Pharmaceutical compositions comprising collagenase I and collagenase II
The invention relates to recombinant nucleic acid and polypeptides encoding collagenase I and collagenase II, methods for the preparation thereof and methods for the use thereof. The invention also encompasses methods related to releasing a composition comprising collagenase prior to therapeutic administration.
1. A pharmaceutical composition comprising,
collagenase I purified from a host cell comprising a vector, the vector comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and a heterologous regulatory sequence operably linked to the polynucleotide; and
collagenase II purified from a host cell comprising a vector, the vector comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 and a heterologous regulatory sequence operably linked to the polynucleotide;
wherein the collagenase I and collagenase II are present in a ratio of about 1 to 1 and the composition is free of hemolytic toxins as measured by a hemolysis assay.
2. The pharmaceutical composition of claim 1 , wherein the heterologous regulatory sequence that is operably linked to the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 is a promoter.
3. The pharmaceutical composition of claim 1 , wherein the recombinant nucleic acid molecule comprising the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and the heterologous regulatory sequence is fused to a marker sequence.
4. The pharmaceutical composition of claim 3 , wherein the marker sequence encodes a polypeptide selected from a glutathione-S-transferase (GST) fusion protein, a hemagglutinin A (HA) polypeptide from influenza, and a hexa-histidine peptide.
5. The pharmaceutical composition of claim 1 , wherein the heterologous regulatory sequence that is operably linked to the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 is a promoter.
6. The pharmaceutical composition of claim 1 , wherein the recombinant nucleic acid molecule comprising the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 and the heterologous regulatory sequence is fused to a marker sequence.
7. The pharmaceutical composition of claim 6 , wherein the marker sequence encodes a polypeptide selected from a glutathione-S-transferase (GST) fusion protein, a hemagglutinin A (HA) polypeptide from influenza, and a hexa-histidine peptide.
8. The pharmaceutical composition of claim 1 , wherein the vector comprising the recombinant nucleic acid molecule that comprises the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and the heterologous regulatory sequence is a plasmid and/or the vector comprising the recombinant nucleic acid molecule that comprises the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 and the heterologous regulatory sequence is a plasmid.
9. The pharmaceutical composition of claim 1 , wherein the host cell from which the collagenase I is purified is selected from a bacterial cell, a fungal cell, an insect cell, a plant cell, and a mammalian cell and/or the host cell from which the collagenase II is purified is selected from a bacterial cell, a fungal cell, an insect cell, a plant cell, and a mammalian cell.
10. The pharmaceutical composition of claim 1 , wherein the host cell from which the collagenase I is purified is E. coli , a Streptomyces species, a Pseudomonas species, Serratia marcescens, Salmonella typhimurium , a yeast cell, a plant cell, a thymocyte, a Chinese hamster ovary cell (CHO), a COS cell, or Lactococcus lactis and/or the host cell from which the collagenase II is purified is E. coli , a Streptomyces species, a Pseudomonas species, Serratia marcescens, Salmonella typhimurium , a yeast cell, a plant cell, a thymocyte, a Chinese hamster ovary cell (CHO), a COS cell, or Lactococcus lactis.
11. A pharmaceutical composition comprising,
collagenase I purified from a host cell comprising a vector, the vector comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and a heterologous regulatory sequence operably linked to the polynucleotide; and
collagenase II purified from a host cell comprising a vector, the vector comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 and a heterologous regulatory sequence operably linked to the polynucleotide;
wherein the collagenase I and collagenase II are present in a ratio of about 1 to 1 and the composition is free of a toxin having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or a combination thereof.
12. The pharmaceutical composition of claim 11 , wherein the heterologous regulatory sequence that is operably linked to the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 is a promoter.
13. The pharmaceutical composition of claim 11 , wherein the recombinant nucleic acid molecule comprising the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and the heterologous regulatory sequence is fused to a marker sequence.
14. The pharmaceutical composition of claim 13 , wherein the marker sequence encodes a polypeptide selected from a glutathione-S-transferase (GST) fusion protein, a hemagglutinin A (HA) polypeptide from influenza, and a hexa-histidine peptide.
15. The pharmaceutical composition of claim 11 , wherein the heterologous regulatory sequence that is operably linked to the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 is a promoter.
16. The pharmaceutical composition of claim 11 , wherein the recombinant nucleic acid molecule comprising the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 and the heterologous regulatory sequence is fused to a marker sequence.
17. The pharmaceutical composition of claim 16 , wherein the marker sequence encodes a polypeptide selected from a glutathione-S-transferase (GST) fusion protein, a hemagglutinin A (HA) polypeptide from influenza, and a hexa-histidine peptide.
18. The pharmaceutical composition of claim 11 , wherein the vector comprising the recombinant nucleic acid molecule that comprises the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and the heterologous regulatory sequence is a plasmid and/or the vector comprising the recombinant nucleic acid molecule that comprises the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 and the heterologous regulatory sequence is a plasmid.
19. The pharmaceutical composition of claim 11 , wherein the host cell from which the collagenase I is purified is selected from a bacterial cell, a fungal cell, an insect cell, a plant cell, and a mammalian cell and/or the host cell from which the collagenase II is purified is selected from a bacterial cell, a fungal cell, an insect cell, a plant cell, and a mammalian cell.
20. The pharmaceutical composition of claim 11 , wherein the host cell from which the collagenase I is purified is E. coli , a Streptomyces species, a Pseudomonas species, Serratia marcescens, Salmonella typhimurium , a yeast cell, a plant cell, a thymocyte, a Chinese hamster ovary cell (CHO), a COS cell, or Lactococcus lactis and/or the host cell from which the collagenase II is purified is E. coli , a Streptomyces species, a Pseudomonas species, Serratia marcescens, Salmonella typhimurium , a yeast cell, a plant cell, a thymocyte, a Chinese hamster ovary cell (CHO), a COS cell, or Lactococcus lactis.