Title: Cellular proteins which mediate herpesvirus entry
United States Patent: 6,641,818
Issued: November 4, 2003
Inventors: Spear; Patricia G. (Chicago, IL); Warner; Morgyn S. (Chicago, IL); Geraghty; Robert J. (Lexington, KY); Martinez; Wanda M. (Chicago, IL); Montgomery; Rebecca I. (Hinsdale, IL); Cohen; Gary H. (Havertown, PA); Eisenberg; Roselyn J. (Haddonfield, NJ); Whitbeck; Charles J. (Glenside, PA); Krummenacher; Claude (Philadelphia, PA)
Assignee: Northwestern University (Evanston, IL)
Appl. No.: 723368
Filed: November 28, 2000
Abstract
A cellular herpesvirus entry protein, or a mutant, a homolog, a derivative, a variant or a biologically active fragment thereof, suspended in a pharmaceutically active carrier in an amount effective to inhibit entry of an alphaherpesvirus into a cell, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily, is provided and method of use thereof
SUMMARY OF THE INVENTION
The invention relates to a cellular herpesvirus entry protein, or a mutant, a homolog, a derivative, a variant or a biologically active fragment thereof, suspended in a pharmaceutically active carrier in an amount effective to inhibit entry of an alphaherpesvirus into a cell, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the alphaherpesvirus is selected from the group consisting of HSV-1, HSV-2, and the animal viruses, pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1).
The invention also relates to a recombinant cell comprising an isolated nucleic acid encoding a cellular herpesvirus entry protein, or a mutant, cL homolog, a derivative, a variant or a biologically active fragment thereof, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily and the herpesvirus is an alphaherpesvirus.
In one aspect, cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the alphaherpesvirus is selected from the group consisting of HSV-1, HSV-2, PRV and BHV-1.
In yet another aspect, the cell is selected from the group consisting of Chinese hamster ovary cells, murine melanoma cells, and swine testes cells.
Also included in the invention is a vector comprising an isolated nucleic acid encoding a cellular herpesvirus entry protein, or a mutant, a homolog, a derivative, a variant or a biologically active fragment thereof, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily and the herpesvirus is an alphaherpesvirus.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the alphaherpesvirus is selected from the group consisting of HSV-1, HSV-2, PRV and BHV-1.
The invention further relates to an anti-cellular herpesvirus protein compound, wherein the compound binds herpesvirus glycoprotein D.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the herpesvirus is selected from the group consisting of HSV-1, HSV-2, PRV and BHV-1.
The invention additionally relates to an anti-cellular herpesvirus protein compound, wherein the compound is selected from the group consisting of an antisense oligonucleotide, an antibody specific for the cellular herpesvirus protein, a peptide and a peptidomimetic.
Also included in the invention is a method of identifying a compound capable of inhibiting entry of an alphaherpesvirus into a cell. The method comprises providing a population of cells which express a cellular herpesvirus entry protein, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily, infecting the cells in the presence or absence of a test compound, and measuring the level of entry of an alphaherpesvirus into the cells, wherein a lower level of entry of the virus into the cells in the presence of the test compound compared with the level of entry of the virus into the cells in the absence of the test compound is an indication that the test compound is an anti-cellular herpesvirus entry protein compound.
The invention also includes an anti-cellular herpesvirus entry protein compound identified by the method of identifying a compound capable of inhibiting entry of an alphaherpesvirus into a cell. The method comprises providing a population of cells which express a cellular herpesvirus entry protein, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily, infecting the cells in the presence or absence of a test compound, and measuring the level of entry of an alphaherpesvirus into the cells, wherein a lower level of entry of the virus into the cells in the presence of the test compound compared with the level of entry of the virus into the cells in the absence of the test compound is an indication that the test compound is an anti-cellular herpesvirus entry protein compound.
The invention also relates to a method of inhibiting entry of an alphaherpesvirus into a cell comprising adding to the cell an anti-cellular herpesvirus entry protein compound thereby inhibiting entry of the virus into the cell.
In addition, the invention relates to a method of treating an alphaherpesvirus infection in an animal comprising administering to the animal an anticellular herpesvirus entry protein compound, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily.
In one aspect, the animal is a human.
In another aspect, the herpesvirus is selected from the group consisting of HSV-1 and HSV-2.
The invention further relates to a composition comprising a soluble alphaherpesvirus glycoprotein D-binding cellular herpesvirus entry protein.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC. In a preferred embodiment, the .HveB and the HveC are truncated.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based upon the discovery of two cellular proteins, HveB and HveC, which are cellular herpesvirus entry proteins and therefore mediate entry of HSV-1 and/or HSV-2 and other herpesviruses into cells. The identification of these proteins facilitates the identification of compounds which serve to inhibit entry of herpesviruses into cells.
HveB and HveC are collectively referred to herein as cellular herpesvirus entry proteins. The nucleotide and corresponding amino acid sequence of HveB and HveC are presented herein in FIGS. 1 and 2, respectively.
The present invention includes a method of screening compounds for their ability to inhibit HveB or HveC mediatedherpesvirus entry into cells. The present invention further includes a compound which is identified using the method of the invention, which compound inhibits herpesvirus entry into cells.
A compound which inhibits HveB or HveC mediated herpesvirus entry into a cell is referred to herein as an "anti-cellular herpesvims entry protein" compound.
In addition, the invention includes a method of inhibiting the entry of a herpesvirus into a cell. The invention further includes a method of treating a herpesvirus infection in a human by administering to the human a compound capable of inhibiting entry of a herpesvirus into a cell.
The cellular proteins, HveB and HveC, which have been discovered according to the present invention to inhibit entry of herpesviruses into cells, are human members of the immunoglobulin superfamily of proteins. HveC is a cellular protein which has been discovered in the present invention to mediate entry of HSV-1, HSV-2, porcine pseudorabiesvirus (PRV) and bovine herpesvirus I (BHV-1) into cells. HveC is a membrane glycoprotein which is highly homologous to the poliovirus receptor related protein 1 (Prr1). As the data presented herein establish, soluble forms of HveC interact directly with the herpesvirus. Further, HveC is expressed in human cells of epithelial and neuronal origin and therefore functions as a coreceptor that facilitates infection of epithelial cells on mucosal surfaces and spread to neuronal cells by both HSV-1and HSV-2.
HveB is a cellular protein which does not exhibit the same versatility as HveC in binding to herpesviruses to facilitate their entry into cells. HveB is highly homologous to poliovirus receptor related protein 2 (Prr2). As the data presented herein establish, this protein mediates entry of certain HSV-1 mutants but does not mediate entry of wild type HSV-1 or BHV-1 into cells. However, entry of HSV-2 and PRV into cells is mediated by HveB. HveB is expressed in some human neuronal cell lines, fibroblastic cells, keratinocytes and primary activated T lymphocytes.
The differences in the ability of HveB and HveC to mediate entry of various herpesviruses into various cell types likely accounts for serotype and strain differences in tissue tropism and pathogenicity of the alphaherpesviruses. However, the discovery that these proteins serve as the gateway for entry of alphaherpesviruses into cells provides the art with heretofore unknown methods of identifying compounds useful for treating herpesvirus infection in both humans and animals.
The invention should not be construed to be limited solely to the use of the specific cellular HveB and HveC disclosed herein, or to the specific nucleotide sequences which encode them. Rather, the invention should be construed to include any and all mutants, homologs, variants and derivatives of HveB or HveC which serve to facilitate entry of a herpesvirus into a cells. An HveB or an HveC cellular protein which mediates entry of an alphaherpesvirus into a cell, as assessed in the cell entry assays described herein, is designated herein as a "biologically active" HveB or HveC protein. Biologically active forms of either HveB or HveC include any and all HveB and HveC proteins having substantial homology with the HveB and HveC proteins disclosed herein so as to facilitate entry of a herpesvirus into a cell.
In addition, the invention should be construed to include any and all mutants, homologs, variants and derivatives of polynucleotides encoding HveB or HveC which serve to facilitate entry of a herpesvirus into a cells. It is well within the skill of those in the art to clone, sequence and otherwise identify such mutants, homologs, variants and derivatives of polynucleotides encoding HveB or HveC, and to use such mutants, etc. to express useful HveB or HveC for use in the methods of the invention.
"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half(e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3' ATTGCC5' and 3' TATGGC share 50% homology.
The invention should be construed to include any form of HveB or HveC having substantial homology to the HveB or HveC proteins disclosed herein. Preferably, a protein which is substantially homologous is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to any one of the HveB or HveC proteins disclosed herein.
As used herein, "homology" is used synonymously with "identity." Percent identity of one polynucleotide or polypeptide with respect to another polynucleotide or polypeptide may be determined using any available algorithm, such as the BLAST program.
Mutants, derivatives and variants of HveB or HveC include any and all HveB and HveC molecules or polynucleotides encoding them, which although they may differ in their primary nucleotide or amino acid sequence from the HveB and HveC disclosed herein, retain the biological activity of HveB and HveC as defined herein. HveB and HveC are considered to be paralogs of each other, in that, although they are encoded by different genes, both exhibit entry activity for selected strains of HSV. When alignments are performed between HveB and HveC amino acid sequences, the percent identity in sequences ranges from about 33-37%. The percent similarity (allowing for conservative amino acid substitutions) between the two proteins is about 47%. Based upon these data, the invention should therefore be construed to include any HveB or HveC polynucleotide molecule which encodes an HveB or and HveC polypeptide having at least about 33-37% identity and at least about 47% similarity with the amino acid sequence of HveB and HveC shown in FIGS. 1 and 2 herein.
Further, the invention should be construed to include biologically active fragments of any homologs, mutants, derivatives or variants of the HveB and HveC proteins disclosed herein. Fragments of HveB or HveC typically may be about 50 amino acids in length in order to retain biological activity of either protein. More typically, biologically active fragments of HveB or HveC will be about 150 amino acids in length.
HveB and HveC proteins or peptide fragments thereof, may themselves be used as agents which block entry of herpesviruses into cells. As the data presented herein establish, these proteins interact directly with the virus. Thus, administration of either of these proteins to an animal, or a mutant, homolog, derivative or variant thereof, is likely to bind to virus thereby preventing virus entry into a cell. Particularly, administration of soluble forms of these proteins to an animal including a human, is a feasible method of treating alphaherpesvirus infection of the same. Soluble forms of HveB and HveC may be generated following the protocols disclosed herein wherein the membrane anchor domain of either protein is modified in some way or is deleted such that the protein is soluble. A preferred soluble form of HveB comprises a deletion in amino acids 357-385 from the full length protein. A preferred soluble form of HveC comprises a deletion in amino acids 349-378 from the full length protein.
It will be appreciated, of course, that the HveB or HveC peptides may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or stabilize the N- and C-termini from "undesirable degradation", a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the biological activity of HveB or HveC, i.e. sequential degradation of the compound at a terminal end thereof.
Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-terminus. Examples of suitable N-terminal blocking groups include C1 -C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (--NH2), and mono-and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methyletijylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without affect on peptide activity.
Other modifications can also be incorporated without adversely affecting virus binding activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the present invention are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.
Acid addition salts of the present invention are also contemplated as functional equivalents. Thus, a peptide in accordance with the present invention treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamie, mandelic, methanesulfonic, ethariesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the peptide is suitable for use as a virus binding agent.
The present invention therefore also provides for analogs of proteins or peptides of HveB and HveC. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups:
glycine, alanine;
valine, isoleucine, leucine;
aspartic acid, glutamic acid;
asparagine, glutamine;
serine, threonine;
lysine, arginine;
phenylalanine, tyrosine.
As noted above, modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.
Thus, the invention should be construed to include substantially pure HveB or HveC, or mutants, homologs, variants or derivatives or modifications thereof, which are useful for inhibiting entry of an alphaherpesvirus into a cell.
Substantially pure protein obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).
The term "substantially pure" as used herein, describes a compound, e.g., a protein or polypeptide which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest: Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.
The invention should also be construed to include an isolated nucleic acid encoding HveB or HveC, or any mutants, homologs, derivatives or variants thereof.
As used herein, a "coding region" of a nucleic acid (i.e., that portion of the nucleic acid which encodes a protein) consists of the nucleotide residues of the coding strand of the nucleic acid and the nucleotides of the non-coding strand of the nucleic acid which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the nucleic acid.
"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
A "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
An "mRNA-coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotide residues of the non-coding strand of the gene which are homologous with or complementary to, respectively, an mRNA molecule which is produced by transcription of the gene. It is understood that, owing to mRNA processing which occurs in certain instances in eukaryotic cells, the mRNA-coding region of a gene may comprise a single region or a plurality of regions separated from one another in the gene as it occurs in the genome. Where the MRNA coding region of a gene comprises separate regions in a genome, "mRNA-coding region" refers both individually and collectively to each of these regions.
In addition, a "coding region" of an mRNA molecule consists of the nucleotide residues of the mRNA molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g. amino acid residues in a protein export signal sequence).
As used herein, an "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which i part of a hybrid gene encoding additional polypeptide sequence.
A "polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
The term "nucleic acid" typically refers to large polynucleotides.
The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."
Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.
The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences."
A "portion" of a polynucleotide means at least at least about five sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.
"Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
"Probe" refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
"Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.
A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
A host cell that comprises a recombinant polynucleotide is referred to as a "recombinant host cell." A gene which is expressed in a recombinant host cell wherein the gene comprises a recombinant polynucleotide, produces a "recombinant polypeptide."
A "recombinant polypeptide" is one which is produced upon expression of a recombinant polynucleotide.
Isolated nucleic acids encoding HveB or HveC may be transfected into cells for the purpose of producing large quantities of these protein, or for the purpose of generating cells which express HveB or HveC for use in drug screening assays. Production of large quantities of HveB or HveC may be accomplished by cloning an isolated nucleic acid encoding either protein into a baculovirus, yeast or bacterial expression vector system and then transfecting the appropriate cells with the vector to facilitate expression of either protein therein. Such technology is described herein and is well known in the art, being described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.) and in Ausubel et al. (1993, Current Protocols in Molecular Biology, Green & Wiley, New York).
Expression of an isolated nucleic acid encoding either HveB or HveC in a cell is accomplished by placing the nucleic acid under the control of a suitable promoter/regulatory sequence such that the nucleic acid encoding the protein is operably linked thereto.
As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for constitutive expression or for tissue-specific, organ specific, or other specific (such as inducible, etc) expression of a nucleic acid operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the nucleic acid in a tissue specific manner.
By describing two polynucleotides as "operably linked" is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region.
A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
Based upon the present discovery, compounds which are useful for inhibiting entry of an alphaherpesvirus into a cell include soluble forms of HveB and HveC as disclosed herein. In addition, compounds useful for inhibiting entry of a hirpesvirus into a cell include polynucleotides which are complementary, i.e., are in an antisense orientation with respect to the coding sequences of HveB or HveC.
"Complementary" as used herein, refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecule, are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).
"Antisense" refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of the coding strand of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
Antisense HveB or HveC polynucleotides include oligonucleotides which are from about five to about one hundred nucleotides in length. The antisense oligonucleotides of the invention preferably comprise between about fourteen and about fifty nucleotides in length. More preferably, the antisense oligonucleotides comprise between about twelve and about thirty nucleotides in length. Most preferably, the antisense oligonucleotides comprise between about sixteen and about twenty-one nucleotides in length. The antisense oligonucleotides of the invention include, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides. Methods for synthesizing oligonucleotides, phosphorothioate oligonucleotides, and otherwise modified oligonucleotides are well known in the art (U.S. Pat. No: 5,034,506; Nielsen et al., 1991, Science 254: 1497). Oligonucleotides which contain at least one phosphorothioate modification are known to confer upon the oligonucleotide enhanced resistance to nucleases. Specific examples of modified oligonucleotides include those which contain phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatomic or heterocyclic intersugar ("backbone") linkages. In addition, oligonucleotides having morpholino backbone structures (U.S. Pat. No: 5,034,506) or polyamide backbone structures (Nielsen et al., 1991, Science 254: 1497) may also be used.
The examples of oligonucleotide modifications described herein are not exhaustive and it is understood that the invention includes additional modifications of the antisense oligonucleotides of the invention which modifications serve to enhance the therapeutic properties of the antisense oligonucleotide without appreciable alteration of the basic sequence of the antisense oligonucleotide.
Antisense oligonucleotides may also be synthesized and expressed in any expression system suitable for this purpose, and may also be contained within a viral delivery vehicle or other vector delivery system suitable for administration to a mammal. Such systems are described herein with respect to antibodies and are equally applicable to the delivery and expression of antisense oligonucleotides.
Antibodies directed against HveB or HveC are also useful compounds for the inhibition of herpesvirus entry into cells. The types of antibodies which may be used include polyclonal antibodies, monoclonal antibodies, phage-derived antibodies, synthetic antibodies, humanized antibodies, and the like. Antibody technology is described in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.). Polyclonal antibodies directed against a herpesvirus entry protein may be made by immunizing any suitable animal and obtaining immune serum from the animal at selected intervals following immunization.
Monoclonal antibodies directed against full length or peptide fragments of a cellular herpesvirus entry protein may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter/regulatory sequence in cells which are suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.
Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and the references cited therein. Further, the antibody may be "humanized" using the technology described in Wright et al., (supra) and in the references cited therein.
To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNTA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (1989. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y. and in Ausubel et al. (Ausubel et al., 1993, Current Protocols in Molecular Biology, Green & Wiley, New York).
Bacteriophage which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage which do not express the antibody will not bind to the cell. Such panning techniques are well known in the art and are described for example, in Wright et al., (supra).
Processes such as those described above, have been developed for the production of human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.
The procedures just presented describe the generation of phage which encode the Fab portion of an antibody molecule. However, the invention should not be construed to be limited solely to the generation of phage encoding Fab antibodies. Rather, phage which encode single chain antibodies (scFv/phage antibody libraries) are also included in the invention. Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHl) of the heavy chain. Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment. An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein. Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al. (1991, J. Mol. Biol. 222:581-597). Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.
The invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1 :837-839; de Kruif et al. 1995, J. Mol. Biol. 248:97-105).
By-the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The invention thus includes an isolated DNA encoding an anti-cellular herpesvirus entry protein antibody or DNA encoding a portion of the antibody.
To isolate DNA encoding an antibody, for example, DNA is extracted from antibody expressing phage obtained as described herein. Such extraction techniques are well known in the art and are described, for example, in Sambrook et al. (supra) and in Ausubel et al. (supra).
By the term "scFv/phage" are used herein, is meant a phage particle which expresses the Fv portion of an antibody as a single chain.
Another form of antibody includes a nucleic acid sequence which -encodes the antibody and which is operably linked to promoter/regulatory sequences which can direct expression of the antibody in vivo. For a discussion of this technology, see, for example, Cohen (1993, Science 259: 1691-1692), Fynan et al. (1993, Proc. Natl. Acad. Sci. 90:11478-11482) and Wolff et al. (1991, Biotechniques 11:474-485) which describe similar the use of naked DNA as antibody/vaccine. For example, a plasmid containing suitable promoter/regulatory sequences operably linked to a DNA sequence encoding an antibody may be directly administered to a patient using the technology described in the aforementioned references.
Alternatively, the promoter/enhancer sequence operably linked to DNA encoding the antibody may be contained within a vector, which vector is administered to the patient. The vector may be a viral vector which is suitable as a delivery vehicle for delivery of the DNA encoding the antibody to the patient, or the vector may be a non-viral vector which is suitable for the same purpose. Examples of viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. USA 94:12744-12746). Examples of viral vectors include, but are not limited to, a recombinant vaccinia virus, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian pox virus, and the like (Cranage et al., 1986, EMBO J. 5:3057-3063; International Patent Application No. W094/17810, published Aug. 18, 1994; International Patent Application No. W094/23 744, published Oct. 27, 1994). Examples of non-viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.
The identity, selection and means for obtaining a desired antibody useful for treatment or prevention of a herpesvirus infection may be performed by the skilled artisan using conventional technology when in possession of the present invention.
The invention should also be construed to include small molecules and/or peptidomimetics which bind HveB or HveC, thereby preventing attachment of HSV thereto and preventing entry of HSV into a cell. The amino acid sequences of HveB and HveC disclosed herein are useful for the generation of peptidomirnetics and other small molecules useful for treatment of herpesvirus infections. Peptidomimetics may be generated using techniques described in PCT/US93/01201 and in U.S. Pat. No. 5,334,702.
Also included in the invention are cells which have been transfected such that they express the cellular herpesvirus entry protein of the invention. Such cells may be transiently or more permanently transfected with DNA encoding biologically active HveB or HveC. Cells transfected are generated using the procedures presented herein in the Examples. Essentially, a plasmid or other vector encoding HveB, HveC or biologically active mutants, homologs, variants or derivatives thereof, is used to transfect cells, usually in conjunction with DNA encoding a selectable marker the expression of which facilitates selection of the desired transfected cells. Transfected cells are selected, cloned and the presence and expression of the desired cellular herpesvirus entry protein is assessed using ordinary molecular biology technology and suitable probes and antibodies and the like. Preferred cells include any cell type which lacks the natural form of the desired herpesvirus entry protein and include, but are not limited to, Chinese hamster ovary cells, murine melanoma cells, and swine testes cells and any mutants thereof.
Cells which express an isolated nucleic acid encoding an herpesvirus entry protein are referred to herein as "recombinant cells." Such cells may be either prokaryotic cells or eukaryotic cells. Typically, prokaryotic cells include those which are useful for propagation of DNA encoding HveB or HveC, and for expression of the protein in a form which is useful in the methods of the invention. For example, a typical prokaryotic cell is E. coli, which is well known in the art as a bacterium useful for the propagation of nucleic acid and expression of the same. The use of such prokaryotic cells is well known in the art and is; described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.); in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York); and in (Gerhardt et al., eds. (1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C.).
Useful eukaryotic cells are those which are documented in the Examples presented herein, and further include any cell type into which HveB or HveC DNA can be introduced, wherein the cell is then useful in the methods of the invention recited herein.
Cells which express HveB or HveC are useful for the identification of additional molecules capable of inhibiting HveB or HveC mediated herpesvirus entry into cells. For example, a simple screening assay may be used to identify such molecules as follows. Essentially, a population of cells which express the desired cellular herpesvirus entry protein is provided. A test compound is administered to an aliquot of the cells either before, after, or concomitantly with infection of the cells with an alphaherpesvirus. Another aliquot of identical cells is not administered the test compound. The ability of the virus to enter the cells in the presence or absence of the test compound is assessed, wherein a lower level of entry of virus into the cells in the presence of the test compound compared with the level of entry of virus into the cells in the absence of the test compound is an indication that the test compound inhibits entry of the virus into the cells. Additional viral replication assays and assays for the initiation and maintenance of or reactivation of the virus from the latent state may also be performed to determine whether the test compound is capable of inhibiting virus infection in a pathologically meaningful manner, i.e., in a manner which either reduces or ablates the pathogenicity of the virus in its natural host.
Test compounds which are identified using the screening methods just described may be any type of molecule, including but not limited to, HveB, HveC, or any homologs, mutants, variants, derivatives or biologically active fragments thereof, antisense oligonucleotides which are complementary to portions of the coding strand of the double stranded encoding HveB or HveC, antibodies which specifically bind to HveB or HveC, small molecules, peptides, a peptidomimetics, and the like which are predicted in any way to inhibit virus entry into a cell. Peptidomimetic compounds whose structure is based upon the known amino acid sequence of HveB or HveC may be designed and produced as described in PCT/US93/01201 and in U.S. Pat. No. 5,334,702.
The anti-cellular herpesvirus entry protein compound of the invention may be formulated in a pharmaceutical composition which is suitable for administration of the compound to an animal or a human patient. It will be appreciated that the precise formulation and dosage amounts will vary depending upon any number of factors, including, but not limited to, the type and severity of the viral disease to be treated, the route of administration, the age and overall health of the animal or human, the nature of the anti-cellular herpesvirus entry protein compound, etc. However, the preparation of a pharmaceutically acceptable composition having an appropriate pH, isotonicity, stability and other characteristics is within the skill of the art. Pharmaceutical compositions are described in the art, for example, in Remington's Pharmaceutical Sciences (Genaro ed., 1985, Mack Publishing Co., Easton, Pa.).
As used herein, the term "pharmaceutically-acceptable carrier" means a chemical composition with which an appropriate anti-cellular herpesvirus entry protein compound may be combined and which, following the combination, can be used to administer the anti-cellular herpesvirus entry protein compound to a patient.
The amount of the anti-cellular herpesvirus entry protein compound administered, whether it is administered as protein or as nucleic acid, is sufficient to prevent, diminish or alleviate the disease state. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between about 1 ng/kg and about 100 mg/kg of patient body-weight. Suitable amounts of the anti-cellular herpesvirus entry protein compound for administration include doses which are high enough to have the desired effect without concomitant adverse effects. When the anti-cellular herpesvirus entry protein compound is a protein or peptide, a preferred dosage range is from about 10 to about 1000 .mu.g of protein or peptide per kg of patient body weight. When the anti-cellular herpesvirus entry protein compound is administered in the form of DNA encoding the same contained within a recombinant virus vector, a dosage of between about 102 and about 1011 plaque forming units of virus per kg of patient body weight may be used. When naked DNA encoding the anti-cellular herpesvirus entry protein compound is to be administered as the pharmaceutical composition, a dosage of between about 10 .mu.g about several mg of DNA per kg of patient body weight may be used.
In the practice of the methods of the invention, a composition containing an anti-cellular herpesvirus entry protein compound is administered to a patient in a sufficient amount to prevent, diminish or alleviate a herpesvirus infection in the animal, preferably, a human.
The frequency of administration of an anti-cellular herpesvirus entry protein compound to an animal or a human patient will also vary depending on several factors including, but not limited to, the type and severity of the infection to be treated, the route of administration, the age and overall health of the animal or human patient, the nature of the anti-cellular herpesvirus entry protein compound, etc. It is contemplated that the frequency of administration of the anti-cellular herpesvirus entry protein compound to the animal or human patient may vary from about once every few months to about once a month, to about once a week, to about once per day, to about several times daily.
Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in parenteral, oral solid and liquid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations. In addition to the appropriate anti-cellular herpesvirus entry protein compound, these pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. Thus, such compositions may optionally contain other components, such as adjuvants, e.g., aqueous suspensions of aluminum and magnesium hydroxides, and/or other pharmaceutically acceptable carriers, such as saline. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer the appropriate anti-cellular herpesvirus entry protein compound to a patient according to the methods of the invention. Oral delivery of antibodies is described in Reilly et al. (1997, Clin. Pharmacol. 32:313-323).
Compounds which are identified using any of the methods described herein may be formulated and administered to a mammal for treatment of herpesvirus infection are now described.
The invention encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of a herpesvirus infection as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.
Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.
Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient.
Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are riot limited to, potato starch and sodium starch glycollate. Known surface active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are riot limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.
Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.
Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.
Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for ex,ample, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gain tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e. about 20oC.) and which is liquid at the rectal temperature of the subject (i.e. about 37oC. in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or gel or cream or a solution for vaginal irrigation.
Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.
As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic: materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65oF. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may (constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).
Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.
The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.
Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalinalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.
Typically dosages of the compound of the invention which may be administered to an animal, preferably a human, range in amount from 1 .mu.g to about 100 g per killogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferably, the dosage of the compound will vary from about 10 mg to about 10 g per killogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per killogram of body weight of the animal.
The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a (lay, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
The invention also includes a kit comprising a composition of the invention and an instructional material which describes adventitially administering the composition to a cell or a tissue of a mammal. In another embodiment, the kit comprises a (preferably sterile) solvent suitable for dissolving or suspending the composition of the invention prior to administering the compound to the mammal.
As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the peptide of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviation the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the peptide of the invention or be shipped together with a container which contains the peptide. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
Claim 1 of 3 Claims
What is claimed is:
1. A cellular herpesvirus entry protein consisting of the polypeptide set forth in SEQ ID NO:4 suspended in a pharmaceutically active carrier.
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