The red fluorescence of rhodamine B and the green fluorescence of dextran FITC from the same Z-section and their merged image are shown in each panel. The enlarged images of a typical cell s are shown in each case in order to show the intracellular distribution of the internalized peptides.
All images were recorded in the same Z-section. The indicated cell lines were treated with DMSO. Figure 16 displays scheme showing the reversible cyclization strategy for delivering linear peptidyl cargos into mammalian cells. GSH, glutathione. Figure 17 displays A Synthesis of disulfide-bond cyclized peptide. B Synthesis of thioether-bond cyclized peptide.
Trt, trityl; Mmt, methoxytrityl. E Structures of Amc 7-aminomethylcourmarin containing caspase. Images in different fluorescence channels were all recorded in 30 the same Z-section. B CAP fluorescence from A after subtraction of background fluorescence untreated cell. Figure 20 displays a comparison of the proteolytic stability of peptides 1 and 2. I, green fluorescence of internalized peptide 8; II, overlay of green peptide fluorescence and blue nuclear stain. D Immunofluorescent staining showing the. P values were calculated from two-tailed t-test.
Figure 23 displays a schematic of the evolution of a cell-permeable PTP1B inhibitor. Figure 25 displays the competitive inhibition of PTP1B by monocyclic peptide inhibitor 2. Figure 27 displays the solid-phase synthesis of inhibitor 4. Figure 28 displays a comparison of the serum stability of monocyclic PTP1B inhibitor 2 and bicyclic inhibitor 4. Figure 29 of global pY protein levels in A cells after treatment with PM inhibitor 4 for 2 h. Figure 30 displays the conversion of impermeable Pin1 inhibitor into a cell- permeable bicyclic inhibitor. Figure 31 displays the FA analysis of the binding of Pin1 inhibitor to Pin1.
Figure 32 displays the competition for binding to Pin1 by inhibitors 5 and 7. Each 15 reaction contained 0. Figure 33 displays the cellular uptake of Pin1 inhibitors. All images were recorded at the same Z-section. After 2 h, the peptide solution was removed, and the cells were washed with DPBS, treated with 0. Finally, the cells were resuspended in the flow cytometry buffer and analyzed by flow cytometry BD FACS Aria , with excitation at nm. The plate was incubated at 37 qC overnight and the absorbance of the formazan product was measured at nm on a Molecular Devices Spectramax M5 plate reader.
Each experiment 5 was performed in triplicates and the cells untreated with peptide were used as control. Figure 36 displays the evolution of bicyclic peptide inhibitors against Pin1. The structural moieties derived from library screening are shown in grey, while the changes made during optimization are shown in light grey. Figure 37 discplays the characterization of peptide MFI, mean fluorescence intensity; none, untreated cells no peptide. The compounds, compositions, and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the30 disclosed subject matter and the Examples and Figures included therein.
Before the present compounds, compositions, and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into 5 this application in order to more fully describe the state of the art to which the disclosed matter pertains.
The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. It will be further understood that the endpoints of 30 each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Thus, the subject can be a human or veterinary patient. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.
Prevent does not require comparison to a 20 control as it is typically more absolute than, for example, reduce. As used herein,. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms.
This term includes active treatment, that is, treatment directed specifically toward the. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, 5 pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
Such amelioration only requires a reduction or alteration, not necessarily elimination. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e. Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.
The cargo moiety can comprise one or more detectable moieties, one or more 5 therapeutic moieties, one or more targeting moieties, or any combination thereof. In some examples, the cell penetrating peptide moiety and cargo moiety together are cyclic. In some examples, the cell penetrating peptide moiety is cyclic and the cargo moiety is appended to the cyclic cell penetrating peptide moiety structure. In some examples, the cargo moiety is cyclic and10 the cell penetrating peptide moiety is cyclic, and together they form a fused bicyclic system.
The cell penetrating peptide moiety can comprise five or more, more specifically six or more, for example, six to twelve, or six to nine amino acids. When there are six to nine amino acids the com n n f F rm l I. Wheren there are more than 9 amino acides, Formula I can have m and p each be 1 and n can be 2 or more, e. In some examples three or more amino acids are 20 arginine and one or more are phenylalanine.
In still other examples one or more amino acids is naphthylalanine or tryptophan. The curved line can be a covalent bond in the backbone of the peptide i. In some examples, the cell penetrating peptide moiety and cargo moiety together are15 cyclic, and the com ounds are of Formula IIa:. In some examples, the cargo moiety is cyclic and the cell penetrating peptide moiety is cyclic, and together they form a fused bicyclic system, and the compounds are of Formula IIc:.
The cell penetratig peptide moeity comprises at least 5, more specifically, at least amino acids, even more specifically from from 6 to 12, from 6 to 9, from 6 to 7, from 7 to 8, from 8 to 9, and more specifally 6, 7, 8, or 9 amino acids. For the endocyclic motif, at least 5 amino acids can be used.
It is also disclosed herein that for the endocyclic structure, some amino acids in the penetrating peptide moiety can also be part of the cargo moity. In this case, the two Arg residues perform dual functions. Thus, in some cases the sequence of the cargo moiety is taken into account when referring to the peptide penetrating moiety.
For the exocyclic motif, at least 6 amino acids can be used with, for example, glutamine being used to attach the cargo. Non-natural amino acids can also be the D-isomer of the natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof.
These, and others, are listed in the Table 1 along with their abbreviations used herein. Table 1. Amino Acid Abbreviations. The amino acids can be coupled10 to the cargo moiety at the amino group, the carboxylate group, or the side chain. In some examples of Formula I, at least one amino acid comprises napthylalanine or tryptophan, or analogues or derivatives thereof.
In some examples of Formula I, at least three of the amino acids independently comprise arginine or an analogue or derivative thereof. In some examples of Formula I, at least one amino acid comprises phenylalanine, phenylglycine, or histidine, or analogues or derivatives thereof. In some examples of Formula I, at least one amino acid comprises glutamine or an analogue or derivative thereof. In some examples, the cell penetrating peptide can be the reverse of any of the sequences listed in Table 2. In some examples, the cell penetrating peptide sequence can be a cyclic form of any of the sequences listed in Table 2.
CPP sequences— linear or cyclic. Peptide variants are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional variants. Deletions are characterized by the removal of one or more amino acid residues from the peptide sequence. Typically, no more than from 1 to 3 residues are deleted at any one site within the peptide.
Amino acid substitutions are typically of single residues, but can occur at a number of different. Deletions or insertions preferably are made in adjacent pairs, i. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a final construct. Substitutional variants are those in which at least one residue has been 20 removed and a different residue inserted in its place.
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Such substitutions generally are made in accordance with the following Table 3 and are referred to as conservative substitutions. Table 3. Amino Acid Substitutions. Ile replaced by leu or val Nal replaced by Trp or Phe Substantial changes in function are made by selecting substitutions that are less conservative than those in Table 3, i.
The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which a a hydrophilic residue, e. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include. Such conservatively substituted variations of each explicitly.
Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level. Derivatives are formed by replacing one or more residues with a modified residue, where the side chain of the residue has been modified. Additional examples are shown in Tables 6 and 18 and include variants thereof.
The cargo moiety can comprise any cargo of interest, for example a linker moiety, a 5 detectable moiety, a therapeutic moiety, a targeting moiety, and the like, or any combination thereof. In some examples, the cargo moiety can comprise one or more additional amino acids e. In some examples the cargo moiety can comprise any of those listed in Table 4, or derivatives or combinations thereof. Detectable moiety. Examples of suitable detectable labels include, but are not limited to, a UV-Vis label, a near-infrared label, a luminescent group, a phosphorescent group, a magnetic spin resonance label, a.
In some embodiments, the label is detectable without the addition of further reagents. In some embodiments, the detectable moiety is a biocompatible detectable moiety, 5 such that the compounds can be suitable for use in a variety of biological applications. Examples of suitable luminophores include, but are not limited to, metal porphyrins; benzoporphyrins; azabenzoporphyrine; napthoporphyrin; phthalocyanine;.
The detectible moiety can be attached to the cell penetrating peptide moiety at the amino group, the carboxylate group, or the side chain of any of the amino acids of the cell 15 penetrating peptide moiety e. The disclosed compounds can also comprise a therapeutic moiety. In some examples, the cargo moiety comprises a therapeutic moiety.
The detectable moiety can be 20 linked to a therapeutic moiety or the detectable moiety can also serve as the therapeutic moiety. Therapeutic moiety refers to a group that when administered to a subject will reduce one or more symptoms of a disease or disorder. The therapeutic moiety can comprise a wide variety of drugs, including antagonists, for example enzyme inhibitors, and agonists, for example a transcription factor which 25 results in an increase in the expression of a desirable gene product although as will be appreciated by those in the art, antagonistic transcription factors can also be used , are all included.
The therapeutic moiety can, for example, comprise an anticancer agent, antiviral agent, antimicrobial agent, anti-inflammatory agent, immunosuppressive agent, anesthetics, or any combination thereof. The therapeutic moiety can comprise an anticancer agent. The therapeutic moiety can also comprise a biopharmaceutical such as, for example, an. In some examples, the therapeutic moiety can comprise an antiviral agent, such as ganciclovir, azidothymidine AZT , lamivudine 3TC , etc.
In some examples, the therapeutic moiety can comprise an antibacterial agent, such as acedapsone; acetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillin 25 pivoxil; amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacin sulfate;. In some examples, the therapeutic moiety can comprise an anti-inflammatory agent. In some examples, the therapeutic moiety can comprise dexamethasone Dex.
In other examples, the therapeutic moiety comprises a therapeutic protein. For example, some people have defects in certain enzymes e. The disclosed cell 5 penetrating peptides have been tested with proteins e. The targeting moiety can comprise, for example, a sequence of amino acids that can target one or more enzyme domains.
In some examples, the targeting moiety can comprise an 10 inhibitor against an enzyme that can play a role in a disease, such as cancer, cystic fibrosis, diabetes, obesity, or combinations thereof. For example, the targeting moiety can comprise any of the sequences listed in Table 5. Table 5. Example targeting moieties. The targeting moitiey and cell penetrating peptide moiety can overlap, that is residues that form the cell penetrating peptide moiety can also be part of the sequence that 5 forms the targeting moiety, and vice a versa.
The therapeutic moiety can be attached to the cell penetrating peptide moiety at the amino group, the carboxylate group, or the side chain of any of the amino acids of the cell penetrating peptide moiety e. In some examples, the therapeutic moiety can be attached to the10 detectable moiety. Ras is a protein that in humans is encoded by the RAS gene. The normal Ras protein 15 performs an essential function in normal tissue signaling, and the mutation of a Ras gene is implicated in the development of many cancers.
Mutated forms of Ras have been implicated in various cancers, including lung cancer, colon cancer, pancreatic cancer, and various 20 leukemias. PTP1B is a negative regulator of the insulin signaling pathway, and is considered a promising potential therapeutic target, in particular for the treatment of type II diabetes.
PIP1B has also been25 implicated in the development of breast cancer.
Pin1 is an enzyme that binds to a subset of proteins and plays a role as a post phosphorylation control in regulating protein function. Pin1 activity can regulate the outcome of proline-directed kinase signaling and consequently can regulate cell. Deregulation of Pin1 can play a role in various diseases. Inhibitors of Pin1 can have therapeutic implications for cancer and immune disorders. Grb2 is an adaptor protein involved in signal transduction and cell communication. The Grb2 protein contains one SH2 domain, which can bind tyrosine phosphorylated sequences.
Grb2 is widely expressed and is essential for multiple cellular functions. Inhibition of Grb2 function can impair developmental processes and can block. Cell , 63, ; Kerem, BS et al. Science , , 10 , by reducing its proteasome-mediated degradation Cushing, PR et al. Thus, disclosed herein is a method for treating a subject having cystic fibrosis by administering an effective amount of a compound or composition disclosed herein. The compound or composition administered to the subject can comprise a therapeutic moiety that can comprise a targeting moiety that can act as an inhibitor against 15 CAL PDZ.
Also, the dcompositions or compositions disclosed herein can be administered with a molecule that corrects the CFTR function. In some examples, the compounds can be of Formula I: In some examples of Formula I, m, n, and p are 0 and the compounds are of Formula I In some examples of Formula I, m is 1, and n and p are 0, and the compounds are of Formula I In some examples of Formula I, m and n are 1, p is 0, and the compounds are of Formula I In some examples of Formula I, m, n, and p are 1, and the compounds are of Formula I In some examples of Formula Ia, m, n, and p are 1, and the compounds are of 5 Formula Ia In some examples, the cell penetrating peptide moiety and cargo moiety together are cyclic, and the com ounds are of Formula IIa:.
In some examples, the cell penetrating peptide moiety is cyclic and the cargo moiety is appended to the cyclic cell penetrating peptide moiety structure, and the compounds are10 of Formula IIb:. In some examples, the cargo moiety is cyclic and the cell penetrating peptide moiety 10 is cyclic, and together they form a fused bicyclic system, and the compounds are of Formula IIc:. Table 7. Previously reported cell penetrating peptides. Also disclosed herein are compositions comprising the compounds described herein. Pharmaceutically-acceptable salts include salts of the disclosed compounds that are prepared with acids or bases, depending on the particular substituents 15 found on the compounds.
Under conditions where the compounds disclosed herein are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts can be appropriate. Examples of pharmaceutically-acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salt. Examples of physiologically-acceptable acid addition salts include hydrochloric,.
Thus, disclosed herein are the hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, 25 tartarate, malonate, ascorbate, alpha-ketoglutarate, alpha-glycophosphate, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of a compound can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
Alkali metal for example, sodium, potassium or lithium or alkaline earth metal for 5 example calcium salts of carboxylic acids can also be made. The compounds described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art.
The compounds described herein can be prepared from readily available starting 10 materials. Optimum reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art. Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be 15 changed.
Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co. Other materials, such as the pharmaceutical carriers disclosed herein can be obtained from commercial sources.
Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be 5 substantially nonreactive with the starting materials reactants , the intermediates, or. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic 10 means, such as nuclear magnetic resonance spectroscopy e.
Suitable protecting groups are 9-fluorenylmethyloxycarbonyl Fmoc , t-butyloxycarbonyl Boc ,. The 9- fluorenylmethyloxycarbonyl Fmoc protecting group is particularly preferred for the synthesis of the disclosed compounds. Other preferred side chain protecting groups are, for 25 side chain amino groups like lysine and arginine, 2,2,5,7,8-pentamethylchromansulfonyl pmc , nitro, p-toluenesulfonyl, 4-methoxybenzene- sulfonyl, Cbz, Boc, and. Suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used.
One method for coupling to the deprotected 4 2',4'-dimethoxyphenyl Fmoc-aminomethyl phenoxy-acetamidoethyl resin is O-benzotriazolyl-N,N,N',N'- tetramethyluroniumhexafluorophosphate HBTU, 1 equiv. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer. Each protected amino acid is then introduced in about 3-fold molar excess, and the coupling is preferably carried out in DMF. At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either in successively or in a single operation.
Removal of the polypeptide and deprotection can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thianisole, water, ethanedithiol and trifluoroacetic acid. Alternatively, the. The protected peptide can be purified at this point or taken to the next step directly. The removal of the side chain protecting groups can be accomplished using the cleavage cocktail described above.
The fully deprotected peptide can be purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin acetate form ; hydrophobic adsorption chromatography on underivitized polystyrene-divinylbenzene for example, Amberlite XAD ; silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; 5 partition chromatography, e.
Also provided herein are methods of use of the compounds or compositions 10 described herein. Also provided herein are methods for treating a disease or pathology in a subject in need thereof comprising administering to the subject an effective amount of any of the compounds or compositions described herein. The methods include administering to a subject an effective amount of one or more 15 of the compounds or compositions described herein, or a pharmaceutically acceptable salt thereof.
The compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating cancer in humans, e. The disclosed methods can optionally include identifying a patient who is or can be in need of treatment of a cancer. The one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be administered in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart.
The administration of the one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be by the same or different routes. When treating with one or more additional agents, the compounds and compositions or pharmaceutically acceptable 10 salts thereof as described herein can be combined into a pharmaceutical composition that includes the one or more additional agents.
For example, the compounds or compositions or pharmaceutically acceptable salts thereof as described herein can be combined into a pharmaceutical composition with an additional anti-cancer agent, such as cis-Retinoic Acid, 2-AminoMercaptopurine, 2- 15 CdA, 2-Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine,. Many tumors and cancers have viral genome present in the tumor or cancer cells.
The compounds disclosed herein can also be used alone or in combination with anticancer or antiviral agents, such as ganciclovir, azidothymidine AZT , lamivudine 3TC , etc.
The compounds disclosed herein can also be used in combination with viral based treatments of oncologic disease. The method includes contacting the tumor cell with an effective amount of a compound or composition as described herein, and optionally includes the step of irradiating the tumor cell with an 10 effective amount of ionizing radiation.
As used herein, the term ionizing radiation refers to radiation comprising particles or photons that have sufficient energy or can produce sufficient energy15 via nuclear interactions to produce ionization. An example of ionizing radiation is x- radiation. An effective amount of ionizing radiation refers to a dose of ionizing radiation that produces an increase in cell damage or death when administered in combination with the compounds described herein.
The ionizing radiation can be delivered according to methods as known in the art, including administering radiolabeled antibodies and. The methods and compounds as described herein are useful for both prophylactic and therapeutic treatment. As used herein the term treating or treatment includes prevention; delay in onset; diminution, eradication, or delay in exacerbation of signs or symptoms after onset; and prevention of relapse. For prophylactic use, a therapeutically 25 effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset e.
Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of an infection. Prophylactic 30 administration can be used, for example, in the chemopreventative treatment of subjects presenting precancerous lesions, those diagnosed with early stage malignancies, and for subgroups with susceptibilities e. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after cancer is diagnosed.
In some examples of the methods of treating of treating, preventing, or ameliorating cancer or a tumor in a subject, the compound or composition administered to the subject can 5 comprise a therapeutic moiety that can comprise a targeting moiety that can act as an. The disclosed subject matter also concerns methods for treating a subject having a metabolic disorder or condition.
In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject having a metabolic 10 disorder and who is in need of treatment thereof. In some examples of the methods of treating of treating, preventing, or ameliorating the metabolic disorder in a subject, the compound or composition administered to the subject can comprise a therapeutic moiety that can comprise a targeting moiety that can act as an inhibitor against PTP1B.
In one particular 15 example of this method the subject is obese and the method comprises treating the subject for obesity by administering a composition as disclosed herein. In one embodiment, an effective amount of one or more compounds or compositions disclosed herein is administered to a subject having an immune 20 disorder and who is in need of treatment thereof. In some examples of the methods of. The disclosed subject matter also concerns methods for treating a subject having 25 cystic fibrosis. In one embodiment, an effective amount of one or more compounds or. In some examples of the methods of treating the cystic fibrosis in a subject, the compound or composition administered to the subject can comprise a therapeutic moiety that can comprise a targeting moiety that can act as an inhibitor against30 CAL PDZ.
In vivo application of the disclosed compounds, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, 5 such as by injection.
Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art. The compounds disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and 10 biodegradable containers.
These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The compounds can also be administered in their salt derivative forms or crystalline forms. The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail 15 in a number of sources which are well known and readily available to those skilled in the art.
Martin describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration 20 of the compound.
The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically- 25 acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents.
To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0. Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried lyophilized condition requiring only the condition of the sterile liquid carrier, for example, water for 5 injections, prior to use.
Extemporaneous injection solutions and suspensions can be. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety.
Another means for delivery of compounds and compositions disclosed herein to a cell comprises attaching the. Patent No. Application Publication Nos. Application Publication No. Compounds can also be incorporated into polymers, examples of which include poly D-L lactide-co-glycolide polymer for intracranial tumors; poly[bis p-carboxyphenoxy propane:sebacic acid] in a molar ratio as used in GLIADEL ; chondroitin; chitin; and chitosan.
For the treatment of oncological disorders, the compounds disclosed herein can be 25 administered to a patient in need of treatment in combination with other antitumor or. These other substances or treatments can be given at the same as or at different times from the compounds disclosed herein. For example, the compounds disclosed herein can be used in combination with mitotic inhibitors such as 30 taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide,.
In certain examples, compounds and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth such as 5 a tumor site or benign skin growth, e. Compounds and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a. They can be enclosed in hard or soft shell gelatin capsules, can be. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.
Oral compositions can be tablets, troches, pills, capsules, and the like, and can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as 20 sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol.
Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, 25 pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active30 compound can be incorporated into sustained-release preparations and devices. Compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection.
Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include 5 sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for 10 example, water, ethanol, a polyol for example, glycerol, propylene glycol, liquid. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
Optionally, the prevention of the action of microorganisms can be 15 brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.
In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of25 the active ingredient plus any additional desired ingredient present in the previously sterile- filtered solutions. For topical administration, compounds and agents disclosed herein can be applied in as a liquid or solid.
However, it will generally be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which 30 can be a solid or a liquid. Compounds and agents disclosed herein can be applied directly to the growth or infection site. Preferably, the compounds and agents are applied to the growth or infection site in a formulation such as an ointment, cream, lotion, solution, tincture, or the like. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area 10 using pump-type or aerosol sprayers, for example.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
The dosage ranges for the administration of the compositions are those large enough 20 to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any.
Dosage can vary, and can be administered in one or more dose. Also disclosed are pharmaceutical compositions that comprise a compound disclosed herein in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an amount 30 of a compound constitute a preferred aspect.
The dose administered to a patient,. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition health of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition. Also disclosed are kits that comprise a compound disclosed herein in one or more containers. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In another embodiment, a kit includes one or more anti- cancer agents, such as those agents described herein.
In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. The following examples are set forth to illustrate the methods and results according to the disclosed subject matter.
These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations which25 are apparent to one skilled in the art. Efforts have been made to ensure accuracy with respect to numbers e. There are numerous variations and combinations of 30 reaction conditions, e. Only reasonable and routine experimentation will be required to optimize such process conditions. Example 1. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the.
Its cargo capacity was examined with a wide variety of molecules including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. The overall delivery efficiency i. The plasma membrane presents a major challenge in drug discovery, especially for 20 biologics such as peptides, proteins and nucleic acids.
Since the initial observation that HIV trans-activator of transcription, Tat, internalizes into mammalian cells and activates viral replication in the late s Frankel, AD and Pabo, CO. Cell, , 25 55, a large number of CPPs consisting of residues have been reported. FEBS Lett. Drug Delivery Rev. Life Sci. Bioconjugate Chem. Nucleic Acids Res. Science, , , , and nanoparticles Josephson, L et al. Bioconjugate 5 Chem. Biomacromolecules, , 2, , into mammalian cells and tissues through either covalent attachment or electrostatic association.
Many CPPs display minimal toxicity and immunogenicity at physiologically relevant concentrations Saar, K et al. Chembiochem, , 5, have been found to increase stability and cytosolic delivery. Despite three decades of investigation, the fundamental basis for CPP activity 15 remains elusive. Two distinct and non-mutually exclusive mechanisms have been proposed for the CPPs whose primary sequences are characterized by having multiple arginine residues. In the first mechanism direct membrane translocation , the arginine guanidinium groups interact with phospholipids of the plasma membrane to generate neutral ion pairs that passively diffuse across the membrane Herce, HD and Garcia, AE.
In the second mechanism, CPPs associate with cell surface glycoproteins and membrane phospholipids, internalize into cells through endocytosis Richard, JP et al. Fittipaldi, A et al. Biochemistry, , 46, and subsequently exit from endosomes into the cytoplasm. Traffic, , 8, However, the mechanism s of entry and the efficiency of uptake may vary with the CPP identity, cargo, cell type, and other factors Mueller, J et al.
Unfortunately, the endosomal membrane has proven to be a significant barrier towards cytoplasmic delivery by these CPPs; often a negligible fraction of the peptides escapes into the cell interior El-Sayed, A et al. Recently, two 10 new types of CPPs with improved endosomal escape efficiencies have been discovered. Appelbaum et al. It has also been found that cyclization of certain arginine-rich CPPs enhances their cellular uptake Qian, Z et al.
ACS Chem. Like the miniature proteins displaying the penta-arginine motif Appelbaum, JS et al. Louis, MO. Cell culture media, fetal bovine serum FBS , penicillin-streptomycin, 0. Lipofectamine were purchased from Invitrogen Carlsbad, CA. Peptide Synthesis and Labeling. Peptides were synthesized on Rink Resin LS 0. The typical coupling reaction contained 5 equiv of Fmoc-amino acid, 5 equiv of 2- 7-aza-1H-benzotriazoleyl -1,1,3,3-tetramethyluronium hexafluorophosphate HATU and 10 equiv of diisopropylethylamine DIPEA and was 5 allowed to proceed with mixing for 75 min.
After the addition of the last N-terminal. The peptides were deprotected and released from the resin by treatment with. The peptides were triturated with cold ethyl ether 3x and purified by reversed-phase HPLC on a C18 column. To generate rhodamine- and Dex-labeled peptides Figure 2 , an N İ - 20 4-methoxytrityl-L-lysine was added to the C-terminus.
After the solid phase peptide. The resin was incubated with Lissamine rhodamine B sulfonyl. The peptides were fully deprotected, triturated with diethyl ether, and purified by HPLC. The peptide was then deprotected, triturated, and purified by HPLC. Stanford, SM et al. Cell Culture and Transfection. SUVs were prepared by 15 modifying a previously reported procedure Magzoub, M et al. Acta, , , A proper lipid mixture was dissolved in chloroform in a test tube.
The lipid mixture was dried gently by blowing argon over the solution, and kept in a desiccator overnight. The suspension was rigorously mixed by vortexing and sonication on ice until it 20 became clear. Brookhaven, CT. The FP values were measured on a Molecular Devices Spectramax M5 spectrofluorimeter, with excitation and emission wavelengths at and nm, respectively.
The FP values were similarly measured, plotted, and analyzed. Image Analysis. Raw images were uniformly modified using imageJ. Genome Biol. Nuclei were distinguished from the Hoescht image via Otsu automatic three-class thresholding, with pixels of the middle intensity class assigned to 10 background. Clumped objects were identified using Laplacian of Gaussian modeling and separated by shape. The translocation ratio was defined as the mean GFP signal inside the nuclear region divided by the mean GFP signal 15 within the cytosolic region measured per cell, and cells from images were captured for each condition tested.
Confocal Microscopy. Biochemistry, , 50, To examine the effect of endocytosis inhibitors, transfected HeLa cells were pretreated for 30 min with clear DMEM containing the inhibitors before incubation with Dex or Dex-peptide conjugates. To examine the internalization of rhodamine-labeled peptides, 5 x 10 4 HEK cells were plated in a 35 mm glass-bottomed microwell dish MatTek. On the day of. To monitor GFP internalization, 5 x 10 4 HEK cells were seeded in a 35 mm glass-bottomed microwell dish and cultured. Flow Cytometry. To quantify the delivery efficiencies of pCAP-containing peptides, 20 HeLa cells were cultured in six-well plates 5 x 10 5 cells per well for 24 h.
Data were analyzed with Flowjo software Tree Star. The cells were washed with DPBS twice, removed from the plate with 0. Whole-cell dextran uptake was analyzed on a BD Accuri C6 flow cytometer using the manufacturer FL1 laser and filter set. The solutions were removed and 5 the cells were washed with cold DPBS twice. After 30 min incubation on ice, the cell lysate was centrifuged at 15, rpm for 25 min in a 10 microcentrifuge.
Serum Stability Test. The stability tests were carried by modifying a previously reported procedure Nguyen, LT et al. PLoS One, , 5, e The final mixture was centrifuged at20 15, rpm for 10 min in a microcentrifuge, and the supernatant was analyzed by reversed- phase HPLC equipped with a C 18 column Waters. Cytotoxicity Assay. Methods, , 65, The plate was incubated at 37 qC for 4 h. The plate was incubated at 37qC for another 4 h. The absorbance of the formazan product was measured at nm using a Molecular Devices Spectramax M5 plate reader.
Each experiment was performed in triplicates and the cells without any peptide added were treated as control. Next, the CPPs were tested for binding to heparan sulfate, which was. Biochemistry, , 46, ; Rusnati, M et al. Biochemistry, , 44, ; Ziegler, A. R9 and Tat both bound to heparan sulfate with high affinity, having EC 50 values of and nM, respectively Figure 5B. These results are in agreement with the previous observations that non-amphipathic cationic CPPs e. Intracellular Delivery of Peptidyl Cargos. The first three peptides were labeled with rhodamine B at a C-terminal lysine side chain Figure 5 2 , and their internalization into HEK cells was examined by live-cell confocal.
In contrast, the fluid phase endocytic marker dextran FITC displayed predominantly punctate fluorescence, indicative of endosomal 10 localization.
The diffuse rhodamine fluorescence suggests that a fraction of the peptides reached the cytosol and nucleus of the cells. This assay detects only the CPP- cargo inside the cytoplasm and nucleus, where the catalytic domains of all known mammalian PTPs are localized Alonso, A et al. Cell, , , To quantify the relative 5 intracellular PCP delivery efficiency, HeLa cells were treated with each peptide and. Kinetic Activities k In recent years, there has been much interest in cyclic peptides as therapeutic agents and biomedical research tools Driggers, EM et al.
Drug Discov. For example, cyclic peptides are effective for inhibition of protein-protein interactions Lian, W et al. ACS Comb. A major obstacle in developing cyclic peptide therapeutics is that they are generally impermeable to the cell membrane Kwon, YU and Kodadek, T. To overcome this limitation, a bicyclic peptide system was explored, in which one ring contains a CPP motif e.
The bicyclic 10 system should in principle be able to accommodate cargos of any size, because the cargo does not change the structure of the CPP ring and should have less impact on its delivery efficiency. The additional rigidity of a bicyclic structure should also improve its metabolic stability as well as the target-binding affinity and specificity. Medicaid Service Coordination.
Cell-penetrating peptides : methods and protocols (eBook, ) [duqehovy.tk]
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