Patent Application
20200362016
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy of the Sequence Listing, which was created on Aug. 5, 2020, is named 11637N-181022.txt and is 70.1 kilobytes in size.
The presently-disclosed subject matter relates to articles and methods for targeted delivery of therapeutic during pregnancy. More particularly, the presently-disclosed subject matter relates to an elastin-like polypeptide (ELP) and methods of use thereof for targeted delivery of therapeutic agents to the placenta in a pregnant subject.
In the U.S. alone, there were 3.98 million births reported in 2015. According to the FDA, at least 50% of these women took at least one medication during pregnancy. However, special considerations must be taken when giving drug therapies to pregnant mothers. Not only must normal concerns of maximizing efficacy while reducing side effects in such subjects be considered, but the effects of the therapeutic agent on the developing fetus must also be taken into account. Many therapeutic agents that are otherwise safe for an adult will cross the placental barrier in pregnant mothers and cause severe adverse effects on the developing fetus.
In view thereof, the ability of different therapeutic agents to cross the placental barrier has been investigated, although the results have been highly variable. For example, while many large molecules such as proteins are prevented from passively crossing the placental barrier, some proteins, such as immunoglobulins, are actively transported across the placental barrier via the neonatal Fc receptor, FcRn, expressed on syncytiotrophoblasts. Transferrin also has receptors on trophoblasts and is actively transported across the placental barrier. The investigation of some high molecular weight (MW) drug carriers for placental transfer has similarly resulted in highly variable results. That is, some types of nanoparticles readily cross the placenta and some do not. Taken as a whole, these studies show that placental transfer isn't restricted by merely the size of macromolecular drug carriers, but is also dependent on the hydrophobicity and charge of the therapeutic agents.
Despite the uncertainty with respect to placental transfer, many of those drugs had no clinical trials in pregnant women. Moreover, pregnancy is often an exclusion criterion for clinical trials due to possible deleterious effects to the fetus. Drug development is additionally hindered because of risk aversion from the pharmaceutical industry and from regulatory bodies. The current strategy for evaluating the safety of drugs that might be used during pregnancy requires initial reproductive toxicity testing in the preclinical and early clinical phase and post-approval monitoring. During preclinical development, studies of developmental and reproductive toxicology (DART) are typically done in mice, rats and rabbits. Additionally, post-approval monitoring in pregnant subjects for drugs not restricted for use during pregnancy is often carried out as part of Phase IV studies. Still, actual patient data on effects of drugs during pregnancy is often scarce. Furthermore, direct drug development for adverse pregnancy-specific conditions is extremely limited. Nevertheless, the need to administer drugs during pregnancy, especially drugs for treatment of pregnancy-related conditions remains high.
Therefore, compositions and methods that reduce the amount of therapeutic agents crossing the placenta in a pregnant subject and which can be used to treat various diseases and disorders during pregnancy are both highly desirable and beneficial.
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
This summary describes several embodiments of the presently-disclosed subject matter, and, in many cases, lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.
In some embodiments, the presently-disclosed subject matter includes a placental region targeting elastin-like polypeptide (ELP) including between 5 and 671 repeat units having the sequence VPGXG, where X in each of the repeat units is individually selected from the group consisting of any amino acid except proline, and where the placental region targeting ELP is selected from the group consisting of a chorionic plate targeting ELP and a chorionic plate and labyrinth/junctional zones targeting ELP. In some embodiments, the ELP is a chorionic plate targeting ELP. In some embodiments, the ELP comprises up to 70 of the repeat units. In one embodiment, the ELP comprises between 5 and 70 of the repeat units. In some embodiments, the ELP comprises a molecular weight of up to 30 kDa. In one embodiment, the ELP comprises a molecular weight of between 3 kDa and 30 kDa. In some embodiments, at least 90% of the ELP accumulates in the chorionic plate.
In some embodiments, the ELP is a chorionic plate and labyrinth/junctional zones targeting ELP. In some embodiments, the ELP comprises at least 95 of the repeat units. In one embodiment, the ELP comprises between 95 and 671 of the repeat units. In some embodiments, the ELP comprises a molecular weight of at least 37 kDa. In one embodiment, the ELP comprises a molecular weight of between 37 kDa and 257 kDa. In some embodiments, at least 15% of the ELP accumulates in labyrinth/junctional zones of the placenta.
In some embodiments, the repeat units include V:G:A in a 1:4:3 ratio. In some embodiments, the ELP further comprising one or more of a group selected from a therapeutic agent or agents, a drug binding domain, a targeting domain, and a cell penetrating peptide.
Also provided herein, in some embodiments, is a method of treating a disease in a pregnant subject, the method including administering a placental region targeting elastin-like peptide (ELP) and a therapeutic drug to a subject in need thereof; where the ELP includes between 5 and 700 repeat units having the sequence VPGXG (SEQ ID NO: 1), where X in each of the repeat units is individually selected from the group consisting of any amino acid except proline; and where the placental region targeting ELP is selected from the group consisting of a chorionic plate targeting ELP, a chorionic plate and labyrinth/junctional zones targeting ELP, and a primarily labyrinth/junctional zones targeting ELP. In some embodiments, the placental region targeting ELP further comprises one or more of a group selected from a drug binding domain, a targeting domain, and a cell penetrating peptide.
Further provided herein, in some embodiments, is a method for decreasing the rate of clearance of an elastin-like polypeptide (ELP) from plasma or a tissue, the method including increasing the number of repeat units in the ELP.
Further features and advantages of the presently-disclosed subject matter will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.
SEQ ID NO: 1 is a ELP amino acid sequence VPGXG, where X can be any amino acid except proline.
SEQ ID NO: 2 is a ELP sequence of 32 repeats of the amino acid sequence VPGXG, where X is Val, Ala, and Gly in a 1:8:7 ratio
SEQ ID NO: 3 is a ELP sequence of 80 repeats of the amino acid sequence VPGXG, where X is Val, Ala, and Gly in a 1:8:7 ratio.
SEQ ID NO: 4 is a ELP sequence of 160 repeats of the amino acid sequence VPGXG, where X is Val, Ala, and Gly in a 1:8:7 ratio.
SEQ ID NO: 5 is a ELP sequence of 40 repeats of the amino acid sequence VPGXG, where X is Gly.
SEQ ID NO: 6 is a ELP sequence of 80 repeats of the amino acid sequence VPGXG, where X is Gly.
SEQ ID NO: 7 is a ELP sequence of 160 repeats of the amino acid sequence VPGXG, where X is Gly.
SEQ ID NO: 8 is a ELP sequence of 32 repeats of the amino acid sequence VPGXG, where X is Val, Ala, or Gly in a 1:4:3 ratio.
SEQ ID NO: 9 is a ELP sequence of 80 repeats of the amino acid sequence VPGXG, where X is Val, Ala, or Gly in a 1:4:3 ratio.
SEQ ID NO: 10 is a ELP sequence of 160 repeats of the amino acid sequence VPGXG, where X is Val, Ala, or Gly in a 1:4:3 ratio.
SEQ ID NO: 11 is a ELP sequence of 40 repeats of the amino acid sequence VPGXG, where X is Lys.
SEQ ID NO: 12 of a ELP sequence of 80 repeats of the amino acid sequence VPGXG, where X is Lys.
SEQ ID NO: 13 is a ELP sequence of 160 repeats of the amino acid sequence VPGXG, where X is Lys.
SEQ ID NO: 14 is a ELP-VEGF amino acid sequence, where a ELP sequence (SEQ ID NO: 4) fused to a C-terminal VEGF121 sequence.
SEQ ID NO: 15 is a SynB1-ELP-p50 amino acid sequence, where a SynB1 peptide fused to N-terminus of a ELP sequence (SEQ ID NO: 4), and a p50 peptide sequence fused to the C-terminus of the ELP sequence.
SEQ ID NO: 16 is a SynB1-ELP-NOX amino acid sequence, where a SynB1 peptide sequence fused to the N-terminus of a ELP sequence (SEQ ID NO: 4), and NOX peptide fused to the C-terminus of the ELP sequence.
SEQ ID NO: 17 is a NF-κB inhibitory peptide amino acid sequence.
SEQ ID NO: 18 is a NADPH oxidase inhibitory peptide amino acid sequence.
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. Further, while the terms used herein are believed to be well-understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
Some of the polynucleotide and polypeptide sequences disclosed herein are cross-referenced to GENBANK® accession numbers. The sequences cross-referenced in the GENBANK® database are expressly incorporated by reference as are equivalent and related sequences present in GENBANK® or other public databases. Also expressly incorporated herein by reference are all annotations present in the GENBANK® database associated with the sequences disclosed herein. Unless otherwise indicated or apparent, the references to the GENBANK® database are references to the most recent version of the database as of the filing date of this Application.
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 the presently-disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are now described.
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
The presently-disclosed subject matter relates to compositions and methods for therapeutic agent delivery during pregnancy. More particularly, the presently-disclosed subject matter relates to elastin-like polypeptides (ELPs), a composition comprising an elastin-like polypeptide (ELP) coupled to a therapeutic agent, and a method of using the composition to reduce the amount of the therapeutic agent crossing the placenta in a pregnant subject. As used herein, the term “elastin-like polypeptide” or “ELP” refers to a synthetic, or genetically engineered, protein containing structural peptide units, which may be repeating units, structurally related to, or derived from, sequences of the elastin protein.
In some embodiments, the ELP includes at least about 5 repeats of the amino acid sequence VPGXG (SEQ ID NO: 1). In some embodiments, the ELP includes up to about 671 repeats of SEQ ID NO: 1 (Table 1). In one embodiment, for example, the ELP sequences comprises about 5 repeats to about 671 repeats of SEQ ID NO: 1. The X position in each repeat of SEQ ID NO: 1 individually includes any amino acid except proline. For example, in one embodiment, the X position in each repeat of SEQ ID NO: 1 includes V, A, or G. In another embodiment, the X in SEQ ID NO: 1 is V, A, and Gin a ratio of 1:4-8:3-7.
Since ELPs are genetically engineered rather than chemically synthesized, the sequence and molecular weight thereof can be precisely controlled. As such, the composition and/or length of the ELP sequence may be modified through know methods, such as, but not limited to, recursive directional ligation. For example, in some embodiments, the composition and/or length of the ELP sequence may be modified to include therapeutic proteins or peptides, targeting proteins or peptides, cell penetrating peptides, reactive sites for chemical attachment of therapeutic agents, or a combination thereof. These modified ELPs form inert and biodegradable macromolecule carriers that have good pharmacokinetic profiles, very low immunogenicity, and can stabilize small proteins, small peptides, and/or small molecule therapeutic agent cargo in systemic circulation. Accordingly, when used as a delivery system for therapeutics, the ELPs disclosed herein provide certain therapeutic advantages to the therapeutic agent(s), such as, but not limited to, comparatively better stability, solubility, bioavailability, half-life, persistence, biological action of the therapeutic proteinaceous component or attached small molecule drug.
In some embodiments, the ELP includes a drug binding domain in place of or in addition to the fused and/or chemically attached therapeutic agent. The drug binding domain facilitates attachment of any suitable known or new small molecule therapeutic agent(s). In some embodiments, the drug binding domain is attached to the ELP carrier via a drug release domain to allow for selective release of the drug under particular environmental conditions or at specific sites within the body. In some embodiments, the drug binding domain improves delivery of the therapeutic agent. For example, the drug binding domain may improve the delivery of therapeutic agents to treat preeclampsia and other pregnancy related disorders, or to treat other diseases that happen to occur during pregnancy such as cancer. Additionally or alternatively, in some embodiments, the ELP coupled therapeutic system includes multiple copies of the therapeutic agent and/or drug binding domain to increase the amount of drug delivered. This may also include the use of two or more different therapeutic agents or different drugs attached to the ELP and/or drug binding domain(s) to achieve combination therapy. Other cases may include both a therapeutic agent/s and a drug binding domain/s to achieve simultaneous delivery of peptide/protein—based therapeutic agents with small molecule drugs.
Furthermore, as opposed to chemically synthesized polymers, the genetically engineered ELPs and ELP-fusion proteins disclosed herein may be expressed in E. coli or other recombinant expression systems. In some embodiments, this facilitates easy production and/or purification of large quantities of the molecules. For example, in some embodiments, the ELPs including the repeats of SEQ ID NO: 1 have a unique physical property called thermal responsiveness, where the polypeptide forms aggregates above a characteristic transition temperature and the aggregates re-dissolve below the transition temperature. When a lysate containing such recombinantly expressed ELPs is heated above the polypeptides' transition temperature the ELPs aggregate. These aggregated ELPs are then collected by centrifugation. Repeated centrifugation above and below the transition temperature leads to large quantities of very pure protein. As will be appreciated by those skilled in the art, the composition and/or length of the ELP sequence may be modified to influence the ELP's transition temperature (Tt), further facilitating ease of purification.
It has now been determined, that ELP does not cross the placenta, and that it can be used as a carrier for therapeutic peptides, antibiotics, and small molecule drugs in a manner that allows pregnant mothers to be treated with a therapeutic agent, while the amount of therapeutic agent crossing the placenta is reduced to thereby protect the developing fetus from damage by the therapeutic agent. Thus, in some embodiments of the presently-disclosed subject matter, the ELP is a therapeutic agent delivery vector that does not cross the placental barrier. As described in further detail below, this therapeutic agent delivery vector is capable of fusion to many types of therapeutic agents, including small molecules, antibiotics, therapeutic peptides, therapeutic proteins, and nucleic acids and allows those therapeutic agents to be stabilized in the maternal circulation, while also preventing them from entering the fetal circulation.
In some embodiments of the presently-disclosed subject matter, a method of delivering a therapeutic agent to a pregnant subject is provided. In some embodiments, an exemplary method includes administering to the pregnant subject an effective amount of a composition comprising ELPs disclosed herein coupled to one or more therapeutic agents. In some embodiments, ELPs with larger molecular weights accumulate in higher amounts in the placenta and/or are cleared slower from the plasma. In some embodiments, the method reduces the amount of the therapeutic agent crossing the placenta in a pregnant subject. Non-limiting examples of ELP which may be used in accordance with the presently-disclosed subject matter include ELPs having: about 32 repeats of SEQ ID NO: 1, where X is Val, Ala, and Gly in a 1:8:7 ratio (e.g., SEQ ID NO: 2); about 80 repeats of SEQ ID NO: 1, where X is Val, Ala, and Gly in a 1:8:7 ratio (e.g., SEQ ID NO: 3); about 160 repeats of SEQ ID NO: 1, where X is Val, Ala, and Gly in a 1:8:7 ratio (e.g., SEQ ID NO: 4); about 40 repeats of SEQ ID NO: 1, where X is Gly (e.g., SEQ ID NO: 5); about 80 repeats of SEQ ID NO: 1, where X is Gly (e.g., SEQ ID NO: 6); about 160 repeats of SEQ ID NO: 1, where X is Gly (e.g., SEQ ID NO: 7); about 32 repeats of SEQ ID NO: 1, where X is Val, Ala, or Gly in a 1:4:3 ratio (e.g., SEQ ID NO: 8); about 80 repeats of SEQ ID NO: 1 where X is Val, Ala, or Gly in a 1:4:3 ratio (e.g., SEQ ID NO: 9); about 160 repeats of SEQ ID NO: 1, where X is Val, Ala, or Gly in a 1:4:3 ratio (e.g., SEQ ID NO: 10); about 40 repeats of SEQ ID NO: 1, where X is Lys (e.g., SEQ ID NO: 11); about 80 repeats of SEQ ID NO: 1, where X is Lys (e.g., SEQ ID NO: 12); and about 160 repeats of SEQ ID NO: 1, where X is Lys (e.g., SEQ ID NO: 13). In some embodiments, the ELP sequence has an amino acid sequence selected from SEQ ID NOS: 2-13.
Additionally or alternatively, in some embodiments, the ELP is targeted to a specific region of the placenta. More specifically, the present inventors have surprisingly and unexpectedly found that specific sized ELP constructs target only particular regions of the placenta, while other sized ELP constructs simultaneously target multiple regions of the placenta. For example, an ELP composition having an ELP protein with a molecular weight of about 30 kDa or less, encoded by DNA containing about 70 or less repeat units of SEQ ID NO: 1, accumulates solely in the chorionic plate region of the placenta following administration. In contrast, an ELP composition having an ELP protein with a molecular weight of 47 kDa or greater, encoded by DNA containing greater than 120 repeat units of SEQ ID NO: 1, accumulates in the chorionic plate and labyrinth and junctional zone regions of the placenta following administration. Further increasing the size of the ELP to more than 200 repeat units of SEQ ID NO: 1, for example, increases the accumulation in labyrinth and junctional zone regions as compared to the ELP with 120 repeat units.
Accordingly, also provided herein are specifically targeted ELPs and methods of use thereof. In some embodiments, the targeted ELP is a chorionic plate targeting ELP. In some embodiments, the chorionic plate targeting ELP has a molecular weight of up to 30 kDa. For example, in one embodiment, the chorionic plate targeting ELP has a molecular weight of up to about 30 kDa, between 3 and about 30 kDa, or any combination, sub-combination, range, or sub-range thereof. In some embodiments, the chorionic plate targeting ELP includes up to 70 repeat units of SEQ ID NO: 1. For example, in one embodiment, the chorionic plate targeting ELP includes up to 70 repeat units, up to 63 repeat units, between 5 and 70 repeat units, between 31 and 63 repeat units of SEQ ID NO: 1, or any combination, sub-combination, range, or sub-range thereof.
In other embodiments, the targeted ELP is a chorionic plate and labyrinth/junctional zones targeting ELP. In some embodiments, the chorionic plate and labyrinth/junctional zones targeting ELP has a molecular weight of at least 40 kDa. For example, in one embodiment, the chorionic plate and labyrinth/junctional zones targeting ELP has a molecular weight of at least about 37 kDa, between about 50 and about 257 kDa, or any combination, sub-combination, range, or sub-range thereof. In some embodiments, the chorionic plate and labyrinth/junctional zones targeting ELP includes at least 95 repeat units of SEQ ID NO: 1. For example, in one embodiment, the chorionic plate and labyrinth/junctional zones targeting ELP includes at least about 95 repeat units, at least about 127 repeat units, between 127 and 671 repeat units of SEQ ID NO: 1, or any combination, sub-combination, range, or sub-range thereof.
Still further provided herein, in some embodiments, is a method of treating a disease or disorder in a pregnant subject, the method including administering one or more of the targeted ELPs to a subject in need thereof. In some embodiments, the method includes administering one or more of the chorionic plate targeting ELPs to a subject in need thereof. In some embodiments, the method includes administering one or more of the chorionic plate and labyrinth/junctional zones targeting ELPs to a subject in need thereof. In some embodiments, the method includes administering one or more of the primarily labyrinth/junctional zones targeting ELPs to a subject in need thereof. In some embodiments, the method includes administering a combination of one or more of the targeted ELPs disclosed herein. As will be appreciated by those skilled in the art, the selection of which targeted ELP will depend upon the specific disease or disorder being treated, as well as the therapeutic agent being used.
Turning now to the therapeutic agents that can be coupled to an exemplary ELP, various therapeutic agents known to those skilled in the art can be used in accordance with the presently-disclosed subject matter. As used herein, the terms “therapeutic agent” and “therapeutic agents” refer to any agent(s) that can be used in the “treatment” of a disease or disorder as defined herein below. In some embodiments, the therapeutic agent is selected from peptides, proteins, nucleic acids, antibodies, and small molecule drugs, or functional analogs thereof.
The terms “polypeptide,” “protein,” and “peptide,” which are used interchangeably herein, refer to a polymer of the 20 protein amino acids, or amino acid analogs, regardless of its size or function. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides, and proteins, unless otherwise noted. The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a gene product. Thus, exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
As used herein, the term “analog” refers to any member of a series of peptides having a common biological activity, including antigenicity/immunogenicity and antiangiogenic activity, and/or structural domain and having sufficient amino acid identity as defined herein.
As noted, in certain embodiments of the presently-disclosed subject matter, the therapeutic agents coupled to ELPs are those therapeutic agents that are desirable for introduction into the maternal circulation, but that should preferably be prevented from crossing the placenta and entering fetal circulation. Thus, in some embodiments, the compositions described herein are useful for delivery of any type of therapeutic agent that is regarded as harmful to fetal development. Such therapeutic agents include, but are not limited to, agents for the treatment of preeclampsia, chemotherapeutics, many drugs for cardiovascular diseases, anti-epileptic drugs, anti-emetic drugs, many immune modulating agents for autoimmune disorders, many drugs for endocrine disorders, certain antibiotics and antivirals, some anti-inflammatory agents, hormonal agents, and some analgesics. A partial list of drugs in pregnancy category X (i.e., drugs with known fetal toxicities) are listed in Table 2 below. In addition, many other drugs in pregnancy categories C or D, which are identified as having some risk in pregnancy can benefit from delivery by coupling the drugs to an exemplary ELP in accordance with the presently-disclosed subject matter.
Additionally, the presently-disclosed subject matter is not limited to delivery of small molecule drugs, but is also useful for delivery of peptide agents, therapeutic proteins, and antibodies. A partial list of such other types of agents that can be improved by ELP delivery during pregnancy is included in Table 3 below.
In some embodiments, the therapeutic agent coupled to the ELP and used in accordance with the presently-disclosed subject matter is a therapeutic agent useful for the treatment of preeclampsia, eclampsia, myocardial infarction, renovascular disease, spinocerebellar ataxia, lupus, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, cancer, Crohn's disease, ankylosing spondylitis, cardiac hypertrophy, plaque psoriasis, hypertension, atherosclerosis, stroke, kidney stones, Alzheimer's disease and other neurodegenerative disorders, prevention of allograft rejection, hepatic fibrosis, schizophrenia, muscular dystrophy, macular degeneration, pulmonary edema, chronic pulmonary hypertension, or other disorders where ROS are deleterious. In some embodiments, the therapeutic agent coupled to the ELP is selected from the therapeutic agents listed in Tables 2 and 3.
In certain embodiments, the therapeutic agent coupled to the ELP is an isoform of vascular endothelial growth factor (VEGF) (
In yet further embodiments, the therapeutic agent is an NF-κB inhibitory peptide (
In still other embodiments, the therapeutic agent coupled to the ELP is a NADPH oxidase inhibitory peptide (
In some embodiments, the therapeutic agent is a small molecule drug, where the size of the small molecule drug is less than 2,000 Dalton. In some embodiments, the small molecule drug is known to cause adverse events during pregnancy. Non-limiting examples of adverse events include teratogenicity, fetal growth restriction, embryotoxicity, or fetal demise. In some embodiments, the small molecules include pregnancy category C, D, or X drugs classified by the US FDA (Federal Register, Vol. 73, No. 104, May 29, 2008; Postmarket Drug Safety Information for Patients and Providers, Index to Drug-Specific Information). In some embodiments, the small molecule drug includes anti-hypertensive drugs. Non-limiting examples of the anti-hypertensive drugs include lovastatin, atorvastatin, pitavastatin, pravastatin, simvastatin, rosuvastatin, fluvastatin, aspirin, captopril, zofenopril, enalapril, ramipril, perindopril, quinapril, lisinopril, cilazapril, trandolapril, benazepril, imidapril, foninopril. In some embodiments, the small molecule drug includes anti-epileptic agents. Non-limiting examples of the anti-epileptic agents include phenytoin, valproate, phenobarbital, valproic acid, trimethadione, paramethadione, topiramate, carbamazepine, levetiracetam, lamotrigine. In some embodiments, the small molecule drug includes anti-emetic drugs. Non-limiting anti-emetic drugs include doxylamine, pyridoxine, prochlorperazine, chlorpromazine, promethazine, trimethobenzamide, ondansetron. In some embodiments, the small molecule drug includes cancer chemotherapeutics. Non-limiting examples of the cancer chemotherapeutics are taxanes including paclitaxel and decetaxel; vinca alkyloids including vinblastine, vincristine, venorelbine, and vinflunin; antimetabolites including methotrexate and 5-fluorouracil; topoisomerase inhibitors including doxorubicin, daunorubicin, epirubicin, etoposide, and camptothecin; cyclophosphamide or related alkylating agents.
Various means of coupling the ELP to therapeutic agents can be used in accordance with the presently-disclosed subject matter and are generally known to those of ordinary skill in the art. Such coupling techniques include, but are not limited to, chemical coupling and recombinant fusion technology. Depending on the particular coupling techniques utilized, in some embodiments, the number of ELPs or therapeutic agents per molecule, and their respective positions within the molecule, can be varied as needed. Further, in some embodiments, the therapeutic agent may further include one or more spacer or linker moieties, which in addition to providing the desired functional independence of the ELP and therapeutic agents, can optionally provide for additional functionalities, such as a protease-sensitive feature to allow for proteolytic release or activation of the therapeutic agent. Moreover, in certain embodiments, the therapeutic agent may be coupled to one or more targeting components such as, for example, a peptide or protein that targets the therapeutic agent to a particular cell type, e.g., a cancer cell, or to a particular organ, e.g., the liver.
To facilitate entry of the peptide compositions described herein into a cell where the therapeutic effect of the compositions can be exerted, in some embodiments, the polypeptide compositions further include a cell-penetrating peptide (CPP) sequence or an organ targeting peptide sequence that is coupled to the ELP.
As used herein, the term “cell penetrating peptide” refers to peptides sequences that facilitate cellular uptake of various agents, such as polypeptides, nanoparticles, small chemical molecules, and fragments of DNA. The function of the CPPs are to deliver the agents into cells, a process that commonly occurs through endocytosis with the cargo delivered to the endosomes of living mammalian cells. In some embodiments, the cell penetrating peptides or organ targeting peptides couple to the ELP carrier either through chemical linkage via covalent bonds or through non-covalent interactions. Non-limiting examples of the cell-penetrating peptide that can be coupled to the therapeutic agent or ELP include penetratin, Tat, SynB1, Bac, polyArg, MTS, Transportan, or pVEC.
The term “organ targeting peptide” refers to peptides designed to have specificity for the vascular beds or other cell types of specific organs. In some embodiments, the organ targeting peptide is selected from kidney targeting peptides, placenta targeting peptides, or brain targeting peptides.
Further provided, in some embodiments of the presently-disclosed subject matter are methods for the treatment of various diseases and disorders using the exemplary ELP-therapeutic agent-containing compositions described herein. In some embodiments, the presently-disclosed subject matter includes a method of treating a disease or disorder in a pregnant subject wherein the pregnant subject is administered an effective amount of a composition comprising an ELP coupled to a therapeutic agent, wherein the ELP is at least 5 repeats of SEQ ID NO: 1. Exemplary diseases or disorders that can be treated in accordance with the presently-disclosed subject matter include, but are not limited to, preeclampsia, eclampsia, myocardial infarction, renovascular disease, spinocerebellar ataxia, lupus, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, cancer, Crohn's disease, ankylosing spondylitis, cardiac hypertrophy, plaque psoriasis, hypertension, atherosclerosis, stroke, kidney stones, Alzheimer's disease and other neurodegenerative disorders, prevention of allograft rejection, hepatic fibrosis, schizophrenia, muscular dystrophy, macular degeneration, pulmonary edema, chronic pulmonary hypertension, or other disorders where ROS are deleterious.
As used herein, the terms “treatment” or “treating” relate to any treatment of a disease or disorder, including but not limited to prophylactic treatment and therapeutic treatment. As such, the terms “treatment” or “treating” include, but are not limited to: preventing a disease or disorder or the development of a disease or disorder; inhibiting the progression of a disease or disorder; arresting or preventing the further development of a disease or disorder; reducing the severity of a disease or disorder; ameliorating or relieving symptoms associated with a disease or disorder; and causing a regression of a disease or disorder or one or more of the symptoms associated with a disease or disorder.
For administration of a therapeutic composition as disclosed herein (e.g., an ELP coupled to a therapeutic agent), conventional methods of extrapolating human dosage based on doses administered to a murine animal model can be carried out using the conversion factor for converting the mouse dosage to human dosage: Dose Human per kg=Dose Mouse per kg×12 (Freireich, et al., (1966) Cancer Chemother Rep. 50: 219-244). Doses can also be given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions. Moreover, body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species as described by Freireich, et al. (Freireich et al., (1966) Cancer Chemother Rep. 50:219-244). Briefly, to express a mg/kg dose in any given species as the equivalent mg/sq m dose, multiply the dose by the appropriate km factor. In an adult human, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/m2.
Suitable methods for administering a therapeutic composition in accordance with the methods of the presently-disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and/or intraarterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper-velocity injection/bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site (see, e.g., U.S. Pat. No. 6,180,082).
Regardless of the route of administration, the compositions of the presently-disclosed subject matter are typically administered in an amount effective to achieve the desired response. As such, the term “effective amount” is used herein to refer to an amount of the therapeutic composition (e.g., en ELP coupled to a therapeutic agent, and a pharmaceutical vehicle, carrier, or excipient) sufficient to produce a measurable biological response. Actual dosage levels of active ingredients in a therapeutic composition of the present invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject and/or application. Of course, the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. Preferably, a minimal dose is administered, and the dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.
For additional guidance regarding formulation and dose, see U.S. Pat. Nos. 5,326,902; 5,234,933; PCT International Publication No. WO 93/25521; Berkow et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, N.J.; Goodman et al., (1996) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Health Professions Division, New York; Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Fla.; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New York; Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed. Mack Pub. Co., Easton, Pa.; and Speight et al., (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland/Philadelphia; Duch et al., (1998) Toxicol. Lett. 100-101:255-263.
In some embodiments of the presently-disclosed subject matter, the compositions described herein have been found to be particularly useful for the treatment of preeclampsia during pregnancy. However, it is contemplated that the exemplary compositions described are also useful not only for the treatment of a number of other diseases and disorders, but also both during pregnancy and in non-pregnant populations. For example, the ELP-delivered VEGF can be useful for treatment of myocardial infarction, renovascular disease, spinocerebellar ataxia, or other disorders in which VEGF levels are reduced. Additionally, the ELP-delivered NF-κB inhibitory peptide (SEQ ID NO: 17) could be useful for a variety of disorders with an inflammatory component, including lupus, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, cancer, Crohn's disease, ankylosing spondylitis, cardiac hypertrophy, plaque psoriasis, or other disorders in which NF-κB plays a central regulatory role. Moreover, the ELP-delivered NOX peptide could be used for hypertension, atherosclerosis, stroke, kidney stones, Alzheimer's disease and other neurodegenerative disorders, prevention of allograft rejection, hepatic fibrosis, schizophrenia, muscular dystrophy, macular degeneration, pulmonary edema, chronic pulmonary hypertension, or other disorders where ROS are deleterious.
In addition to the advantageous properties and uses described above, and without wishing to be bound by any particular theory, it is believed that the fusion of therapeutic agents to the ELP carrier provides many other advantages as well. For instance, in certain embodiments, ELP fusion increases the plasma half-life of therapeutic agents as many small molecule drugs, peptides, and therapeutic proteins are typically rapidly cleared from circulation by renal filtration. As another example, in some embodiments, ELP fusion increases the solubility of therapeutics as ELP fusion has been shown to increase the solubility of many poorly soluble therapeutics. As yet another example, in some embodiments, ELP fusion protects labile peptide therapeutics from degradation in vivo as ELP fusion provides a large sized carrier for labile therapeutics that protects them from enzymes that would degrade them (Bidwell G L, et al., 2013; Bidwell G L, 3rd, et al., 2012). Further, in some embodiments, ELP fusion decreases the immunogenicity of therapeutics that may be otherwise recognized as foreign by the immune system as ELP has been shown to be non-immunogenic and to decrease the immunogenicity of attached therapeutics.
As an additional example of the advantageous use of an ELP, in some embodiments, the ELP sequence can be easily modified to carry any desired protein or peptide, or to incorporate labeling sites for attachment of small molecules. Indeed, when an ELP is genetically encoded, and its coding sequence is inserted into a plasmid vector, doing so allows manipulation of the ELP sequence, and fusions of peptides and therapeutic proteins can be made by molecular biology techniques (Bidwell G L, 2012; Bidwell G L, et al., 2005; Bidwell G L, et al., 2010; Bidwell G L, 3rd, Wittom A A, et al., 2010; Massodi I, et al., 2005; Massodi I, et al., 2009; Meyer D E, 1999; Moktan S, et al., 2012; Moktan S, et al., 2012). Moreover, ELP can be purified after recombinant expression in bacteria. The genetically encoded nature of ELP also allows for expression in bacteria. Large amounts of ELP or ELP fusion proteins can be expressed recombinantly using E. coli-based expression systems. Additionally, ELP has the property of being thermally responsive. Above a characteristic transition temperature, ELP aggregates and precipitates, and when the temperature is lowered below the transition temperature, ELP re-dissolves. Therefore, purification of ELP after expression in bacteria can include heating the bacterial lysate above the transition temperature and collecting ELP or ELP fusion proteins by centrifugation. Repeated centrifugations above and below the transition temperature then results in pure ELP (Meyer D E, et al., 1999).
Furthermore, in some embodiments of the presently-disclosed subject matter, by using ELPs, ELPs can be targeted to desired tissues in vivo using targeting agents or peptides. As noted above, because of the ease of generating ELP fusions, ELP can be conjugated with any targeting agent, be it a peptide, small molecule, or antibody. Indeed, fusion with CPPs or organ targeting peptides can be used to not only increase cell and tissue uptake of ELPs, but also to direct ELP to specific tissues in vivo and even to specific intracellular compartments within a particular subject (Bidwell G L, et al., 2013; Bidwell G L, et al., 2009).
As used herein, the term “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided in accordance with the presently disclosed subject matter. As such, the presently-disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.
The practice of the presently-disclosed subject matter can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Polynucleotide Synthesis, M. J. Gait, ed., 1984; Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984; Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984; Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; See Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987; Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987; Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., 1986.
The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples.
ELP sequences were made by recursive directional ligation. A synthetic nucleotide cassette containing the coding sequence for 5 to 10 VPGXG (SEQ ID NO: 1) repeats with the desired amino acids at the X position and flanked by PflMI and BlgI restriction sites was cloned into the pUC19 vector at the EcoRI and HinDIII sites. The sequence of this construct was confirmed by DNA sequencing using standard M13 forward and reverse primers. Once one block of 5 to 10 VPGXG (SEQ ID NO: 1) repeats was inserted and confirmed, it was excised from pUC19 using PflMI and BglI restriction digestion and purified using agarose electrophoresis. A second aliquot of pUC19 containing the VPGXG (SEQ ID NO: 1) repeated sequence was linearized by digestion with PflMI only, and the gel purified cassette was ligated into the PflMI restriction site. This resulted in an in-frame fusion of the block of 5-10 VPGXG (SEQ ID NO: 1) repeats with a second block of 5 to 10 VPGXG (SEQ ID NO: 1) repeats, effectively doubling the number of ELP repeats. This process was repeated, doubling the ELP repeat number each time, until the desired molecular weight was reached. If necessary, smaller blocks (such as the original 5-10 repeat block) were used to increase the ELP repeat size in 5 to 10 block increments until the exact desired VPGXG (SEQ ID NO: 1) repeat number was achieved. The final ELP sequence was then excised from pUC19 using PflMI and BglI and inserted into a modified pET25b expression vector at an engineered SfiI site for recombinant protein expression.
N- and C-terminal modifications of ELP were made by cloning desired N- and/or C-terminal peptide or protein coding sequence into the pET25b expression vector between the NdeI and BamHI restriction sites. In all cases, the N- and/or C-terminal modifications were separated by an SfiI restriction site for later insertion of ELP. For peptide modification (such as CPPs, the NADPH oxidase inhibitory peptide (SEQ ID NO: 18), or the NF-κB inhibitory peptide (SEQ ID NO: 17)), the coding sequence for the peptides was generated as a synthetic oligonucleotide cassette with ends compatible with the desired restriction sites. For larger protein insertions, such as VEGF, the coding sequence was either commercially synthesized with E. coli-optimized codons and flanked by the desired restriction sites, or the coding sequence was amplified from human cDNA by PCR with custom primers used to add any necessary N- or C-terminal amino acids and to add the desired restriction sites. The intermediate constructs containing only the N- and/or C-terminal modifications in the pET25b vector were confirmed by DNA sequencing using the standard T7 promoter and T7 terminator primers. The desired ELP coding sequence was extracted from pUC19 using PflMI and BglI digestion and cloned into the modified pET25b vector at the engineered SfiI site. This resulted in in-frame fusions of ELP with the desired N- and/or C-terminal peptide or protein modifications. The final constructs were again confirmed by DNA sequencing.
ELPs and ELP fusion proteins were expressed and purified from E. coli BLR (DE3) or Rosetta2®(DE3) (for constructs resulting from human cDNA containing human-optimized codons). Briefly, 500 mL of TB Dry liquid culture media (MoBio) was inoculated with the expression strain and cultured at 37° C. with 250 rpm agitation for 16-18 h. In the absence of the pLysS lysozyme-expressing plasmid, the pET expression system allows for leaky production of the recombinant protein even without inducing agents. Bacteria were harvested by centrifugation and lysed by sonication (10×10 sec pulses, 75% amplitude, Fisher Sonic Dismembrator). Cell debris was removed by centrifugation, and nucleic acids were precipitated with 10% polyethylene imine and removed by centrifugation. NaCl was added to the soluble bacterial lysate to lower the ELP transition temperature (4 g/30 mL), and the lysate was heated to 42° C. to induce aggregation of the ELP-containing polypeptides. Polypeptides were collected by centrifugation at 42° C., the supernatant containing other soluble proteins was discarded, then the ELPs or ELP-fusion proteins were re-solubilized in ice cold PBS. Any remaining debris was removed after re-dissolving the ELP-containing proteins by centrifugation at 4° C. This heat-induced aggregation process was repeated two to three times to achieve purified ELP or ELP-fusion proteins. Purity of the resulting polypeptides was confirmed by SDS-PAGE analysis.
In order to test the hypothesis that ELP-fused therapeutics do not cross the placenta, an experiment was performed using the unmodified ELP carrier. Pregnant Sprague Dawley rats on day 14 of gestation were injected with fluorescently labeled ELP (100 mg/kg IV). Four hours after injection, which is about one half-life for this polypeptide, the rats were sacrificed and the placentas, pups, and major organs were removed for examination. Placentas and pups were dissected from the amniotic sacks and imaged ex vivo using an IVIS Spectrum animal imager to detect and quantitate the ELP levels. As shown in
This example also examined whether the addition of a CPP to ELP would affect its penetration across the placenta. SynB1-ELP was labeled with Alexa633 and injected as described above. For comparison, animals were injected with saline control or ELP-Alexa633 at an equivalent dose. Four hours after injection, placental, fetal, and organ levels were determined by ex vivo fluorescence imaging. As shown in
When using fluorescently labeled proteins, it is imperative that the label be stably bound in order to get accurate pharmacokinetic and biodistribution data. To determine the stability of the rhodamine label attached to the proteins via maleimide chemistry, the labeled protein is incubated in plasma from pregnant rats for various times at 37° C. After incubation, all proteins were precipitated using a 1:1 mixture with 10% trichloroacetic acid, and the fluorescence of the remaining supernatant was measured and compared to the pre-precipitation fluorescence. As shown in
In addition to measuring dye release in vitro, the degradation of the protein in plasma samples in vivo is also examined. Plasma from the pharmacokinetic experiment above was analyzed by SDS-PAGE using direct fluorescence imaging to detect the labeled protein. As shown in
The ex vivo whole organ analysis shown in
The placental tissue is also examined microscopically with a cytokeratin counterstain to detect trophoblast cells. Low magnification revealed that both ELP and SynB1-ELP accumulated highly at the chorionic plate (
Ex vivo whole organ and quantitative histological fluorescence analysis revealed that ELP and SynB1-ELP accumulate highly in the placenta but are excluded from the fetus four hours after bolus administration on GD14. Whether the fetal exclusion held after five days of continuous infusion of the polypeptides is also examined. ELP or SynB1-ELP was administered continuously from GD14 to GD19 using an IP minipump. As shown in
Ex vivo whole organ fluorescence analysis of the placentas is performed, pups, and organs on GD19 following five days of continuous polypeptide infusion. Relative to the acute experiment, the placental levels of the polypeptides were lower, which resulted from the difference in dose (100 mg/kg in the bolus dosing versus 30 mg/kg/day in the chronic infusion). However, similar to the acute data, the polypeptides accumulated at high levels in the placenta but were undetectable over autofluorescence in the pups (
In summary, this work has shown that the ELP and CPP-ELP carrier do not cross the placental barrier, even after five days of continuous infusion. These data demonstrate that a CPP can be used to direct intracellular delivery of the drug carrier within the placenta without affecting the penetration into the fetus.
The coding sequence for VEGF was amplified from a human cDNA for VEGF-A. The sequence was modified by addition of C-terminal amino acids to generate a sequence identical to VEGF121 in and to add restriction sites for cloning into the ELP expression vector. The coding sequence was cloned in frame with the ELP coding sequence to generate the ELP-VEGF chimeric construct. ELP-VEGF was expressed in E. coli BL21-Rosetta cells using the pET expression system with IPTG induction, and ELP-VEGF was purified by three to five rounds of inverse transition cycling (Bidwell G L, 3rd, et al., Mol Cancer Ther, 2005; Meyer D E, et al., 1999), taking advantage of the thermally responsive nature of ELP. The result was a 73 kDa protein that was very pure (
This example demonstrates that the ELP-VEGF was active and that ELP fusion did not alter the potency of VEGF. Proliferation of human umbilical vein endothelial cells (HUVECs) is stimulated when the cell are exposed to VEGF. As shown in
In addition to examining the ELP-VEGF activity in vitro, the pharmacokinetics (PK) and biodistribution of ELP-VEGF in comparison to free VEGF121 is also determined. Both free VEGF121 and ELP-VEGF were fluorescently labeled, and their PK and biodistribution were determined in mice after bolus intravenous administration. Free VEGF121 had a very rapid plasma clearance (
Whether ELP-VEGF was effective for lowering blood pressure in a rat model of preeclampsia is texted next. Pregnant rats at gestational day 14 (GD14) were subjected to surgery to reduce the blood flow to the placentas. It has previously been shown that this model, achieved by partially restricting the ovarian arteries and the dorsal aorta, results in a preeclampsia-like syndrome in the rat. The effects mirror human preeclampsia in that the rats develop hypertension, proteinuria, reduced renal function, fetal growth restriction, and some fetal loss. The model also induces molecular markers that mirror the human syndrome, including elevated sFlt-1 levels, increased pro-inflammatory cytokines, and increased placental reactive oxygen species. The hypertension associated with this model can be seen in
This investigation has developed an ELP-fused peptide inhibitor of activated NF-κB. NF-κB activation upon extracellular signaling is mediated by phosphorylation and release of the natural inhibitor I-κB from the NF-κB p50/p65 heterodimer. I-κB release exposes a nuclear localization sequence (NLS) on the p50 subunit of NF-κB, and once exposed, this NLS mediates nuclear import of NF-κB. Once inside the nucleus, NF-κB binds to response elements on its target genes and regulates gene expression. A synthetic cell permeable peptide containing the p50 NLS is capable of blocking the nuclear import of NF-κB upon stimulation in a variety of cell lines (Lin Y Z, et al., 1995). A copy of the p50 NLS is fused to the SynB1-ELP carrier and validated its activity using an in vitro NF-κB activation assay. Stimulation of cultured HUVECs with TNF-α leads to rapid activation of the NF-κB pathway, and this can be detected by monitoring nuclear localization of NF-κB (
TNFα stimulation also leads to the secretion of the vasoactive peptide endothelin-1 by HUVECs. This endothelin release contributes to the hypertension associated with the pro-inflammatory environment in preeclampsia. As shown in
To test whether the NF-κB inhibitory polypeptide had any effect on proliferation of normal tissue cell types, were determined its effects on proliferation of endothelial and chorionic cells. As shown in
Using pregnant Sprague Dawley rats, the pharmacokinetics and biodistribution of the SynB1-ELP-delivered p50 peptide with the free p50 peptide is determined. Rats were given a single bolus dose of 100 mg/kg of rhodamine-labeled SynB1-ELP-p50 or free p50, blood was sampled intermittently for four hours, and organs, placentas, and pups were removed for ex vivo fluorescence analysis. As shown in
The cell penetrating NADPH oxidase inhibitory polypeptide was generated by modifying the coding sequence for ELP with the addition of the coding sequence for the SynB1 CPP at its N-terminus and with the coding sequence for the NOX inhibitory peptide at its C-terminus. A DNA cassette encoding the SynB1 and NOX peptides separated by an SfiI restriction site and containing sticky ends compatible with NdeI and BamHI restriction sites was synthesized (Integrated DNA Technologies). The cassette was cloned into pET25b between the NdeI and BamHI restriction sites. The coding sequence for ELP was restricted from its pUC19 host vector using PflMI and BglI, the DNA was gel purified, and it was ligated into the SfiI site of the modified pET25b vector. The result was an in-frame fusion of SynB1, ELP, and the NOX peptide (SynB1-ELP-NOX). The final construct was confirmed by DNA sequencing and transformed into the BLR (DE3) expression strain (Novagen). A construct containing the SynB1 peptide fused to the N-terminus of ELP, but lacking the NOX peptide (SynB1-ELP) was generated in a similar manner. Polypeptides were purified by three to five rounds of inverse transition cycling.
It is confirmed that the SynB1-ELP-NOX polypeptide was internalized by cells. Both endothelial cells (HUVECs) and chorionic cells (BeWo choriocarcinoma cells) were exposed to fluorescently labeled SynB1-ELP-NOX for 1 h. The cells were then washed and given fresh media for 24 h. Internalization was confirmed by fluorescence microscopy as shown in
Next, the ability of the SynB1-ELP-NOX polypeptide to block ROS production in placental chorionic villous explants is demonstrated. Chorionic villous explants were cut on GD19 and cultured ex vivo on Matrigel coated wells with complete cell culture medium. After equilibration, culture medium was replaced with medium containing SynB1-ELP-NOX or the SynB1-ELP control polypeptides at 20 or 50 μM, and explants were incubated at 6% O2 (representing a healthy placenta) or 1% O2 (representing a preeclamptic placenta). After 48 h exposure to hypoxia, detection of ROS was performed using the dihydroethidium (DHE) assay. As shown in
For various diseases, it is often beneficial to deliver therapeutics to specific organs of interest. Organ targeting can increase the efficacy of the delivered therapeutic, and it can reduce off-target side effects. For treatment of preeclampsia, which mediated by factors produced in the placenta which act in systemic vascular beds and in the kidney, it would be beneficial to deliver pro-angiogenic, anti-inflammatory, or anti-oxidant therapeutics to both the placenta and the kidneys. ELP naturally accumulates at high levels in the kidneys, and
Examples 8-11 are directed to demonstrating the effects of molecular weight on the pharmacokinetics, biodistribution, and placenta deposition of ELP and defining the molecular weights best suited for placental drug delivery depending on the intra-placenta target. More specifically, Examples 8-11 show that in addition to an increased half-life and tissue accumulation with an increase in their molecular weights, specific sized ELP constructs were surprisingly and unexpectedly found to target only specific regions of the placenta. Alternatively, other sized ELP constructs simultaneously target multiple regions of the placenta. Beyond the specific application to placental drug delivery, these Examples also provide a detailed characterization of how ELP chain length affects the protein's pharmacokinetics and biodistribution during pregnancy, which is critical information when developing ELPs as drug carriers for other diseases and conditions of pregnancy.
ELP is a genetically engineered polypeptide consisting of repeated units of a five amino-acid motif, and it has a unique physical property called thermal responsiveness. Above a characteristic transition temperature, the polypeptide forms aggregates, while below the transition temperature, the aggregates re-dissolve. There are many advantages of using ELP for drug delivery. First, ELPs are genetically encoded rather than chemically synthesized. This means the user has absolute control over the ELP sequence and molecular weight (MW), and it allows the addition of targeting peptides and therapeutic peptides. Second, ELP and ELP-fusion proteins can be expressed in E. coli or other recombinant expression systems, allowing large quantities of the molecules to be purified easily because the polypeptide is thermally responsive. Purification of ELP-fusion proteins is achieved by heating a lysate containing the recombinantly expressed ELP above the polypeptides' transition temperature. This induces ELP aggregation, and it is collected by centrifugation. Repeated centrifugation above and below the transition temperature leads to large quantities of very pure protein. The third advantage of using ELP for drug delivery is that it is a large, non-immunogenic macromolecule. Therefore, ELP fusion can stabilize small protein or peptide or small molecule therapeutic agent cargo in systemic circulation, and targeting agents can be used to direct the ELP-fused therapeutics' biodistribution.
Starting with ELP, it was coupled to the therapeutic agent that may be a peptide or protein or protein fragment or small molecule drug known to have therapeutic activity in preeclampsia or other pregnancy related disease or condition. In addition to altering the physical properties of the ELP carrier itself, other attributes of the ELP coupled therapeutic agent are designed. To optimize the drug delivery to the placenta, in vivo targeting may be accomplished by the inclusion of targeting sequences or peptides on the ELP carrier coupled to the targeting agent. The targeting agent may be a peptide, protein, or small molecule with a specific molecular target in the placenta. Further, it also may also contain a cell penetrating peptide, other peptide, or protein capable of penetrating the cellular membrane.
Other modifications of the drug delivery system included a drug binding domain to allow attachment of known or new small molecule therapeutic agents to improve their delivery to treat preeclampsia and other pregnancy related disorders or to treat other diseases that happen to occur during pregnancy such as cancer. The drug binding domain may be attached to the ELP carrier via a drug release domain to allow for selective release of the drug under particular environmental conditions or at specific sites within the body. In other delivery vehicles, the ELP coupled therapeutic system includes multiple copies of the therapeutic agent and/or drug binding domain to increase the amount of drug delivered. This may also include the use of 2 or more different therapeutic agents or different drugs attached to the drug binding domain/s to achieve combination therapy. Other cases may include both a therapeutic agent/s and a drug binding domain/s to achieve simultaneous delivery of peptide/protein-based therapeutic agents with small molecule drugs.
The prior patent application described the use of the ELP drug delivery system for maternally sequestered drug delivery and for prevention of fetal drug exposure. In these embodiments, a library of ELPs with varying MW were evaluated (Table 4). The predicted molecular weight in the table are based on the primary amino acid sequence (the number following “ELP” in the protein name represents the number of repeats of SEQ ID NO: 1), as calculated by the ExPASy ProtParam tool. Transition temperature was measured by turbidity analysis. Hydrodynamic radius was measured by dynamic light scattering (adapted from Kuna, et al., Scientific Reports, 2018 May 21; 8(1):7923). The MW of the ELP protein has strong effects on the polymer's hydrodynamic radius, pharmacokinetics, and biodistribution.
A pharmacokinetic study was conducted in Sprague Dawley timed pregnant rats on gestational day 14 (GD14) to determine the effects of MW on plasma clearance of ELPs. This time point was chosen as it represents the first day of the third week of the rodent gestation, which is a commonly used time point to deliver therapeutics in rat models of preeclampsia drug development studies.
Animal studies were approved by the Animal Care and Use Committee of the University of Mississippi Medical Center and conducted according to the guidelines of the Guide for the Care and Use of Laboratory Animals. For pharmacokinetic and biodistribution experiments, three ELPs ranging in MW from 25 kDa (63 repeats of SEQ ID NO: 1) to 86 kDa (223 repeats of SEQ ID NO: 1) were used. Sprague Dawley timed pregnant rats on GD14 (Charles River) were anesthetized with isoflurane (1-3%, to effect) and injected with rhodamine-labeled polypeptides (1.5 μmol/kg) by intravenous injection into the femoral vein. Blood was sampled by tail prick at various time points for 4 hours, collected in Greiner Bio-One MiniCollect capillary blood collection tubes (Greiner Bio-One), and plasma was collected after centrifugation. Plasma samples were analyzed for concentration of the polypeptides using quantitative fluorescence analysis.
Plasma ELP levels were assessed with a two-way repeat measures ANOVA for factors of polypeptide treatment and time with a post hoc Tukey's multiple comparison. Organ biodistribution was assessed with a two-way ANOVA for factors of polypeptide treatment and organ type with post hoc Tukey's multiple comparison. The relationship between MW and organ levels was determined by Pearson correlation coefficient. Urine free dye and rhodamine levels were assessed by a one-way ANOVA for differences in polypeptide treatment with post hoc Tukey's multiple comparison. In all analyses, a p value of <0.05 was considered statistically significant. All statistical analysis and curve fitting was done using GraphPad Prism (Version 7.04).
The preparation of ELP proteins for the study was performed as follows. The synthesis of ELP expression constructs was performed by recursive directional ligation. pET25b+vectors encoding ELP proteins were transformed into E. coli BLR (DE3). All proteins were purified by inverse transition cycling. Fluorescent labeling of ELP proteins was accomplished by labeling each ELP protein on its N-terminal cysteine residue using a maleimide conjugate of rhodamine.
After preparation, three ELPs with MWs of 25, 50, and 86 kDa were administered by bolus IV injection. Blood was sampled at various time points up to 4 h after bolus i.v. injection. Based on direct fluorescence measurement of plasma, an increase in MW of these proteins resulted in slower plasma clearance (
The trends in plasma clearance are consistent with our observations for these proteins in non-pregnant mice, in which we measured 48 hours of plasma clearance data. In
For tissue biodistribution studies, the same animals from the pharmacokinetic study were euthanized while still under anesthesia. The placenta, pups and major organs were collected and measured by whole organ fluorescence imaging and biodistribution analysis (n=4 rats per agent). Major organs and placentae and their associated pups (n>5 per animal) were imaged ex vivo using an IVIS Spectrum. Total organ fluorescence was quantified and fit to standard curves of the appropriate ELP to correct for any differences in labeling levels among polypeptides.
Representative images of major organs, placentae, and pups for each of the treatment groups are shown in
Tissue biodistribution of ELP constructs in a rat pregnancy model was examined as follows. The change in fluorescence levels in the tissue accumulation of labeled ELPs having varying MW was determined using data was quantified relative to standard curves of the injection protein using the images in
The ELP proteins used in the study accumulated strongly in the kidneys regardless of their MW, followed by the liver and placenta (
ELP levels in individual major organs was examined as a function of MW were fit by linear regression using GraphPad Prism. In
Urine samples collected prior to euthanasia four hours after protein injection were analyzed by quantitative fluorescence analysis. The fluorescence intensity of 2 μl of urine was measured in a plate reader before and after trichloroacetic acid (TCA) precipitation. Post-precipitation levels were corrected for dilution and compared to pre-precipitation fluorescence to calculate percentage of free dye. Urine samples were analyzed for creatinine levels using QuantiChrom Creatinine Assay Kit (DICT-500, BioAssay Systems) per Manufacturer's instructions. Urine samples were also analyzed by SDS-PAGE. 10 μl of each urine sample was prepared with Bolt LDS sample buffer 4× (Novex, Thermo Fisher Scientific), analyzed on a Bolt 4-12% Bis-tris Plus gel, and visualized by direct fluorescence imaging using an IVIS Spectrum (PerkinElmer) using 535-nm excitation and 580-nm emission filters and small binning, followed by Coomassie Brilliant Blue staining.
Urine was collected from the animals at the time of organ harvest, four hours after bolus i.v. injection of fluorescently labeled proteins before and after precipitation of the proteins with TCA. Urine fluorescence was fit to a standard curve, corrected for rhodamine and creatinine concentration, and assessed by one-way ANOVA with post hoc Tukey's multiple comparison (F(2, 9)=5.706, p=0.0251). Rhodamine levels in the urine corrected by creatinine were significantly different between ELP-127 and ELP-223, as assessed by one-way ANOVA with post hoc Tukey's multiple comparison (
Analysis of fluorescence before and after TCA precipitation revealed that the urine contained 78, 72, and 69% free fluorophore for ELP-63, ELP-127, and ELP-223 (
SDS-PAGE analysis of urine samples with direct fluorescence imaging is shown in
Gel electrophoresis with direct fluorescence detection revealed that no intact ELP was present in the urine (
To investigate the localization of different MW ELP proteins in the placenta after administration, sections of intact feto-amnio-placental units were imaged by confocal microscopy. One amniotic sac, containing a pup and its placenta, was removed from each animal and kept intact. These feto-amnio-placental units were then embedded in freezing medium (Tissue-Plus O.C.T Compound) and flash frozen in isopentane on dry ice. Placentae and pups were cut into 20 μm sections with a cryostat. Kidneys were also embedded, flash frozen and cut into 14 μm sections. Slides were equilibrated to room temperature, and unprocessed tissue sections were imaged by confocal microscopy image stitching using a 561-nm laser and a 10× magnification objective. The same imaging settings were maintained for all samples. Brightness was adjusted equally among all groups to allow for better visualization while still maintaining quantitative differences in fluorescence levels.
In
It is demonstrated that, due to an increase in ELP plasma half-life combined with the large blood pool in the placenta, accumulation of ELP carriers in maternal organs, especially in the placenta, is increased with an increase in ELP MW. Surprisingly, as shown in
For quantitative measurements, intra-placental ELP levels were determined from whole-slice imaging of intact feto-amnio-placental units. Not only were the overall placental levels dependent on the size of the ELP protein, the distribution within the placenta also varied by ELP size. As shown in
In conclusion, all of the ELPs had significant accumulation at the chorionic plate, however the ELP-63 was found to be predominantly localized in the chorionic plate and not present throughout the other placenta regions. For the delivery of at least 90% of therapeutics to the chorionic plate of the placenta, an ELP including 70 repeat units of SEQ ID NO: 1 or less, about 30 kDaltons in size, is required. In contrast, larger MW ELPs, ELP-127 and ELP-223, were distributed throughout the labyrinth and junctional zone rather than being only in the chorionic plate region. The largest MW ELP, ELP-223, having the highest placental level was broadly distributed throughout the labyrinth and junctional zone, indicating that larger MW ELP have increasing levels in the labyrinth and junctional zone. To provide a therapeutic of least 15% distribution to the labyrinth and junctional zones of the placenta, the preferred ELP requires 95 repeat units of SEQ ID NO: 1 or a size of about 37 kDaltons or greater. The differences will play a major role in delivering adequate amounts of the therapeutic to the critical areas of the placenta.
Throughout this document, various references are mentioned. All such references are incorporated herein by reference, including the references set forth in the following list.
This application claims priority to U.S. Provisional Application Ser. No. 62/826,447, filed Mar. 29, 2019, and is a continuation-in-part of U.S. patent application Ser. No. 16/104,037, filed Aug. 16, 2018, which claims priority from U.S. patent application Ser. No. 14/917,460, filed Mar. 8, 2016, which is a national phase application of International Patent Application No. PCT/US2014/058640, filed Oct. 1, 2014, which claims priority to U.S. Provisional Application Ser. No. 61/885,300, filed Oct. 1, 2013, the entire disclosures of which are incorporated herein by this reference.
This presently-disclosed subject matter was made with government support under grant numbers NIH R01HL121527 and R01HL137791 awarded by National Institutes of Health. The government has certain rights in it.
| Number | Date | Country | |
|---|---|---|---|
| 62826447 | Mar 2019 | US | |
| 61885300 | Oct 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14917460 | Mar 2016 | US |
| Child | 16104037 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16104037 | Aug 2018 | US |
| Child | 16835041 | US |
