JP2006526022A - Use of interferon for the treatment of severe acute respiratory syndrome and other viral infections - Google Patents

Abstract

The present invention relates to Severe Acute Respiratory Syndrome (SARS) comprising the step of administering to a subject an amount of an interferon polypeptide effective to reduce the concentration of SARS-related coronavirus particles in the subject. A method for treating a subject suffering from a disease is provided. The present invention further provides a method of treating a subject infected with a particular virus and a method of reducing the risk of viral infection in a subject, comprising administering an interferon polypeptide to the subject. The invention further provides a method of treating a subject afflicted with influenza or reducing the risk of influenza virus infection in a subject comprising administering to the subject an interferon polypeptide.

Description

Field of Invention

  This application claims priority from US Provisional Application No. 60 / 473,134 filed May 23, 2003, the contents of which are incorporated herein by reference.

  Various references are cited throughout this application, the contents of each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION Interferons (IFNs) are a well-known family of cytokines that are secreted when various eukaryotic cells are exposed to various stimuli (Zoon KC: Human Interferons: Structure and Function. P. 1- 12. In:
Interferon 8. Academic Press, London, 1987; Walter et al., Cancer Biotherm Radiopharm 1998 June; 13 (3): 143-54; Pestka, S., Biopolymers 2000; 55 (4): 254-287; Pestka , S., Methods in Enzymology,
78, 1981; Pestka, S., Methods in Enzymology, 79, 1981; Pestka, S., Methods in
Enzymology, 119, 1986). Interferons have been classified according to their chemical and biological characteristics. Interferons are divided into two groups called type I and type II interferons (Pestka, S .; Langer; JA; Zoon, KC; Samuel, CE Ann Rev Biochem)
1987, 56, 727-777; Pestka, S., Biopolymers 2000; 55 (4): 254-287). IFN-γ, also known as immune interferon, is the only type II interferon, while type I human interferons are IFN-α, IFN-β, IFN-ω, IFN-κ, and IFN-τ, It consists of five classes. There is only one human IFN-β and human IFN-ω, but there is a family of multiple IFN-α species. IFN-τ is found only in ungulates and there is no human IFN-τ. IFN exhibits antiviral activity, immunomodulatory activity and antiproliferative activity. The clinical potential of interferon is recognized.

Interferon was discovered by Isaac and Lindenman (Proc. Royal Soc. London, Ser B 147, 258, 1957). Attempts to purify and characterize human leukocyte interferon prepare a homogeneous leukocyte interferon (now called IFN-α) derived from normal and leukemic (chronic myeloid leukemia or “CML”) donor leukocytes (Pestka,
S., Biopolymers 2000; 55 (4): 254-287; Rubinstein, M .; Levy, WP; Moschera, JA; Lai, C.-Y .; Hershberg,
RD; Bartlett, RT; Pestka, S. Arch Biochem Biophys 1981, 210, 307-318.). Homogeneous fibroblast interferon (now called IFN-β) was also purified to homogeneity (Friesen, H.-J .; Stein, S .; Evinger, M .; Familletti, PC; Moschera,
J .; Meienhofer, J .; Shively, J .; Pestka, S. Arch Biochem Biophys 1981, 206,
432-450). These interferons are a family of proteins characterized by a strong ability to bring a virus resistance state to their target cells. In addition, interferons can inhibit cell proliferation, regulate immune responses, and alter protein expression.
These properties spur clinical use of leukocyte interferon as a therapeutic agent for the treatment of viral infections and malignant tumors.

  During the past decades, a large number of human and animal interferons have been produced, identified, purified and cloned. Some of the interferon formulations were prepared for clinical trials, both in crude form and in purified form. Several individual recombinant interferon-α species have been cloned and expressed. Thus, the protein has been purified by a variety of techniques and formulated for clinical use in a variety of formulations. Most of the interferons approved by various legal agencies around the world for clinical use are mixtures of human alpha interferon (Hu-IFN-alpha) or individual species. In some countries, Hu-IFN-β and γ have also been approved for clinical trials and in some cases their therapeutic use. The main theory underlying the clinical use of these interferons was that they are natural molecules produced by normal individuals. In fact, the specific theory is that all interferons prepared for clinical use were naturally produced by normal humans, regardless of whether they were natural or recombinantly produced products. Is a point. This is true not only for numerous interferons, but also for the specific growth factors, lymphokines, cytokines, hormones, clotting factors and other proteins that have been produced.

The present invention relates to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42. 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or a fragment thereof A nucleic acid comprising a polynucleotide encoding at least a portion of an interferon polypeptide comprising the amino acid sequence shown in at least one of the above. Thus, one aspect of the present invention provides (a) a nucleotide, sequence encoding an interferon polypeptide comprising at least one amino acid sequence of these SEQ ID NOs; (b) at least one of these SEQ ID NOs: A nucleotide, sequence encoding a biologically active fragment of the polypeptide shown in Figure 3; and (c) a nucleotide sequence complementary to at least one of the nucleotide sequences of (a) or (b) above: A polynucleotide comprising a nucleotide sequence selected from the group consisting of: Further embodiments of the invention include at least 95% identical, and more preferably at least 96% identical, and even more preferably at least at least one of the nucleotide sequences of (a), (b), or (c) above. A polynucleotide having a nucleotide sequence that is 97% identical, and even more preferably at least 98% identical, and even more preferably at least 99.25% identical, or a polynucleotide of (a), (b), or (c) above And preferably SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, As shown in at least one of 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85 Polynucleotides that hybridize to stringent polynucleotides under stringent hybridization conditions Kureochido, there is a nucleic acid that contains the. Such fragments include biologically active fragments of interferon polypeptides. Sequence number 1-26 is the arrangement | sequence of a cat interferon. SEQ ID NOs: 27-36 are rhesus monkey interferon sequences. SEQ ID NOs: 37-86 are human interferon sequences.

“Stringent hybridization conditions” are intended to include stringent washing conditions, and those skilled in the art will be able to determine such high conditions, such as low salt and high temperature. An example of a low salt is 0.1 X SSC. An example of a high temperature is 65-68 ° C. One embodiment comprises 50% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5 x Denhart's solution, 10% dextran sulfate, and 20
After overnight incubation at 42 ° C. in a solution containing ug / ml denatured sheared salmon sperm DNA:
Including washing in SSC. As used herein, “high stringency” means (i) using low ionic strength and high temperature for post-hybridization washing, eg, 0.1 times SSC and 0.1% (w / v) SDS at 50 ° C. Or (ii) during hybridization conditions, for example, the hybridization temperature is 25 ° C., which is lower than the duplex melting temperature of the polynucleotide to be hybridized, for example 1.5 times SSPE at 65 ° C., 10 % (w / v), polyethylene glycol 6000, 7% (w / v), SDS, 0.25 mg / ml using fragmented herring sperm DNA; or (iii) 0.5M sodium phosphate, pH 7.2, eg, 70 ° C. 5 mM EDTA, 7% (w / v), SDS (28), and 0.5% (w / v), use BLOTTO; or (iv) formamide during hybridization, eg 50% (v / v) formamide , Etc., 5 times SSC at 42 ° C, 50 mM Use with sodium acid (pH 6.5) and 5 × Denharz solution; or (v) 50% (v / v), eg 42 ° C., formamide, 5 × SSC, 50 mM sodium phosphate (pH 6.8) , 0.1% (w / v), sodium pyrophosphate, 5-fold Denharz solution, sonicated salmon sperm DNA (50 .mu.g / ml), and 10% dextran sulfate.

  With respect to polypeptide, peptide and protein fragments, these may be biologically active fragments and of any length shorter than the full length of the sequence described in the sequence identification element provided herein. Also good. In certain embodiments, the fragment is at least 10 amino acids in length. In another embodiment, the fragment is at least 20 amino acids in length. In other embodiments, the fragment may be at least 30, 40, 50, 60, 70, 80, 90 or 100 amino acids long. In further embodiments, the fragment may be at least 110, 120, 130, 140, 150, 160, or 170 amino acids long.

  With respect to nucleic acid fragments, these include those that would be useful as diagnostic probes and primers as discussed herein. Such sequences may specifically identify the interferon nucleotide sequences described herein. And they can be of any length. For example, they include those that are at least about 15 nucleotides, and more preferably at least about 20 nucleotides, even more preferably at least about 30 nucleotides, and even more preferably at least about 40 nucleotides in length. Of course, larger fragments, such as those 50-300 nucleotides in length, as well as fragments corresponding to most if not all of the nucleotide sequences shown in at least one of the SEQ ID NOs provided herein, Useful according to the invention. Fragment is a fragment that is intended to be at least 20 nucleotides in length including, for example, 20 or more consecutive bases from at least one of the nucleotide sequences as set forth in at least one of the SEQ ID NOs provided herein. It is. Fragments that are 50-300 nucleotides in length include, for example, those that are about 50, 60, 70, 80, 90 or 100 bases long. These will also include fragments that are about 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 bases in length. These will also include fragments that are about 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 bases long.

  A polynucleotide that hybridizes to a “portion” of a polynucleotide includes at least about 15 nucleotides of the reference polynucleotide, and more preferably at least about 20 nucleotides, including any intermediate length (eg, 50 bases). And even more preferably a polynucleotide (either DNA or RNA) that hybridizes to at least about 30 nucleotides, and even more preferably about 30-70 nucleotides. These are useful as diagnostic probes and primers as discussed above and as detailed below.

In another aspect, the invention provides an amino acid set forth in at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a fragment thereof. Provided is a nucleic acid comprising a polynucleotide encoding at least a portion of a feline interferon polypeptide comprising a sequence. Thus, one aspect of the invention provides: (a) a nucleotide sequence encoding a feline interferon polypeptide comprising at least one amino acid sequence of these SEQ ID NOs; (b) of these SEQ ID NOs: A nucleotide sequence encoding a biologically active fragment of the polypeptide shown in at least one of: and (c) a nucleotide sequence complementary to at least one of the nucleotide sequences of (a) or (b) above, A polynucleotide comprising a nucleotide sequence selected from the group consisting of: Further embodiments of the invention include at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably any of the nucleotide sequences described herein. A polynucleotide having a nucleotide sequence that is at least 98% identical, and even more preferably at least 99.25% identical, or a polynucleotide sequence described herein, and preferably SEQ ID NOs: 1, 3, 5, 7, 9, There is a nucleic acid comprising a polynucleotide that hybridizes under stringent hybridization conditions to the polynucleotide sequence shown in at least one of 11, 13, 15, 17, 19, 21, 23 or 25. .

The present invention further provides a polynucleotide encoding at least a part of a rhesus monkey interferon polypeptide comprising the amino acid sequence shown in SEQ ID NO: 28, 30, 32, 34, 36 or at least one fragment thereof. The nucleic acid containing is provided.
Thus, one aspect of the invention provides (a) a nucleotide sequence encoding a rhesus monkey interferon polypeptide comprising at least one amino acid sequence of these SEQ ID NOs; (b) of these SEQ ID NOs: A nucleotide sequence encoding a biologically active fragment of at least one of the polypeptides shown; and (c) a nucleotide sequence complementary to any of the nucleotide sequences of (a) or (b) above A polynucleotide comprising a selected nucleotide sequence is provided. In further embodiments, at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical to any of the nucleotide sequences of (a), (b), or (c) above And even more preferably at least 98% identical, and even more preferably at least 99.25% identical polynucleotides having a nucleotide sequence, or (a), (b), or (c) nucleotides, and preferably the sequence There is a nucleic acid comprising a polynucleotide that hybridizes under stringent hybridization conditions to the polynucleotide shown in at least one of the numbers 27, 29, 31, 33 or 35.

The present invention further relates to SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80. , 82, 84, 86 or a fragment thereof, a nucleic acid comprising a polynucleotide encoding at least a portion of a human interferon polypeptide comprising the amino acid sequence set forth in at least one of its fragments. Thus, one aspect of the present invention provides: (a) a nucleotide sequence encoding a human interferon polypeptide comprising at least one amino acid sequence of these SEQ ID NOs; (b) at least of these SEQ ID NOs: A nucleotide sequence encoding a biologically active fragment of the polypeptide shown in one; and (c) a nucleotide sequence complementary to any of the nucleotide sequences of (a) or (c) above. An isolated polynucleotide comprising the nucleotide sequence to be provided is provided. Further embodiments of the invention include at least 95% identical, and more preferably at least 96% identical, and even more preferred to any of the nucleotide sequences of (a), (b), or (c) above. Is a polynucleotide having a nucleotide sequence that is at least 97% identical, and even more preferably at least 98% identical, and even more preferably at least 99.25% identical, or a polynucleotide of (a), (b), or (c) above Nucleotides, and preferably SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, There is an isolated nucleic acid comprising a polynucleotide that hybridizes under stringent hybridization conditions to a polynucleotide set forth in at least one of 79, 81, 83 or 85.

  In another aspect, any of the nucleic acids of the invention that encode an interferon polypeptide includes, but is not limited to, the amino acid sequence of the complete polypeptide by itself; and the complete polypeptide coding sequence Or additional sequences such as those encoding additional secretory leader sequences such as pre-, or pro- or pre-pro-protein sequences may be included.

  In addition, it plays a role in transcription and mRNA processing including, but not limited to, splicing and polyadenylation signals such as introns and non-coding 5 'and 3' sequences, for example, ribosome binding and stability of mRNA. Sequences described here, along with additional non-coding sequences, including additional coding sequences that encode additional amino acids, such as those that are transcribed but not translated; and those that provide additional functionality Nucleic acids are also provided.

Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In some preferred embodiments of this aspect of the invention, the marker amino acid sequence is, among other things, a pQE vector, many of which are commercially available (91311, CA, Chatsworth, Eaton Avenue 9259, Qiagen). The hexa-histidine peptide, such as the tag provided in. For example, the hexa-histidine described by Gentz et al. Is useful for convenient purification of fusion proteins (Gentz et al. (1989), Proc. Natl. Acad. Sci. USA 86: 821-824). The “HA” tag is another peptide useful for purification and corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) Cell 37: 767). Other examples of fusion tags include: (1) MBP tag is part of maltose binding protein (vector is available from Roche ^TM (example vector designation: pIVEX MBP), New England Biological (Example vector designation: pMAL-p2X); (2) HA tag is part of hemagglutinin from human influenza virus (vector is available from Roche (example vector designation: pIVEX HA -Tag), BD Biosciences (pCMV-HA); (3) FLAG is a special 8-amino acid epitope (vector is designated by Sigma Chemical as an example vector: gWiz / GFP); (4) CBP tag is part of calmodulin binding protein (vector is available from Stratagene: example vector designation: pDual); and (5) GFP is green fluorescent (Vector is available from Stratagene: example vector designation pDual) Other common epitope tags for creating fusion proteins: c-myc, GST, AU1 , AU5, DDDDK, E epitope, E2 tag, Glu-Glu, S1, KT-3, T7 epitope tag, V5 epitope tag, VSV-G, BFP, CFY, YFP, as discussed below Such fusion proteins include interferons fused to Fc at the N or C terminus.

  Furthermore, the present invention provides a recombinant vector comprising the nucleic acid of the present invention, a host cell containing the recombinant vector, a method for producing such a vector and host cell, an interferon polypeptide or Their use for making peptides is also provided.

The present invention further relates to (a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86 An amino acid sequence of an interferon polypeptide comprising the acid sequence shown in FIG. 1; and (b) an amino acid sequence of a biologically active fragment of the polypeptide shown in at least one of these SEQ ID NOs. An interferon polypeptide comprising the amino acid sequence is provided. Further, the present invention provides at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical to the polypeptides described herein, and Even more preferably, a polypeptide having an amino acid sequence at least 99.25% identical is also included. Also, at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity to those described herein. And even more preferably, a polypeptide having an amino acid sequence of at least 99.25% similarity is also provided. Also provided are polypeptides having amino acid sequences that are at least 95%, 96%, 97%, 98% or 99.25% homologous to those described herein. A polynucleotide encoding such a polypeptide is also provided.

  Additional embodiments of this aspect of the invention relate to peptides or polypeptides comprising the amino acid sequence of the epitope-bearing portion of an interferon polypeptide having the amino acid sequence described herein. The peptide or polypeptide having the amino acid sequence of the epitope-bearing portion of the interferon polypeptide of the present invention has at least 6 or 7, preferably at least 9, and more preferably at least about 30 amino acids to about 50. A portion of such a polypeptide consisting of the following amino acids is included, but any length of epitope-bearing polypeptide that includes, as a maximum, the entire amino acid sequence of the polypeptide of the present invention described above is also included in the present invention.

  In another embodiment, the present invention provides an antibody that specifically binds to an interferon polypeptide having an amino acid sequence described herein. Such an antibody may be a monoclonal antibody. It may also be an antigen binding fragment such as a Fab or Fab 'fragment. The antibody may be a chimeric, humanized or fully human antibody. The antibody may be a feline antibody or a rhesus monkey antibody. The present invention further provides a method for isolating an antibody that specifically binds to an interferon polypeptide having the amino acid sequence described herein. Such antibodies are useful therapeutically as described below.

  In another aspect, the present invention further comprises the amino acid sequence shown in at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26. A cat comprising an amino acid sequence selected from: (b) an amino acid sequence of a biologically active fragment of the polypeptide shown in at least one of these SEQ ID NOs: An interferon polypeptide is provided. The invention also provides that the polypeptide described herein is at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and even more A polypeptide having an amino acid sequence that is preferably at least 99.25% identical is also included. Further, at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity to those described herein, and Even more preferably, a polypeptide having an amino acid sequence with at least 99.25% similarity is also provided. Further provided are polypeptides having amino acid sequences with at least 95%, 96%, 97%, 98% or 99.25% homology to those described herein. A polynucleotide encoding such a polypeptide is also provided.

In another aspect, the invention provides (a) an amino acid sequence of an interferon polypeptide comprising an acid sequence set forth in at least one of SEQ ID NOs: 28, 30, 32, 34 or 36; and (b),
Also provided are rhesus monkey interferon polypeptides comprising an amino acid sequence selected from the amino acid sequences of biologically active fragments of the polypeptides set forth in at least one of these SEQ ID NOs: Further, the present invention provides that the polypeptides described herein are at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and even more A polypeptide having an amino acid sequence that is preferably at least 99.25% identical is also included. Further, at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity to those described herein, and Even more preferably, a polypeptide having an amino acid sequence of at least 99.25% similarity is also provided. Further provided are polypeptides having amino acid sequences that are at least 95%, 96%, 97%, 98% or 99.25% homologous to those described herein. A polynucleotide encoding such a polypeptide is also provided.

  In another aspect, the present invention further provides (a) SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 , 72, 74, 76, 78, 80, 82, 84 or 86, the amino acid sequence of an interferon polypeptide comprising the acid sequence shown in at least one of; and (b), shown in these SEQ ID NOs Also provided is a human interferon polypeptide comprising an amino acid sequence selected from the amino acid sequence of at least one biologically active fragment of the polypeptides: Furthermore, the present invention provides that the polypeptides described herein are at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and even more Polypeptides having an amino acid sequence that is preferably at least 99.25% identical are also included. Further, what is described herein includes at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity, And even more preferably, a polypeptide having an amino acid sequence of at least 99.25% similarity is also provided. Further provided are polypeptides having amino acid sequences that are at least 95%, 96%, 97%, 98%, or 99.25% homologous to those described herein. A polynucleotide encoding such a polypeptide is also provided.

  In another aspect, the present invention further provides an interferon polynucleotide or interferon as described herein for administration to an in vitro cell, to an ex vivo cell, and to an in vivo cell, or to a multicellular organism. Also provided are compositions comprising any of the polypeptides. In some particularly preferred embodiments of this aspect of the invention, the composition comprises an interferon polynucleotide for expressing the interferon polypeptide in a host organism to treat the disease. Particularly preferred in this regard is expression in human patients for treating dysfunction associated with loss or deficiency of endogenous interferon activity.

Furthermore, the present invention may be used to treat or prevent immune system related abnormalities such as viral infection, parasitic infection, bacterial infection, cancer, autoimmune disease, multiple sclerosis, lymphoma and allergic, etc. Pharmaceutical compositions and methods comprising the polypeptides are provided. Also provided are methods of treating an individual in need of an interferon polypeptide. In some preferred embodiments, the pharmaceutical composition is a veterinary composition for administration to a non-human animal, preferably a non-human primate. Examples of possible conditions of treatment with interferon include, but are not limited to, viral infection. Without limitation, treatment with interferon may be used to treat conditions that would benefit from inhibiting replication of interferon-sensitive viruses. Viral infections that could be treated according to the present invention include severe acute respiratory syndrome (SARS), smallpox virus, cowpox virus, monkeypox virus, West Nile virus, vaccinia, coronavirus, hepatitis A, hepatitis B, Hepatitis C, other non-A / N hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes simplex virus (HHV-6), papilloma virus , Including poxvirus, picornavirus, adenovirus, rhinovirus, type 1 and type 2 human T lymphocyte-favorable virus (HTLV-1 / -2), human rotavirus, rabies, human immunodeficiency virus (HIV) Retrovirus, encephalitis, arterivirus, filovirus, reovirus, papovavirus, hepadnavirus, astrovirus, coxsacki -Viruses, orthomyxoviruses (influenza viruses), paramyxoviruses, echoviruses, enteroviruses and respiratory rash viruses. Of particular relevance is the treatment of SARS, which is believed to be caused by a new coronavirus, named SARS-associated coronavirus (Peiris et al.
al., Lancet, April 19, 2003; 361 (9366): 1319-25; Ksiazek et al., New England Journal of Medicine, April
30, 2003 under PubMed ID # 12690092; Drosten et al., New England Journal of Medicine, April 10, 2003, under PubMed
ID # 12690091; Marra et al., Science,
epublication on May 1, 2003 with PubMed ID # 12730501. PubMed of the article can be accessed by http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? CMD = Search & DB = PubMed), in ID number.

  Furthermore, the methods of the invention can also be used to modify a variety of immune responses. The interferon polypeptides described here may be used to treat SARS and viral infections caused by the viruses described here. These interferon polypeptides may also be used in a prophylactic manner, for example, preventing infection or preventing the subject from showing symptoms associated with infection. Subjects that would benefit from such prophylactic treatment include those at high risk of infection, such as subjects who have been exposed to the virus, or individuals who have been infected or exposed to the virus.

  In certain embodiments, interferon can be used as an antiviral agent. Interferon is used in the treatment of acquired immune abnormalities, chronic hepatitis B, hepatitis C, viral hepatitis including hepatitis D, papilloma virus, herpes zoster, viral encephalitis, and in the prevention of rhinitis and respiratory infections. It has been used above.

In another embodiment, interferon can be used as an antiparasitic agent. Interferon, for example Cryptosporidium-Palbumu (Original: Cryptosporidium
parvum) may be used to treat infections. In yet another embodiment, interferon can be used as an antimicrobial agent. Interferon has been used clinically for antibacterial treatment. For example, interferon can be used to treat multidrug resistant pulmonary tuberculosis.

  In yet another embodiment, interferon can be used as part of an immunotherapy protocol. The interferon of the present invention is clinically used for immunotherapy, or more specifically to prevent graft-versus-host rejection, or to suppress the progression of autoimmune diseases such as arthritis, multiple sclerosis, or diabetes. May be used.

  In another embodiment, interferon can be used as part of an allergic treatment program. In yet another embodiment, interferon can be used as a vaccine adjuvant. Interferon may be used as an adjuvant or co-adjuvant to enhance or stimulate the immune response in the case of prophylactic or therapeutic vaccination.

  In addition to general animal treatment, the present invention also specifically contemplates the use of interferon for the treatment of primates as part of a veterinary protocol. In some embodiments, the interferon is rhesus monkey interferon. The present invention further specifically contemplates the use of interferon for the treatment of cats as part of a veterinary protocol. In certain embodiments, the interferon is feline interferon.

  In some embodiments, interferons are used to treat cats for viral infection. For example, cats with feline immunodeficiency virus (FIV) require supportive care to maintain normal health. Interferon can be used as part of the treatment of cats infected with FIV. Similarly, interferon can be used as part of the treatment of cats infected with feline leukemia virus (FeLV). Feline leukemia virus (FeLV) is responsible for the most important complex lethal infectious diseases of most American domestic cats today.

  Interferon can be used to treat feline panleukopenia. Feline infectious enteritis, feline “distemper” and feline panleukopenia, also known as cat ataxia or inability to coordinate, are sudden onset, fever, loss of appetite (loss of appetite), dehydration, depression, vomiting, blood leukocytes It is a feline highly infectious viral disease characterized by a reduced number (leukopenia) and often high mortality. Cerebellar hypoplasia (cerebellar hypoplasia) manifested as a result of intrauterine infection (intrauterine), miscarriage, stillbirth, early neonatal death, and inability to coordinate kittens (ataxia) starting at 2-3 weeks of age , May occur. All of the feline (feline) family members can suffer from feline panleukopenia (FPV) infection, as do raccoons, raccoons, and cacomisles.

Interferon can be used to treat cats infected with feline infectious peritonitis. Interferon can be used to treat cats infected with rabies. In other embodiments relating to the treatment of cats, interferon can be used in treating inflammatory airway disease (IAD). In yet another embodiment, interferon can be used to treat dogs or other domestic pets.
(de Mari K, Maynard L, Eun HM,
Lebreux B. Vet Rec. (2003) 152: 105-8). In yet another embodiment, interferon can be used to treat livestock. In yet another embodiment, interferon can be used to treat humans. The present invention is a method for treating a subject suffering from severe acute respiratory syndrome, comprising administering to the subject an amount of an interferon polypeptide effective to reduce the concentration of SARS-related coronavirus particles in the subject, A method comprising the step of treating the subject is provided.

  The present invention includes coronavirus, smallpox virus, cowpox virus, monkeypox virus, west nile virus, vaccinia virus, respiratory rash virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, retrovirus, papova virus , Herpes virus, pox virus, hepadna virus, astrovirus, coxsackie virus, paramyxovirus genus, orthomyxovirus genus, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, banyan virus, arenavirus A method for treating a subject infected with a virus selected from the group consisting of Bornavirus, Adenovirus, Parvovirus, and Flavivirus, wherein the concentration of virus particles in the subject is reduced By administering to the subject an effective amount of interferon polypeptide, in which the method comprising the step of treating the subject.

  The present invention provides any of the therapies described herein as a method of further preventing a subject from morbidity or infection, or as a method of reducing the risk of morbidity or infection of a subject, or certain viruses Also considered is a method of protecting the subject from abnormalities / conditions associated with, or a method of preventing the subject from exhibiting symptoms associated with a viral infection. For example, the methods described above for treating a subject infected with a virus may be further used to prevent a subject's viral infection or to reduce the risk of a subject's viral infection.

  The present invention provides a method for reducing the risk of viral infection in a subject comprising administering an interferon polypeptide to the subject. In certain embodiments, the method includes preventing the subject from viral infection. In certain embodiments, the method includes preventing the subject from exhibiting symptoms associated with a viral infection. In some embodiments, the method includes protecting the subject from abnormalities / conditions associated with a particular virus. This protection may be provided by preventing or reducing the severity of the anomaly / condition caused by the infection. In another embodiment, the protection may be provided by reducing the spread of the infection to others by reducing the severity of the abnormality / condition caused by the infection in the patient. In another embodiment, the protection or risk reduction is made by reducing the subject's susceptibility to infection. The methods and embodiments described herein are not necessarily mutually exclusive. Virus infections include, but are not limited to, coronavirus, smallpox virus, cowpox virus, monkeypox virus, West Nile virus, vaccinia virus, respiratory rash virus, rhinovirus, arterivirus, filovirus, picornavirus, leo Virus, retrovirus, papova virus, herpes virus, pox virus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus genus, orthomyxovirus genus, echo virus, enterovirus, cardiovirus, togavirus, rhabdovirus , Bunyaviruses, arenaviruses, bornaviruses, adenoviruses, parvoviruses, and flaviviruses.

  The present invention is a method of treating a subject afflicted with influenza (orthomyxovirus), comprising administering to the subject an amount of an interferon polypeptide effective to reduce the concentration of influenza virus particles in the subject. Wherein the interferon polypeptide is at least 95% identical, and more preferably at least 96% identical, and further to at least one of the SEQ ID NOs described herein. More preferably at least 97% identical, and even more preferably at least 98% identical, and even more preferably at least 99.25% identical amino acid sequences, thus treating the subject.

The interferon polypeptides referred to herein include, but are not limited to, IFN-α polypeptides, IFN-β polypeptides, IFN-γ polypeptides, and IFN-ω polypeptides, eg, for therapeutic and / or preventive purposes. (Zoon KC:
Human Interferons: Structure and Function. P. 1-12. In: Interferon 8. Academic
Press, London, 1987; Walter et
al., Cancer Biotherm Radiopharm 1998 June; 13 (3): 143-54; Pestka, S., Biopolymers 2000; 55 (4): 254-287; Biopolymers 2000; 55 (4): 254-287; Pestka, S., Methods in Enzymology,
78, 1981; Pestka, S., Methods in Enzymology, 79, 1981; Pestka, S., Methods in
Enzymology, 119, 1986; Pestka, S., Langer; JA, Zoon, KC, Samuel, CE Ann
Rev Biochem 1987, 56, 727-777). These further include, but are not limited to, human IFN, mouse IFN, feline IFN, and rhesus monkey IFN.

In certain embodiments of the interferon polypeptides described herein, the interferon polypeptide is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, At least 95% identical to one of 78, 80, 82, 84 or 86, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical; And even more preferably, it comprises an amino acid sequence that is at least 99.25% identical. Furthermore, at least 95% similarity to the above, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity, and even more preferred Also provided is a polypeptide having an amino acid sequence with at least 99.25% similarity. Further provided are polypeptides having amino acid sequences having at least 95%, 96%, 97%, 98% or 99.25% homology to those described herein.

  In certain embodiments of the interferon polypeptides described herein, the interferon polypeptide is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, An amino acid sequence selected from the group consisting of 78, 80, 82, 84 and 86. In certain embodiments of the interferon polypeptides described herein, the interferon polypeptide is encoded by a nucleic acid that is encoded by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 , 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85. In certain embodiments of the interferon polypeptides described herein, the interferon polypeptide is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, It is encoded by a nucleic acid that hybridizes under high stringency to a nucleic acid comprising a sequence selected from the group consisting of 77, 79, 81, 83 and 85. In certain embodiments of the interferon polypeptides described herein, the interferon polypeptide is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, It is encoded by a nucleic acid that hybridizes under high stringency to a nucleic acid complementary to a sequence selected from the group consisting of 77, 79, 81, 83 and 85.

  The present invention provides a method of preventing a subject from suffering from severe acute respiratory syndrome, comprising administering to the subject an interferon polypeptide. The method provides a method of reducing the risk that the subject will suffer from severe acute respiratory syndrome, comprising administering to the subject an interferon polypeptide.

  The invention further comprises administering to the subject an amount of an interferon polypeptide, wherein the subject is a coronavirus, smallpox virus, cowpox virus, monkeypox virus, west nile virus, vaccinia virus, respiratory rash virus, rhinovirus. Virus, arterivirus, filovirus, picornavirus, reovirus, retrovirus, papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackievirus, paramyxovirus genus, orthomyxovirus genus, echo Selected from the group consisting of viruses, enteroviruses, cardioviruses, togaviruses, rhabdoviruses, bunyaviruses, arenaviruses, bornaviruses, adenoviruses, parvoviruses, and flaviviruses How that prevents suffering from syndromes caused by-virus is also provided.

  The present invention provides a method of preventing a subject from suffering from influenza comprising the step of administering to the subject an amount of an interferon polypeptide as described herein. The present invention provides a method of reducing a subject's risk of suffering from influenza, comprising administering to the subject an amount of an interferon polypeptide as described herein. In certain embodiments of the methods described herein, the subject is a human. Such subjects include, but are not limited to, dogs, cats, monkeys, and livestock.

  The present invention provides the use of the interferon polypeptides described herein for the preparation of a medicament for treatment, prevention, prevention, and any of the methods of reducing risk described herein.

  The present invention provides a pharmaceutical package comprising an interferon composition together with instructions for administering the composition to a subject. The instructions may include written and / or drawing instructions. The instructions can be for any of the methods of treatment, prevention, prevention, and risk reduction described herein.

  The present invention further contemplates functional antagonists that, for example, one or more amino acid residues are different from wild-type interferon and inhibit one or more biological activities of the wild-type interferon. Such antagonists can be used to treat abnormalities caused by abnormal overexpression or other activation of endogenous interferon. The functional antagonist may be formulated into a pharmaceutical formulation.

  Furthermore, the present invention also provides a screening method for identifying a compound capable of enhancing or inhibiting the biological activity of an interferon polypeptide. The screening method comprises a receptor enhanced by an interferon polypeptide. The step of contacting the candidate compound in the presence of an interferon polypeptide, the step of assaying antiviral activity, etc. in the presence of the candidate compound and the interferon polypeptide, and comparing the activity with a standard level of activity. And wherein said standard is assayed when said receptor is contacted with interferon in the absence of said compound. In this assay, an increase in activity when compared to the standard indicates that the candidate compound is an agonist of interferon activity, and a decrease in activity when compared to the standard indicates that the compound exhibits interferon activity. It shows that it is an antagonist.

  A further aspect of the invention relates to a method of treating an animal in need of increasing interferon activity levels in the body, said method comprising such an animal with an isolated interferon polypeptide of the invention. Or administering a composition comprising a therapeutically effective amount of an agonist thereof.

  A further aspect of the invention is a method of treating an animal in need of reducing interferon activity levels in the body, the method comprising a therapeutically effective amount of an interferon antagonist in such animal. Administering. A preferred antagonist for use in the present invention is an interferon specific antibody.

  The above dosage may be administered every other day, but preferably once or twice a week. The dose is usually administered by injection over at least 24 weeks.

  Administration of the dose may be intravenous, subcutaneous, intramuscular, or any other acceptable systemic method. Based on the judgment of the attending physician, the amount of drug to be administered, the treatment plan to be used, of course, the age, sex and medical calendar of the patient to be treated, the severity of neutrophil count (eg neutropenia) ), Depending on the severity of the particular disease state and patient tolerance to treatment, as evidenced by local toxicity and systemic side effects. Dosage and frequency may be determined during the initial screening for neutrophil count.

Conventional pharmaceutical formulations can also be prepared using the interferon composition of the present invention. Such formulations comprise a therapeutically effective amount of an interferon polypeptide together with a pharmaceutically acceptable carrier. For example, if necessary, adjuvants, diluents, preservatives and / or solubilizers may be used in the practice of the present invention. Interferon pharmaceutical compositions, including those of the present invention, include diluents, carriers (eg, human serum albumin) of various buffers (eg, Tris-HCl, acetate, phosphate) having a range of pH and ionic strength. ), Solubilizers (eg, polyoxyethylene sorbitan or TWEEN ^™ polysorbate), and preservatives (eg, thimerosal, benzyl alcohol). See, for example, US Pat. No. 4,496,537.

  The therapeutic amount of the interferon composition administered to treat the above conditions is based on the interferon activity of the composition. It is an amount effective to significantly affect a positive clinical response. In the case of treatment of a viral infection, a positive clinical response will be indicated by a reduction in the concentration of viral particles in the subject, or more generally a reduction in the symptoms of the infection. In the case of prophylactic treatment, a positive clinical response is, for example, the absence of viral particles in a subject, a decrease in the concentration of viral particles in a subject, or a threshold in which a subject indicates a viral infection symptom. It will be indicated by keeping it below the concentration. As used herein, prophylactic treatment includes preventing a subject from suffering from an abnormality caused by a virus.

  Clinical doses may cause some side effects in patients, but the maximum dose for mammals, including humans, is such that there is no untoward clinically significant side effect The highest dose. For the purposes of the present invention, such clinically significant side effects include severe cold-like symptoms, central nervous system depression, severe gastrointestinal abnormalities, hair loss, severe pruritus or rash. It is necessary to stop treatment as the cause. Substantial white blood cells and / or red blood cells and / or liver enzyme abnormalities or anemia-like conditions can also limit dosage.

Of course, the dosage of interferon will vary somewhat depending on the formulation chosen. In general, however, interferon compositions are based on the mammal's condition,
Administered in amounts ranging from about 100,000 to about millions of IU / m ² per day. The above ranges are exemplary and one of ordinary skill in the art will determine the optimal dosage of the selected interferon based on clinical experience and therapeutic indices.

  The pharmaceutical composition may be in the form of a solution, suspension, tablet, capsule, lyophilized powder, etc., prepared according to methods known in the art. Furthermore, administration of such compositions will be primarily by parenteral administration, although it is contemplated that oral or inhalation routes may also be used depending on the needs of those skilled in the art.

  The term “isolated” as used herein with respect to nucleic acids such as DNA or RNA refers to molecules that are separated from other DNA or RNA, respectively, that are present in the natural source of the macromolecule. The term isolated further substantially entails cellular material or media when made by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. No nucleic acid or peptide, polypeptide or protein. Furthermore, an “isolated nucleic acid” is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state.

A preferred method of determining the best overall match between the query sequence and the subject sequence, also referred to as global sequence alignment, is the FASTDB computer program Brutlag
It can be determined based on the algorithm of et al. (Comp. App. Biosci., 6: 237-245 (1990)). The term “sequence” includes nucleotide and amino acid sequences. In sequence alignment, the query sequence and the subject sequence are both nucleotides or both are amino acid sequences. The results of the global sequence alignment are presented as percent identity. The preferred parameters used in FASTDB searches of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, miss pairing penalty = 1, joining penalty = 30, randomized group length = 0 and cut-off score = 1, gap penalty = 5, gap size penalty 0.05, and window size = 500
Or the query sequence length in nucleotide bases, whichever is shorter. The preferred parameters used to calculate percent identity and amino acid alignment similarity are: Matrix = PAM 150, k-tuple = 2, miss pairing penalty = 1, joining penalty = 20, randomized group Length = 0, cut-off score = 1, gap penalty = 5, gap size penalty = 0.05, and window size = 500 or query sequence length with amino acid residues, whichever is shorter.

Protein and / or nucleic acid sequence homology may be assessed using any of a variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are in no way limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl.
Acad. Sci. USA 85 (8): 2444-2448;
Altschul et al., 1990, J. Mol. Biol. 215 (3): 403-410; Thompson et al., 1994, Nucleic Acids Res. 22 (2): 4673-4680; Higgins et al., 1996, Methods
Enzymol. 266: 383-402; Altschul et al., 1990, J. Mol. Biol. 215 (3): 403-410; Altschul et al., 1993, Nature
Genetics 3: 266-272).

In certain particularly preferred embodiments, protein and nucleic acid sequence homology is assessed using a basic local alignment search tool (“BLAST”) known in the art (eg, Karlin).
and Altschul, 1990, Proc. Natl. Acad Sci. USA 87: 2267-2268; Altschul et al.,
1990, J. Mol. Biol. 215: 403-410; Altschul et al., 1993, Nature Genetics
3: 266-272; Altschul et al., 1997, Nuc. Acids Res. 25: 3389-3402). In particular, the following tasks are performed using five specific BLAST programs:
(1) Compare amino acid query sequence to protein sequence database with BLASTP and BLAST3;
(2) Compare nucleotide query sequence to nucleotide sequence database with BLASTN;
(3) Compare the 6-frame hypothetical translation product of the query nucleotide sequence (both strands) to the protein sequence database with BLASTX;
(4) Compare the query protein sequence with TBLASTN to the nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX with a six-frame translation of the nucleotide query sequence Compare with 6-frame translation of sequence database.

The BLAST program identifies homologues by identifying "high score segment pairs" and similar segments referred to herein between query amino acid or nucleic acid sequences and preferably test sequences obtained from protein or nucleic acid sequence databases. Identify the sequence. High score segment pairs are preferably identified (ie, aligned), many of which are by a scoring matrix known in the art. Preferably, the scoring matrix used is a BLOSUM62 matrix (Gonnet et al.
al., 1992, Science 256: 1443-1445; Henikoff and Henikoff, 1993, Proteins
17: 49-61). PAM or PAM250 matrix may then preferably be used (eg Schwartz and Dayhoff, eds.,
1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence
and Structure, Washington: see National Biomedical Research Foundation).

The BLAST program evaluates all statistical significance of the identified high-scoring segment pairs, but preferably meets the significance threshold specified by the user, such as the percent homology specified by the user. A segment may be selected. Preferably, the statistical significance of high-scoring segment pairs is determined by Karlin (eg Karlin and
Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268).

  The parameters used in the above algorithm may be adapted according to the length of sequence studied and the degree of homology. In some implementations, the parameter may be a default parameter that the algorithm uses when there is no instruction from the user.

In some embodiments, Brutlag et
The FASTDB algorithm described in al. Comp. App. Biosci. 6: 237-245, 1990 is used. In such an analysis, the parameters may be selected as follows: Matrix = Unitary, k-tuple = 4, Miss pairing penalty = 1, Joining penalty = 30, Randomized group length = 0 Cut-off score = 1, gap penalty = 5, gap size penalty = 0.05, window size = 500, or the length of the sequence hybridizing to the probe, whichever is shorter.

Using the above methods and algorithms, such as FASTA, with parameters depending on the length of sequence to be studied and the degree of homology, e.g. default parameters that the algorithm uses when there is no instruction from the user, A protein having at least 99.25%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80% or at least 75% homology to a protein encoded by a nucleic acid The encoding nucleic acid can be obtained. In some embodiments, the homology level is determined by “default” opening penalty and “default” gap penalty, and a scoring matrix such as PAM250 (standard scoring matrix; Dayhoff et al., In: Atlas of Protein Sequence and Structure, Vol. 5,
Supp. 3 (1978)).

Alternatively, the level of polypeptide homology
Determination may be made using the FASTDB algorithm described in et al. Comp. App. Biosci. 6: 237-245, 1990. In such an analysis, the parameters may be selected as follows: Matrix = PAM 0, k-tuple = 2, Miss pairing penalty = 1, Joining penalty = 20, Randomized group length 0, cut-off score = 1, window size = sequence length, gap penalty = 5, gap size penalty = 0.05, window size = 500, or length of homologous sequence, whichever is shorter.

  The invention further encompasses sequences that have a lower degree of identity than those described herein, but have sufficient similarity to perform one or more of the same functions. Similarity is determined by conservative amino acid substitutions. Such a substitution replaces one amino acid in a polypeptide with another amino acid of similar characteristics. Conservative substitutions are likely to be phenotypically silent. Typically found as conservative substitutions are substitutions where one of the aliphatic amino acids Ala, Val, Leu, and Ile replaces another, substitution between the hydroxyl residues Ser and Thr, acidic residues Exchange of Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr. Guidance on which amino acid changes are likely to be phenotypically silent can be found in Bowie et al., Science 247: 1306-1310 (1990).

  In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (eg, gaps for the first and second amino acids or for optimal alignment). Non-homologous sequences that can be introduced into one or both of the nucleic acid sequences can be ignored for purposes of comparison). In certain preferred embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for purposes of comparison. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as in the corresponding position in the second sequence, then the molecules are identical at that position (as used herein). Amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is the common identity between these sequences, taking into account the number of gaps that need to be introduced in order to optimally align the two sequences, and the length of each gap. It is a function of the number of positions.

Comparison of sequences between two sequences and determination of percent identity and similarity can be done using mathematical algorithms (Computational Molecular Biology, Lesk, AM, ed., Oxford University
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis primer, Gribskov, M. and Devereux, J., eds., M
Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined by the Needleman and Wansch (J. ed.) Incorporated into the GAP program in the GCG software package (available at http://www.gcg.com). Mol. Biol. (48): 444-453 (1970)) and using either Blossum 62 matrix or PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or Set to 4 and set the length weight to 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12 (1): 387 (1984)), ( (available at http://www.gcg.com), using the NWSgapdna.CMP matrix, with a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, Judge as 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined by the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) incorporated into the ALIGN program (version 2.0). Using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as “query sequences” to search a sequence database to identify other family members or related sequences, and the like. Such a search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215: 403-10 (1990)). A BLAST nucleotide search can be performed with the NBLAST program, score = 100, word length = 12, to obtain a nucleotide sequence homologous to the nucleic acid molecule of the present invention. When a BLAST protein search is performed using the XBLAST program with a score of 50 and a word length of 3, an amino acid sequence homologous to the protein of the present invention can be obtained. Gapped BLAST can be used as described in Altschul et al. (Nucleic Acids Res. 25 (17): 3389-3402 (1997)) for gapped alignment for comparison purposes. When using BLAST and Gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used. In practicing the present invention, unless otherwise indicated, conventional techniques known to those skilled in the art of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology are used. Will be used. Such techniques are explained in the literature. For example
Molecular Cloning A
Laboratory Manual, 2nd Ed., Ed. By Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Oligonucleotide, synthesis (MJ Gait ed., 1984); Mullis et al. US Patent No:
4,683,195; Nucleic Acid Hybridization
(BD Hames & SJ Higgins eds. 1984); Transcription And
Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press,
Inc., NY); Gene Transfer Vectors For Mammalian Cells
(JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. Eds.) Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and
CC Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
1986).

  In the nucleotide and amino acid sequences described herein in the sequence listing, “n” or “x” may refer to any nucleotide with respect to the nucleotide sequence, whereas “n” or “x” with respect to the protein sequence. May refer to any amino acid.

  Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

Detailed Description of the Invention
I. In another aspect according to formulation examples , the present invention provides a pharmaceutical formulation comprising an interferon, an interferon agonist or an interferon antagonist. The interferon, interferon agonist and / or interferon antagonist for use in the present method is, for example, water, buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), or a suitable mixture thereof, etc. It can be appropriately formulated for administration with a biologically acceptable medium. The optimum concentration of the active ingredient in the selected medium can be determined empirically according to techniques known to medical chemists. “Biologically acceptable medium” as used herein includes any solvent, dispersion medium, etc. that may be suitable for the desired route of administration of the pharmaceutical formulation. The use of such media for pharmaceutically active substances is known in the art. Except where any conventional media and agents are incompatible with the activity of the composition of the present invention, its use in the pharmaceutical formulation of the present invention is contemplated. Suitable vehicles containing other proteins and their formulations are described, for example, in the book Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985). These vehicles include injectable “depot formulations”.

  The pharmaceutical formulations of the present invention further include veterinarians such as pharmaceutical formulations of the compositions of the present invention suitable for veterinary use, such as for the treatment of domestic animals, non-human primates, or pets such as dogs and cats. Compositions can also be included.

  A refillable or biodegradable instrument may be used as the introduction method. In recent years, a variety of delayed release polymer devices have been developed and controlled in vivo for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biodegradable polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release at specific target sites.

  The formulations of the present invention may be administered orally, parenterally, topically, intrathecally / intraventricularly (ICV), intracranial, directly into the central nervous system (intracavity) or rectally. These are of course administered in a form suitable for each route of administration. For example, they are in tablet or capsule form, administered by injection, inhalation, ointment, ointment, suppository, controlled release patch, etc., administered by injection, infusion or inhalation; topical by eye drop or ointment; and administered rectally by suppository. In some embodiments, oral and topical administration may be preferred. In certain embodiments, the interferon is delivered directly to the nasopharyngeal mucosa. In certain embodiments, the interferon is delivered directly to the lung epithelium. This would make these cells resistant to the virus, rather than using these tissues as a vehicle for systemic delivery. In this case, systemic interferon will be minimized, so that side effects will be minimized or eliminated.

  The terms “parenteral administration” and “administer parenterally” as used herein mean administration forms other than enteral and topical administration, usually by injection, including but not limited to intravenous Intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, intrathecal, intrathecal, and intrasternal injection And infusion.

  The terms “systemic administration”, “systemic administration”, “peripheral administration” and “peripheral administration” as used herein refer to, for example, subcutaneous administration, which enters the patient's whole body, thus metabolism and other similar By administration of a compound, drug or other substance other than directly to the central nervous system, bladder or other compartment of the body, such as undergoing a process.

  These compounds are administered by any suitable route of administration, including oral, spray, etc. nostril, rectal, vaginal, parenteral, in-bath and powder, ointment or topical by mouth and sublingual drops. It may be administered to humans and other animals for treatment.

  Regardless of the chosen route of administration, the compounds of the present invention and / or the pharmaceutical compositions of the present invention that may be used in a suitable hydrated form are as described below or known in the art. Is formulated into a pharmaceutically acceptable dosage form by other conventional methods.

  The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is effective to achieve the desired therapeutic response for a particular patient, composition and dosage form without becoming toxic to the patient. Changes may be made to obtain quantities.

  The selected dosage level is combined with the particular compound of the invention used, or the activity of the ester, salt or amide thereof, route of administration, administration time, excretion rate of the particular compound used, duration of treatment, particular composition used Will depend on a variety of factors including factors known in the medical industry, such as other drugs, compounds and / or substances used, the age, sex, weight, condition, general health and previous medical calendar of the patient being treated .

  A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian starts the dose of the compound of the invention used in the pharmaceutical composition at a level that is less than that required to achieve the desired therapeutic effect, and until the desired effect is achieved. This dosage may be gradually increased.

  In general, a suitable daily dose of a compound of the invention will be the amount of compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors discussed above.

  If necessary, an effective daily dose of the active compound may be administered two, three, four times, five times, six times or more at appropriate intervals throughout the day, optionally in unit dosage form. The dose may be subdivided into the above times and administered separately.

  The term “treatment” is intended to encompass prevention, therapy, cure, and prevention of the spread of infection.

  Patients receiving this treatment are primates, especially humans, and other mammals such as horses, cows, pigs, rodents and sheep; and any necessary animals or animal vectors, including common poultry and pets. (Virus source).

  The compounds of the present invention can be administered alone, or as a mixture with pharmaceutically acceptable and / or sterile carriers, and can be administered in conjunction with other agents. Non-limiting examples of such agents include antibacterial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides. Thus, in combination therapy, the active compounds may be administered sequentially, simultaneously and separately, in such a way that the therapeutic effect of the first dose is not completely eliminated when the next dose is administered. included.

II. Pharmaceutical Compositions While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The compositions of the invention may be formulated for administration in any convenient manner for use in human or veterinary medicine. In some embodiments, the compound included in the pharmaceutical formulation may be active on its own, or it may be a prodrug, eg, it can be converted to an active compound under physiological conditions.

Thus, another aspect of the present invention is to formulate a therapeutically effective amount of one or more of the above compounds together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. And a pharmaceutically acceptable composition. As described in detail below, the pharmaceutical composition of the present invention comprises the following: (1) Oral administration, such as a drug (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, Paste for application to the tongue; (2) Parenteral administration by subcutaneous, intramuscular or intravenous injection as a sterile solution or suspension; (3) Cream, ointment or spray applied to the skin Or (4) specially formulated for administration in solid or liquid form, including those adapted for intravaginal or rectal, such as pessaries, creams or foams. However, in some embodiments, the compound may simply be dissolved or suspended in sterile water.
In some embodiments, the pharmaceutical formulation is an apyrogenic source, i.e. does not increase the patient's body temperature.

  The term “therapeutically effective amount” as used herein is the amount of a compound, substance, or composition comprising a compound of the invention that is applicable to any medical treatment. Means an amount effective to produce any desired therapeutic effect in at least one sub-population of cells of the animal, and thus block the biological consequences of the pathway in the treated cells, at a reasonable benefit / risk ratio .

  The term “medically acceptable” is within the scope of sound medical judgment and is a reasonable benefit / risk ratio for humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications. Is used herein to refer to compounds, substances, compositions, and / or dosage forms suitable for use in contact with other tissues.

  The term “pharmaceutically acceptable carrier” as used herein refers to a liquid or solid filler that is involved in transporting or transporting the agonist of interest from one organ or body part to another organ or body part. Means a pharmaceutically acceptable substance, composition or vehicle, such as a diluent, pharmaceutical additive, solvent or encapsulant. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) eg carboxy Cellulose and its derivatives such as sodium methylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) pharmaceutical additives such as cocoa butter and suppository waxes; (9) Fats and oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; 12) Ethyl oleate and lauric acid Esters such as chill; (13) Agar; (14) Buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Non-pyrogenic water; (17) Isotonic saline; (18) (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic compatible materials used in pharmaceutical formulations.

III. Formulation Examples Other molecules, molecular structures such as liposomes, polymers, receptor-targeting molecules, oral, rectal, topical or other formulations, etc. Or it may be mixed, encapsulated, bound or otherwise combined with a mixture of compounds. The interferon can also be provided in a formulation that also includes a penetration enhancer, carrier compound and / or transfection agent.

  Furthermore, the compositions of the invention can be any pharmaceutically capable of providing a biologically active metabolite or residue thereof (directly or indirectly) when administered to an animal, including a human. Also included are acceptable salts, esters or salts of such esters, or any other compound. Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts and other biological equivalents.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium and the like. Examples of suitable amines are N, NI-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci.,
1977, 66, 1-19). The base addition salt of the acidic compound is prepared in the conventional manner by contacting the free acid form with a sufficient amount of the desired base to form a salt. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ somewhat from their respective salt forms in some physical properties, such as solubility in polar solvents, but in other respects the salts are for purposes of the present invention. , Equivalent to their respective free acid forms. As used herein, “pharmaceutical additive salt” includes a pharmaceutically acceptable salt of one acid form of the components of the composition of the present invention. These include amine organic or inorganic acid salts. Suitable acid salts are hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are known to those skilled in the art, and include basic salts of a variety of inorganic and organic acids.

A. Supramolecular complex In some embodiments, the formulation is part of a “supermolecular complex”. To further illustrate, the interferon is brought into contact with at least one polymer to form a composite, and then the composite polymer is combined with a supramolecular composite containing the interferon and a multidimensional polymer network. It can be treated under conditions sufficient to form a body. The polymer molecule may be linear or branched. Thus, a group of two or more polymer molecules may be linear, branched, or a mixture of linear and branched polymers. The composite may be prepared by any suitable means known in the art. For example, the composite may be formed by simply contacting, mixing or dispersing the interferon with the polymer. Composites could also be prepared by polymerizing monomers, which can be the same or different, that can form a linear or branched polymer in the presence of the expression construct. The composite may be further modified with at least one ligand, such as to direct intracellular uptake of the expression construct, or to allow tissue or cellular distribution of the expression construct in vivo. The composite may take any suitable form, but is preferably in the form of particles.

  In some preferred embodiments, the interferon is formulated with a cationic polymer. Examples of cationic polymers include poly (L), lysine (PLL), and polyethyleneimine (PEI). In some preferred embodiments, the expression construct is formulated with a β-cyclodextrin-containing polymer (βCD-polymer). βCD-polymers can form nucleic acids and polyplexes and can be transfected into cultured cells. βCD-polymers can be synthesized, for example, by condensing diamino-cyclodextrin monomer A with diimidate comonomer B. Cyclodextrins are naturally occurring D (+), a cyclic polysaccharide containing α- (1,4) -glucopyranose units in the bond. The most common cyclodextrins are α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin, which contain 6, 7 or 8 glucopyranose units, respectively. Examples of cyclodextrin delivery systems that can be easily adapted for delivery of the present interferon are described, for example, in Gonzalez et al PCT application WO00 / 01734 and Davis PCT application WO00 / 33885.

  In some embodiments, the supramolecular complex is agglomerated into particles, such as a formulation of particles having an average diameter between 20 and 5000 nanometers (nm). In another embodiment, the particles have an average diameter between 20 and 200 nm. In another embodiment, the particles have an average diameter between 2 and 10 microns. The use of particle sizes between 2 and 10 microns may be used, for example, for delivery to the lung.

B. Other Cationic, Non-lipid Formulations In some embodiments, the interferon is provided in a cationic, non-lipid vehicle and formulated for use in aerosol delivery through the respiratory tract. Formulations using poly (ethyleneimine) and macromolecules such as dsRNA and dsRNA-coding plasmids can increase lung transfection levels and increase stability during nebulization. PEI-nucleic acid formulations can also show high specificity for the lung.

  In addition to formulating interferon with PEI, the present invention further contemplates the use of cyclodextrin-modified polymers such as cyclodextrin-modified poly (ethyleneimine). In some embodiments, the polymer has the formula:

Where R is independently H, lower alkyl, cyclodextrin moiety, or

Represents; and
m individually represents an integer of 2 to 10,000, preferably 10 to 5,000, or 100 to 1,000.

In some embodiments, R
Is at least about 1%, more preferably at least about 2%, or at least about 3% of the nitrogen atom that would be a primary amine (ie having two Rs representing H) apart from the cyclodextrin moiety, and Represents up to about 5% or even 10% cyclodextrin.

  In some embodiments, the cyclodextrin moiety comprises by weight at least about 2%, 3% or 4%, up to 5%, 7%, or even 10% by weight of the cyclodextrin-modified polymer.

In some embodiments, at least about 2%, 3% or 4%, up to 5%, 7%, or even 10% by weight of the ethyleneimine subunit in the polymer is modified with a cyclodextrin moiety. ing.

  Poly (ethyleneimine) copolymers with nucleophilic amino substituents susceptible to derivatization with cyclodextrin moieties can also be used to prepare cyclodextrin-modified polymers within the scope of the present invention.

  Examples of cyclodextrin moieties include cyclic structures consisting essentially of 7 to 9 sugar moieties such as cyclodextrin and oxidized cyclodextrin. The cyclodextrin moiety optionally has a cyclic structure, preferably an alkyl chain such as a dicarboxylic acid derivative (eg, glutaric acid derivative, succinic acid derivative, etc.), a heteroalkyl chain such as an oligoethylene glycol chain, etc. It contains a linker moiety that forms a covalent bond with a polymer backbone that has 20 atoms in the chain.

C. Liposome Formulation In some embodiments, the present invention provides a composition comprising interferon encapsulated or bound to liposomes. Liposomes as used herein refer to lipid vesicles having an outer lipid shell typically formed on one or more lipid bilayers enclosing an aqueous interior. In certain preferred embodiments, the liposome is composed of about 20 to 80 mole percent cationic vesicle-forming lipid and the remaining neutral vesicle-forming lipid and / or other components. It is. “Vesicle-forming lipid” as used herein refers to a parent having a hydrophobic and polar head group, such as phospholipid, and capable of spontaneously forming a bilayer vesicle in itself in water. Says a lipid lipid. Suitable vesicle-forming lipids are diacyl chain lipids, such as phospholipids, whose acyl chains typically have a length of about 14 to 22 carbon atoms and are unsaturated to varying degrees.

  Cationic vesicle-forming lipids are those whose polar head groups have a net positive charge at the pH at which they work, such as pH 4-9. Typical examples include phospholipids, such as phosphatidylethanolamine, whose polar head group is derivatized with a positive moiety such as lysine, such as lipid DOPE derivatized with L-lysine (LYS-DOPE) (Guo, et al., 1993). This class further includes glycolipids such as cerebrosides and gangliosides that have a cationic polar head group.

  Another cationic vesicle-forming lipid that may be used is cholesterolamine and related cationic sterols. Examples of cationic lipids include 1,2-dioleoyloxy-3- (trimethylamino) propane (DOTAP); N- [1- (2,3, -ditetradecyloxy) propyl] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N- [1- (2,3, -dioleyloxy) propyl] -N, N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA); 3β [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol ); And dimethyl dioctadecyl ammonium (DDAB).

  The rest of the liposomes may contain a small percentage of neutral vesicle-forming lipids, i.e. lipids that have no net charge or a negative charge on the polar head group. Formed from vesicle-forming lipids. This class of lipids includes phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), cholesterol, cholesterol derivatives, and other uncharged substances. Of sterols.

  The above lipids can be obtained commercially or can be prepared according to published methods. Other lipids that can be included in the present invention are glycolipids such as cerebroside and ganglioside.

  In one embodiment of the invention, the interferon-liposome complex comprises a liposome having a surface coating of hydrophilic polymer chains that is effective to extend the blood circulation time of the plasmid / liposome complex. Suitable hydrophilic polymers include cyclodextrin (CD), polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinyl-pyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide , Polydimethylacrylamide, and derivatized cellulose, such as hydroxymethylcellulose or hydroxyethyl-cellulose. A preferred hydrophilic polymer chain is polyethylene glycol (PEG), preferably as a PEG chain having a molecular weight of 500 to 10,000 daltons, more preferably 1,000 to 5,000 daltons. The hydrophilic polymer will be soluble in non-aqueous solvents such as water and chloroform.

  The coating is preferably prepared by including vesicle-forming lipids containing phospholipids or other diacyl chain lipids derivatized with polymer chains at their head groups. Methods for preparing such lipids and for forming polymer-coated liposomes with such lipids are described in US Pat. Nos. 5,013,556 and 5,395,619, the contents of which are incorporated herein by reference. Has been.

  The hydrophilic polymer is stably bound to the lipid, or circulates in the bloodstream, or after localization to the target site, the hydrophilic property of the plasmid-liposome complex coated with the polymer is increased. It will be understood that the polymer coating can also be attached via labile bonds such as releasing or “releasing” the polymer coating. Attachment of hydrophilic polymers, particularly polyethylene glycol (PEG), to vesicle-forming lipids via a bond effective to release the polymer chain in response to a stimulus is hereby incorporated by reference. WO 98/16202, WO 98/16201 and Kirpotin, D. et a. (FEBS Letters, 388: 115-118 (1996)).

  In certain embodiments, the releasable bond is cleaved by administration of a suitable free agent, or chemically releasable that is cleaved under selective physiological conditions, such as in the presence of an enzyme or a reducing agent. It is a simple bond. For example, ester and peptide bonds are cleaved by esterase or peptidase enzymes. The disulfide bond is cleaved by administration of a reducing agent such as glutathione or ascorbate, or by a reducing agent present in vivo, such as cysteine present in plasma and cells.

  Other releasable bonds include pH sensitive bonds and bonds that are cleaved upon exposure to glucose, light or heat. For example, when the hydrophilic polymer chain is attached to a liposome by a pH-sensitive bond, the plasmid-liposome complex has a pH effective to cleave the bond and release the hydrophilic chain. A site such as a tumor area can be targeted. Examples of pH sensitive linkages are acyloxyalkyl ether, acetal and ketal linkages. As another example, the cleavable bond is a disulfide bond that is widely intended herein to refer to a sulfur-containing bond. Sulfur-containing bonds can be synthesized to achieve a selected degree of instability, among which are disulfide bonds, mixed sulfide-sulfone bonds, and sulfide-sulfoxide bonds. Of these three bonds, disulfide bonds are least susceptible to thiolysis, and sulfide-sulfoxide bonds are most likely to occur.

  Such releasable linkages are useful for controlling the rate of release of the hydrophilic polymer moiety from the liposome complex. For example, very labile disulfide bonds can be used for blood cell or endothelial cell targeting. This is because these cells are easily reachable and it is sufficient that the liposomes have a short circulating life. In the opposite case, longer lasting times when tumor tissue or other organs are the target, which generally require a longer circulating circulation life of the liposomes to reach the desired target. Or a strong disulfide bond can be used.

  In vivo, the releasable bond that attaches the hydrophilic polymer chain to the liposome is typically a small amount in vivo, typically in a region of the tumor tissue or a site with reducing conditions such as a hypoxic tumor. When the liposome reaches a specific site having a low pH, it is cleaved as a result of an environmental change. In vivo reducing conditions can also be caused by the administration of reducing agents such as ascorbate, cysteine or glutathione. The cleavable bond may also be broken in response to an external stimulus such as light or heat.

  In another embodiment, the liposome complex comprises an affinity moiety or targeting ligand that is effective to specifically bind to a target cell for therapeutic purposes. Such a moiety can be attached to the surface of the liposome or it can be attached to the distal end of the hydrophilic polymer chain. Examples of moieties include antibodies, ligands that specifically bind to target cell surface receptors, and the like, as described in PCT applications WO US94 / 03103, WO 98/16202, and WO 98/16201. The moiety may further be a hydrophobic moiety that facilitates fusion of the complex with a target cell.

  The polycationic condensing agent used to condense the interferon may be a multiply charged cationic polymer, preferably a specific histone such as spermidine, spermine, polylysine, protamine, total histone, H1, H2, H3, H4, etc. Fractions and other biopolymers such as other polycationic polypeptides may be included, but biocompatible polymers such as polymyxin B may also be included. It will be appreciated that these polycationic condensing agents can be used in free base or salt form, such as, for example, protamine sulfate and polylysine hydrobromide. In certain preferred embodiments, the polycationic condensing agent is a histone referred to herein as comprising all histones or a specific histone fraction.

  In some embodiments, the hydrophobic moiety in the polymer-lipid conjugate is a hydrophobic polypeptide sequence. Preferably, the polypeptide sequence consists of about 5-80, more preferably 10-50, most preferably 20-30 nonpolar and / or aliphatic / aromatic amino acid residues. These sequences are active in inducing the fusion of certain enveloped viruses to host cells, including parainfluenza viruses such as Sendai, Simian Virus-5 (SV5), measles virus , Newcastle disease virus (NDV), and respiratory rash virus (RSV). Other examples include the human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, which infects cells by fusion of human retroviruses, eg, viral envelopes, to the cell membrane of host cells. Fusion occurs at physiological (ie, neutral), pH, followed by injection of viral genetic material (nucleocapsid) into the cytoplasmic compartment of the host cell.

D. Ligand directed formulations In some embodiments, the present invention is effective for binding polymer complexes and liposomes, such as supramolecular complexes, to specific cell surface proteins or matrices on target cells. Binding to one or more ligands can facilitate the targeting of the complex to the target cell and, in some cases, can facilitate the uptake of interferon by the cell. By way of example only, examples of ligands suitable for use in targeting the supramolecular complexes and liposomes of the present invention to specific cell types are listed in Table 1 below.

  The present invention further contemplates derivatization of such polymer and liposome complexes with ligands that facilitate transcellular transport of the complexes. To further illustrate, in order to promote intracellular localization of interferon, a polymer complex such as a supramolecular complex can be covalently bound to an internalizing peptide that induces translocation across the cell membrane of the complex. . In this case, the internalizing peptide itself can cross the cell membrane at a relatively high speed by transcellular transport or the like. The internalizing peptide is bound to the polymer as, for example, a covalent side group.

  In one embodiment, the internalizing peptide is obtained from a Drosophila antepennepedia protein or a homologue thereof. The 60 amino acid homeodomain of the homeoprotein Antepenepedia has been demonstrated to translocate across biological membranes and can facilitate translocation of heterologous polypeptides to which it binds. See, for example, Derossi et al. (1994) J Biol Chem 269: 10444-10450; and Perez et al. (1992) J Cell Sci 102: 717-722. Recently, it has been demonstrated that a fragment consisting of as few as 16 amino acids of this protein is sufficient to induce internalization. See Derossi et al. (1996) J Biol Chem 271: 18188-18193. Of the antepenepedia proteins (or homologs thereof) sufficient to increase the transmembrane transport of the modified complex by a statistically significant amount compared to the unmodified complex. Consider interferon complexes modified at least in part.

Another example of an internalizing peptide is HIV transactivator (TAT), a protein. This protein appears to be divided into four domains (Kuppuswamy
et al. (1989) Nucl. Acids
Res. 17: 3551-3561). Purified TAT protein is taken up by cells in tissue culture (Frankel and Pabo, (1989) Cell 55: 1189-1193) and peptides such as fragments corresponding to residues 37-62 of TAT can be obtained in vitro. (Green and Loewenstein, (1989) Cell 55: 1179-1188). This highly basic region mediates internalization of the internalization moiety into the nucleus and targeting (Ruben et al., (1989) J. Virol. 63: 1-8). Peptides or analogs containing sequences present in the above highly basic regions, such as CFITKALGISYGRKKRRQRRRPPQGS, are attached to the polymer to aid in the internalization of such complexes to intracellular mileus and targeting.

Another example of a transcellular polypeptide can be made to contain a sufficient portion of mastoparan to increase transmembrane transport of the interferon complex (T. Higashijima et al., (1990) J. Biol Chem. 265: 14176).

Other suitable internalizing peptides are, for example, histone, insulin, transferrin, basic albumin, prolactin and insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), or other growth factors It can produce using all or one part. For example, an insulin fragment that has an affinity for the insulin receptor on capillary cells, which is less effective than insulin in reducing blood glucose,
It has been found that transmembrane transport by receptor-mediated transcellular transport can be performed and thus serves as an internalizing peptide for the transcellular polypeptide of interest. Suitable growth factor derived internalization peptides include EGF (epidermal growth factor) derived peptides such as CMHIESLDSYTC and CMYIEALDKYAC; TGF-beta (transforming growth factor beta) derived peptides; PDGF (platelet derived growth factor), or PDGF- Peptides derived from 2; peptides derived from IGF-1 (insulin-like growth factor) or IGF-II; and peptides derived from FGF (fibroblast growth factor).

  Another class of translocation / internalizing peptides is one that exhibits pH-dependent membrane binding. In the case of an internalizing peptide that adopts a helical conformation at acidic pH, the internalizing peptide acquires amphipathic properties, for example, it has both a hydrophobic and a hydrophilic interface. More specifically, within the pH range of approximately 5.0-5.5, the internalizing peptide forms an alpha helix, an amphiphilic structure that facilitates insertion of this portion into the target membrane. An alpha helix-inducible acidic pH environment may be found, for example, in a low pH environment present within cell endosomes. Such internalizing peptides can be used to facilitate transport of interferon-complexes taken up by endocytotic mechanisms from the endosomal compartment to the cytoplasm.

  Still other suitable internalizing peptides include peptides of apolipoproteins A-1 and B; peptide toxins such as melittin, bonbolitin, deltahemolysin and pardaxin; antibiotic peptides such as alamethicin; peptide hormones such as calcitonin, corti There are cotrophin releasing factors, beta-endorphin, glucagon, parathyroid hormone, pancreatic polypeptide; and peptides corresponding to the signal sequences of many secreted proteins. In addition, the exemplified internalizing peptide may be modified by attaching a substituent that enhances the alpha helix nature of the internalizing peptide at acidic pH.

  Yet another class of internalizing peptides suitable for use within the present invention has hydrophobic domains that are “hidden” at physiological pH but exposed in the low pH environment of the target cell endosome. . When pH-induced denaturation occurs and this hydrophobic domain is exposed, this moiety binds to the lipid bilayer and performs the translocation of the covalently bound complex to the cytoplasm. Such internalizing peptides could be modeled after sequences identified with, for example, Pseudomonas exotoxin A, clathrin, or diphtheria toxin.

  Proteins or peptides that form pores will also serve as internalizing peptides here. The protein or peptide forming the pore may be obtained from or derived from, for example, C9 complement protein, cytolytic T cell molecule or NK cell molecule. These portions form a ring-like structure in the membrane, and transport the attached complex to the inside of the cell through the membrane.

  In some cases, mere membrane insertion of an internalizing peptide may be sufficient to permeate the complex through the cell membrane and translocate. However, attachment of an internalizing peptide to a substrate of an intracellular enzyme (ie, an auxiliary peptide) will improve translocation. Of the internalizing peptides, it is preferable to attach the auxiliary peptide to a portion protruding from the cell membrane to the cytoplasmic side. It may be advantageous to attach this auxiliary peptide to one end of the translocation / internalization moiety or anchor peptide. The auxiliary part of the present invention may contain one or more amino acid residues. In certain embodiments, the auxiliary moiety may be a substrate for intracellular phosphorylation (eg, the auxiliary moiety may contain a tyrosine residue).

An exemplary auxiliary moiety in this regard would be a peptide substrate for N-myristoyltransferase, such as GNAAAARR (Eubanks et al., In: Peptides. Chemistry and Biology , Garland
Marshall (ed.) ESCOM, Leiden, 1988, pp. 566-69). In this construct, an internalizing peptide will be attached to the C-terminus of the auxiliary peptide. This is because the N-terminal glycine is important for the activity of the auxiliary moiety. When this hybrid peptide is attached to a polymer complex, it is N-myristylated and further tethered to the target cell membrane, for example it serves to increase the local concentration of the complex at the cell membrane.

Suitable auxiliary peptides include peptides that are kinase substrates, peptides with a single sex charge, and peptides that contain sequences that are glycosylated by membrane-bound glycotransferases. Auxiliary peptides that are glycosylated by membrane-bound glycotransferases include the sequence x-NLT-x, where “x” may be another peptide, amino acid, coupling agent or hydrophobic molecule, etc. . When this hydrophobic tripeptide is incubated with a microsomal vesicle, it permeates the membrane of the vesicle, is glycosylated on the luminal side, and is trapped within the vesicle due to its hydrophilicity (C. Hirschberg et al. , (1987)
Ann. Rev. Biochem. 56: 63-8). Thus, an auxiliary peptide containing the sequence x-NLT-x will enhance the target cell retention of the corresponding complex.

As described above, the internalizing peptide and the auxiliary peptide are each individually linked to an interferon complex or liposome with chemical cross-linking or non-covalent interactions (eg, streptavidin-biotin conjugate, His ₆ -Ni interaction). Use of action etc.). In certain cases, an unstructured polypeptide linker can be included between the peptide moiety and the polymer complex or liposome.

  Furthermore, it is also conceivable that such an internalizing peptide and auxiliary peptide can be directly linked to interferon through, for example, a covalent bond to a hydroxyl group on the backbone of the protein. In some embodiments, the bond is susceptible to cleavage under physiological conditions, such as exposure to an esterase or a simple hydrolysis reaction. Such compositions can be used alone or can be formulated into polymer complexes or liposomes.

E. Respiratory Interferon Another aspect of the present invention provides an aerosol for delivering interferon to the respiratory tract. The respiratory tract includes the upper airway including the oropharynx and larynx, followed by the lower airway including the trachea that leads to the bronchi and bronchioles by bifurcations. The upper and lower airways are called guided bronchi. Thus, the terminal bronchiole is divided into respiratory bronchioles, which in turn lead to the final respiratory zone, the alveoli, the deep lung.

  Here, administration by inhalation may be oral and / or nasal, intratracheal (through a trans-anastomosis or tracheostomy tube), or through a respiratory aid such as a ventilator. Examples of pharmaceutical devices for aerosol delivery include fixed dose inhalers (MDI), dry powder inhalers (DPI), and air jet nebulizers. Examples of delivery systems by inhalation that can be readily adapted for delivery of such interferons include, for example, US Pat. Nos. 5,756,353; 5,858,784; and PCT applications WO98 / 31346; WO98 / 10796; WO00 / 27359; WO01 / 54664 Described in WO02 / 060412. Other aerosol formulations that may be used to deliver interferon are described in US Pat. Nos. 6,294,153; 6,344,194; 6,071,497, and PCT applications WO02 / 066078; WO02 / 053190; WO01 / 60420; WO00 / 66206. It is explained.

The human lung can remove or rapidly degrade hydrolysable deposited aerosol in minutes to hours. In the upper respiratory tract, the ciliated epithelium contributes to “mucus ciliary removal” in which particles are swept out of the airway toward the mouth. Pavia, D., "LungMucociliary Clearance," in Aerosols and
the Lung: Clinical and Experimental Aspects , Clarke, SW and Pavia, D.,
Eds., Butterworths, London, 1984. In the deep lung, alveolar macrophages can phagocytose them immediately after the particles are deposited. Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and Brain, JD,
"Physiology and Pathophysiology of Pulmonary Macrophages," in The
Reticuloendothelial System , SM Reichard and J. Filkins, Eds., Plenum,
New. York, pp. 315-327, 1985. Deep lung, or alveoli, are important targets for inhaled therapeutic aerosols for systemic delivery of interferon.

  In a preferred embodiment, the aerosolized interferon is formulated as microparticles, particularly if systemic administration of interferon is desired. Microparticles having a diameter between 0.5 and 10 microns can penetrate the lungs and pass through most of the natural barriers. A diameter of less than 10 microns is necessary to bypass the throat; a diameter of 0.5 microns or more is necessary to avoid being swept out of exhaled breath.

  In some preferred embodiments, the interferon is a supramolecular complex as described above, having a diameter of 0.5 to 10 microns and capable of agglomerating into particles having a diameter of 0.5 to 10 microns. Blend into.

  In other embodiments, the interferon is provided in liposomes or supramolecular complexes that are suitably (eg, as described above) formulated for pulmonary delivery.

(I) Polymer for forming microparticles In addition to the supramolecular complex described above, a number of other polymers can be used to form microparticles. As used herein, the term “microparticle” includes microspheres (uniform spheres), microcapsules (having a core and an outer layer of polymer), and atypically shaped particles.

  The polymer is preferably over a period of time during which it is desired to liberate interferon, or relatively soon, generally in the range of one year, more typically months, and more typically days to weeks. It should be biodegradable. Biodegradation can be said to be either the degradation of the microparticles, ie the dissociation of the polymer forming the microparticles and / or the degradation of the polymer itself. This can occur as a result of a pH change from the carrier in which the particles are contained to the pH of the free site, as in the case of diketopiperazine, or as in the case of poly (hydroxy acids). As a result, or as a result of diffusion of ions such as calcium from the microparticle, as in the case of microparticles formed by ionic bonds of polymers such as alginate, and in the case of many polysaccharides and proteins. As a result of enzyme action. In some cases, linear release may be most useful, while pulsed release or “bulk release” may yield more effective results.

Typical synthetic materials are: diketopiperazine, poly (lactic acid), poly (glycolic acid), and poly (hydroxy acids) such as copolymers, polyanhydrides, polyesters such as polyorthoesters, polyamides, polycarbonates, polyethylenes Polyalkylene such as polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), polyvinyl alcohol, polyvinyl ether, polyvinyl ester, halogenated polyvinyl, polyvinyl pyrrolidone, polyvinyl acetate, and polyvinyl chloride Compound,
Polystyrene, polysiloxane, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly ( Phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate), polyurethane and acrylic and methacrylic acid polymers including these copolymers, alkyl cellulose, hydroxyalkyl cellulose, Cellulose ether, cellulose ester, nitrocellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydro Cellulose, poly (butyric acid), including xy-propylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethylcellulose, cellulose triacetate, and cellulose sulfate sodium salt poly (butic acid), poly (valeric acid), and poly (lactide-co-caprolactone).

  Natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, albumin and other hydrophilic proteins, zein and other prolamins and hydrophobic proteins, copolymers and mixtures thereof. As used herein, these chemical derivatives refer to, for example, substitution, addition, hydroxylation, oxidation, and other modifications commonly made by those skilled in the art, such as alkyl, alkylene, and the like.

Bioadhesive polymers include H.
Bioerodible hydrogels, polyhyaluronic acid, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginic acid described in S. Sawhney, CP Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587 There are salts, chitosan, and polyacrylates.

To further illustrate, the matrix can be formed from the polymer by solvent evaporation, spray drying, solvent extraction, and other methods known in the art. Methods developed to create microspheres for drug delivery are described in Mathiowitz and Langer, J. Controlled Release 5, 13-22 (1987); Mathiowitz, et al., Reactive
Polymers 6, 275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988). The choice of method is, for example, Mathiowitz,
et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45, 125-134 (1992); and Benita, et al., J. Pharm. Sci. 73, As described in 1721-1724 (1984), it depends on the choice of polymer, desired size, external morphology, and crystallinity.

Mathiowitz,
In solvent extraction methods such as described in et al., (1990), Benita, and Jaffe, US Pat. No. 4,272,398, a polymer is dissolved in a volatile organic solvent. Interferon, either soluble or dispersed as fine particles, is added to the polymer solution and the mixture is suspended in an aqueous phase containing a surfactant such as poly (vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent has evaporated and a solid microsphere remains.

  In general, the polymer can be dissolved in methylene chloride. Several different polymer concentrations can be used, for example, between 0.05 and 0.20 g / ml. After adding the drug to the solution, the solution is suspended in 200 ml of well-stirred distilled water containing 1% (w / v) poly (vinyl alcohol) (St. Louis, MO, Sigma Chemical Co.). After stirring for 4 hours, the organic solvent has evaporated from the polymer and the resulting microspheres will be washed with water and dried overnight in a lyophilizer.

Various sizes (1-1000
Microspheres, but less than 10 microns for aerosol applications) and morphology, can be obtained by this method useful for relatively stable polymers such as polyesters and polystyrene. However, unstable polymers such as polyanhydrides will degrade upon exposure to water. For these polymers, hot melt encapsulation and solvent removal may be preferred.

  In hot melt encapsulation, the polymer is first dissolved and then mixed with solid interferon particles, preferably sieved to the appropriate size. This mixture is suspended in an immiscible solvent such as silicone oil and heated to 5 ° C. above the melting point of the polymer with continued stirring. When the emulsion is stable, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decanting with petroleum ether to obtain a free flowing powder. Microspheres with a diameter of 1 to 1000 microns can be obtained by this method. The outer surface of spheres prepared with this technique is usually smooth and dense. This approach is useful for water labile polymers, but is limited to use with polymers having a molecular weight of 1000 to 50000.

  In spray drying, the polymer is dissolved in an organic solvent such as methylene chloride (0.04 g / ml). A known amount of interferon is suspended in the polymer solution (if insoluble) or co-dissolved (if soluble). The solution or dispersion is then spray dried. Microspheres in the range of 1 to 10 microns in diameter can be obtained in a form depending on the choice of polymer.

  A hydrogel microsphere consisting of a gel-type polymer such as alginate or polyphosphadine or other dicarboxylic acid polymer dissolves the polymer in an aqueous solution, suspends the substance to be incorporated into the mixture, and is equipped with a nitrogen gas jet. It can be prepared by extruding this polymer mixture through a droplet former. As described in Salib, et al., Pharmazeutische Industrie 40-111A, 1230 (1978), the resulting microspheres slowly fall into the stirring ion curing tank. The advantage of this system is that, as described in Lim, et al., J. Pharm. Sci. 70, 351-354 (1981), the surface of the microsphere is made with a polycationic polymer such as polylysine after production. The possibility of further modification by coating. For example, in the case of alginate, a hydrogel can be formed by ionically crosslinking the alginate with calcium ions and then crosslinking the outer surface of the microparticles with a polycation such as polylysine after manufacture. The particle size of the microspheres will be controlled using various sized extruders, polymer flow rates and gas flow rates.

  Chitosan microspheres can be prepared by dissolving the polymer in an acidic solution and cross-linking with tripolyphosphate. For example, carboxymethylcellulose (CMC) microspheres are prepared by dissolving a polymer in an acid solution and precipitating the microspheres with lead ions. Alginate / polyethyleneimine (PEI) can be prepared to reduce the amount of carboxyl groups on the alginate microcapsules.

(Ii) Pharmaceutical Composition The microparticles can be suspended in a suitable pharmaceutical carrier such as saline for administration to a patient. In the most preferred embodiment, the microparticles will be stored in dry or lyophilized form until just prior to administration. They can thus be suspended in a sufficient solution, such as an aqueous solution for administration as an aerosol, or administered as a dry powder.

(Iii) Targeted administration The microparticles can be delivered to specific cells, particularly phagocytic cells and organs. Phagocytic cells within Peyer's patches appear to selectively take up orally administered microparticles. Phagocytic cells of the reticuloendothelial tissue system also take up microparticles when administered intravenously. The microparticle endocytosis by macrophages in the lung can be used to target the microparticle to the spleen, bone marrow, liver and lymph nodes.

  Microparticles can also be targeted by attachment of ligands, such as those described above, that bind specifically or non-specifically to a particular target. Examples of such ligands include antibodies and fragments that contain variable regions, lectins and hormones, or other organic molecules that have receptors on the target cell surface.

(Iv) Storage of microparticles In a preferred embodiment, the microparticles are stored in a lyophilized state. The dosage is determined by the amount of interferon encapsulated, the release rate within the pulmonary system, and the pharmacokinetics of the compound.

(V) Microparticle delivery Microparticles range from direct administration to the nasal passages where some of the particles reach the pulmonary system, to the use of powder instillation devices, to the use of a catheter or tube to reach the lung tube. It can be delivered using a method. Dry powder inhalers are commercially available, but those that use hydrocarbon propellants are no longer used and rely on patient respiratory uptake and can be in various doses. Examples of suitable propellants include, for example, 1,1,1,2-tetrafluoroethane (CF3CH2F) (HFA-134a), and 1,1,1,2,3,3,3-heptafluoro-n- There are hydrofluoroalkane propellants such as propane (CF3CHFCF3) (HFA-227), perfluoroethane, monochloroleufluoromethane, 1,1 difluoroethane, and combinations thereof.

F. Medical Device Coating for Interferon Release Another aspect of the present invention relates to a coated medical device. For example, in some embodiments, the present invention provides a medical device having a coating adhered to at least one surface, wherein the coating comprises the polymer matrix and an interferon. . Such coatings can be applied to surgical instruments such as screws, plates, washers, sutures, prosthetic anchors, scissors, staples, electrical leads, valves, membranes and the like. The device includes a catheter, an implantable vascular access port, a blood storage bag, a blood tube material, a central venous catheter, an arterial catheter, a vascular graft, an intra-aortic balloon pump, a heart valve, a cardiovascular suture,
It may be an artificial heart, a pacemaker, a ventricular assist pump, an extracorporeal device, a blood filter, a hemodialysis device, an adsorptive blood purification device, a plasma phlebotomy device, and a filter adapted to be installed in a blood vessel.

  In some embodiments according to the present invention, the monomer for forming the polymer is blended with the interferon and mixed to uniformly disperse the interferon in the monomer solution. This dispersion is then applied to a stent or other device according to conventional coating methods, after which the crosslinking process is initiated with a conventional starting material such as ultraviolet light. In another embodiment according to the present invention, the polymer composition is blended with interferon to form a dispersion. The dispersion is then applied to the surface of the medical device and the polymer is crosslinked to form a solid coating. In another embodiment according to the present invention, the polymer and interferon are combined in a suitable solvent to form a dispersion, which is then applied to the stent in a conventional manner. The solvent is then removed by a conventional process such as thermal evaporation, so that the polymer and interferon (both forming a sustained release drug delivery system) remain on the stent as a coating. A similar process may be used when interferon is dissolved in the polymer composition.

  In some embodiments according to the present invention, the system includes a relatively rigid polymer. In other embodiments, the system includes a soft and malleable polymer. In yet another embodiment, the system includes an adhesive polymer. The hardness, elasticity, adhesion and other characteristics of the polymer can vary widely depending on the specific final physical shape of the system, as will be described in further detail below.

  Embodiments of the system according to the present invention take many different forms. In some embodiments, the system consists of a suspension or dispersion of interferon in a polymer. In some other embodiments, the system consists of interferon and a semi-solid or gel polymer adapted to be injected into the body with respect to the syringe barrel. In another embodiment according to the present invention, the system consists of interferon and a soft flexible polymer adapted to be inserted or implanted into the body by a suitable surgical method. In a further embodiment according to the invention, the system consists of a rigid solid polymer adapted to be inserted or implanted into the body by suitable surgical methods. In a further embodiment, the system includes a polymer having interferon suspended or dispersed therein, wherein the interferon and polymer mixture is on a surgical instrument such as a screw, stent, pacemaker, or the like. Form a coating. In a specific embodiment according to the present invention, the instrument is a rigid solid polymer molded in the form of a surgical instrument such as a surgical screw, plate, stent, or any part thereof. In another embodiment according to the present invention, the system includes a polymer in the form of a suture having interferon dispersed or suspended therein.

  In some embodiments according to the invention, a medical device is provided that includes a substrate having a surface, such as an external surface, and a coating on the external surface. The coating includes a polymer and interferon dispersed in the polymer, where the polymer can permeate the interferon or biodegrade to liberate the interferon. In some embodiments according to the invention, the instrument comprises interferon suspended or dispersed in a suitable polymer, wherein the interferon and polymer are coated over a substrate such as a surgical instrument. The Such coating could be achieved by spray coating or dip coating.

  In another embodiment according to the invention, the device comprises an interferon and a polymer suspension or dispersion, where the polymer is rigid and is one of the devices to be inserted or implanted into the body. It is a component part. For example, in certain embodiments according to the present invention, the instrument is a surgical screw, stent, pacemaker, etc. coated with interferon suspended or dispersed in a polymer. In some other embodiments according to the present invention, the polymer in which the interferon is suspended forms the tip or head of a surgical screw, or a portion thereof. In other embodiments according to the present invention, polymers that suspend or disperse interferon in surgical materials such as surgical tubing (eg, colon fistulas, peritoneal dialysis, catheters, and intravenous tubing). Coat on instrument. In a further embodiment according to the present invention, the device is an intravenous needle having a polymer and interferon coated thereon.

As discussed above, the coating according to the present invention comprises a bioerodible or non-bioerodible polymer.
The choice of bioerodible versus non-bioerodible polymer is made based on the intended end use of the system or device. In some embodiments according to the present invention, the polymer is advantageously bioerodible. For example, if the system is a coating on a surgically implantable device such as a screw, stent, pacemaker, etc., it is advantageous if the polymer is bioerodible. In another embodiment according to the invention, where the polymer is advantageously bioerodible, a device that is an implantable, inhalable, or injectable suspension or dispersion of interferon into the polymer. In this case, no further elements (for example screws or anchors) are used.

  In some embodiments according to the present invention where the polymer is poorly permeable and bioerodible, the polymer remains in place for a substantial time after the interferon is released, but ultimately Advantageously, the rate of polymer bioerosion is sufficiently slower than the rate of interferon release so that it is bioerodible and reabsorbed into the surrounding tissue. For example, if the device is a bioerodible suture that includes interferon suspended or dispersed in a bioerodible polymer, the rate of bioerosion of the polymer is such that the interferon is released linearly over about 3 to about 14 days. However, it is advantageous if the sutures are sufficiently slow so that they last for about 3 weeks to about 6 months. A similar instrument according to the present invention is a surgical staple comprising interferon suspended or dispersed in a bioerodible polymer.

  In another embodiment according to the present invention, the bioerosion rate of the polymer is advantageously the same as the rate of interferon release. For example, if the system includes interferon suspended or dispersed in a polymer coated on a surgical instrument such as an orthopedic screw, stent, pacemaker, or non-bioerodible suture, Advantageously, the polymer bioerodes at a rate such that the surface area of the interferon that is directly exposed to surrounding body tissue is substantially constant over a period of time.

  In other embodiments according to the invention, the polymer vehicle is permeable to water in surrounding tissues, such as in plasma. In such cases, the aqueous solution will penetrate the polymer and contact the interferon. The rate of dissolution will be governed by complex combinations of variables such as polymer permeability, interferon solubility, physiological fluid pH, ionic strength, and protein composition.

  In some embodiments according to the present invention, the polymer is non-bioerodible. A non-bioerodible polymer is coated on a surgical instrument adapted to be permanently or semi-permanently inserted or implanted into the body, or a component of the surgical instrument. Contains a polymer that is intended to form. Examples of instruments in which the polymer advantageously forms a permanent coating on the surgical instrument include orthopedic screws, stents, prosthetic joints, prosthetic valves, permanent sutures, pacemakers, and the like.

  There are a variety of stents that may be used after percutaneous transluminal angioplasty. Any number of stents may be used in accordance with the present invention, but for simplicity, a limited number of stents will be described in the embodiments of the present invention. One skilled in the art will recognize that any number of stents may be used in the context of the present invention. In addition, as described above, other medical devices may be used.

  Stents are commonly used as tubular structures that are left in the lumen of a conduit to reduce occlusion. Usually, the stent is expanded non-expanded into the lumen and then expands autonomously or with the help of a second in situ instrument. A typical method of dilation is the use of an angioplasty balloon attached to a catheter that shears and breaks the occlusion associated with the vessel wall component and expands within a constricted blood vessel or internal pain relief to enlarge the lumen. Done through.

  The stent of the present invention may be manufactured using any number of methods. For example, the stent may be manufactured from a hollow or molded stainless steel tube that may be machined using laser, electrical discharge milling, chemical etching or other means. The stent is inserted into the body and placed at the desired site in an unexpanded form. In one exemplary embodiment, the balloon catheter may be expanded intravascularly, where the final diameter of the stent is a function of the diameter of the balloon catheter used.

  It should be understood that stents according to the present invention may be implemented with shape memory materials including, for example, suitable alloys of nickel and titanium or stainless steel.

  A structure formed from stainless steel could be made self-expanding by configuring the stainless steel in a predetermined manner, for example by twisting it into a braided shape. In this embodiment, after forming the stent, the insertion means may be compressed to occupy a small enough space so that it can be inserted into a blood vessel or other tissue, in which case the insertion means includes: Suitable catheters or flexible rods are included.

  The stent may be formed to expand to the desired shape upon exiting the catheter, where expansion is automatic or triggered by changes in pressure, temperature or electrical stimulation. .

To provide effective dosage to the lesion area, regardless of the stent design,
It is preferable to apply interferon with sufficient specificity and sufficient concentration. In this regard, the “reservoir size” of the coating is preferably sized to properly apply the desired interferon in the desired location and amount.

  In an alternative embodiment, the entire inner and outer surfaces of the stent may be coated with a therapeutic dosage of interferon. However, it is important to note that the coating technique will vary depending on the interferon in question. The coating technique will also vary depending on the material including the stent or other intraluminal medical device.

  The intraluminal medical device includes a sustained release drug delivery coating. The interferon coating may be applied to the stent according to conventional coating processes such as, for example, impregnation coating, spray coating or dip coating.

  In certain embodiments, the endoluminal medical device includes a long radially expandable tubular stent having an inner lumen surface extending along a longitudinal stent axis and an opposing outer surface. The stent may include permanent, implantable stents, implantable implantable stents, or temporary stents, said temporary stents being expandable within a blood vessel, and It is defined as a stent that can then be retracted from the blood vessel. The shape of the stent includes a coil stent, a memory coil stent, a nitinol stent, a mesh stent, a scaffold stent, a sleeve stent, a permeable stent, a stent having a temperature sensor, and a porous stent. Will be included. In addition, the stent may be mounted according to conventional methods, such as an inflatable balloon catheter, a self-attaching mechanism (after release from the catheter), or other suitable means. The elongate radially expandable tubular stent may be an implantable stent, wherein the implantable stent is a composite device having a stent inside or outside the implant. The graft may be a vascular graft, such as an ePTFE graft, a biological graft, or a woven graft.

  Interferon may be incorporated into or secured to the stent in a number of ways. In an exemplary embodiment, interferon is incorporated directly into the polymer matrix and sprayed onto the outer surface of the stent. The interferon elutes from the polymer matrix over time and enters the surrounding tissue. The interferon preferably remains on the stent for at least 3 days to approximately 6 months, and more preferably for 7 to 30 days.

  In some embodiments, a polymer according to the invention is a biologically tolerant polymer that is permeable to interferon and has a permeability such that it is not a major rate determinant of the rate of release of interferon from the polymer. including.

  In some embodiments according to the present invention, the polymer is non-bioerodible. Examples of non-bioerodible polymers useful in the present invention include poly (ethylene-co-vinyl acetate) (EVA), polyvinyl alcohol and polyurethanes such as polycarbonate-based polyurethanes. In another embodiment of the invention, the polymer is bioerodible. Examples of bioerodible polymers useful in the present invention include polyanhydrides, polylactic acid, polyglycolic acid, polyorthoesters, polyalkylcyanoacrylates, or derivatives and copolymers thereof. One skilled in the art will recognize that the choice of polymer bioerodibility and non-bioerodibility depends on the final physical form of the system, as described in further detail below. Other exemplary polymers include poly-silicone and polymers derived from hyaluronic acid. One skilled in the art understands that a polymer according to the present invention is prepared under conditions suitable to have permeability such that it is not a major rate determinant in the release of interferon from the polymer. Let's be done.

  In addition, suitable polymers are naturally compatible (collagen, hyaluronic acid, etc.) that are biologically compatible with bodily fluids and mammalian tissues and are essentially insoluble in bodily fluids to which they come into contact. ) Or synthetic materials. In addition, suitable polymers are basically those that prevent interaction between interferon dispersed / suspended in the polymer and proteinaceous components in body fluids. The use of rapidly dissolving polymers, highly soluble polymers in body fluids, or those that allow interaction between interferons and proteinaceous components must be avoided in some cases. This is because the dissolution of the polymer or the interaction with the proteinaceous component can affect the steadiness of the drug release.

  Other suitable polymers include polypropylene, polyester, polyethylene vinyl acetate (PVA or EVA), oxidized polyethylene (PEO), oxidized polypropylene, polycarboxylic acid, polyalkyl acrylate, cellulose ether, silicone, poly (dl-lactide-copolymer). Glycolide), various Eudragrits (eg, Eudragrits) (eg NE30D, RS PO and RL PO), polyalkyl-alkyl acrylate copolymers, polyester-polyurethane block copolymers, polyether-polyurethane block copolymers, polydioxanone, poly (Β-hydroxybutyrate), polylactic acid (PLA), polycaprolactone, polyglycolic acid, and PEO-PLA copolymer.

The coating of the present invention may be formed by mixing one or more suitable monomers and a suitable interferon and then polymerizing the monomers to form a polymer system. In this way, interferon is dissolved or dispersed in the polymer. In other embodiments, after interferon is mixed with a liquid polymer or polymer dispersion, the polymer is further processed to form the coating of the present invention. Suitable further processing includes crosslinking with suitable crosslinkable interferons, further polymerisation of liquid polymers or polymer dispersions, copolymerization with suitable monomers, block copolymerization with suitable polymer blocks Etc. would be included.
Further processing is to capture the interferon in the polymer so that it is suspended or dispersed in the polymer vehicle.

  Any number of non-erodible polymers may be used with the interferon. Film-forming polymers that can be used for coatings in this application may be absorbable or non-absorbable but must be biocompatible to reduce irritation to the vessel wall. The polymer may be biostable or bioabsorbable depending on the desired release rate or the desired degree of polymer stability, although bioabsorbable polymers will be preferred. This is because, unlike biostable polymers, it does not exist for a long time after transplantation and does not cause an adverse chronic local response. In addition, bioabsorbable polymers are a further problem after long periods of time, even after the stent has been removed from the coating and the stent has been encapsulated in the tissue due to the stress of the biological environment that has been lost. Does not pose a risk of causing

Suitable film-forming bioabsorbable polymers that may be used include aliphatic polyesters, poly (amino acids), copoly (ether-esters), polyalkylene oxalates, polyamides, poly (iminocarbonates), polyorthoesters. There are polymers selected from the group consisting of: polyoxaesters, polyamide esters, polyoxaesters containing amide groups, poly (anhydrides), polyphosphazenes, biomolecules and mixtures thereof. For the purposes of the present invention, aliphatic polyesters include lactide homopolymers and copolymers (including lactate d-, l- and meso-lactide), ε-caprolactone, glycolide (including glycolic acid), hydroxybutyrate, hydroxyvale. Rate, para-dioxanone, trimethylene carbonate (and its alkyl derivatives), 1,4-dioxepan-2-one, 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxane-2- There are ON and a mixture of these polymers. Poly (iminocarbonates) for the purposes of the present invention include Kemnitzer and Kohn, in the Handbook of Biodegradable Polymers,
edited by Domb, Kost and Wisemen, Hardwood Academic Press, 1997, pages 251-272. Copoly (ether-esters) for the purposes of the present invention include Journal of Biomaterials Research,
Vol. 22, pages 993-1009, 1988 by Cohn and Younes and Cohn, Polymer Preprints
There are copolyester-ethers described in (ACS Division of Polymer Chemistry), Vol. 30 (1), page 498, 1989 (eg PEO / PLA). Polyalkylene oxalates for the purposes of the present invention include US Pat. Nos. 4,208,511; 4,141,087; 4,130,639; 4,140,678; 4,105,034; and 4,205,399 (incorporated herein by reference). . Allcock in The Encyclopedia of Polymer Science, Vol. 13, pages
31-41, Wiley Intersciences, John Wiley & Sons, 1988 and by Vandorpe,
Schacht, Dejardin and Lemmouchi in the Handbook of Biodegradable Polymers,
edited by Domb, Kost and Wisemen, Hardwood Academic Press, 1997, pages 161-182, L-lactide, D, L-lactide, lactic acid, glycolide, glycolic acid, para-dioxanone, trimethylene carbonate and Polyphosphazene, co-, ter- and higher mixed monomer based polymers made from ε-caprolactone. Type _{HOOC-C 6 H 4 -O- (} CH 2) m -O-C6H4-COOH is the polyanhydrides derived from diacids (though Shikichu, m up to a certain and up to 12 integer of 2 to 8 Its copolymer with aliphatic alpha-omega diacids consisting of carbon. Polyoxaester polyoxaamides and polyoxaesters containing amine and / or amide groups are described below in US Pat. Nos. 5,464,929; 5,595,751; 5,597,579; 5,607,687; No. 5,618,552; No. 5,620,698; No. 5,645,850; No. 5,648,088; No. 5,698,213 and No. 5,700,583. Heller in Handbook of Biodegradable Polymers, edited by Domb, Kost
Polyorthoesters such as those described by and Wisemen, Hardwood Academic Press, 1997, pages 99-118. Film-forming polymer biomolecules for the purposes of the present invention include naturally occurring substances that are enzymatically degraded in the human body or hydrolytically unstable in the human body, such as fibrin, fibrinogen, collagen, elastin, And absorbable biocompatible polysaccharides such as chitosan, starch, fatty acids (and their esters) glucoso-glycans and hyaluronic acid.

Suitable film-forming biostable polymers with relatively low chronic tissue response, such as polyurethane, silicone, poly (meth), acrylate, polyester, polyalkyl oxide (polyethylene oxide), polyvinyl alcohol, polyethylene glycol and polyvinyl pyrrolidone, Hydrogels such as those formed from cross-linked polyvinyl pyrrolidone and polyester could also be used. Other polymers could be used as long as they can be dissolved, cured or polymerized on the stent. These include polyolefins, polyisobutylene and ethylene-alpha olefin copolymers; acrylic polymers (including methacrylates) and copolymers, halogenated vinyl polymers and copolymers, such as salts or polyvinyls; polyvinyl ethers, such as polyvinyl methyl ether; halogenated polyvinylidenes, For example, polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketone; polyvinyl aromatics such as polystyrene; polyvinyl esters such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins such as ethylene-methyl methacrylate copolymers, acrylonitrile Styrene copolymers, ABS resins and ethylene-vinyl acetate copolymers; polyamides such as nylon 66 and polycaprolactam; Polycarbonate; Polyoxymethylene; Polyimide; Polyether; Epoxy resin, Polyurethane; Rayon; Rayon-triacetate, cellulose, cellulose acetate, cellulose acetate butyrate; Cellophane; Cellulose nitrate; Cellulose propionate; Cellulose ether (ie, carboxy) Methyl cellulose and hydroxyalkyl cellulose) and combinations thereof. Polyamides for the purposes of this application are of the form —NH— (CH ₂ ) _n —CO— and NH— (CH ₂ ) _x —NH—CO— (CH ₂ ) _y —CO, where n is preferably Is an integer from 6 to 13; x is an integer ranging from 6 to 12; and y is an integer ranging from 4 to 16. The above list is exemplary and not limiting.

  The polymer used for the coating may be a film-forming polymer having a sufficiently high molecular weight so as not to become waxy or sticky. The polymer must also adhere to the stent and, after being deposited on the stent, it must not be so deformable that it can be displaced by hemodynamic stress. The molecular weight of the polymer is high enough to provide sufficient strength to prevent the polymer from being scraped off during manipulation or placement of the stent, and must not tear during stent expansion. In some embodiments, the polymer has a melting temperature greater than 40 ° C, preferably greater than about 45 ° C, more preferably greater than about 50 ° C, and most preferably greater than about 55 ° C.

  The coating could be formulated by mixing one or more of the therapeutic interferons with the coating polymer in the coating mixture. The interferon may be present in a liquid, a finely divided solid, or any other suitable physical form. Optionally, the mixed solution may contain one or more additives such as a diluent, a simple substance, a pharmaceutical additive, a non-toxic auxiliary substance such as a stabilizer. Other suitable additives may be formulated with the polymer and interferon. For example, a hydrophilic polymer selected from the previously described list of biocompatible film-forming polymers may be added to the biocompatible hydrophobic coating to alter the release profile (or alternatively, the hydrophobic polymer The release profile may be modified in addition to the hydrophilic coating). An example would be to add a hydrophilic polymer selected from the group consisting of polyethylene oxide, polyvinyl pyrrolidone, polyethylene glycol, carboxymethyl cellulose, hydroxymethyl cellulose and combinations thereof to the aliphatic polyester coating to alter the release profile. . A suitable relative amount can be determined by observing the in vitro and / or in vivo release profile of the therapeutic interferon.

  The thickness of the coating can determine the rate at which the interferon elutes from the matrix. Basically, interferon elutes from the matrix by diffusion through the polymer matrix. Because the polymer is permeable, solids, liquids and gases can escape from it. The total thickness of the polymer matrix is in the range of about 1 micron to about 20 microns or more. It is important to note that the primer layer and metal surface treatment may be used prior to securing the polymer matrix to the medical device. For example, acid washing, alkali (base) washing, salt treatment and parylene precipitation may be used as part of the overall process described.

  To further illustrate, poly (ethylene-co-vinyl acetate), polybutylmethacrylate and interferon solutions may be incorporated into or onto a stent in a number of ways. For example, the solution may be sprayed onto the stent, or the stent may be immersed in the solution. Other methods include spin coating and RF plasma polymerization. In one exemplary embodiment, the solution is sprayed onto the stent and then dried. In another exemplary embodiment, the solution is charged to one polarity and the stent is charged to the opposite polarity. In this way, the solution and stent will be attracted to each other. Using this type of spraying process would reduce waste and allow more precise control of the coating thickness.

In another exemplary embodiment, the interferon comprises an amount of a first portion selected from the group consisting of polymerized vinylidene fluoride and polymerized tetrafluoroethylene, and an amount of other than the first portion, A polyfluoro copolymer may be made by incorporating into a film-forming polyfluoro copolymer comprising a first portion and a second portion to be copolymerized, wherein the second portion is tough to the polyfluoro copolymer. Or can provide elasticity,
Also, the relative amounts of the first and second portions provide the coating and resulting film with properties that are useful for use in treating implantable medical devices.

  In certain embodiments according to the present invention, the outer surface of the expandable tubular stent of the intraluminal medical device of the present invention comprises a coating according to the present invention. This outer surface of the stent with the coating is a tissue contacting surface and is biocompatible. “Surface coated with a sustained release interferon delivery system” is synonymous with “coated surface”, which surface is coated or covered by a sustained release interferon delivery system according to the present invention, or Impregnated. Examples of delivery systems (controlled release, sustained release, delayed release) are US serial numbers 10 / 193,654 and WO02 published as Applicant's US2003 / 0017169, the contents of which are incorporated herein by reference. It is described in PCT / US01 / 50355 published as / 053174.

  In an alternative embodiment, the inner lumen surface or the entire surface (ie, inner and outer surfaces) of the long radially expandable tubular stent of the endoluminal medical device of the present invention has a coated surface. The inner lumen surface with the sustained release interferon delivery system coating of the present invention is also a fluid contact surface and is biocompatible and blood compatible.

IV. Variant Interferon Polypeptides It is anticipated that certain variants (or variants) of the interferon polypeptides of the present invention will act as agonists or antagonists. While not wishing to be bound by any particular theory, it is known that mutated protein signaling factors can bind to their appropriate receptors but still cannot activate this receptor . Such mutant proteins act as antagonists by displacing the wild-type protein and blocking normal receptor activation. In addition, many proteins are known to be able to make one or more amino acid substitutions to increase their activity compared to the wild-type protein. Such agonists will have, for example, increased half-life, binding affinity, stability, or activity compared to the wild-type protein. There are a number of known methods for obtaining a variant (or variant) with the desired activity.

  Methods for generating large pools of mutant / atypical proteins are known in the art. In certain embodiments, the present invention contemplates using interferon polypeptides made by combinatorial mutagenesis. Such methods are convenient for creating both point and truncation variants, as is known in the art, and in particular identify potential variant sequences (eg, homologues) that are functional in the assay. Would be useful for. The purpose of screening such a combinatorial library is, for example, to create an interferon variant or homologue that can act as either an agonist or an antagonist. Thus, combinatorial variants can be made to have improved potency compared to naturally occurring types of proteins. Similarly, binding to other extracellular matrix components (eg, receptors) can be mimicked, but still does not induce any biological response and thus inhibits the action of interferon polypeptides or interferon agonists. It is possible with this combinatorial method to create an interferon variant that acts as an antagonist in that it can. In addition, manipulation of several domains of interferon according to this method can provide domains that are more suitable for use in fusion or chimeric proteins, such as domains that have been demonstrated to have certain useful properties.

To further illustrate the state of the art of combinatorial mutagenesis, see Gallop et al. (1994), J
Note that the review literature in Med Chem 37: 1233 describes the general state of the art of combinatorial libraries as of the early 1990s. Specifically, Gallop et al., Page 1239, “Screening similar libraries determines the minimum size of active sequences and identifies residues that are important for binding and cannot tolerate substitution. It helps. ” In addition, Ladner et al. PCT Gazette
WO90 / 02809, Goeddel et al., US Pat. No. 5,223,408, and Markland et al. PCT
Publication WO92 / 15679 describes specific techniques that one skilled in the art would be able to use to create a library of variants that can be screened at high speed to identify variants / fragments with specific activities. ing. These techniques are exemplary to those skilled in the art and demonstrate that if a large library of related variants / cleaved variants is generated and tested, specific variants can be isolated without undue experimentation. is doing. Gustin et al. (1993)
Virology 193: 653, and Bass et al. (1990) Proteins: Structure,
Function and Genetics 8: 309-314 also describes other exemplary techniques in the art that can be adapted as a means of generating mutagenic variants of the interferon polypeptides of the present invention.

  In fact, without extensive knowledge of which residues are important for biological function, large-scale mutagenesis of interferon proteins can produce a wide array of variants with equal biological activity. This is evident from combinatorial mutagenesis techniques. Alternatively, such methods can be used to create a wide array of variants with higher or antagonist activity. In fact, the ability of combinatorial techniques is to screen billions of different variants with high-throughput analysis that eliminates the need to know or know important residues in advance.

V. Antibody antagonists It is anticipated that some antibodies can act as interferon antagonists. An antibody may have surprising affinity and specificity for a particular epitope. Binding of an antibody to its epitope on a protein can be achieved by competitively or non-competitively inhibiting the interaction of the protein with other proteins necessary to perform its proper function. It will antagonize the function of

  Antibodies with interferon antagonist activity can be identified in much the same way as other interferon antagonists. For example, a candidate antibody can be administered to a cell expressing a reporter gene, and thus an antibody that causes a decrease in reporter gene expression is an antagonist.

  In one variation, an antibody of the invention may be a single chain antibody (scFv) comprising variable antigen binding domains linked by a polypeptide linker. Single chain antibodies are expressed as a single polypeptide chain, but can be expressed in bacteria and as part of a phage display library. In this method, a phage that expresses a suitable scFv will have interferon antagonist activity. The nucleic acid encoding the single chain antibody can then be recovered from this phage and used to generate large quantities of scFv. The construction and screening of scFv libraries has been extensively described in a variety of published literature (US Pat. Nos. 5,258,498; 5,482,858; 5,091,513; 4,946,778; 5,969,108; 5,871,907; 5,223,409; No. 5,225,539).

G. Human IFN-α alleles and protein products Table 2 below shows common alleles of human interferon α family genes / proteins, Pestka, S. (1983) Arch Biochem Biophys 221: 1-37; Diaz, MO , Pomykala, HM,
Bohlander, SK, Maltepe, E., Malik, K., Brownstein, B., and Olopade, OI (1994) Genomics 22: 540-52; and Pestka, S. (1986) Meth. Enzymol 119: 3-14 Built on the basis of Krause, CD, Lunn, CA, Izotova, LS, Mirochnitchenko, O.,
Reviewed by Kotenko, SV, Lundell, DJ, Narula, SK, and Pestka, S. (2000) J Biol Chem. 275: 22995-3004.

Allele variants and interferon-α variants of the human interferon-α gene have been reported in the following applications: WO 2002/095067; WO
WO 02/101048; WO 02/095067; WO 02/083733; WO
WO 08/086156; WO 2002/083733; WO 03/000896; WO 02/101048; WO 02/079249; WO
03/000896; WO 2004/022593; WO 2004/022747; WO 03/023032;
WO 2004/022593. See also: (1) Kim et al, Cancer Lett. 2003 Jan 28; 189 (2): 183-8; (2) Hussain et al., J Interferon Cytokine Res. 2000 Sep; 20 ( 9): 763-8; (3) Hussain et al., J Interferon
Cytokine Res. 1998 Jul; 18 (7): 469-77; (4) Nyman et al., Biochem J. 1998 Jan 15; 329 (Pt 2): 295-302; (5) Golovleva et al, J Interferon
Cytokine Res. 1997 Oct; 17 (10): 637-45; (6) Hussain et al, J Interferon Cytokine Res. 1997 Sep; 17 (9): 559-66; (7) Golovleva, I., Saha, N ., Beckman,
L. Hum Hered. 1997 Jul-Aug; 47 (4): 185-8; (8) Golovleva et al, 1997 Apr; 18 (4): 645-7; (9) Kita et al, J Interferon Cytokine
Res. 1997 Mar; 17 (3): 135-40; (10) Golovleva et al, Am J Hum Genet. 1996 Sep; 59 (3): 570-8; (11) Hussain et al, J Interferon Cytokine
Res. 1996 Jul; 16 (7): 523-9; (12) Linge et al, Biochim Biophys Acta. 1995 Dec 27; 1264 (3): 363-8; (13) Gewert et al, J Interferon Cytokine
1995 May; 15 (5): 403-6; (14) Lee et al, J Interferon Cytokine Res. 1995 Apr; 15 (4): 341-9; (15) Kaluz et al, Acta Virol. 1994
Apr; 38 (2): 101-4; (16) Emanuel et al, J Interferon Res.
1993 Jun; 13 (3): 227-31; (17) Kaluz et al, Acta
Virol. 1993 Feb; 37 (1): 97-100; (18) Shekhter et al, Dokl Akad Nauk SSSR. 1990; 314: 998-1001; (19) Li et
al, Sci China B. 1992 Feb; 35 (2): 200-6.

EXAMPLES Having generally described the invention, it will be readily understood by reference to the following examples, which are merely for purposes of illustration of certain aspects and embodiments of the invention. It is included and is not intended to limit the present invention.

Example 1 : Isolation of a feline IFNα clone A feline IFNα clone was isolated by PCR amplification of genomic DNA from a feline lung cell line (AKD) using standard methods. Nine individual sequences were isolated, Fe-IFN-αA (SEQ ID NO: 9), Fe-IFN-αB (SEQ ID NO: 11), Fe-IFN-αC (SEQ ID NO: 13), Fe-IFN-αD (sequence) No. 15), Fe-IFN-αE (SEQ ID NO: 17), Fe-IFN-αF (SEQ ID NO: 19), Fe-IFN-αG (SEQ ID NO: 21), Fe-IFN-αH (SEQ ID NO: 23), and Fe -IFN-αI (SEQ ID NO: 25) was designated. Amino acid sequences corresponding to each of these are also provided: Fe-IFN-αA (SEQ ID NO: 10), Fe-IFN-αB (SEQ ID NO: 12), Fe-IFN-αC (SEQ ID NO: 14), Fe-IFN -αD (SEQ ID NO: 16), Fe-IFN-αE (SEQ ID NO: 18), Fe-IFN-αF (SEQ ID NO: 20), Fe-IFN-αG (SEQ ID NO: 22), Fe-IFN-αH (SEQ ID NO: 24) ), And Fe-IFN-αI (SEQ ID NO: 26).

PCR was performed using standard techniques. Genomic DNA was amplified twice. The flanking primers used to amplify the cat sequence are:
5 'primer: 5'-CTCTTCCTTCTTGGTGGCCCTG-3'
3 'Primer: 5'-GTGATGAGTCAGTGAGAATCATTTC-3'
It is.

Example 2: Antiviral activity of feline IFNα species The antiviral activity of the relevant interferon species was measured using a cytopathic effect assay (CPE). Briefly, serial dilutions of interferon were incubated with test cells for 1-4 hours at 37 ° C. Virus was then added to the cells and incubated for 16 hours at 37 ° C. Surviving cells were visualized by uptake of crystal violet dye, and the dilution of interferon that approximately 50% of the cells survived after viral infection was determined.

Figure 1 shows that cat IFN-αA, IFN-αB, IFN-αC, IFN-αD, IFN-αE, IFN-αF, IFN-αG, and IFN-αI have antiviral activity when measured by CPE. It summarizes the results of these experiments that demonstrate their possession.
The activity of feline IFN-αH was not determined by this assay. In this specific experiment, the test cells were AKD cat lung cells and the virus was vesicular stomatitis virus (VSV).

Example 3: Isolation of Rhesus monkey IFNα clones Rhesus monkey IFNα clones were isolated from genomic DNA from rhesus monkey kidney cell line (LLCMK-2) by PCR amplification using standard methods. The sequence was amplified using two separate primer pairs. Using the first primer pair, one sequence was isolated and designated Rh-IFN-α4b (SEQ ID NO: 29). The amino acid sequence corresponding to the Rh-IFN-α4b nucleic acid sequence was designated SEQ ID NO: 30. PCR was performed using standard techniques. Amplification from genomic DNA was performed twice. The flanking primers used to amplify this rhesus monkey sequence are:
5 'Primer: 5'-CTTCAGAGAACCTGGAGCC-3'
3 '
Primer: 5'-AATCATTTCCATGTTGAACCAG-3 '
It is.

  Three additional rhesus IFNα clones were prepared using standard methods and a second primer pair: Rh-IFN-αD1 (SEQ ID NO: 31), Rh-IFN-αD2 (SEQ ID NO: 33), and Rh-IFN-αD3 ( It was isolated by PCR amplification of genomic DNA from rhesus monkey kidney cell line (LLCMK-2) using SEQ ID NO: 35). The amino acid sequences corresponding to each of these are also provided: Rh-IFN-αD1 (SEQ ID NO: 32), Rh-IFN-αD2 (SEQ ID NO: 34), and Rh-IFN-αD3 (SEQ ID NO: 36).

PCR was performed using standard techniques. Amplification from genomic DNA was performed twice. The flanking primers used to amplify these rhesus monkey sequences are:
5 'Primer: 5'-AGAAGCATCTGCCTGCAATATC-3'
3 '
Primer: 5'-GCTATGACCATGATTACGAATTC-3 '
It is.

Example 4 : Antiviral activity of Rhesus monkey IFNα species The antiviral activity of the relevant interferon species was measured using a cytopathic effect assay (CPE). Briefly, serial dilutions of interferon were incubated with test cells for 1-4 hours at 37 ° C. Virus was then added to the cells and incubated for 16 hours at 37 ° C. Surviving cells were visualized by uptake of crystal violet dye to determine the dilution of interferon in which 50% of the cells survived after virus infection.

  FIG. 2 summarizes the results of experiments demonstrating that rhesus monkey IFN-α4b has antiviral activity as measured by antiviral activity assay (CPE). The activity of rhesus monkey IFN-αD1, IFN-αD2, and IFN-αD3 was not determined by this assay. This assay was performed using either Madin-Darby bovine kidney endothelial cells (MDBK) or African green monkey kidney cells (Vero) infected with VSV as test cells.

Example 5: Isolation of human IFNα clones 18 human interferon-α species were isolated according to the procedure described in US Pat. Nos. 5,789,551, 5,869,293 and 6,001,589. Briefly, human genomic DNA was analyzed by PCR using standard methods. The primers used in this analysis are illustrated in FIGS.

  Eighteen human interferon-α species identified using this approach are: hu-IFN-α001 (SEQ ID NO: 37), hu-IFN-α002 (SEQ ID NO: 39), hu-IFN-α003 (SEQ ID NO: 41) , Hu-IFN-α004 (SEQ ID NO: 43), hu-IFN-α005 (SEQ ID NO: 45), hu-IFN-α006 (SEQ ID NO: 47), hu-IFN-α007 (SEQ ID NO: 49), hu-IFN-α008 (SEQ ID NO: 51), hu-IFN-α009 (SEQ ID NO: 53), hu-IFN-α010 (SEQ ID NO: 55), hu-IFN-α011 (SEQ ID NO: 57) hu-IFN-α012 (SEQ ID NO: 59), hu -IFN-α013 (SEQ ID NO: 61), hu-IFN-α014 (SEQ ID NO: 63), hu-IFN-α015 (SEQ ID NO: 65), hu-IFN-α016 (SEQ ID NO: 67), hu-IFN-α017 ( SEQ ID NO: 69) and hu-IFN-α018 (SEQ ID NO: 71). Amino acid sequences corresponding to each of these are also provided: IFN-α001 (SEQ ID NO: 38), hu-IFN-α002 (SEQ ID NO: 40), hu-IFN-α003 (SEQ ID NO: 42), hu-IFN-α004 (SEQ ID NO: 44), hu-IFN-α005 (SEQ ID NO: 46), hu-IFN-α006 (SEQ ID NO: 48), hu-IFN-α007 (SEQ ID NO: 50), hu-IFN-α008 (SEQ ID NO: 52), hu-IFN-α009 (SEQ ID NO: 54), hu-IFN-α010 (SEQ ID NO: 56), hu-IFN-α011 (SEQ ID NO: 58), hu-IFN-α012 (SEQ ID NO: 60), hu-IFN-α013 ( SEQ ID NO: 62), hu-IFN-α014 (SEQ ID NO: 64), hu-IFN-α015 (SEQ ID NO: 66), hu-IFN-α016 (SEQ ID NO: 68), hu-IFN-α017 (SEQ ID NO: 70), hu -IFN-α018 (SEQ ID NO: 72).

  Furthermore, hu-IFN-α001 and hu-IFN-α012 are back-translated using the optimal E. coli codon, and hu-IFN-α001-BT (SEQ ID NO: 73) and hu-IFN-α012-BT (sequence) No. 75). The amino acid sequences corresponding to each of these are also provided: hu-IFN-α001-BT (SEQ ID NO: 74) and hu-IFN-α012-BT (SEQ ID NO: 76).

Example 6 : Isolation of human IFNα variants The following clones containing mutations were made while constructing an expression vector containing the human IFNα species detailed above. These IFNα variants can be tested for activity. By including a silent substitution in the IFNα variant, it can have the same activity as the wild-type IFNα species. Alternatively, the variant may include substitutions that alter the activity of the polypeptide. Such substitutions may increase, enhance or enhance the activity in question and thus may be IFNα agonists. In addition, the substitution may reduce or interfere with the activity in question and thus may be an IFNα antagonist.

  Provide nucleic acid sequences of atypical species: hu-IFN-α019 (SEQ ID NO: 77), hu-IFN-α020 (SEQ ID NO: 79), hu-IFN-α021 (SEQ ID NO: 81), hu-IFN-α022 (SEQ ID NO: 81) 83), and hu-IFN-α023 (SEQ ID NO: 85). Amino acid sequences corresponding to each of these are also provided: hu-IFN-α019 (SEQ ID NO: 78), hu-IFN-α020 (SEQ ID NO: 80), hu-IFN-α021 (SEQ ID NO: 82), hu-IFN -α022 (SEQ ID NO: 84) and hu-IFN-α023 (SEQ ID NO: 86).

Example 7 : Antiviral activity of human IFNα species Antiviral activity of human IFNα species was also determined using the CPE assay as outlined in detail above. This assay was performed using a combination of the following test cells and viruses: MDBK test cells with VSV; human epithelial squamous (HEP-2) cells with VSV; mouse connective tissue fibroblasts with EMC (L929) Human lung squamous (H226) cells with VSV; and human lung fibroblasts with influenza virus.

  Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Various published documents are cited throughout this application. The contents of these published documents are incorporated herein by reference. See FIG. 10 and Example 13 for more details.

Example 8 : Interferon Inhibition of SARS Coronavirus Cytopathic Effect by Interferon Materials and Methods: To evaluate the anti-SARS-CoV activity of interferon, it was placed in MEM medium supplemented with 10% fetal bovine serum (FBS, Invitrogen). FRhk-4 cells (5 × 10 ³ per well) were seeded in 96-well cell culture plates and cultured overnight. Cells were then incubated for 1 hour with various concentrations of various forms of interferon dissolved in 100 μl of MEM medium. Next, these were infected with SARS-CoV (GZ50 strain) and cultured. Twenty-four hours after SARS-CoV infection, the degree of protection provided to cells against SARS-CoV virus cytopathic effect was determined by observing and recording cell morphology with a phase contrast microscope. Each experiment was performed in triplicate and repeated at least three times. Non-infected cells became flat, while SARS-CoV infected cells became photorefractive and rounded. SARS-CoV-induced cytopathic effects range from 0 (no CPE); ± 1 (weak CPE); ± 2 (moderate CPE); ± 3 (strong CPE, no protective agent added) Graded.

  Cells were seeded in 96-well dishes 16 hours before infection. The conditioned medium of infected cells was diluted 10-fold continuously with MEM supplemented with 1% FBS and used to infect cells. Ten wells were used for each dilution and two wells were used as controls without virus infection. CPE was recorded at 24 hours post infection under a phase contrast microscope and infectious virus titer was calculated according to standard protocol (50% tissue culture infectious dose).

Results: See FIG. The efficacy of IFN α2a and the novel interferon was tested in vitro in an assay that measures the inhibition of cytopathic effects of SAR CoV isolate GZ50 on FRhk-4 monkey kidney cells. As can be seen in Figure X, IFN alpha 2b at a concentration of 20 ng / ml does not completely inhibit the lethality of SARS coronavirus to these cultured cells. IFN α012 is more active in protecting these cells, with complete protection occurring at 20 ng / ml and an IC50 between 0.8 and 0.16 ng / ml. IFN α023 is even more potent, with full protection occurring at 4 ng / ml and an IC50 between 0.8 and 0.16 ng / ml.

Example 9 Inhibition of SARS Cov Replication by Interferon Materials and Methods: Cells and Virus: African green monkey kidney cells (Vero 76) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were routinely grown in minimal basal medium (MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Logan, Utah, USA). For antiviral assays, serum was diluted to 2% and gentamicin was added to the medium to a final concentration of 50 μg / ml.

  Severe acute respiratory syndrome coronavirus (SARS CoV) strain Urbani (200300592) was obtained from CDC and passaged routinely in Vero 76 cells.

Cytopathic effect (CPE) inhibition assay. Barnard et al. 2001 (Barnard,
DL, Stowell, VD, Seley, KL, Hegde, VR, Das, SR, Rajappan, VP,
Schneller, SW, Smee, DF, & Sidwell, RW 2001, Antiviral Chemistry
& Chemotherapy 12: 221-230). Compounds are tested in this assay once or twice at various concentrations (4 single log10 or 7 1/2 log10 dilutions) and then cell viability is then neutral red (NR) on the same plate Confirmed by spectroscopic analysis by uptake assay (see below). Virus (multiplicity of infection [MOI] = 0.001) and compound were added in equal volumes to a monolayer of 80-90% confluent cells in 96-well tissue culture plates. In wells evaluating interferon, these cells were pretreated with interferon for 24 hours prior to virus exposure. The MOI used was that 100% of the cells in virus control showed cytopathic effect (CPE) within 3-5 days. The plates were incubated at 37 ° C. until cells in the virus control wells showed complete viral CPE when observed with a light microscope. Each concentration of drug was assayed in triplicate for viral CPE inhibition and in duplicate for cytotoxicity. Six wells per microplate were set aside as uninfected untreated cell controls, and six wells were given virus per microplate in medium alone and served as controls for virus replication.
Alferon was included as a positive control drug for each set of compounds tested. For all CPE-based assays, the 50% effective concentration of interferon (IC50) was calculated by linear regression analysis of the median CPE grade expressed as a percentage of untreated uninfected controls for each concentration.

Neutral red (NR) uptake assay for CPE inhibition and compound cytotoxicity. This assay was performed on the same CPE inhibition test plate described above to confirm the inhibitory activity and cytotoxicity observed by visual observation. The normal correlation between our visual and NR tests was over 95% (Barnard, DL, Stowell, VD, Seley, KL, Hegde, VR, Das, SR,
Rajappan, VP, Schneller, SW, Smee, DF, & Sidwell, RW 2001,
Antiviral Chemistry & Chemotherapy 12: 221-230). This NR test is based on Barnard et al. (Barnard, DL, Sidwell, RW, Xiao, W., Player,
MR, Adah, S. & Torrence, PF (1999) Antiviral Research 41: 119-134), as described in Cavenaugh et al. (Cavenaugh, PR Jr., Moskwa, PS, Donish, WH,
Pera, PJ, Richardson, D. & Andrese, AP (1990) Investigational New Drugs
8: 347-354).

  Briefly, media was removed from each well of a plate, 0.034% NR was added to each well of the plate, and the plate was incubated for 2 hours at 37 ° C. in the dark. The NR solution was removed from the wells, rinsed and the remaining dye extracted using ethanol buffered with Sorenson's citrate buffer. Absorbance at 540 nm / 450 nm was read with a microplate reader (Bio-Tek EL 1309; Winooski, Vermont, Bio-Tech Instruments). Absorbance values were expressed as a percentage of untreated controls and IC50 values were calculated as described above.

Results: See FIG. The effectiveness of IFN α2a and the novel interferon in inhibiting SARS CoV was tested in vitro with another assay for SARS CoV inhibition. In these experiments, different virus strains Urbani and the cell line African green monkey cells (Vero 76) were used. As measured by neutral red uptake, only IFN α012 was more effective at protecting cells than IFNα 2a. In visual measurements, all three of the novel IFNs shown (IFNα003, α023 and α012) were more effective than IFN α2a, and again IFN α012 showed the highest antiviral activity.

Example 10 : Interferon inhibition of respiratory rash virus replication Materials and methods: Flat bottom 96-well plates containing human embryonic diploid fibroblasts and Eagle's MEM with 5% fetal bovine serum (FBS) Grow until confluent. Cell monolayers were then infected with various clinical isolates of RSV at low MOI (about 0.01) and the virus was allowed to adsorb for 1 hour at room temperature. Virus was removed and monolayers were washed with PBS and replaced with MEM with 2% FBS containing serial dilutions of various interferons, including drug-free controls. After incubating at 36 ° C. for 3 days, the cells were fixed in 0.05% glutaraldehyde / PBS and blocked with 5% BSA / PBS for 1 hour. After washing, diluted mouse monoclonal antibodies (cross-reactive with A & B subtype) were administered to the monolayer and incubated at 36 ° C. for 1 hour. After washing, the monolayer was incubated with the recommended concentration of protein-A-HRP conjugate for 1 hour at 36 ° C. After washing, the substrate was added and the reaction was stopped by adding dilute sulfuric acid. Absorbance values were measured at 450 nm. Data was tabulated and 50% endpoint was determined.

Results: See FIG. The efficacy of IFN α2a and the novel interferon was tested in an in vitro model of respiratory rash virus (RSV) infection. For most of the isolates, IFN α012 was more potent than IFNα2a, with the exception of VS1511, where IFN α023 was absolutely more potent against all strains tested. IFN α003 and α001 were not very effective in this assay system.

Example 11 : Effectiveness of interferon in protecting human lung fibroblasts from influenza virus infection Materials and methods: To determine the appropriate virus titer to be used in this assay, a virus susceptibility assay was performed. Plate 100 μl cells in duplicate in 96 well microtiter plates to a concentration of 2.0E + 5 human lung fibroblasts (HLF) cells / ml. The microtiter plate is placed at 37 ° C. overnight with 5.0% CO ₂ . On the second day, influenza is titrated on another microtiter plate. A 1: 2 serial dilution is performed on the entire plate, for a total of 24 dilutions. Next, 50 μl of these virus dilutions are added to the microtiter plate of cells and placed in the incubator overnight. On day 3, the plate is stained with crystal violet and dried. To obtain the virus dilution to be used in this assay, use the endpoint of the third well to the right of the last well where 100% of the cells are killed. In this case, the 1:20 influenza dilution of the virus stock we used was determined to be the best dilution.

To find the interferon titer, first dilute the interferon with 90% DMEM, 10% FBS to approximately 8000 U / ml. Next, 10 μl of this solution is added to 190 μl of medium on a 96-well microtiter plate. Two-fold dilutions are made across the plate to make a total of 12 interferon dilutions. A known concentration of human IFN alpha A laboratory standard is also diluted 1:20 in the first well and serially diluted throughout the plate. HLF cells are counted and diluted to a concentration of 2.0E + 5 cells / ml. Next, 100 μl of these cells are added to each well. The microtiter plate is then placed on a platform rocker and shaken for 10 minutes. The plate is then placed in the incubator (37 ° C., 5.0% CO ₂ ) overnight.
On day 2, influenza is diluted to a predetermined concentration, here 1:20, and 50 μl is added to each well. Again, place the plate in the incubator overnight. The plate is then stained with crystal violet and dried. To determine the interferon titer, determine the endpoint at which 50% death occurred. This endpoint is then compared to the endpoint of the human IFN alpha A experimental standard.

Results: See FIG. The efficacy of the novel interferon was examined in vitro in an influenza model using the virus (H1N1) strain WSN / A and normal human lung fibroblasts (HLF).
The IFN concentration required to inhibit 50% of the viral cytopathic effect is determined and expressed in relative potency. In this assay, IFN α003 was equivalent to IFN α2a, but IFN α001 was less potent. IFNα012 was more than 20 times higher than the efficacy of IFNα2a.

Example 12 Effectiveness of Interferon in Protecting Hepatocytes from Yellow Fever Virus Infection Materials and Methods: Flat bottom 96 well microtiter plates coated with 0.1% collagen type 1 diluted 1 in 40 in PBS HepG2 cells were seeded. The cells were grown to subconfluent monolayers and then putative antiviral agents were added. The compound was added to wells B2 to G2 in 6 replicates, and 3-fold dilutions were prepared across the plate (B2 to B10, C2 to C10, etc.). No compound was added to column 11.

  The compound was incubated for 27 hours in the presence of cells. Yellow fever virus strain 17D (stock 6) was added to cells in rows B, C and D at 50 μl per well to give 500 pfu / well. When sufficient lethality occurred in virus-infected cells, viable cells were measured by adding 25 μl per well of XTT and PMS to each well and the plates were incubated for 1 hour at 37 ° C. The plate was allowed to cool to room temperature for 10 minutes before applying a plate sealer and analyzing the plate for spectral absorbance at 450 nm.

Results: See FIG. The effectiveness of the novel interferon in protecting HepG2 hepatocytes from lethality by yellow fever virus was compared to IFN α2a. IFN α012 was slightly more effective than IFN α2a, while IFN α003 and α023 were significantly more effective than IFN α2a. Since YFV is closely related to the hepatitis C virus, this will serve as a model for the treatment of hepatitis C.

Example 13 Comparative Materials and Methods for IFN α2a and Novel IFN in Various Cell Types Infected with Various Viruses: Antiviral Assay Protocol:
HEp-2 cells / VSV
To determine the appropriate virus titer to be used in the assay, a virus susceptibility assay was first performed. Plate 100 μl cells in duplicate in 96 well microtiter plates to a concentration of 3.0E + 5 HEp-2 cells / ml. Place the microtiter plate at 37 ° C. overnight with 5.0% CO ₂ . On the second day, VSV is titrated on another microtiter plate. 1: 2 serial dilutions are made across the plate to make a total of 24 dilutions. Next, 50 μl of these virus dilutions are added to the cell microtiter plate and placed in the incubator overnight. On the third day, the plates are stained with crystal violet and dried. To obtain the actual dilution of virus to be used in this assay, use the endpoint of the third well to the right of the last well where 100% cell death occurs. In this case, a 1: 150 dilution of VSV from our viral working stock was determined as the best dilution.

To determine the interferon IC50, first dilute the interferon with 90% DMEM and 10% FBS to approximately 8000 U / ml. Next, 10 μl of this solution is added to 190 μl of medium on a 96-well microtiter plate. A two-fold dilution is performed on the entire plate for a total of 12 interferon dilutions. A known concentration of human IFN alpha A experimental standard is also diluted to 1:20 in the first well and serially diluted throughout the plate. HEP-2 cells are counted and diluted to a concentration of 3.0E + 5 cells / ml. Next, 100 μl of these cells are added to each well. Then, get the microtiter plate on the platform rocker and rock for 10 minutes. The plate is then placed in an incubator (37 ° C., 5.0% CO ₂ ) overnight. On day 2, VSV is diluted to a predetermined concentration, here 1: 150, and 50 μl is added to each well. Again, place the plate in the incubator overnight. The plate is then stained with crystal violet and dried. To determine the interferon titer, determine the endpoint at which 50% death occurred. This endpoint is then compared to the endpoint of the human IFN alpha A experimental standard.

H226 cells / VSV
To determine the appropriate virus titer to be used in this assay, a virus susceptibility assay was first performed. Plate 100 μl cells in duplicate in 96 well microtiter plates to a concentration of 2.5E + 5 H226 cells / ml. Place the microtiter plate at 37 ° C. overnight with 5.0% CO ₂ . On the second day, titrate VSV on another microtiter plate. 1: 2 serial dilutions are performed on the entire plate for a total of 24 dilutions. Next, 50 μl of these virus dilutions are added to the cell microtiter plate and placed in the incubator overnight. On day 3, the plate is stained with crystal violet and dried. To obtain the actual dilution of virus to be used in this assay, use the endpoint of the third well to the right of the last well where 100% of the cells are killed. In this case, a 1: 1000 dilution of VSV from our virus working stock was determined as the best dilution.

To determine the interferon IC50, the interferon is first diluted with 90% RPMI and 10% FBS to approximately 8000 U / ml. Next, 10 μl of this solution is added to 190 μl of medium on a 96-well microtiter plate. A two-fold dilution is performed on the entire plate for a total of 12 interferon dilutions. A known concentration of human IFN alpha A experimental standard is also diluted to 1:20 in the first well and serially diluted throughout the plate. H226 cells are counted and diluted to a concentration of 2.5E + 5 cells / ml. Next, 100 μl of these cells are added to each well. Then, get the microtiter plate on the platform rocker and rock for 10 minutes. The plate is then placed in an incubator (37 ° C., 5.0% CO ₂ ) overnight. On day 2, influenza is diluted to a predetermined concentration, here 1: 1000, and 50 μl is added to each well. Again, place the plate in the incubator overnight. The plate is then stained with crystal violet and dried. To determine the interferon titer, determine the endpoint at which 50% death occurred. This endpoint is then compared to the endpoint of the human IFN alpha A experimental standard.

HLF cells / influenza A virus susceptibility assay was first performed to determine the appropriate virus titer to be used in this assay. Plate 100 μl of cells in duplicate in a 96-well microtiter plate to a concentration of 2.0E + 5 HLF cells / ml. Place the microtiter plate at 37 ° C. overnight with 5.0% CO ₂ . On the second day, influenza is titrated on another microtiter plate. 1: 2 serial dilutions are performed on the entire plate for a total of 24 dilutions. Next, 50 μl of these virus dilutions are added to the cell microtiter plate and placed in the incubator overnight. On day 3, the plate is stained with crystal violet and dried. To obtain the actual dilution of virus to be used in this assay, use the endpoint of the third well to the right of the last well where 100% of the cells are killed. In this case, a 1:20 dilution of influenza from our viral working stock was determined as the best dilution.

To determine the interferon IC50, first dilute the interferon with 90% DMEM and 10% FBS to approximately 8000 U / ml. Next, 10 μl of this solution is added to 190 μl of medium on a 96-well microtiter plate. A two-fold dilution is performed on the entire plate for a total of 12 interferon dilutions. A known concentration of human IFN alpha A experimental standard is also diluted to 1:20 in the first well and serially diluted throughout the plate. HLF cells are counted and diluted to a concentration of 2.0E + 5 cells / ml. Next, 100 μl of these cells are added to each well. Then, get the microtiter plate on the platform rocker and rock for 10 minutes. The plate is then placed in an incubator (37 ° C., 5.0% CO ₂ ) overnight. On day 2, influenza is diluted to a predetermined concentration, here 1:20, and 50 μl is added to each well. Again, place the plate in the incubator overnight. The plate is then stained with crystal violet and dried. To determine the interferon titer, determine the endpoint at which 50% death occurred. This endpoint is then compared to the endpoint of the human IFN alpha A experimental standard.

L cell / EMCV
To determine the appropriate virus titer to be used in this assay, a virus susceptibility assay was first performed. Plate 100 μl of cells in duplicate in a 96 well microtiter plate to a concentration of 3.0E + 5 L cells / ml. Place the microtiter plate at 37 ° C. overnight with 5.0% CO ₂ . On the second day, EMCV is titrated on another microtiter plate. 1: 2 serial dilutions are performed on the entire plate for a total of 24 dilutions. Next, 50 μl of these virus dilutions are added to the cell microtiter plate and placed in the incubator overnight. On day 3, the plate is stained with crystal violet and dried. To obtain the actual dilution of virus to be used in this assay, use the endpoint of the third well to the right of the last well where 100% of the cells are killed. In this case, a 1: 2000 dilution of EMCV from our viral working stock was determined as the best dilution.

To determine the interferon IC50, first dilute the interferon with 90% MEM, 10% FBS to approximately 8000 U / ml. Next, 10 μl of this solution is added to 190 μl of medium on a 96-well microtiter plate. A two-fold dilution is performed on the entire plate for a total of 12 interferon dilutions. A known concentration of human IFN alpha A experimental standard is also diluted to 1:20 in the first well and serially diluted throughout the plate. HEP-2 cells are counted and diluted to a concentration of 3.0E + 5 cells / ml. Next, 100 μl of these cells are added to each well. Then, get the microtiter plate on the platform rocker and rock for 10 minutes. The plate is then placed in an incubator (37 ° C., 5.0% CO ₂ ) overnight. On day 2, EMCV is diluted to a predetermined concentration, here 1: 2000, and 50 μl is added to each well. Again, place the plate in the incubator overnight. The plate is then stained with crystal violet and dried. To determine the interferon titer, determine the endpoint at which 50% death occurred. This endpoint is then compared to the endpoint of the human IFN alpha A experimental standard.

Results: See FIG. The effectiveness of the novel interferon in protecting numerous cell lines from viral infection was determined. The cell lines were bovine strain MDBK, mouse strain L cells, and three human strains HEP-2, H226 and HLF. IFN α12 and α023 were better in all human strains tested than IFN α2a (IFN α023 was not tested in the HLF / Flu test). In addition, IFN α012 showed significant activity (˜70 times more potent than IFN α2a) in mouse cells where IFN α2a had little effect. This makes it possible to use a mouse model of infection more easily with this IFN than with others.

  Differences in the relative efficacy of these IFNs in various virus / cell pairs suggests that the selected IFN or combination of selected IFNs may be suitable for the treatment of specific viral infections. ing. For example, IFN α003 is significantly more potent than IFN α2a in protecting HEP-2 cells from VSV infection, while IFN α003 has a potency similar to that of IFN α2a in the HLF / Flu virus assay.

Furthermore, although IFN α021 and IFN α018 did not show strong antiviral activity in the assay introduced here, it is clear that other virus / cell combinations would be highly responsive to these IFNs Please note that. Alternatively, these IFNs would be useful as IFN antagonists under certain conditions that ensure modulation of endogenous IFN activity.

FIG. 1 shows the antiviral activity of the feline IFNα species. FIG. 2 shows the antiviral activity of Rhesus monkey IFNα species. FIG. 3 shows the sequence of the PCR primer used to amplify human IFNα species. FIG. 4 shows the primer pairs (detailed in FIG. 3) used to identify each of the human IFNα species. FIG. 5 shows the inhibition of SARS coronavirus cytopathic effect by interferon. Interferon inhibits SARSCoV's cytopathic effect in a dose-dependent manner. This assay was performed by infecting monkey kidney FRhk-4 cells with SARS clinical isolate (GZ50). 3 points indicate complete viral lethality of the cells by the virus (and therefore no protection by interferon) and 0 points indicate no cell death by the virus (complete protection by interferon). See Example 8. FIG. 6 shows inhibition of SARSCoV replication by interferon. Efficacy of IFN α2a and a novel interferon in inhibiting SARS CoV (Urbani) cytopathic effect on African green monkey cells (Vero76). The IC50 for virus inhibition was determined by direct visual assessment of hepatocytes or by neutral red staining of hepatocytes. See Example 9. FIG. 7 shows inhibition of respiratory rash virus replication by interferon. The ability of IFN to inhibit replication of two clinical isolates of group B respiratory rash virus (RSV) and two clinical isolates of group A RSV was tested in vitro. IC50 was determined by serial dilution of IFN, and the concentration at which viral replication was inhibited by 50% was determined by immunoassay for viral proteins. For samples where the bar extends to the top of the graph, the IC50 was calculated to exceed 2500 pg / ml and could not be easily determined by this assay. See Example 10. FIG. 8 shows the effectiveness of interferon in protecting human lung fibroblasts from influenza virus infection. Efficacy of interferon in inhibiting influenza growth in human lung fibroblasts in vitro. The IC50 for each interferon was determined and the IC50 value obtained for IFN α2a was set to 1. The ratio of IC50 novel IFNα / IC50 IFN α2a was determined. In this graph, a higher number indicates that interferon is more potent than IFN α2a. See Example 11. FIG. 9 shows the protection of hepatocytes from yellow fever virus infection: The effectiveness of interferon in protecting hepatocytes from YFV infection as a model for hepatitis C treatment was determined using HepG2 hepatocytes . IC50 was determined by a colorimetric method. Two separate decisions were made. See Example 12. FIG. 10 shows a comparison of IFN α2a and novel IFN in various cell types infected with various viruses. The efficacy of IFN α2a and the novel interferon was tested with 5 different cell types and 3 different viruses. In either case, if the activity of IFN α2 is set to 1 and IFN is more potent, this number is greater than 1 and less than 1 if less potent. See Examples 7 and 13.

[Sequence Listing]

Claims (12)

A method of treating a subject infected with a virus or reducing the risk of viral infection in a subject comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 , 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74 , 76, 78, 80, 82, 84 or 86, comprising administering to the subject an interferon polypeptide comprising an amino acid sequence that is at least 95% identical to one of said, and is not a naturally occurring interferon allele The virus is coronavirus, influenza, coronavirus, smallpox virus, cowpox virus, west nile virus, vaccinia virus, respiratory rash virus, rhinovirus, arterivirus, filovirus associated with severe acute respiratory syndrome. , Picornavirus, les Virus, retrovirus, papova virus, herpes virus, pox virus, hepadna virus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echo virus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunya Selected from the group consisting of viruses, arenaviruses, bornaviruses, adenoviruses, parvoviruses, and flaviviruses, thereby treating a subject infected with the virus or reducing the risk of viral infection in the subject, Method. The interferon polypeptide is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42. 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 86 The method of claim 1 comprising an amino acid sequence. The interferon polypeptide is encoded by a nucleic acid, and the nucleic acid is SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31. 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 The method of claim 1, comprising a nucleotide sequence selected from the group consisting of: The interferon polypeptide is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41. , 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85 The method of claim 1, wherein the method is encoded by a nucleic acid that hybridizes under high stringency to a nucleic acid comprising a sequence comprising: The interferon polypeptide is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41. , 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85 2. The method of claim 1, wherein the method is encoded by a nucleic acid that hybridizes under high stringency to a nucleic acid complementary to the sequence. The method according to claim 1 or 2, wherein the subject is a human, a non-human primate, a cat, a dog, or a domestic animal. 3. The method of claim 1 or 2, wherein the interferon is administered through the nostril, buccal, parenteral, topical, rectal by injection, inhalation, eye drops, ointment, suppository, controlled release patch, infusion or inhalation. 3. The method of claim 1 or 2, wherein the interferon is administered to the subject's nasopharyngeal mucosa or lung epithelium. 3. The method of claim 1 or 2, wherein the amount of interferon polypeptide administered is an amount effective to reduce the concentration of viral particles in the subject and treat the subject. 3. The method of claim 1 or 2, wherein the amount of interferon polypeptide administered is an amount effective to prevent or reduce the increase in virus particle concentration in the subject and to treat the subject prophylactically. A pharmaceutical package comprising an interferon composition together with instructions for administering said composition to a subject, wherein (i) said instructions treat a subject infected with a virus or of a subject's viral infection To reduce risk, said virus is coronavirus associated with severe acute respiratory syndrome; influenza, coronavirus, smallpox virus, cowpox virus, west nile virus, vaccinia virus, respiratory rash virus, rhinovirus, Arteri virus, filovirus, picornavirus, reovirus, retrovirus, papova virus, herpes virus, pox virus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus, enterovirus , Cal Selected from the group consisting of oviruses, togaviruses, rhabdoviruses, bunyaviruses, arenaviruses, bornaviruses, adenoviruses, parvoviruses, and flaviviruses, and (ii) the interferon polypeptide is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68. A pharmaceutical package comprising an amino acid sequence that is at least 95% identical to one of 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86. The interferon composition is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86 The pharmaceutical package according to claim 11, comprising interferon.

Images