WO1988004666A2

WO1988004666A2 - Lung cytotoxic peptides

Info

Publication number: WO1988004666A2
Application number: PCT/US1987/003399
Authority: WO
Inventors: Samuel Solomon
Original assignee: Ivax Laboratories, Inc.
Priority date: 1986-12-23
Filing date: 1987-12-23
Publication date: 1988-06-30
Also published as: WO1988004666A3

Abstract

A method of reducing lung tumor population in a cancer inflicted patient which comprises contacting lung carcinoma cells in said patient with a peptide selected from the group consisting of Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys or Ala-Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys. Anticarcinoma compositions are disclosed including an isotonic solution and an aerosol spray, each containing an effective amount of said peptide. Also disclosed is method of producing substantially pure peptide mixture of each of said peptides. Compositions including the compound Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys are provided.

Description


  
 



   LUNG CYTOTOXIC PEPTIDES
 DETAILED DESCRIPTION OF THE INVENTION
 Various macromolecular peptides have been proposed as antitumor agents such as the interleukins. It has been discovered that an effective anticarcinoma agent is
Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg
Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys or Ala-Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg
Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe
Cys-Cys or a mixture of the two compounds. Hereinafter, the term Lung Cytotoxic Peptide (I) will sometimes hereinafter be used to collectively refer to either or both of these two compounds, which differ in the "1" position
Alanyl group of the latter compound being absent in the first compound.



   In a first aspect of the invention there is provided a method of reducing lung tumor population in a cancer inflicted patient which comprises contacting lung carcinoma cells in said patient with Cys-Try-Cys-Arg-Ile-Pro-Ala
Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln
Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys. In this first aspect, the existing lung carcinoma cells in said patient are killed by contact with said peptide. Studies indicate that this is one mechanism whereby the tumor is treated.



   Also in this first aspect, the DNA synthesis in lung carcinoma cells is inhibited by contact with said peptide.



  This also contributes to successful treatment of the cancer and to reduction of the tumor population.



   In one preferred embodiment, said peptide is administered via lavage. In a second embodiment, said peptide is administered via a tracheal spray.



   In accordance with a second aspect of the present invention, there is provided an anticarcinoma composition suitable for application to lung cells which comprises an  isotonic solution containing an effective amount of a peptide capable of reducing the population of lung carcinoma cells upon contact with said peptide; and pharmaceutically inert ingredients, said peptide being a member selected from the group consisting of Cys-Try-Cys
Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr
Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys, Ala-Cys   
Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-      Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys    and mixtures thereof. An isotonic solution of the invention may contain in addition to said peptide, water and salt, also conventional ingredients such as glucose.



   In accordance with a third aspect of the present invention, there is provided an anticarcinoma composition suitable for application to lung cells which comprises an aerosol spray containing an effective amount of a peptide capable of reducing the population of lung carcinoma cells upon contact with said peptide; and pharmaceutically inert ingredients, said peptide being a member selected from the group consisting of Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile   
Ala-Gly-Gl u-Arg-Arg-Try-Gly-Thr- Cys -Ile-Try-Gln-Gly-Arg-   
Leu-Trp-Ala-Phe-Cys-Cys, Ala-Cys-Try-Cys-Arg-Ile-Pro-Ala   
Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-   
Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and mixtures thereof.



  Typical aerosol spray excipients and propellant agents are contemplated insofar as they do not interfere with the desired biological activity.



   In a further aspect of the invention there is provided a method of producing substantially pure peptides Cys-Try   
Cys-Arg-Ile-Pro-Ala-Cys-Ile-Al a-Gly-Glu-Arg-Arg-Try-Gly-      Thr-Cys-Il e-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys    and   
Ala- Cys -Try-Cys -Arg-Il e-Pro-Ala-Cys -Ile-Ala-Gly-Glu-Arg-
Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-   
Cys-Cys which comprises  
 (a) extracting lung carcinoma tissue in an acid
 medium;
 (b) separating protein from said peptides via
 chromatography;
 (c) sequentially submitting the extract to a series
 of column extractions which contain respectively
 (i) trifluoroacetic acid;
 (ii) heptafluorobenzoic acid; and
 (iii) trifluoroacetic acid;

   whereby a substantially pure mixture of Cys-Try-Cys-Arg   
Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-   
Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and Ala-Cys   
Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-   
Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys is obtained.

  In one subcombination of this aspect of the invention, there is provided a method of producing substantially pure peptides Cys-Try-Cys-Arg-Ile-Pro-Ala   
Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-   
Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and Ala-Cys-Try-Cys-Arg   
Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-   
Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys which comprises sequentially submitting a proteinaecious mixture to a series of column extractions which contain respectively
 (i) trifluoroacetic acid;
 (ii) heptafluorobenzoic acid; and
 (iii) trifluoroacetic acid;

   whereby a substantially pure mixture of Cys-Try-Cys-Arg   
Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-   
Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and Ala-Cys   
Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-      Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys    is obtained.



   In a further aspect of the invention there is provided a composition comprising the peptide Cys-Try-Cys-Arg-Ile-Pro  
Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try
Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys in a form substantially free from ingredients which would interfere with the anticarcinoma properties of said peptide. It is contemplated that in one preferred embodiment of the invention, the compound Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys   
Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-   
Arg-Leu-Trp-Ala-Phe-Cys-Cys will be in admixture with Ala   
Cys-Try- Cys-Arg- Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-
Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-   
Cys.

  The terminology "a form substantially free from ingredients which would interfere with the anticarcinoma properties of said peptide" refers to the exclusion from the scope of the present invention of the tumor mass from which the peptide is obtained, which would be unsuitable for treatment of carcinomas.

 

   The method of application to the lung carcinoma is not of primary importance, it being understood that any method that will provide the Lung Cytotoxic Peptide (I) in a sufficiently large concentration to the carcinoma cells will be satisfactory. Intravenous delivery of peptides is well known as a method of introducing peptides into the bloodstream without destruction in either the gastrointestinal tract or the liver. As practical matter, there are well recognized problems with the oral delivery of peptides, although an oral form that would be capable of delivery of the Lung Cytotoxic Peptide (I) to the bloodstream in a sufficient quantity would be contemplated as an aspect of the invention.



   An effective amount of Lung Cytotoxic Peptide (I) in an isotonic solution to be introduced by lavage is from about 0.5 to about 100 mmolar, and preferably about 2 to about 5 mmolar.  



   EXAMPLE I
 The Lung Cytotoxic Peptide (I) is produced by extraction procedures designed to optimize recovery of peptides rather than proteins. 1.35 g of surgically removed human lung tumor and undiseased lung tissue is stored at   -40"C    for 24 hours, and homogenized in 20 ml cold acetone. The resultant mixture as then centrifuged at 200 g for 15 minutes; the supernatant is discarded.



   The resultant dried pellet from the centrifugation process is homogenized in 25 ml of an extraction medium which comprises
 1 M HCl
 5% formic acid
 1% NaCl
 1% trifluoroacetic acid and centrifuged at 2000 g for 15 minutes. The pellet is reextracted and the pooled supernatant passed through 3 ODS cartridges (Sep-Pak C18 cartridges, Waters Associates,
Massachusetts), and the peptides eluted from each cartridge with 4 ml of 80% acetonitrile containing 0.1% trifluoroacetic acid.



   Reverse phase HPLC purification of this extract is accomplished with a C-18   UBondapak    column using a three hour gradient form 0% to 80% acetonitrile in   0.1%    trifluoroacetic acid at 1.5 ml/min. The peak fractions eluting at 68 min are further purified using a second 60 min linear gradient of   0%    to 65% acetonitrile in 0.13% heptafluorobenzoic acid at 1.5 ml/min. The purified peak material is then rechromatographed using a solution of
 1 M HCl
 5% formic acid
 1% NaCl
 1% trifluoroacetic acid to yield a homogenous peak.

  The Lung Cytotoxic Peptide (I)  produced in this example is a mixture of the two compounds, the first peptide Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala
Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu
Trp-Ala-Phe-Cys-Cys being present in an amount of approximately one part per three parts of the c ompound Ala-Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly   
Glu-Arg-Arg-Try-Gly-Thr- Cys- Il e-Try-Gln-Gly-Arg-teu-Trp-   
Ala-Phe-Cys-Cys.



   EXAMPLE II
 Using the Lung Cytotoxic Peptide (I) produced in Example
I, a solution is prepared for use in a lavage for lung administration. Sufficient amount of (I) is introduced into an isotonic solution to create a 3 mmolar (I). For each lavage dosage unit, 15 ml. of the resultant solution is provided.



   EXAMPLE III
 This example demonstrates one mode of administration of the invention by lavage. Administration is effected upon rising and retiring each day. The administration comprises utilization of a lavage, in which a tube having a diameter of about 0.5 mm is introduced into the lung. The solution of Example II is placed into the lavage, which typically has a long plunger. The plunger first pushes out the drugladen solution into the lungs, and after permitting the solution to remain in the lungs for about two minutes, the drug is dissolved into the lung cells. Thereafter, the plunger is withdrawn, thereby taking up the excess fluid from the lung cavity.  



   EXAMPLE   IV   
 A tracheal spray composition is provided using the Lung
Cytotoxic Peptide (I) prepared in accordance with Example
I. A typical aerosol composition substitutes this peptide for the medicinally active ingredient of known aerosol sprays for tracheal administration, to provide a four times per day dosage form that can be easily applied by the patient. Unlike the less convenient lavage form of administration, a patient can more readily administer the drug to himself through use of an aerosol spray.

 

   EXAMPLE V
 The tracheal spray composition of Example IV is administered once every six hours to provide a dosage four times per day. Each dosage unit has about 3 mm Lung
Cytotoxic Peptide (I).



   In addition to the dosage forms for direct injection to the lung cavity via lavage and through a tracheal spray, any other forms which would provide the Lung Cytotoxic
Peptide (I) to the bloodstream of a patient are contemplated. For example, an oral dosage form which would permit the Lung Cytotoxic Peptide (I) to enter the bloodstream without substantial destruction in the gastrointestinal tract or the liver would also be contemplated as an embodiment of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of reducing lung tumor population in a cancer inflicted patient which comprises contacting lung carcinoma cells in said patient with a peptide selected from the group consisting of Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg- Leu-Trp-Ala-Phe-Cys-Cys or Ala-Cys-Try-Cys -Arg-Ile-Pro-Ala Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys.

2. A method of claim 1 wherein existing lung carcinoma cells in said patient are killed by contact with said peptide.

3. A method of claim 1 wherein the DNA synthesis in lung carcinoma cells is inhibited by contact with said peptide.

4. A method of claim 1 wherein said peptide is administered via lavage.

5. A method of claim 1 wherein said peptide is administered via a tracheal spray.

6. An anticarcinoma composition suitable for application to lung cells which comprises an isotonic solution containing an effective amount of a peptide capable of reducing the population of lung carcinoma cells upon contact with said peptide; and pharmaceutically inert ingredients, said peptide being a member selected from the group consisting of Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg- Leu-Trp-Ala-Phe-Cys-Cys, Ala-Cys-Try-Cys-Arg-Ile-Pro-Ala Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys-Ile-Try-Gln Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and mixtures thereof.

7. An anticarcinoma composition suitable for application to lung cells which comprises an aerosol spray containing an effective amount of a peptide capable of reducing the population of lung carcinoma cells upon contact with said peptide; and pharmaceutically inert ingredients, said peptide being a member selected from the group consisting of Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg- Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys- Cys, Ala-Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-G1y-Glu- Arg-Arg-Try-Gly-Thr- Cys- Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala- Phe-Cys-Cys and mixtures thereof.

8. A method of producing substantially pure peptides Cys Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and Ala-Cys-Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg- Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe- Cys-Cys which comprises (a) extracting lung carcinoma tissue in an acid medium; (b) separating protein from said peptides via chromatography; (c) sequentially submitting the extract to a series of column extractions which contain respectively (i) trifluoroacetic acid; (ii) heptafluorobenzoic acid; and (iii) trifluoroacetic acid;

whereby a substantially pure mixture of Cys-Try-Cys-Arg Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and Ala-Cys Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try- Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys is obtained.

9. A method of producing substantially pure peptides Cys Try-Cys-Arg-Il e-Pro-Al a-Cys-Ile-Al a-G1y-Glu-Arg-Arg-Try- Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and Ala-Cys-Try-Cys-Arg-I le-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg- Arg-Try-Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe- Cys-Cys which comprises sequentially submitting a proteinacious mixture to a series of column extractions which contain respectively (i) trifluoroacetic acid; (ii) heptafluorobenzoic acid; and (iii) trifluoroacetic acid;

whereby a substantially pure mixture of Cys-Try-Cys-Arg Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-Gly-Thr-Cys- Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys and Ala-Cys Try-Cys-Arg-Ile-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try Gly-Thr-Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys is obtained.

10. A composition comprising the peptide Cys-Try-Cys Arg-IIe-Pro-Ala-Cys-Ile-Ala-Gly-Glu-Arg-Arg-Try-ly-Thr- Cys-Ile-Try-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys-Cys in a form substantially free from ingredients which would interfere with the anticarcinoma properties of said peptide.