shows chemical names for compounds and materials reported herein.

[0032] Figure 2A show data on the low dose screening polymers with cargo after nebulization of in mice lung collection using IVIS imaging.

[0033] Figure 2B shows data on the degree of branching in P81 was calculated from proton NMR. Degree of branching = (2D) / (2D+L), here D is the branching moiety peak integration values at 0.89 ppm and 1.86 ppm, and L is the linear amine values at 1.19 ppm. Peak integration values per proton at 2.26 ppm and 3.66 ppm were used to calculate the same for P76, respectively.

[0034] Figure 3 shows polyplex formation data and polymer to RNA ratio effect of mRNA translation across mRNA cargos.

[0035] Figure 4A shows data on buffering capacity. Hypotonic solutions increase uptake at mucosal surfaces. This data indicates that isothiourea end-capped polymers have strong protonation and maintain a constant pH irrespective of buffer concentration, which means that these polymers do not require a high buffering salt concentration (do not require a buffer having higher buffering capacity) to maintain a constant pH.

[0036] Figure 4B This shows data indicating that isothiourea end-capped polymers have strong protonation and maintain a constant pH irrespective of buffer concentration, which means that these polymers do not require a high buffering salt concentration to maintain a constant pH. At lower buffer salts, some polymers in polyplex could deprotonate, self-assemble and pull out the loaded mRNA. Since weakly protonated polymers will self-assemble in diluted buffers, buffer exchangeability of its polyplexes is not possible. Intrinsic self-assembly of the polymers in polyplex at low buffer salts destabilizes the polyplexes.

[0037] Figure 4C shows data indicating P81 expresses approximately 35-80 times more protein than P76 (Ij here) across a range of mRNA sizes using 100 ug of mRNA in the nebulizer.

[0038] Figure 4D shows data on the ratio of polymer to mRNA. The results indicate that a minimum 3-4:1 ratio of polymer to mRNA is sufficient to lead to 100% encapsulation of RNA. mRNA encoding for mAb-Nluc, Casl3-Nluc and VPR-Nluc were formulated with P81 at different polymer to mRNA ratios from 1:1 to 20: 1 and a Ribogreen™ assay was performed to quantify the percentage of free RNA in each condition.

[0039] Figure 4E shows data indicating improved mRNA delivery and protein production in lungs (mAb) in mouse. P81 admin 100 ug / animal in H2O. P76 admin 100 ug / animal in NaAc. Treated with mRNA encoding an mAb by nebulization to the lung. After 60 hr lungs were collect and analyzed by ELISA.

[0040] Figure 5A shows an experiment with an influenza challenge in a mouse by nebulization using P81 containing RNA encoding INFL-3 and Casl3.

[0041] Figure 5B shows data after infection.

[0042] Figure 6A illustrates an experiment to determine whether P81 is feasible for a severe Pneumonia aeruginosa infection. The subject animal is administered the bacteria. A nebulizer is used to administer P81 containing mRNA cargo to express cytosolic nLuc.

[0043] Figure 6B shows data indicating 100 pg neat formulation is not statistically different between PNA-infected and sham control groups. DETAILED DESCRIPTION

[0044] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution. "Embodiments" refer to an example, and it is contemplated that the embodiments are not necessarily limited to the example.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0046] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0047] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0048] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0049] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. Also, the term "or" is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term "or" means one, some, or all of the elements in the list.

[0050] As used herein, the term "about" is synonymous with the term "approximately." Illustratively, the use of the term "about" indicates that a value includes values slightly outside the cited values. Variation may be due to conditions such as experimental error, manufacturing tolerances, variations in equilibrium conditions, and the like. In some embodiments, the term "about" includes the cited value plus or minus 5% or 10%. In all cases, where the term "about" has been used to describe a value, it should be appreciated that this disclosure also supports the exact value.

[0051] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0052] "Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel character! stic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

[0053] A "subject" refers to any animal, preferably a human patient, livestock, rodent, monkey, or domestic pet.

[0054] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0055] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

[0056] The terms “amino” or “amine” refer to the chemical group -NH- or -NR- or -NH2 wherein R is any variety of chemical groups such as a hydrogen or alkyl group optionally substituted.

[0057] The terms “thiol” or “thio” or “mercapto” refer to the chemical group -SH or -S-.

[0058] A “beta-amino-ester” refers to a -(C=O)-CH2CH2-NR- chemical linkage and “beta-thiol-ester” refers to a -(C=O)-CH2CH2-S- chemical linkage. A “beta-amino-ester and beta-thiol-ester” polymer, or “beta-amino-thiol-ester” polymer refers to a polymer comprising monomers of both linkages.

[0059] The terms, a "linking group" or “linker” refers to any variety of molecular arrangements that can be used to bridge to molecular moi eties together. An example formula may be -Rn- wherein R is selected individually and independently at each occurrence as: -CRnRn-, -CHRn-, -CH-, -C-, -CH2-, -C(0H)Rn, -C(0H)(0H)-, -C(0H)H, -C(Hal)Rn-, -C(Hal)(Hal)-, -C(Hal)H-, -C(N3)Rn-, -C(CN)Rn-, -C(CN)(CN)-, -C(CN)H-, -C(N3)(N3)-, -C(N3)H-, -O-, -S-, -N-, -NH-, -NRn-, -(C=O)-, -(C=NH)-, -(C=S)-, -(C=CH2)-, which may contain single, double, or triple bonds individually and independently between the R groups. If an R is branched with an Rnit may be terminated with a group such as -CH3, -H, -CH=CH2, -CCH, -OH, -SH, -NH2, -N3, -CN, or -Hal, or two branched Rs may form a cyclic structure. It is contemplated that in certain instances, each “n” may be individually and independently at each occurrence 1, 2, 3, 4, 5, or 6. It is contemplated that in certain instances, the total Rs or “n” may be less than 100 or 50 or 25 or 10. Examples of linking groups include bridging amide groups, alkyl groups, polycyclic groups, aromatic rings, alkoxy groups, alkoxyalkyl groups, and combinations thereof.

[0060] A “hydrophobic group” is a chemical arrangement highly insoluble in water. A linking group is considered highly insoluble in water when the points of connections on the linker are replaced with hydrogen and the resulting compound has a solubility of less than 0.63 x 10'4% w / w (at 25 °C) in water, which is the percent solubility of octane in water by weight. See Solvent Recovery Handbook, 2ndEd, Smallwood, 2002 by Blackwell Science, page 195. Examples of naturally occurring hydrophobic linking groups include saturated or unsaturated, branched, or unbranched, hydrocarbon chains, mono or polycyclic aromatic or non-aromatic rings, or combinations thereof. The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule may be multiply substituted. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, or aryl.

[0061] As used herein, the term “small molecule” refers to any variety of covalently bound molecules with a molecular weight of less than 900 or 1000. Typically, the majority of atoms include carbon, hydrogen, oxygen, nitrogen, and to a lesser extent sulfur and / or a halogen. Examples include steroids, di- or tri-peptides, mono or polycyclic aromatic or non-aromatic, heterocyclic compounds.

[0062] The term "nucleic acid" refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements.

[0063] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates or provide additional features important to synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence (with T replaced by U) and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0064] As used herein, an “RNA” refers to a polymer of ribonucleic acid that may be naturally or non-naturally occurring. For example, an RNA may include modified and / or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. RNAs may be selected from the nonlimiting group consisting of small interfering RNA (siRNA), microRNA (miRNA), Dicersubstrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0065] The term “nucleobase polymer” refers to a polymer comprising nitrogen containing aromatic or heterocyclic bases that bind to naturally occurring nucleic acids through hydrogen bonding otherwise known as base pairing containing at least one chemical modification such that it is not naturally occurring. A typical nucleobase polymer is a nucleic acid, RNA, DNA, or chemically modified form thereof. A nucleobase polymer may contain DNA or RNA or a combination of DNA or RNA nucleotides or may be single or double stranded or both, e.g., they may contain overhangs, hairpins, bends, etc. Nucleobase polymers may contain naturally occurring or synthetically modified bases and backbones. In certain embodiments, a nucleobase polymer contains less than 50 or 100 bases.

[0066] Nucleobase polymers may be chemically modified, e.g., within the sugar backbone or on the 5’ or 3’ ends. The nucleobase polymers can be modified, for example, with 2'-amino, 2'-O-allyl, 2'-fluoro, 2'-O-methyl, 2'-methyl, 2'-H of the ribose ring, or a locked nucleic acid. Locked nucleic acid (LNA) refers to oligonucleotides that contain one or more nucleobases in which an extra methylene bridge fixes the confirmation sugar moiety, e.g., in the C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) conformation of ribose. Using locked nucleic acids within a nucleobase polymer typically increases the specific binding between a double stranded complex. In certain embodiments, the nucleobase polymer comprises locked monomers of l-(hydroxymethyl)-2,5-dioxabicyclo[2.2. l]heptan-7-ol or phosphorodiamidate morpholino oligomers (PMO) or a peptide nucleic acid (PNA).

[0067] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression systems. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In certain embodiments, this disclosure contemplates a vector encoding a peptide disclosed herein in operable combination with a heterologous promoter.

[0068] In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen (or any cell or organism attached to the antigen) and thereby targeting the antigen for degradation or elimination. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0069] In humans, from a structural standpoint, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. Alternatively, other animals produce antibodies from nucleic acids that encode a single protein. In humans, the heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs." “Bifunctional antibodies” refer to antibody that are bispecific, with each arm binding to a different antigen, and may be produced by biological or chemical methods.

[0070] "Single chain antibodies" refer to a single peptide containing naturally or non-naturally occurring sequences, including synthetically modified peptide sequences, derived from an antibody variable region that specifically binds an antigen of interest. Single chain antibodies are sometimes fragments or variants of naturally occurring mammalian antibodies. Such antibodies are sometimes referred to as single-domain antibodies (sdAbs or VHHs), or camelid single-domain antibodies, e.g., when derived from an animal of Camelidae family, e.g., lamas, camels. Other animals produce peptides based single chain antibodies that contain leucine rich repeats, e g. lamprey antibodies.

[0071] Beta-amino-ester and beta-thiol-ester polymers

[0072] Disclosed herein are beta-amino-thiol-ester polymers. In certain embodiments, the beta-amino-thiol-ester polymers are the reaction products of a multi-acrylated polyol comprising a hydrophobic linker, a branched compound having two or three or more terminal thiol groups, a compound comprising a primary amine and acetal or a primary amine and a morpholine group, and a compound comprising two amine groups, e.g., a primary amine and a secondary amine group. In certain embodiments, the previous components are reacted with a compound comprising a primary amine and a carbamimidothioate group or a compound comprising a first primary amine and a second primary amine group. In certain embodiments, methods of delivering a therapeutic agent to the lungs are done using said polymers, particles, or related materials. In certain embodiments, the beta-amino-thiol-ester polymers are made by the process of contacting: a first compound comprising a first acrylate polyol group and a second acrylate polyol group; a second compound comprising a first thiol group and a second thiol group; a third compound comprising a primary amine and a second group, wherein the second group is an acetal group or a morpholine; a fourth compound comprising two amine groups, e.g., a primary amine and a secondary amine group; and a fifth end-capping compound comprising a primary amine and a second chemical group, wherein the second chemical group is a carbamimidothioate group or the second chemical group is a second amine group; providing a beta-amino-thiol-ester polymer. In certain embodiments, the first through fourth compounds are heated prior to contacting with the fifth end-capping compound.

[0073] In certain embodiments, the first compound comprises a hydrophobic group linking the first acrylate polyol group, and the second acrylate polyol group. In certain embodiments, the hydrophobic group is a propane-2, 2-diyldibenzene group. In certain embodiments, the first compound is ((propane-2, 2-diylbis(4, l-phenylene))bis(oxy))bis(2-hydroxypropane-3, 1-diyl) di acrylate.

[0074] In certain embodiments, the second compound is 2-ethyl-2-(((3-mercaptopropanoyl)oxy)methyl)propane- 1,3 -diyl bis(3-mercaptopropanoate). In certain embodiments, the second compound is ethane- 1,2-dithiol. In certain embodiments, the second compound comprises a third thiol group.

[0075] In certain embodiments, the third compound comprises a primary amine and an acetal group. In certain embodiments, the acetal is a diethyl acetal. In certain embodiments, the third compound is 4,4-diethoxybutan-l -amine. In certain embodiments, the third compound comprises a primary amine and a second group, wherein the second group is a glycol, cyclic glycol, or morpholino group. In certain embodiments, the third compound is 2-morpholinoethane-l -amine.

[0076] In certain embodiments, the fourth compound comprises a primary amine and a secondary amine. In certain embodiments, the fourth compound is Is -m ethylpropane- 1,3 -diamine.

[0077] In certain embodiments, the fifth compound comprises a primary amine and a carbamimidothioate group. In certain embodiments, the fifth compound is 2-aminoethyl carb amimi dothi oate . In certain embodiments, the fifth compound comprises a first primary amine and a secondary primary amine group. In certain embodiments, the fifth compound is 2-methylpentane-1,5-diamine.

[0078] In certain embodiments, the contacting is mixing 0.5 to 1.5 equivalents of the first compound, 0.05 to 0.20 equivalents of the second compound, 0.3 to 0.5 equivalents of the third compound, 0.1 to 0.3 equivalents of the fourth compound, and 1.5 to 7 equivalents of the fifth compound.

[0079] In certain embodiments, the polymer further comprises a therapeutic agent such as nucleobase polymer, peptide, an antibody, or small molecule drug. In certain embodiments, the nucleobase polymer is RNA, mRNA, or DNA. In certain embodiments, the RNA or DNA encode a therapeutic protein. In certain embodiments, the RNA is an aptamer.

[0080] In certain embodiments, the polymers are loaded with a therapeutic agent. In certain embodiments, methods of delivering a therapeutic agent to the lungs are done using said polymers, particles, or related materials. In certain embodiments, the therapeutic agent is a nucleic acid, antisense oligonucleotide, aptamer, DNA, RNA, mRNA or vector encoding a therapeutic protein, antibody, single chain antibody, or bispecific antibody.

[0081] In certain embodiments, this disclosure relates to methods of treating a subject for a disease or condition by administering or implanting a polymer reported herein comprising a nucleobase polymer, peptide, or small molecule drug a to a subject in need thereof. In certain embodiments, the polymer is administered through the mouth to the lungs. In certain embodiments, the disease or conditions is a pulmonary or vascular disease, pathogenic, bacterial, viral infection, cancer, lung cancer or hematological cancer.

[0082] In certain embodiments, administration is by a nebulizer. In certain embodiments, is a jet nebulizer driven by compressed air. In certain embodiments, the nebulizer is an ultrasonic nebulizer having a piezoelectric transducer for creating droplets from a liquid reservoir. In certain embodiments, the nebulizer is vibrating mesh nebulizer having perforated membranes actuated by an annular piezo element that vibrates in resonant bending mode.

[0083] In certain embodiments, administration is by inhalation of an aerosol having a polymer reported herein in the pulmonary airway. In certain embodiments, administration is by inhalation of a polymer reported herein through the mouth and / or nose. In certain embodiments, administration is by a metered-dose inhaler. In certain embodiments, administration is by a single or multiple dose dry powder inhaler.

[0084] In certain embodiments, administration is by intratracheal instillation using a syringe. In certain embodiments, administration is by inhalation of a polymer reported herein through a nostril or mouth.

[0085] In certain embodiments, a polymer reported herein is administered daily or twice daily for more than one, two, three, four, five, or six week(s), or more than two months.

[0086] In certain embodiments, the subject is diagnosed with a bacterial infection or viral infection.

[0087] In certain embodiments, this disclosure relates to a container comprising a polymer disclosed herein and a propellant.

[0088] In some embodiments, the compounds are administered to the pulmonary tract (i.e., via pulmonary administration). In one specific embodiment, pulmonary administration comprises inhalation of the compounds, typically in the form of particles or droplets, such as by nasal, oral inhalation, or both. The particles or droplets can be administered in two or more separate administrations (doses).

[0089] In certain embodiments, particles may be formulated as an aerosol (e.g., liquid droplets of a stable dispersion or suspension of particles which include one or more of the compounds described herein in a gaseous medium). Particles delivered by aerosol may be deposited in the airways by gravitational sedimentation, inertial impaction, and / or diffusion. Any suitable device for generating the aerosol may be used, including but not limited to pressured meter inhalers (pMDI), nebulizers, dry powder inhalers (DPI), and soft-mist inhalers.

[0090] In certain embodiments, contemplated methods include inhalation of particles including one or more of the compounds described herein aerosolized via nebulization. Nebulizers generally use compressed air or ultrasonic power to create inhalable aerosol droplets of the particles or suspensions thereof. In this embodiment, the nebulizing results in pulmonary delivery to the subject of aerosol droplets of the particles or suspension thereof.

[0091] In certain embodiments, the subject to be treated is diagnoses with or is at risk of an adenovirus, herpesvirus, papilloma virus, polyomavirus, hepadnavirus, parvovirus, astrovirus, calcivirus, picornavirus, coronavirus, flavivirus, togavirus, hepevirus, retrovirus, orthomyxovirus, arenavirus, bunyaviruses, filovirus, paramyxovirus, rhabdovirus, reovirus, or poxvirus. In certain embodiments, the subject to be treated has or is at risk of human immunodeficiency virus (HIV), human herpesviruses varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and herpes simplex virus 1 (HSV-1), hepatitis C virus (HCV), influenza A and B viruses (IAV, IBV), Ebola virus, and SARS-CoV-2 virus.

[0092] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with an anti-viral agent such as abacavir, galidesivir, ganciclovir, vidarabine, acyclovir, adefovir, amantadine, ampligen, amprenavir, atazanavir, atripla, baloxavir marboxil, bictegravir, boceprevir, bulevirtide, cidofovir, cobicistat, combivir, daclatasvir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ibacitabine, ibalizumab, idoxuridine, imiquimod, imunovir, indinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, norvir, oseltamivir, penciclovir, peramivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, taribavirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, fumarate tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, umifenovir, valaciclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, zidovudine, or combinations thereof.

[0093] In embodiments, the infectious disease is a bacteria associated disease (e.g., tuberculosis, which is caused by Mycobacterium tuberculosis). Non-limiting bacteria associated diseases include pneumonia, which may be caused by bacteria such as Streptococcus and Pseudomonas; or foodbome illnesses, which can be caused by bacteria such as Shigella, Campylobacter, and Salmonella. Bacteria associated diseases also includes tetanus, typhoid fever, diphtheria, syphilis, and leprosy. In embodiments, the disease is Bacterial vaginosis (i.e. bacteria that change the vaginal microbiota caused by an overgrowth of bacteria that crowd out the Lactobacilli species that maintain healthy vaginal microbial populations) (e.g., yeast infection, or Trichomonas vaginalis); Bacterial meningitis (i.e. a bacterial inflammation of the meninges); Bacterial pneumonia (i.e. a bacterial infection of the lungs); Urinary tract infection; Bacterial gastroenteritis; or Bacterial skin infections (e.g. impetigo, or cellulitis). In embodiments, the infectious disease is a Campylobacter jejuni, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitides, Staphylococcus aureus, Streptococcus pneumonia, or Vibrio cholera infection.

[0094] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with an antibacterial agent such as 2,4-diaminopyrimidines, including baquiloprim, brodimoprim, iclaprim, ormetoprim, pyrimethamine, tetroxoprim, trimethoprim; aminocoumarins, including novobiocin; aminocyclitols, including spectinomycin; aminoglycosides, including amikacin, apramycin, arbekacin, bekanamycin, butirosin, dibekacin, dihydrostreptomycin, etimicin, fortimicins, astromicin, framycetin, gentamicin, hygromycin B, isepamicin, kanamycin, micronomicin, neomycin, netilmicin, paromomycin, plazomicin, ribostamycin, sisomicin, streptomycin, tobramycin; aminomethylcyclines, including omadacycline; amphenicols, including azidamfenicol, chloramphenicol, florfenicol, thiamphenicol; ansamycins, including rifabutin, rifampicin, rifapentine, rifaximin; antitubercular agents, including cycloserine, delamanid, ethambutol, ethionamide, morinamide, p-aminosalicylic acid (PAS), protionamide, pyrazinamide, terizidone, thioacetazone; bacteriocins, including nisin; b-lactam carbapenems, including loracarbef biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, sulopenem, tebipenem, tomopenem; b-lactam cephalosporins, including cefacetrile, cefaclor, cefadroxil, cefalexin, cefalonium, cefaloridine, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefcapene, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefoselis, cefotaxime, cefotiam, cefovecin, cefozopran, cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin, ceftaroline, ceftazidime, cefteram, ceftezole, ceftibuten, ceftiofur, ceftizoxime, ceftobiprole, ceftolozane, ceftriaxone, cefuroxime, cefuzonam; b-lactam cephamycin, including cefbuperazone, cefmetazole, cefotetan, cefoxitin; b-lactam monobactams, including aztreonam, carumonam, tigemonam; b-lactam oxacephems, including flomoxef, latamoxef, moxalactam; b-lactam penicillins, including amdinocillin, amoxicillin, ampicillin, apalcillin, azidocillin, azlocillin, bacampicillin, carbenicillin, ciclacillin, clemizole penicillin, cloxacillin, cyclacillin, dicloxacillin, epicillin, fenbenicillin, floxacillin, hetacillin, mecillinam, metampicillin, methicillin sodium, mezlocillin, nafcillin, oxacillin, penamecillin, penethamate hydroiodide, penicillin G, penicillin G benzathine, penicillin G procaine, penicillin N, penicillin O, penicillin V, phenethicillin potassium, piperacillin, pivampicillin, pivmecillinam, propicillin, quinacillin, sultamicillin, talampicillin, temocillin, ticarcillin; b-lactam in combination with b- lactamase inhibitors, including avibactam, clavulanic acid, relebactam, sulbactam, tazobactam, vaborbactam, zidebactam; bicyclomycins, including bicozamycin; bis-benzimidazoles including ridinilazole; cyclic esters, including fosfomycin; fluoroquinolones, including avarofloxacin, balofloxacin, besifloxacin, cinoxacin, ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin, finafloxacin, fleroxacin, flumequine, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, marbofloxacin, miloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin, prulifloxacin, rosoxacin, rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin, zabofloxacin; fusidane-type, including helvolic acid, fusidic acid and cephalosporin Pl; glycolipodepsipeptides, including ramoplanin; glycopeptides, including avoparcin, dalbavancin, norvancomycin, oritavancin, teicoplanin, telavancin, vancomycin; glycyl cy clines, including tigecycline; hybrids, oxazolidinone-quinolone, TD-1792 (glycopeptide-cephalosporin); lincosamides, including clindamycin, lincomycin, pirlimycin; lipopeptides, including daptomycin, surotomycin; macrolides, including azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, fidaxomicin, flurithromycin, gamithromycin, josamycin, kitasamycin, leucomycin, miocamycin, oleandomycin, primycin, rokitamycin, rosaramicin, roxithromycin, solithromycin, spiramycin, telithromycin, tildipirosin, tilmicosin, troleandomycin, tulathromycin, tylosin, tylvalosin; nitrofurans, including furaltadone, furazidin, furazolidone, nifuroxazide, nifurtoinol, nifurzide, nitrofural, nitrofurantoin, nitrofurazone; nitroimidazoles, including dimetridazole, metronidazole, omidazole, ronidazole, secnidazole, tinidazole; oligosaccharides, including avilamycin, everninomicin; polymyxins, including polymyxin E (colistin), polymyxin B; polypeptides, including amphomycin, bacitracin, capreomycin, enduracidin, enramycin, gramicidin(s), ristocetin, tuberactinomycin, tyrocidine, tyrothricin, viomycin; pseudomonic acids including mupirocin; quinoxalines including carbadox, olaquindox; sulphonamides including acetyl sulfamethoxypyrazine, chloramine-B, chloramine-T, dichloramine T, mafenide, sulfacetamide, sulfadiazine, sulfadimidine, sulfamethazine, sulfamethizole, sulfapyridine, sulfathiazole, sulfisomidine, sulfisoxazole; sulfones, including dapsone sodium, sulfanilic acid; tetracycline including chlortetracycline, clomocycline, demeclocycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, rolitetracycline, tetracycline; tri azaacenaphthylene. In certain embodiments, the subject to be treated is diagnoses with cancer, lung cancer or hematological cancer. In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with and anti cancer agent such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfdzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa-2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5 -fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

[0095] In certain embodiments, a polymer reported herein optionally comprises or comprises a nucleic acid encoding or is administered in combination with an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).

[0096] In certain embodiments, the subject is diagnosed with pulmonary fibrosis, bronchiectasis, or cystic fibrosis.

[0097] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with another active agent, such as a bronchodilator, corticosteroid, antimuscarinic, antibiotic, nintedanib, pirfenidone, or combinations thereof.

[0098] In certain embodiments, the bronchodilator is a beta-2 agonist, such as salbutamol, salmeterol, formoterol and vilanterol or an anticholinergic, such as ipratropium, tiotropium, aclidinium, or glycopyrronium, or an antimuscarinic such as atropine or scopolamine, or theophylline.

[0099] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with a bronchodilator such as albuterol, formoterol, or levalbuterol or salts thereof.

[0100] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with a mucolytic agent such as bromhexine or salts thereof.

[0101] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with is administered in combination with an anti-inflammatory agent such as a corticosteroid, fluticasone, or salts thereof.

[0102] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with an antibiotic agent such as macrolides, azithromycin, antipseudomonal, fluoroquinolones, ciprofloxacin, levofloxacin, ceftazidime, piperacillin and tazobactam, imipenem, aminoglycosides, aztreonam, tobramycin, colistin, colistimethate sodium, or salt thereof. In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with a cystic fibrosis drug such as lumacaftor, elexacaftor, ivacaftor, tezacaftor, cavosonstat, olacaftor, posenacaftor, galicaftor, navocaftor, deutivacaftor, nesolicaftor, or combinations thereof

[0103] In certain embodiments, a polymer reported herein optionally comprises or is administered in combination with other pharmaceutically active agents. These compounds include but are not limited to analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxiolytics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics, and anti-narcoleptics.

[0104] Specific examples of the pharmaceutically active agents that can be included in the polymer or adjunctively administered include, but are not limited to, aceclofenac, acetaminophen, atomoxetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amlodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine, beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone, bicifadine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicylate, citalopram, clomipramine, clonazepam, clonidine, clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine, corticosterone, cortisone, cyclobenzaprine, cyproheptadine, demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine, diazepam, dibenzepin, diclofenac sodium, difluni sal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimetacrine, divalproex, dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine, ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan, gabapentin, galantamine, gepirone, granisetron, haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone, hydroxyzine, ibuprofen, imipramine, indiplon, indomethacin, indoprofen, iprindole, ipsapirone, ketanserin, ketoprofen, ketorolac, lesopitron, levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline, mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine, meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone, methamphetamine, methocarbamol, methyldopa, methylphenidate, methyl salycylate, metoclopramide, mianserin, mifepristone, milnacipran, minaprine, mirtazapine, moclobemide, molindone, morphine, morphine hydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone, neurontin, nomifensine, nortriptyline, olanzapine, olsalazine, ondansetron, opipramol, orphenadrine, oxaflozane, oxaprozin, oxazepam, oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine, pemoline, pentazocine, pepsin, perphenazine, phenacetin, phendimetrazine, phenmetrazine, phenylbutazone, phenytoin, phosphatidyl serine, pimozide, pirlindole, piroxicam, pizotifen, pizotyline, pramipexole, prednisolone, prednisone, pregabalin, propranolol, propizepine, propoxyphene, protriptyline, quazepam, quinupramine, reboxetine, reserpine, risperidone, ritanserin, rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, salsalate, sertraline, sibutramine, sildenafil, sulfasalazine, sulindac, sumatriptan, tacrine, temazepam, tetrabenazine, thiazides, thioridazine, thiothixene, tiapride, taziprinone, tizanidine, tofenacin, tolmetin, toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine, trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid, venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone, zolmitriptan, zolpidem, zopiclone, and combinations thereof.

[0105] Protocol for making P81 and other examples P77, P78, P79, and P80

[0106]

[0107] The above drawing is illustrative of possible connections using components disclosed herein for preparing P81; however, the alternative connections are possible. In the example, R is a linking group containing a hydrophobic linker, i.e., propane-2, 2-diyldibenzene para substituted with an alkane polyol, propane-polyol (1,2,3-triol), wherein the terminal hydroxyl groups form the R esters. Preparation of P81 is described in detail below. P77, P78, P79, and P80 were prepared similarly by substituting the appropriate starting materials as illustrated in Figures 1A-1D.

[0108] A 25 mL flask was equipped with a magnetic stir bar and rubber septum. Bisphenol A glycerolate (1 glycerol / phenol) diacrylate (BDA) (567.5 mg, 1.17 mmol, 1 equiv) in a tube was add to 4.375 mL of anhydrous DMF and vortex to dissolve completely. The BDA solution was transferred into the flask. The compounds 4-aminobutyraldehyde diethyl acetal (76 mg (89 uL), 0.468 mmol, 0.4 equiv), and N1-methylpropane- 1,3 -diamine (20.625 mg (25 uL), 0.234 mmol, 0.2 equiv.) were add to the flask containing BDA solution. The mixture was stirred for 10 minutes at 40 °C under a nitrogen balloon. Under stirring, trimethylolpropane tri s(3 -mercaptopropionate) (46.6 mg (38.5 uL), 0.117 mmol, 0.1 equiv.) was added dropwise. The reaction mixture was stirred at 40 °C for 4 h and then stirred at 90 °C for 48 h under a nitrogen balloon. After 48 h, the reaction mixture was cooled to 40 °C.

[0109] Neutralized S-(2-aminoethyl)isothiourea dihydrobromide (842 mg, 7.02 mmol, 6 equiv) was added and the reaction mixture and was stirred at 40 °C for another 24 h for end-capping. 2-(2-Aminoethyl)isothiourea dihydrobromide was neutralized with triethyl amine in CHCh. 2-(2-Aminoethyl)isothiourea dihydrobromide (1.972 g), 7.02 mmol, 6 equiv) powder was mixed with triethyl amine (2.12 mL, 15.21 mmol, 13 equiv) in a 50 mL tube, and vortexed to mix well. CHCh, (10 mL) of was added, vortexed for 5-10 minutes, and allowed to settle. The product, 2-(2-ami noethyl )isothiourea, was separated as a lower layer from CHCh. The top CHCh layer was removed and transferred to the 2-(2-aminoethyl)isothiourea oil in a separating funnel. CHCh and 2-(2-aminoethyl)isothiourea were wash 7 times with (each time 10 mL). The final pale pink viscous liquid S-(2-aminoethyl)isothiourea was used for end-capping.

[0110] The polymers were isolated by dropwise precipitation into cold anhydrous diethyl ether containing 0.1% glacial acetic acid, vortexed and centrifuged at 1,000 x g for 1 min to pellet the polymer. The supernatant was discarded, and the polymer was washed thrice with fresh diethyl ether (free from glacial acetic acid) and dried under a vacuum for 6 h. The polymer was dissolved in 5 mL of methanol, and centrifuged (2100 x g). The methanol solution was transferred into a dialysis tubing (MWCO = 3.5kDa) and dialyzed against methanol for 24 h, (dialysis tubing was immersed in a 250 mb beaker containing methanol, 7-times menthol in the beaker was replaced with fresh methanol). The dialysate was added dropwise into diethyl ether, precipitated P81 was washed thrice with diethyl ether, dried under vacuum for 48h. The P81 obtained as white powder and stored at -20 °C.

[0111] The structures of the leading polymer candidates were consistent with proton NMR. The monomer composition in the P81 was used to calculate its degree of branching (Figure 2B). The degree of branching calculated from NMR shows that P81 has 1.6 times more branching than P76 due to P -thiopropionate bonds added from the tri-thiol monomer.

[0112] Discovering P81

[0113] PBAE (P76) was identified as a desirable non-viral vector to facilitate deliver mRNA to the lungs by nebulization with high expression, low toxicity and desirable degradability. However, a relatively low percentage of mRNA was delivered via nebulization. Using P76 also necessitated including acidic buffers to formulate and deliver the polyplexes resulting in eye, skin and respiratory tract irritation. Experiments were performed to identify an improved delivery vector that can be used in typical aqueous buffered conditions for efficient delivery.

[0114] Identifying chemically diverse ionizable monomers that can be incorporation into the P76 backbones by Michael addition reactions has led to the identification of improved pulmonary delivery polymer constructs. A polymer library was prepared by synthesizing chemically distinct polymers from combinations of different monomers and at different ratios. The pulmonary delivery potentials of the polymers were assessed by preparing the polymers with mRNA encoding aNLuc in 0.1 M sodium acetate buffer of pH 5 and nebulizing to the mice lungs. Based on the screening, newly identified polymer constructs show significantly better pulmonary delivery when compared to P76. Those leading polymer candidates are sequentially represented as P77, P78, P79, P80, and P81.

[0115] Multi-armed alkyl thiols cores were used to introduce intracellular pH-sensitive acid degradable P-thiopropionate linkages in the polymers. A tri-thiol monomer trimethylolpropane tris(3 -mercaptopropionate) construct P77 initially showed a 2.2-fold increase in pulmonary delivery than P76. Preparation of a pH-sensitive monomer 4-aminobutyraldehyde diethyl acetal (polymer P78 acetal) provided a 1.1 -fold increase. Although it is not intended that embodiments of this disclosure be limited by any particular mechanism, it is contemplated that the acetal has the ability to deoxygenate under intracellular acidic conditions by pH-sensitive hydrolysis thereby helping to pull out the loaded cargoes by breaking mRNA-polymer backbone interactions. The acetal can deoxygenate under intracellular acidic conditions by pH-sensitive hydrolysis. Although it is not intended that embodiments of this disclosure be limited by any particular mechanism, it is contemplated that intracellular pH-responsive 0 -thiopropionate branches and acetal linkages increase the intracellular degradability, such fragmentation-associated structural changes could pull out the loaded cargoes by breaking mRNA-polymer backbone interactions.

[0116] The polymer step-growth was terminated by end-capping agents. Screening lead to the identification of an end-capping monomer S-(2-aminoethyl)isothiourea which has chemical similarity to the guanidine sidechain of arginine, which is a constituent of cell-penetrating peptides. The S-(2-aminoethyl)isothiourea end-capped polymer P79 shows a 4.0-fold increase in pulmonary delivery. Combining the changed in P77 and P78 provide polymer P80, which showed a 5.5-fold increase. Replacing the end-capping agent of P80 with the one used in P79, (S-(2-aminoethyl)isothiourea), gave a salient hyperbranched polymer P81. Compared to P76, the harmony of monomers in P81 gives an unprecedented 46.2-fold increase in pulmonary delivery of aNLuc mRNA. P81 also showed an 18-fold improvement for ADI-14442 aNLuc, 29.6-fold Casl3 NLuc, and 18.1-fold VPRNLuc pulmonary delivery enhancement over P76, respectively.

[0117] P81 is an improvement over P76

[0118] P81 has 3 distinct chemical changes from P76, that synergize to dramatically improve delivery providing higher expression or the RNA cargo. These advantages were not predicted but found via screening expression in vivo in mice lungs. Both P81 and P79 have a desirable buffering capacity allowing polyplexes to be dissolved in hypotonic solutions (0.1 mM sodium acetate in water) and retaining a desirable pH. This is especially important for lung delivery as mucosal delivery is greatly enhanced using hypotonic solutions. Other advantages include improved nebulization and decreasing nebulization times to approximately 1 mg / min. The chemical combination and use in hypotonic solutions greatly improved the properties of the polymer.

[0119] The polyplexes can be formulated by hand or via microfluidic mixing and are compatible with tangential agents. An assay was developed to measure free RNA and total RNA which are used during manufacturing of the polyplexes (containing the drug substance). The polyplexes had stability at 4C for greater than 1 month, provide rapid sustained expression over 72 hours in the lungs, are compatibility with both jet and vibrating mesh nebulizers. Experiments indicate that more RNA is retained in the lung over time when compared to the P76 polymer.

[0120] Organ level expression was observed in mice, hamsters, and swine and tissue level mRNA expression in mice, hamsters and non-human primates (NHP), and expression occurs in human air-liquid-interface human cultures and in the presence of a pathogen (bacterial infection).

Claims

CLAIMS1. A beta-amino-ester and beta-thiol -ester containing polymer comprisingmonomers of hydrophobic linker disubstituted with alkane polyol;monomers of tri-branched terminal thiol alkanoate; andmonomers of an alkyl diamine.

2. The polymer of claim 1 further comprising monomers of an amine alkyl acetal.

3. The polymer of claim 1 further comprising monomers of an aminoalkyl carbamimidothioate.

4. The polymer of claim 1, wherein the hydrophobic linker is propane-2, 2-diyldibenzene.

6. The polymer of claim 1, wherein the alkane polyol is propane-polyol.

7. The polymer of claim 1, wherein the tri-branched terminal thiol alkanoate is tri-branched 2-ethyl-2-(((3-mercaptopropanoyl) oxy )methyl)propane- 1,3 -diyl bis(3-mercaptopropanoate).

8. The polymer of claim 1, wherein the alkyl diamine is N1-methylpropane- 1,3 -diamine.

9. The polymer of claim 1, wherein the amine alkyl acetal is 4,4-diethoxybutan-l -amine.

10. The polymer of claim 1, wherein the aminoalkyl carbamimidothioate is 2-aminoethyl carbamimidothioate.

11. A beta-amino-ester and beta-thiol-ester polymer made by process of contacting:a first compound comprising a first acrylate polyol group and a second acrylate polyol group;a second compound comprising a first thiol group and a second thiol group;a third compound comprising a primary amine and a second group, wherein the second group is an acetal group;a fourth compound comprising a primary amine and a secondary amine group; and a fifth compound comprising a primary amine and a carbamimidothioate group; providing a beta-amino-thiol-ester polymer.

12. The polymer of claim 11, wherein the first compound comprises a hydrophobic group linking the first acrylate polyol group, and the second acrylate polyol group.

13. The polymer of claim 12, wherein the hydrophobic group is a propane-2, 2-diyldibenzene group.

14. The polymer of claim 11, wherein the first compound is ((propane-2,2-diylbis(4,l-phenylene))bis(oxy))bis(2-hydroxypropane-3,l-diyl) diacrylate.

15. The polymer of claim 14, wherein in the second compound comprises a third thiol group.

16. The polymer of claim 15, wherein in the second compound is 2-ethyl-2-(((3-mercaptopropanoyl)oxy)methyl)propane- 1,3 -diyl bis(3-mercaptopropanoate).

17. The polymer of claim 15, wherein the third compound is an acetal.

18. The polymer of claim 17, the third compound is 4,4-diethoxybutan-l -amine.

19. The polymer of claim 11, wherein the third compound is 2-morpholinoethane-l -amine.

20. The polymer of claim 11, wherein the fourth compound is N'-methylpropane- 1 ,3-diamine.

21. The polymer of any of claims 11-20, wherein the fifth compound is 2-aminoethyl carbamimidothioate.

22. The polymer of claim 11, whereinthe first compound is ((propane-2,2-diylbis(4,l-phenylene))bis(oxy))bis(2-hydroxypropane-3, 1-diyl) diacrylate;the second compound is 2-ethyl-2-(((3-mercaptopropanoyl)oxy)methyl)propane- 1,3 -diyl bi s(3 -mercapt opropanoate);the third compound is 4,4-diethoxybutan-l -aminethe fourth compound is N^methylpropane- 1,3 -diamine; andthe fifth compound is 2-aminoethyl carbamimidothioate.

23. The polymer of any of claims 11-22, wherein contacting is mixing0.5 to 1.5 equivalents of the first compound,0.05 to 0.20 equivalents of the second compound,0.3 to 0.5 equivalents of the third compound,0.1 to 0.3 equivalents of the forth compound, and1.5 to 7 equivalents of the fifth compound.

24. The polymer of any of claims 1-23 further comprising a nucleobase polymer, peptide, small molecule drug, or other therapeutic agent.

25. The polymer of claim 24, wherein the nucleobase polymer is RNA or DNA.

26. The polymer of claim 25, wherein the RNA or DNA encode a protein.

27. A method of treating a disease or condition comprising administering or implanting a polymer as in any of claims 1-26 comprising a therapeutic agent to a subject in need thereof.

28. The method of claim 27, wherein the polymer is administered to the lungs.

29. The method of claim 27, wherein administration is by nebulization or aerosolization.

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