Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
  • C07K14/003—Peptide-nucleic acids (PNAs)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention is directed to compositions comprising a peptide nucleic acid (PNA) which is conjugated to a lipophilic group and incorporated into liposomes.
• PNA peptide nucleic acid
• the PNA is composed of naftirally-occurring nucleobases or non-naturally-occurring nucleobases which are covalently bound to a polyamide backbone.
• the PNA compositions of the present invention may further comprise a PNA modified by an amino acid side chain.
• the PNA compositions of the present invention exhibit enhanced cellular uptake and distribution. PNA compositions which were incorporated into liposomes demonstrated increased cellular uptake and more diffuse distribution than PNA compositions without liposomes.
• mRNA messenger RNA
• mRNA messenger RNA
• tRNAs transfer R As
• Oligonucleotides and their analogs have been developed and used as diagnostics, therapeutics and research reagents.
• One example of a modification to oligonucleotides is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
• Other modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphoro- dithioates, and 2'-O-methyl ribose sugar moieties.
• Other oligonucleotide modifications include those made to modulate uptake and cellular distribution.
• Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for the treatment of various disease states. Although some improvements in diagnostic and therapeutic uses have been realized with these oligonucleotide modifications, there exists an ongoing demand for improved oligonucleotide analogs.
• nucleic acid analogs having nucleobases bound to backbones other than the naturally-occurring ribonucleic acids or deoxyribonucleic acids. These nucleic acid analogs have the ability to bind to nucleic acids with complementary nucleobase sequences.
• the peptide nucleic acids (PNAs) as described, for example, in WO 92/20702, have been shown to be useful as therapeutic and diagnostic reagents. This may be due to their generally higher affinity for complementary nucleobase sequence than the corresponding wild-type nucleic acids.
• PNAs are useful surrogates for oligonucleotides in binding to DNA and RNA. Egholm et al, Nature, 1993, 365, 566, and references cited therein. The current literature reflects the various applications of PNAs. Hyrup et al, Bioorganic & Med. Chem., 1996, 4, 5; and Nielsen, Perspectives Drug Disc. Des., 1996, 4, 76.
• PNAs are compounds that are analogous to oligonucleotides, but differ in composition.
• the deoxyribose backbone of oligonucleotide is replaced by a peptide backbone.
• Each subunit of the peptide backbone is attached to a naturally-occurring or non- naturally-occurring nucleobase.
• One such peptide backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds.
• PNAs More recent advances in the structure and synthesis of PNAs are illustrated in WO 93/12129 and U.S. Patent 5,539,082, issued July 23, 1996, the contents of both being herein incorporated by reference. Further, the literature is replete with publications describing synthetic procedures, biological properties and uses of PNAs. For example, PNAs possess the ability to effect strand displacement of double- stranded DNA. Patel, Nature, 1993, 365, 490. Improved synthetic procedures for PNAs have also been described. Nielsen etal, Science, 1991, 254, 1497; and Egholm, J. Am. Chem. Soc, 1992, 114, 1895. PNAs form duplexes and triplexes with complementary DNA or RNA.
• PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes.
• the resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (T m ).
• T m melting temperatures
• This high thermal stability of PNA/DNA(R A) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes.
• Another advantage of PNA/DNA(RNA) duplexes is that T m is practically independent of salt concentration. DNA/DNA duplexes, on the other hand, are highly dependent on the ionic strength.
• the new pyrimidine strand is oriented parallel to the purine Watson-Crick strand in the major groove of the DNA and binds through sequence-specific Hoogsteen hydrogen bonding.
• the sequence specificity is derived from thymine recognizing adenine (T:A-T) and protonated cytosine recognizing guanine (C:G-C). Best et al, J. Am. Chem. Soc, 1995, 117, 1187. In a less well-studied triplex motif, purine-rich oligonucleotides bind to purine targets of double-stranded DNA.
• the orientation of the third strand in this motif is anti-parallel to the purine Watson-Crick strand, and the specificity is derived from guanine recognizing guanine (G:G-C) and thymine or adenine recognizing adenine (A:A-T or T: A-T).
• G:G-C guanine recognizing guanine
• A:A-T or T: A-T adenine recognizing adenine
• Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA) 2 T)NA(RNA) triplexes of high thermal stability. Egholm et al., Science, 1991, 254, 1497; Egholm et al, J. Am. Chem.
• Peptide nucleic acids have been shown to have higher binding affinities (as determined by their Tm's) for both DNA and RNA than that of DNA or RNA to either DNA or RNA. This increase in binding affinity makes these peptide nucleic acid oligomers especially useful as molecular probes and diagnostic agents for nucleic acid species. In addition to increased affinity, PNAs have increased specificity for DNA binding.
• a further advantage of PNAs compared to oligonucleotides, is that the polyamide backbone of PNAs is resistant to degradation by enzymes.
• oligonucleotides and oligonucleotide analogs that bind to complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics.
• the oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express their activity.
• PCT/EP/01219 describes novel PNAs which bind to complementary DNA and RNA more tightly than the corresponding DNA. It is desirable to append groups to these PNAs which will modulate their activity, modify their membrane permeability or increase their cellular uptake property.
• One method for increasing amount of cellular uptake property of PNAs is to attach a lipophilic group.
• Liposomes are microscopic spheres composed of an aqueous core and a lipid bilayer enveloping the core. Procedures for preparation of liposomes are available in the literature.
• WO 96/24334 published August 15, 1996, describes lipid constructs having an aminomannose-derivatized cholesterol moiety for the delivery of drugs to the cytoplasm of cells, particularly to vascular smooth muscle tissues.
• WO 96/40627 published December 19, 1996, describes cationic lipid-containing liposome formulations which are useful in the delivery of biomolecules such as oligonucleotides, nucleic acids, peptides and other agents.
• the present invention provides peptide nucleic acids (PNAs) conjugated to a lipophilic group and having a modified backbone wherein an amino acid side chain is attached to the backbone.
• the present invention also provides liposomal compositions comprising a peptide nucleic acid (PNA) conjugated to a lipophilic group which is incorporated into liposomes.
• PNAs of the present invention comprise nucleobases covalently bound to a polyamide backbone. Representative nucleobases include the four major naturally-occurring DNA nucleobases (i.e., thymine, cytosine, adenine and guanine), other naturally-occurring nucleobases (e.g.
• nucleobases e.g., bromothymine, azaadenines and azaguanines.
• Preferred peptide nucleic acids of the invention have the general formula (I):
• each L is, independently, a naturally-occurring nucleobase or a non-naturally- occurring nucleobase
• each R 7 is hydrogen or the side chain of a naturally-occurring or non-naturally- occurring amino acid, at least one R 7 being the side chain of an amino acid
• R h is OH, NH 2 , or NHLysNH 2
• each of R 1 and R J is, independently, a lipophilic group or an amino acid labeled with a fluorescent group; or R 1 and R J , together, are a lipophilic group
• n is an integer from 1 to 30.
• PNAs having formula (I) wherein R 1 is D-lysine labeled with a fluorescent group and
• R J is an adamantoyl group are preferred. Even more preferred are PNAs of formula (I) wherein R 1 is D-lysine labeled with fluorescein and R J is an adamantoyl group. Also preferred are PNAs having formula (I) wherein R 1 and R J , together, are an adamantoyl group. Further preferred are PNAs of formula (I) wherein at least one of said R 7 is the side chain of D-lysine.
• the carbon atom to which substituent R 7' is attached is stereochemically enriched.
• stereochemically enriched means that one stereoisomer predominates over the other stereoisomer in a sufficient amount as to provide a beneficial effect.
• one stereoisomer predominates by more than 50%. More preferably, one stereoisomer predominates by more than 80%. Even more preferably, one stereoisomer predominates by more than 90%. Still more preferably, one stereoisomer predominates by more than 95%. Even more preferably, one stereoisomer predominates by more than 99%. Still even more preferably, one stereoisomer is present substantially quantitatively.
• the present invention also provides liposomal compositions comprising a peptide nucleic acid incorporated in a liposome, said peptide nucleic acid having formula (I) wherein: each L is, independently, a naturally-occurring nucleobase or a non-naturally- occurring nucleobase; each R 7 is hydrogen or the side chain of a naturally-occurring or non-naturally- occurring amino acid;
• R h is OH, NH 2 , or NHLysNH 2 each of R' and R J is, independently, a lipophilic group or an amino acid labeled with a fluorescent group; or R' and R J , together, are a lipophilic group; n is an integer from 1 to 30.
• PNAs having formula (I) wherein R' is D-lysine labeled with a fluorescent group and R J is an adamantoyl group are preferred. Even more preferred are PNAs of formula (I) wherein R 1 is D-lysine labeled with fluorescein and R J is an adamantoyl group. Also preferred are PNAs having formula (I) wherein R 1 and R J , together, are an adamantoyl group. Further preferred are PNAs of formula (I) wherein at least one of said R 7 is the side chain of D-lysine.
• the carbon atom to which substituent R 7 is attached is stereochemically enriched.
• the PNAs of the present invention are synthesized by adaptation of standard peptide synthesis procedures, either in solution or on a solid phase.
• the present invention further provides methods for enhancing the cellular uptake and distribution of peptide nucleic acids by incorporation of amino acid side chains into PNA backbones, conjugating lipophilic groups with PNAs and introducing PNAs into liposomes.
• FIG. 1 shows structures of some lipophilic groups.
• peptide nucleic acids and liposomal compositions exhibiting enhanced cellular uptake and distribution are provided.
• the peptide nucleic acids (PNAs) of the invention are assembled from a plurality of nucleobases which are attached to a polyamide backbone by a suitable linker.
• the PNAs are conjugated to a lipophilic group.
• conjugating refers to attaching a lipophilic group to a PNA of the invention.
• the polyamide backbone of PNAs of the invention is derivatized.
• liposomal compositions of the present invention comprise peptide nucleic acids of the invention that are incorporated into liposomes.
• the liposomal compositions of the present invention comprise PNAs which are encapsulated by liposomes.
• the PNAs and liposomal compositions of the present invention exhibit enhanced cellular uptake and distribution.
• nucleobase L is a naturally-occurring nucleobase attached at the position found in nature, i.e., position 9 for adenine or guanine, and position 1 for thymine or cytosine, a non-naturally-occurring nucleobase (nucleobase analog) or a nucleobase-binding moiety.
• Representative nucleobases include the four major naturally-occurring DNA nucleobases (i. e., thymine, cytosine, adenine and guanine), other naturally-occurring nucleobases (e.g.
• nucleobases e.g., bromothymine, azaadenines and azaguanines.
• the PNAs of formula (I) include one or more amino acid moieties within their structure. These amino acids may be naturally-occurring or non-naturally-occurring. Naturally-occurring amino acids include ⁇ -amino acids where the chiral center has a D- configuration. Such naturally-occurring amino-acids may be either essential or non-essential amino acids. Non-naturally-occurring amino acids used in the PNAs of the present invention of formula (I) include ⁇ -amino acids with chiral centers bearing an L-configuration.
• Non- naturally-occurring amino acids also include amino acids bearing unusual side chains that do not exist in nature and are prepared synthetically, such as halo- and cyano- substituted benzyl, tetrahydroisoquinolylmethyl, cyclohexylmethyl, and pyridylmethyl.
• Other synthetic amino- acids include ⁇ -amino acids.
• the amino acids may be introduced into the PNAs of formula (I) either as part of the monomer used or at the terminal ends of the PNA. Any of the abovementioned amino acids could be incorporated into the monomeric building blocks used in PNA synthesis.
• the amino acid used is glycine, where R 7 is H.
• R 7 can also be methyl, ethyl, benzyl, isopropyl, ?-hydroxybenzyl, halobenzyl, carboxymethyl, tetrahydroisoquinolinylmethyl, or aminohexanoyl.
• Amino acids may also be attached at the C-terminus of PNAs such that the terminal R h -CO- group represents an amino acyl group derived from any naturally- or non- naturally-occurring amino acid, ⁇ - or ⁇ - amino acid, and with a D- or L-configuration at the ⁇ -chiral center.
• the C-terminal amino acid is lysine.
• Amino acids may also be incorporated at the N-terminal end of the PNA of structure (I) where each of R' and R J may, independently, be an amino acyl group derived from any naturally- or non-naturally-occurring amino acid, ⁇ - or ⁇ - amino acid, and with a D- or L-configuration at the ⁇ -chiral center.
• the N-terminal amino acid is lysine.
• Lipophilic groups attached to PNA' s of formula (I) of the present invention include natural and synthetic fatty acids, fatty alcohol derivatives and diacylglycerol derivatives such as adipic acid, palmitic acid, decanoic acid, octadecanoic acid, oleic acid, elaidic acid, linoleic acid, bile acids, heptylsuccinic acid, palmitylsuccinic acid, polyglycolic acid, dioctadecylglycerol phosphatidic acid, dioleoylglycerol phosphatidic acid, adamantoyl, octadecyloxycarbonyl, and decalinoyl.
• lipophilic groups may be attached at any suitable location in the PNA molecule of formula (I).
• the lipophilic group is attached to the N-terminus of the PNA of the invention wherein each of R 1 and R J may, independently, be a lipophilic group. More preferably, R' and R j , together, are an adamantoyl group.
• the PNAs of the present invention have the formula (I) wherein labels, such as fluorescent groups, are incorporated so as to allow a convenient means by which to detect the PNA.
• Fluorescent groups include, but are not limited to, dyes such as fluorescein, rhodamine, pyrenyl, cyanine dyes, Cy5TM (Biological Detection Systems, Inc., Pittsburgh, PA), and derivatives of such dyes. These may be incorporated into the PNA of formula (I) at any suitable position in the PNA.
• each of R' is a chemical moiety to which is attached a fluorescent group. It is more preferred that R 1 is an amino acid that has been derivatized with a fluorescent group. It is further more preferred that R' is a lysine with an ⁇ -fluoresceinyl group.
• Liposomal compositions of the invention comprise PNAs of the invention which are incorporated into liposomes.
• the liposomal compositions exhibit enhanced cellular uptake and distribution.
• Liposomes are a colloidal dispersion system, and constitute a stable delivery system which protects the incorporated PNA from the environment while being transported to target areas. Liposomes represent a stable delivery vehicle to enhance the in vitro and in vivo stability of the PNAs of the invention.
• the liposomal compositions of the present invention, comprising PNAs of the invention incorporated into liposomes can be formulated as pharmaceutical compositions according to standard techniques known by the art-skilled using suitable and acceptable carriers and adjuvants.
• Liposomes that may be used include small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). It has been shown that LUVs, which range in size from 0.2-0.4 ⁇ m, can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules (e.g., RNA, DNA and intact virions can be encapsulated within the aqueous interior and delivered to brain cells in a biologically active form: Fraley et al, Trends Biochem. Sci., 1981, 6, 77).
• SUVs small unilamellar vesicles
• LUVs large unilamellar vesicles
• MLVs multilamellar vesicles
• the composition of the liposome is usually a combination of lipids, particularly phospholipids, in particular, high phase transition temperature phospholipids, usually in combination with one or more steroids, particularly cholesterol.
• lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides.
• Particularly useful are diacyl phosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated (lacking double bonds within the 14-18 carbon atom chain).
• Illustrative phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
• the targeting of liposomes can be either passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system in organs that contain sinusoidal capillaries. Active targeting, by contrast, involves modification of the liposome by coupling thereto a specific ligand such as a viral protein coat (Morishita et al, Proc Natl Acad. Sci. U.S.A., 1993, 90, 8474), monoclonal antibody (or a suitable binding portion thereof), sugar, glycolipid or protein (or a suitable oligopeptide fragment thereof), or by changing the composition and/or size of the liposome in order to achieve distribution to organs and cell types other than the naturally occurring sites of localization.
• a specific ligand such as a viral protein coat (Morishita et al, Proc Natl Acad. Sci. U.S.A., 1993, 90, 8474), monoclonal antibody (or a suitable binding portion thereof),
• the surface of the targeted colloidal dispersion system can be modified in a variety of ways.
• lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in close association with the lipid bilayer.
• Various linking groups can be used for joining the lipid chains to the targeting ligand.
• the targeting ligand which binds a specific cell surface molecule found predominantly on cells to which delivery of the oligonucleotides of the invention is desired, may be, for example, (1) a hormone, growth factor or a suitable oligopeptide fragment thereof which is bound by a specific cellular receptor predominantly expressed by cells to which delivery is desired; or (2) a polyclonal or monoclonal antibody, or a suitable fragment thereof (e.g., Fab; F(ab') 2 ) which specifically binds an antigenic epitope found predominantly on targeted cells.
• Two or more bioactive agents e.g., a PNA and a conventional drug, or two PNAs
• the PNAs of the present invention may be used for gene modulation (e.g., gene targeted drugs), diagnostics, biotechnology and other research purposes.
• the PNAs may also be used to target RNA and single-stranded DNA (ssDNA) to produce both antisense-type gene regulating moieties and as hybridization probes, e.g., for the identification and purification of nucleic acids.
• ssDNA single-stranded DNA
• the PNAs may be modified in such a way that they form triple helices with double stranded DNA (dsDNA).
• dsDNA double stranded DNA
• Compounds that bind sequence- specifically to dsDNA have applications as gene targeted drugs. These compounds are extremely useful drugs for treating various diseases, including cancer, acquired immune deficiency syndrome (AIDS) and other virus infections and genetic disorders. Furthermore, these compounds may be used in research, diagnostics and for detection and isolation of specific nucleic acids.
• Gene-targeted drugs are designed with a nucleobase sequence
• the gene-targeted drugs bind to the promoter and prevent RNA polymerase from accessing the promoter. Consequently, no mRNA, and thus no gene product (protein), is produced. If the target is within a vital gene for a virus, no viable virus particles will be produced. Alternatively, the target region could be downstream from the promoter, causing the RNA polymerase to terminate at this position, thus forming a truncated mRNA/protein which is nonfunctional.
• a preferred method for PNA synthesis employs aminomethyl as the initial functionality.
• Aminomethyl is particularly advantageous as a "spacer” or “handle” group because it forms amide bonds with a carboxylic acid group in nearly quantitative amounts.
• spacer- or handle-forming bifunctional reagents have been described. Barany et al, Int. J. Peptide Protein Res., 1987, 30, 705.
• N-protecting groups are tert-butyloxycarbonyl (BOC) (Carpino, J. Am. Chem. Soc, 1957, 79, 4427; McKay, et al, J. Am. Chem. Soc, 1957, 79, 4686; and Anderson et al, J. Am. Chem. Soc, 1957, 79, 6180), 9-fluorenylmethyloxycarbonyl (FMOC) (Carpino et al. , J. Am. Chem. Soc , 1970, 92, 5748 and J. Org. Chem. , 1972, 37, 3404), Adoc (Hass et al,J. Am. Chem.
• amino-protecting groups particularly those based on the widely-used urethane functionality, prohibit racemization (mediated by tautomerization of the readily formed oxazolinone (azlactone) intermediates (Goodman et al, J. Am. Chem. Soc, 1964, 86, 2918)) during the coupling of most ⁇ -amino acids.
• nonurethane-type of amino-protecting groups are also applicable when assembling PNA molecules.
• side chain protecting groups in general, depends on the choice of the amino-protecting group, because the side chain protecting group must withstand the conditions of the repeated amino deprotection cycles. This is true whether the overall strategy for chemically assembling PNA molecules relies on, for example, different acid stability of amino and side chain protecting groups (such as is the case for the above-mentioned "BOC- benzyl” approach) or employs an orthogonal, that is, chemoselective, protection scheme (such as is the case for the above-mentioned "FMOC-t-Bu” approach).
• BOC- benzyl an orthogonal, that is, chemoselective, protection scheme
• a temporary protecting group, such as BOC or FMOC, on the last coupled amino acid is quantitatively removed by a suitable treatment, for example, by acidolysis, such as with trifluoroacetic acid in the case of BOC, or by base treatment, such as with piperidine in the case of FMOC, so as to liberate the N-terminal amine function.
• a suitable treatment for example, by acidolysis, such as with trifluoroacetic acid in the case of BOC, or by base treatment, such as with piperidine in the case of FMOC, so as to liberate the N-terminal amine function.
• the next desired N-protected amino acid is then coupled to the N-terminal of the last coupled amino acid.
• This coupling of the C-terminal of an amino acid with the N-terminal of the last coupled amino acid can be achieved in several ways. For example, it can be achieved by providing the incoming amino acid in a form with the carboxyl group activated by any of several methods, including the initial formation of an active ester derivative such as a phthalimido ester (Nefkens et al, J. Am. Chem. Soc, 1961, 83, 1263), a pentafluoro- phenyl ester (Kovacs et al, J. Am. Chem.
• the carboxyl group of the incoming amino acid can be reacted directly with the N-terminal of the last coupled amino acid with the assistance of a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan et al. , J. Am. Chem. Soc, 1955, 77, 1067) or derivatives thereof.
• a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan et al. , J. Am. Chem. Soc, 1955, 77, 1067) or derivatives thereof.
• BOP Benzotriazolyl N-oxy- trisdi ethylan inophosphonium hexafluorophosphate
• Castro's reagent see Rivaille et al, Tetrahedron, 1980, 36, 3413
• the next step will normally be deprotection of the amino acid moieties of the PNA chain and cleavage of the synthesized PNA from the solid support. These processes can take place substantially simultaneously, thereby providing the free PNA molecule in the desired form.
• a strong acid (e.g., anhydrous HF) deprotection method may produce very reactive carbocations that may lead to alkylation and acylation of sensitive residues in the PNA chain. Such side reactions are only partly avoided by the presence of scavengers such as anisole, phenol, dimethyl sulfide, and mercaptoethanol.
• stepwise chain building of achiral PNAs such as those based on aminoethylglycyl backbone units can start either from the N-terminus or the C-terminus.
• synthesis commencing at the C-terminus typically employ protected amine groups and free or activated acid groups
• syntheses commencing at the N-terminus typically employ protected acid groups and free or activated amine groups.
• HSV herpes simplex virus
• HPV human papillomavirus
• HMV human immunodeficiency virus
• Candida albicans influenza virus
• CMV cytomegalovirus
• IAM intercellular adhesion molecules
• 5- lipoxygenase (5-LO) 5- lipoxygenase
• PKA 2 protein kinase C
• PKC protein kinase C
• Potential treatment of such targeting include ocular, labial, genital, and systemic herpes simplex I and II infections; genital warts; cervical cancer; common warts; Kaposi's sarcoma; AIDS; skin and systemic fungal infections; flu; pneumonia; retinitis and pneumonitis in immunosuppressed patients; mononucleosis; ocular, skin and systemic inflammation; cardiovascular disease; cancer; asthma; psoriasis; cardiovascular collapse; cardiac infarction; gastrointestinal disease; kidney disease; rheumatoid arthritis; osteoarthritis; acute pancreatitis; septic shock; and Crohn's disease.
• a patient suspected of requiring such therapy is administered a PNA or liposomal composition of the present invention, commonly in a pharmaceutically acceptable carrier, in amounts and for periods of time which will vary depending upon the nature of the particular disease, its severity and the patient's overall condition.
• the PNAs and liposomal compositions of the invention can be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like.
• Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics and the like, in addition to the peptide nucleic acids.
• the pharmaceutical composition may be administered in a number of ways depending upon whether local or systemic treatment is desired, and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection or intrathecal or intraventricular administration.
• Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection or intrathecal or intraventricular administration.
• Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
• Conventional pharmaceutical carriers, nucleic acid carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable in certain circumstances.
• Coated condoms, gloves and the like may also be useful.
• Topical administration also includes delivery of the PNAs and liposomal compositions of the invention into the epidermis of an animal by electroporation. Zewart et al, WO 96/39531, published December 12, 1996.
• compositions for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
• Intravitreal injection for direct delivery of the PNAs and liposomal compositions of the invention to the vitreous humor of the eye of an animal is described in U.S. Patent 5,595,978, issued January 21, 1997, the contents of which are herein incorporated by reference.
• Intraluminal administration for direct delivery of PNAs and liposomal compositions of the invention to an isolated portion of a tubular organ or tissue (e.g., artery, vein, ureter or urethra) may be desired for the treatment of patients with diseases or conditions afflicting the lumen of such organs or tissues.
• a catheter or cannula is surgically introduced by appropriate means.
• the PNA or liposomal composition of the invention is infused through the catheter or cannula.
• the infusion catheter or cannula is then removed, and flow within the tubular organ or tissue is restored by removal of the ligatures which effected the isolation of a segment thereof.
• Intraventricular administration for direct delivery of PNAs or liposomal compositions of the invention to the brain of a patient, may be desired for the treatment of patients with diseases or conditions afflicting the brain.
• a silicon catheter is surgically introduced into a ventricle of the brain, and is connected to a subcutaneous infusion pump (Medtronic, Inc., Minneapolis, MN) that has been surgically implanted in the abdominal region.
• Medtronic, Inc., Minneapolis, MN subcutaneous infusion pump
• the pump is used to inject the PNA or liposomal composition, and allows precise dosage adjustments and variation in dosage schedules with the aid of an external programming device.
• the reservoir capacity of the pump is 18-20 mL, and infusion rates may range from 0.1 mL/hour to 1 mL/hour.
• the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by percutaneous puncture of the self-sealing septum of the pump.
• Compositions for intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
• Intrathecal administration for the direct delivery of PNAs or liposomal compositions of the invention into the spinal column of a patient, may be desired for the treatment of patients with diseases of the central nervous system.
• a silicon catheter is surgically implanted into the L3-4 lumbar spinal interspace of the patient, and is connected to a subcutaneous infusion pump which has been surgically implanted in the upper abdominal region. Luer and Hatton, The Annals of Pharmacotherapy, 1993, 27, 912; Ettinger et al, Cancer, 1978, 41, 1270; and Yaida et al, Regul Pept., 1995, 59, 193.
• the pump is used to inject the PNA or liposomal composition, and allows precise dosage adjustments and variations in dose schedules with the aid of an external programming device.
• the reservoir capacity of the pump is 18-20 mL, and infusion rates may vary from 0.1 mL/hour to 1 mL/hour.
• the pump reservoir may be refilled at 3-10 week intervals. Refilling of the pump is accomplished by a single percutaneous puncture to the self-sealing septum of the pump.
• Compositions for intrathecal administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
• the silicon catheter may be configured to connect the subcutaneous infusion pump to, e.g., the hepatic artery, for delivery to the liver.
• the subcutaneous infusion pump e.g., the hepatic artery
• Infusion pumps may also be used to effect systemic delivery. Ewel et al, Cancer Research, 1992, 52,
• compositions for parenteral, intrathecal or intraventricular administration, or liposomal systems may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
• Optimum dosages may vary depending on the relative potency of individual PNAs, and can generally be estimated based on EC 50 s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years.
• the monomer subunits preferably are synthesized by a general scheme that commences with the preparation of either the methyl or ethyl ester of (BOC- aminoethyl)glycine, via a protection/deprotection procedure, as described in Examples 1 and 2.
• the synthesis of thymine monomer is described in Examples 4 and 5, and the synthesis of protected cytosine monomer is described in Example 6.
• the synthesis of a protected adenine monomer involves alkylation of adenine with ethyl bromoacetate (Example 7) and verification of the position of substitution (i.e. position 9) by X-ray crystallography.
• the N 6 -amino group is then protected with the benzyloxy- carbonyl group by the use of the reagent N-ethyl-benzyloxycarbonylimidazole tetrafluoroborate (Example 8).
• Simple hydrolysis of the product ester (Example 9) gave N 6 - benzyloxycarbonyl-9-carboxymethyl adenine (Examples 10 and 11), which was used in the standard PNA oligomer synthesis.
• the starting material 2-amino-6- chloropurine
• 2-amino-6- chloropurine was alkylated with bromoacetic acid (Example 12), and the 6-chloro group was then substituted with a benzyloxy group (Example 13).
• the resulting acid was coupled to the (BOC-aminoethyl)glycine methyl ester (from Example 2) with agent PyBropTM being used as a coupling agent, and the resulting ester was hydro lyzed (Example 14) to afford the protected G monomer.
• the O 6 -benzyl group was removed in the final HF-cleavage step following synthesis of the PNA-oligomer.
• NMR spectra were recorded on JEOL FX 90Q spectrometer or a Bruker 250 MHz with tetramethylsilane as an internal standard. Mass spectrometry was performed on a MassLab VG 12-250 quadropole instrument fitted with a VG FAB source and probe. Melting points were recorded on a Buchi melting point apparatus and are uncorrected. N,N- Dimethylformamide was dried over 4 A molecular sieves, distilled and stored over 4 A molecular sieves. Pyridine (HPLC quality) was dried and stored over 4 A molecular sieves.
• Thin layer chromatography was performed using the following solvent systems: (1) chloroform:triethyl amine:methanol, 7:1:2; (2) methylene chloride:methanol, 9:1; (3) chloroform:methanol:acetic acid 85:10:5. Spots were visualized by UV (254 nm) and/or spraying with a ninhydrin solution (3 g ninhydrin in 1000 mL of 1-butanol and 30 mL of acetic acid), after heating at 120° C for 5 minutes and, after spraying, heating again.
• a ninhydrin solution 3 g ninhydrin in 1000 mL of 1-butanol and 30 mL of acetic acid
• the carboxyl terminal (C terminus) end of PNA oligomers can be substituted with a variety of functional groups. One way this is performed is through the use of different resins.
• the amino terminal (N terminus) end of PNA oligomers can also be capped with a carboxylic acid-based capping reagent for the final PNA monomer in the final coupling step, or substituted with a variety of conjugate groups. Representative examples of the types of C and N terminal groups are shown below.
• Aminoethyl glycine (52.86 g, 0.447 mol) was dissolved in water (900 mL) and dioxane (900 mL) was added. The pH was adjusted to 11.2 with 2N NaOH. While the pH was kept at 11.2, tert-butyl-p-nitrophenyl carbonate (128.4 g, 0.537 mol) was dissolved in dioxane (720 mL) and added dropwise over the course of 2 hours. The pH was kept at 11.2 for at least three more hours and then allowed to stand overnight, with stirring. The yellow solution was cooled to 0°C and the pH was adjusted to 3.5 with 2 N HCl.
• the oil was dissolved in absolute ethanol (600 mL) and was added 10% palladium on carbon (6.6 g) was added. The solution was hydrogenated at atmospheric pressure. After 4 hours, 3.3 L was consumed out of the theoretical 4.2 L. The reaction mixture was filtered through celite and evaporated to dryness, in vacuo, affording 39.5 g (94%) of an oily substance. A 13 g portion of the oily substance was purified by silica gel (SiO 2 , 600 g) chromatography. After elution with 300 mL of 20%) petroleum ether in methylene chloride, the title compound was eluted with 1700 mL of 5% methanol in methylene chloride.
• 3-BOC-amino-l,2-propanediol 144.7 g, 0.757 mol was suspended in water (750 mL) and potassium periodate (191.5 g, 0.833 mol) was added. The mixture was stirred under nitrogen for 2.5 h and the precipitated potassium iodate was removed by filtration and washed once with water (100 mL). The aqueous phase was extracted with chloroform (6x400 mL).
• the title compound was prepared by the above procedure with glycine ethyl ester hydrochloride substituted for glycine methyl ester hydrochloride. Also, the solvent used was ethanol. The yield was 78%.
• N'-BOC-aminoethylglycine ethyl ester (13.5 g, 54.8 mmol), DhbtOH (9.84 g, 60.3 mmol) and 1-carboxymethyl thymine (11.1 g, 60.3 mmol) were dissolved in DMF (210 mL).
• Example 4 The material from Example 4 was suspended in THF (194 mL, gives a 0.2 M solution), and 1 M aqueous lithium hydroxide (116 mL) was added. The mixture was stirred for 45 minutes at ambient temperature and then filtered to remove residual DCU. Water (40 mL) was added to the solution which was then washed with methylene chloride (300 mL). Additional water (30 mL) was added, and the alkaline solution was washed once more with methylene chloride (150 mL). The aqueous solution was cooled to 0°C and the pH was adjusted to 2 by the dropwise addition of 1 N HCl (approx. 110 mL).
• N'-BOC-aminoethyl glycine ethyl ester (5 g, 20.3 mmol), DhbtOH (3.64 g, 22.3 mmol) and N 4 -benzyloxycarbonyl-l-carboxymethyl cytosine (6.77 g, 22.3 mmol) were suspended in DMF (100 mL). Methylene chloride (100 mL) was added. The solution was cooled to 0°C and DCC (5.03 g, 24.4 mmol) was added. The ice bath was removed after 2 h and stirring was continued for another hour at ambient temperature. The reaction mixture then was evaporated to dryness, in vacuo.
• the ester was then suspended in THF (100 mL), cooled to 0°C, and 1 N LiOH (61 mL) was added. After stirring for 15 minutes, the mixture was filtered and the filtrate was washed with methylene chloride (2x150 mL). The alkaline solution then was cooled to 0°C and the pH was adjusted to 2.0 with 1 N HCl. The title compound was isolated by filtration and was washed once with water, leaving 11.3 g of a white powder after drying. The material was suspended in methylene chloride (300 mL) and petroleum ether (300 mL) was added. Filtration and wash afforded 7.1 g (69%) after drying.
• IR Frequency in cm 1 . 3423, 3035, 2978, 1736, 1658, 1563, 1501 and 1456.
• Adenine (10 g, 74 mmol) and potassium carbonate (10.29 g, 74 mmol) were suspended in DMF and ethyl bromoacetate (8.24 mL, 74 mmol) was added. The suspension was stirred for 2.5 h under nitrogen at room temperature and then filtered. The solid residue was washed three times with DMF (10 mL). The combined filtrate was evaporated to dryness, in vacuo. Water (200 mL) was added to the yellowish-orange solid material and the pH adjusted to 6 with 4 N HCl. After stirring at 0 °C for 10 minutes, the solid was filtered off, washed with water, and recrystallized from 96% ethanol (150 mL).
• 9-carboxymethyladenine ethyl ester can be prepared by the following procedure. To a suspension of adenine (50 g, 0.37 mol) in DMF (1100 mL) in 2 L three- necked flask equipped with a nitrogen inlet, a mechanical stirrer and a dropping funnel, was added 16.4 g (0.407 mol) of hexane-washed sodium hydride-mineral oil dispersion. The mixture was stirred vigorously for 2 hours, after which ethyl bromoacetate (75 mL, 0.67 mol) was added dropwise over the course of 3 hours. The mixture was stirred for one additional hour, after which tic indicated complete conversion of adenine.
• N 6 -Benzyloxycarbonyl-9-carboxymethyladenine ethyl ester (3.2 g, 9.01 mmol) was mixed with methanol (50 mL) cooled to 0 °C.
• Sodium hydroxide solution (2 N, 50 mL) was added, whereby the material quickly dissolved.
• the alkaline solution was washed with methylene chloride (2x50 mL).
• the pH of the aqueous solution was adjusted to 1 with 4 N HCl at 0°C, whereby the title compound precipitated.
• the yield after filtration, washing with water, and drying was 3.08 g (104% ⁇ ).
• the product contained salt, and the elemental analysis reflected that.
• N'-BOC-aminoethylglycine ethyl ester (2 g, 8.12 mmol), DhbtOH (1.46 g, 8.93 mmol) and N 6 -benzyloxycarbonyl-9-carboxymethyl adenine (2.92 g, 8.93 mmol) were dissolved in DMF (15 mL). Methylene chloride (15 mL) was then added. The solution was cooled to 0°C in an ethanol/ice bath. DCC (2.01 g, 9.74 mmol) was added. The ice bath was removed after 2.5 h and stirring was continued for another 1.5 hour at ambient temperature.
• the precipitated DCU was removed by filtration and washed once with DMF (15 mL), and twice with methylene chloride (2x15 mL). To the combined filtrate was added more methylene chloride (100 mL). The solution was washed successively with dilute sodium hydrogen carbonate (2x100 mL), dilute potassium hydrogen sulfate (2x100 mL), and saturated sodium chloride (1x100 mL). The organic phase was evaporated to dryness, in vacuo, which afforded 3.28 g (73%) of a yellowish oily substance. HPLC of the raw product showed a purity of only 66% with several impurities, both more and less polar than the main peak.
• N 6 -Benzyloxycarbonyl-l-(BOC-aeg)adenine ethyl ester (1.48 g, 2.66 mmol) was suspended in THF (13 mL) and the mixture was cooled to 0°C. Lithium hydroxide (8 mL, 1 N) was added. After 15 minutes of stirring, the reaction mixture was filtered, extra water (25 mL) was added, and the solution was washed with methylene chloride (2x25 mL). The pH of the aqueous solution was adjusted to 2 with 1 N HCl. The precipitate was isolated by filtration, washed with water, and dried, affording 0.82 g (58%) of the product.
• N 6 -(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetic acid (3.6 g, 10.5 mmol) in DMF (150 mL) was added N,N-diisopropylethylamine (2.75 mL, 21 mmole), and N-(BOC- aminoethyl)- ⁇ -(2-chlorobenzyloxycarbonyl)-lysine allyl ester hydrochloride (7.31 gm, 15.8 mmol).
• Example 24 The procedure used for the guanine monomer in Example 22 above was followed for the synthesis of the adenine monomer using N6-benzyl-9-carboxymethylene-adenine.
• Example 24 The procedure used for the guanine monomer in Example 22 above was followed for the synthesis of the adenine monomer using N6-benzyl-9-carboxymethylene-adenine.
• the resulting residue was purified by silica gel flash column chromatography using dichloromethane:hexanes: methanol (8:2:1) to give 2.4 g (85%) of the cytosine attached to the aminoethyl-lysine backbone as the allyl ester.
• the allyl ester is converted to the active monomer by deprotection using palladiun following the procedure used in Example 22 above to give 1.05 g (46%) of the title compound.
• the thymine monomer was prepared following the procedure of Example 24 above.
• BOC-Taeg-A(Z)aeg-[Taeg] 8 -Lys(ClZ)-MBHA resin was assembled by in situ DCC coupling (single) of the A(Z)aeg residue utilizing 0.19 M of BOC-A(Z)aeg-OH together with 0.15 M DCC in 2.5 mL of 50% DMF/CH 2 C1 2 and a single coupling with 0.15 M BOC- Taeg-OPfp in neat CH 2 C1 2 ("Synthetic Protocol I").
• the synthesis was monitored by the quantitative ninhydrin reaction, which showed about 50% inco ⁇ oration of A(Z)aeg and about 96%) inco ⁇ oration of Taeg.
• BOC-[Taeg] 5 -Lys(ClZ)-MBHA resin was placed in a 5 mL SPPS reaction vessel.
• BOC-[Taeg] 2 -A(Z)aeg-[Taeg] 5 -Lys(ClZ)-MBHA resin was assembled by in situ DCC coupling of both the A(Z)aeg and the Taeg residues utilising 0.15 M to 0.2 M of protected PNA monomer (free acid) together with an equivalent amount of DCC in 2 mL neat CH 2 C1 2 ("Synthetic Protocol II").
• the synthesis was monitored by the quantitative ninhydrin reaction which showed a total of about 82%> inco ⁇ oration of A(Z)aeg after coupling three times (the first coupling gave about 50% inco ⁇ oration; a fourth HOBt-mediated coupling in 50%) DMF/CH 2 C1 2 did not increase the total coupling yield significantly) and quantitative inco ⁇ oration (single couplings) of the Taeg residues.
• Wash B 2 M Collidine in 20% DMSO in NMP Deblock: 5% w-Cresol, 95% TFA
• Neutralizer 1 M DIEA in 20% DMSO in NMP Cap: 0.5 M Acetic Anhydride, 1.5 M Collidine in 20% DMSO in NMP
• Activator 0.2 M HATU in DMF
• the PNA-resin is washed with 5 mL of MeOH and dried under vacuum. The dried resin is emptied into a 1.5 mL Durapore ultrafree filter unit. Thioanisole (25 ⁇ L), 25 ⁇ L of m-Cresol, 100 ⁇ L of TFA and 100 ⁇ L of TFMSA is added to the resin, vortexed for about 30 seconds and allowed to stand for 2 h. The reaction mixture is then centrifuged for 5 minutes at 10 K and the inner tube with resin is removed. Approximately 1.5 mL of ether is added to the TFA solution to precipitate the product. The TFA solution is vortexed, followed by centrifugation at 10 K for 2 minutes. The ether is removed in vacuo.
• Ether precipitation and centrifugation are repeated an additional 2 times.
• the dry pellet is heated in a heat block (55°C) for 15 to 30 minutes to remove excess ether and redissolved in 200 ⁇ L of H 2 O.
• Solvent is added to 100 mg of Dowex Acetate Resin in a 1.5 mL Durapore ultrafree filter unit, vortexed, allowed to stand for 30 minutes and centrifuged at 10 K for 2 minutes. Characterization: The absorbance of a 1 ⁇ L sample in 1 mL of H 2 O is measured at 260 nm. Isopropanol (50%) in H 2 O with 1%> Acetic acid (100 ⁇ L) is added to 4 ⁇ L of the sample. This sample is characterized by electrospray mass spectrometry.
• NMP N-methyl pyrrolidinone
• TFA Trifluoroacetic acid
• HATU O-(7-azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate
• / presence of liposomes
• x absence of liposomes
• Tk is thymine attached to an aminoethyl-lysine backbone
• Lys is D-Lysine
• Ada is adamantyl
• Fl is fluoresceinyl lysine.
• Linolenic acid (40 ⁇ moles) was dissolved in coupling solvent (100 ⁇ L) (0.5 M DIE A in 20% DMSO/NMP),to which HATU (90 ⁇ L of 0.4 M) was added and the solution was mixed. After a 2 minute activation period, the solution was mixed with protected PNA resin (15.4 mg, 2 ⁇ moles). After 1 hour, the resin was washed with 20% DMSO/NMP, CH 2 C1 2 and MeOH (about 3 mL each). The resulting linolenyl-conjugated PNA was cleaved from the solid support and characterized according to the procedure described in Example 28.
• Oleic acid (40 ⁇ moles) was dissolved in coupling solvent (100 ⁇ L ) (0.5 M DIEA in 20% DMSO/NMP),to which HATU (90 ⁇ L of 0.4 M) was added and the solution was mixed. After a 2 minute activation period, the solution was mixed with protected PNA resin
• Caproyl-gly (40 ⁇ moles) was dissolved in coupling solvent (100 ⁇ L) (0.5 M DIE A in 20%) DMSO/NMP), to which HATU (90 ⁇ L of 0.4 M) was added and the solution was mixed. After 2 minutes of activation, the solution was mixed with protected PNA resin (15.4 mg, 2 ⁇ moles). After 1 hour, the resin was washed with 20% DMSO/NMP, CH 2 C1 2 and MeOH (about 3 mL each). The resulting PNA was cleaved from the solid support and characterized according to the procedure described in Example 28.
• PNA of sequence GGT-GCT-CAC-TGC-GGC-Lys-NH 2 was synthesized following standard PNA synthesis protocols (as in examples 27 and 28) and commencing with lysine- derivatized synthesis resin.
• the N-terminal BOC group of the PNA bound to resin was deprotected using:
• the BOC group was cleaved and then the PNA (Fl-GGT-GCT-CAC-TGC-GGC-Lys-NH 2 ) was cleaved and purified as in examples 27 and 28.
• the PNA is left attached to the resin and used for derivatization with a lipophilic group as in example 35.
• Example 35
• the resulting PNA concentration in the liposome mix was 25 mM.
• the PNA-liposome mix 40 mL was added to OptiMEMTM (1 mL) and fed to cells. The final concentration of PNA was 1 mM.
• Liposome transfection reagents Four commercially available transfection liposome reagents were employed: LipofectinTM (Gibco BRL), Lipofectamine (Cftbco BRL), Tfx-50 (Promega) and DOTAPTM (Boehringer Mannheim). Each liposome reagent was mixed with conjugated PNA 1118 to give a final PNA concentration of 1 mM in the culture medium (1 mL). The optimal concentration of each liposome reagent in terms of PNA cell uptake was determined. The table below shows the amount of reagent used per mL of OptiMEM. LipofectinTM LipofectamineTM Tfx-50TM DOTAPTM
• the human carcinoma cell line HeLa was grown in RPMI 1640 medium containing GlutamaxTM, penicillin, streptomycin and fetal bovine serum. On the day preceding the experiment, the cells were plated at a density of 2 x 10 5 cells per dish in 35 mm dishes containing coverslips. The following day the cells were washed once with OptiMEM, then fed with 1 mL OptiMEM containing 1 ⁇ M PNA or PS-ODN, either alone, mixed with one of the 4 liposome reagents or inco ⁇ orated in DOPE/DDAB liposomes, as described above.
• the PNA-treated cells were fixed in 3% formaldehyde/0.2% glutaraldehyde on ice.
• the coverslips were then mounted on objective glasses and the cells observed by fluorescence microscopy on a Leits Diaplan microscope. Micrographs were taken with Kodak Ektacrome 1600 ASA film.
• the lipophilic groups (R) investigated include adamantoyl, decanoyl, heptyl-succinyl and palmityl-succinyl groups (as shown in Figure 1).
• Stock solutions of the four conjugated PNAs were prepared by dissolving the PNAs in DMSO. Dilutions of these stock solutions were made in either water or OptiMEM (Gibco BRL).
• the human carcinoma cell line HeLa was grown in RPMI 1640 medium containing GlutamaxTM, penicillin, streptomycin and fetal calf serum (10% v/v).
• the cells were plated at a density of 2x10 5 cells per dish in 35 mm dishes containing coverslips. The next day the cells were rinsed once with OptiMEM, then fed with 3 ⁇ M PNA in 1 mL of OptiMEM and further incubated. In order to visualize PNA uptake, the coverslips were washed twice with PBS and the cells were fixed for 15 minutes in 3%o formaldehyde/0.2% glutaraldehyde on ice. After washing twice with PBS, the coverslips were mounted on objective glasses using 90% glycerol in PBS, and the cells were observed by fluorescent microscopy on a Leitz Diaplan Microscope. Micrographs were taken with Kodak Ektachrome 1600 ASA film.
• the four conjugated PNAs were tested for uptake into human cells in culture.
• the PNAs were added directly to the cell culture medium.
• HeLa cells grown on coverslips were incubated with PNA (3 ⁇ m) in serum free medium overnight, then fixed and examined by fluorescence microscopy.
• Both the palmityl-succinyl and the heptyl-succinyl conjugated PNAs showed punctate and spotted fluorescence in all cells. Generally, the spots were evenly distributed over the cell with a tendency of an enhanced staining at the edges of the cells, probably the cell membrane.
• the adamantoyl- and decanoyl-conjugated PNAs showed much less cell-associated fluorescence with large fluorescent aggregates seen outside the cells.
• the palmityl-succinyl PNA conjugate was further studied by confocal microscopy to determine the exact location and distribution of the PNA conjugate inside the cell.
• a cell was selected from the above study and further scanned through 12 sections. The images confirm that the PNA conjugate was indeed taken up by the cells and distributed in spots throughout the cytoplasm. There was, apparently, no fluorescence in the nucleus. This pattern is indicative of the endocytotic pathway of uptake, implying that the PNA conjugates end up in endosomes.
• the palmityl-succinyl PNA conjugate was also observed in a time course experiment.
• Cells were incubated for different lengths of time in the presence of 3 ⁇ M of PNA.
• the uptake of the PNA conjugate by the cells increased with time up until 24 hours of incubation when the PNA-containing medium was replaced with fresh serum containing medium.
• intracellular PNA was concentrated in compartments of the cells, probably secondary lysosomes. After 72 hours there was virtually no PNA left inside the cells.
• PNA having the sequence R-Lys(Fluorescein)-TTT-AGC-TTC-CTT-AGC-Lys-NH 2 (SEQ ID NO:3) is complementary to the 15 nucleotides immediately 5' to the AUG start codon in CAT mRNA and the corresponding unconjugated PNA has previously been shown to be able to inhibit translation of CAT in vitro.
• the four conjugated PNAs of Example 38 (SEQ ID NO:2) were tested in this assay. All four conjugated PNAs specifically inhibited CAT translation at similar concentrations as the unconjugated PNA.
• Liposome constructs were prepared using two of the conjugated PNAs having SEQ ID NO:2.
• the adamantoyl- and decanoyl-conjugated PNAs were combined with liposomes by a modification of the ethanol injection method described by Campbell in Biotechniques, 1995, 18, 1027. Following this method, 13.4 ⁇ mole of DOPE (dioleyl-L- ⁇ -phosphatidyl- ethanolamine) and 6.6 ⁇ mole of DDAB (dimethyldioctadecylammonium bromide) were dissolved in 1 mL of absolute ethanol. A solution of PNA (10 ⁇ L, 3 mM PNA/DMSO) was combined with 40 ⁇ L of the lipid mixture.
• DOPE dioleyl-L- ⁇ -phosphatidyl- ethanolamine
• DDAB dimethyldioctadecylammonium bromide
• reaction mixture was then rapidly added to 1 mL of sterile distilled H 2 O while vortex mixing.
• the PNA concentration in the liposome mixture was thus 30 ⁇ M.
• 60 ⁇ L of the PNA- liposome mixture was added to 1 mL of OptiMEM and fed to the cells.
• COS-7 green monkey kidney derived cells
• NIH 3T3, mouse fibroblast cells When other cell lines were used (COS-7, green monkey kidney derived cells; and NIH 3T3, mouse fibroblast cells) identical uptake patterns were observed.
• the cellular uptake of an adamantyl-PNA (prepared according to Examples 35 or 36) was determined, and was also compared to the uptake of a phosphorothioate oligonucleotide.
• the adamantyl-conjugated PNA and oligonucleotide were added directly to subconfluent HeLa cells at 1 ⁇ M concentrations and left over night. The cells were next fixed and uptake visualized by fluorescence microscopy.
• the oligonucleotide exhibited fine punctate fluorescence, mainly confined to clusters in the cytoplasm of the cells and absent from the nuclei. With the PNA, punctate fluorescence was similarly observed. However, the spots were somewhat larger and present both in the cytoplasm and on the cell membrane.
• the PNA was combined with various commercially available cationic liposomes normally used for transfection of DNA.
• PNA-containing liposomes composed of the lipids DOPE and DDAB were also prepared.
• the hydrophobic adamantyl-group of the PNA should insert into the lipid layer of the liposomes and thus entrap the PNA.
• the liposomes were prepared by a simple ethanol injection technique, which was reported to be efficient for the transport of plasmid DNA into cells.
• the different PNA-liposome mixtures were fed to cells with a final PNA concentration of 1 ⁇ M and incubated over night.

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