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EP-4041881-B1 - ENHANCEMENT AND STABILISATION OF PROTEOLYTIC ACTIVITY OF PROTEASES

EP4041881B1EP 4041881 B1EP4041881 B1EP 4041881B1EP-4041881-B1

Inventors

  • TRIGIANTE, GIUSEPPE

Dates

Publication Date
20260506
Application Date
20201008

Claims (8)

  1. A method for producing a composition comprising one or more cysteine proteases with proteolytic activity that has been enhanced and/or stabilised, wherein the method comprises (i) contacting the one or more cysteine proteases with cysteine, wherein an active site cysteine residue of the one or more cysteine proteases is maintained in a reduced state, (ii) removing substantially all oxygen gas from the area surrounding the one or more cysteine proteases and (iii) combining the one or more cysteine proteases and a carbomer, such that the one or more cysteine proteases are non-covalently bound to the carbomer, thereby producing a composition comprising one or more cysteine proteases with proteolytic activity that has been enhanced and/or stabilised.
  2. The method of claim 1, wherein oxygen gas is removed by degassing the preparation.
  3. A composition comprising one or more cysteine proteases, cysteine and a carbomer, wherein (i) an active site cysteine residue of the one or more cysteine proteases is maintained in a reduced state, (ii) the composition is substantially oxygen free and (iii) the one or more cysteine proteases are non-covalently bound to the carbomer such that the proteolytic activity of the one or more cysteine proteases has been enhanced and/or stabilised.
  4. The composition of claim 3, wherein oxygen gas is removed by degassing the composition.
  5. A composition for use as a medicament comprising the composition of either one of claims 3 or 4 optionally together with a pharmaceutically acceptable carrier, diluent, excipient, surfactant and/or adjuvant.
  6. Use of a composition of either one of claims 3 or 4 together with a cosmetically acceptable carrier, diluent, excipient, surfactant and/or adjuvant in cosmetic applications.
  7. A composition according to any one of claims 3 to 5, for use in the treatment of wounds, for use in debridement, or for use in treating burns, ulcers or gangrene.
  8. The composition for use of claim 7, wherein the composition is applied topically.

Description

Field of the Invention The present invention relates to methods for producing compositions comprising proteases with proteolytic activity that has been enhanced and stabilised, compositions comprising proteases with proteolytic activity that has been enhanced and stabilised obtained or obtainable by the aforementioned methods, use of such compositions in the manufacture of medicaments and cosmetics, use of such compositions in the treatment of diseases and disorders including wounds, and in cosmetic applications, and associated kits. Background to the Invention Papain and bromelain are proteases used in pharmaceutical products for debridement and in cosmetic/cosmeceutical products for exfoliation and skin lightening. It is the proteolytic activity of papain and bromelain that brings about such debridement, exfoliation and skin lightening. However, neither papain nor bromelain preparations have fulfilled their potential due to the instability of their proteolytic activity. Considerable effort has previously been invested in developing stable compositions that promote wound healing through the removal of dead and damaged tissue such as that found in wounds, such as burns and chronic ulcers. Efficient debridement is essential since dead and dying tissue is an excellent culture medium for opportunistic infections. Septicemia resulting from infections is the major cause of death in severely burned patients. One previous approach has been to use proteolytic enzymes such as papain, trypsin and bromelain. In particular, NexoBrid™, which is a concentrate of proteolytic enzymes enriched in bromelain, was approved in Europe in 2012 for the removal of eschar (i.e. debridement) in adults with deep partial- and full-thickness thermal burns. However, the European Medicines Agency confirmed in its "Assessment report - Nexobrid - Concentrate of proteolytic enzymes enriched in bromelain" 20 September 2012 under section 2.2.3, page 14, that a common problem with compositions of proteolytic enzymes is the poor stability of proteolytic activity, stating that: "New compatibility (in-use stability) studies at 25°C and 37°C have been performed demonstrating that NexoBrid degrades within hours after mixing. The applicant's conclusion that the product should be used immediately after mixing is therefore supported." Accordingly, NexoBrid™ is typically supplied as a lyophilised powder which is reconstituted prior to use with a gel vehicle and must be used within 15 minutes of formulation. It would therefore be advantageous to provide compositions such as NexoBrid™ in a ready-for-use form and without the need for reconstitution. However, to do so would require a significant enhancement of the stability of the debriding activity in order to achieve an acceptable shelf life. Similarly, in cosmetic products containing papain and/or bromelain that are marketed as having exfoliating and/or skin lightening properties, the exfoliating activity is often severely reduced in a short period of time due to the loss of activity of the papain and/or bromelain in the cosmetic products. Methods have previously been devised to address the problem of loss of proteolytic activity of proteases in pharmaceutical products, including storing the enzymes at a pH where the loss of activity is low or absent (in the case of papain, an acidic pH has been used) or in a solid form. These treatments, however, require expert end-user processing to obtain a viable product. This limits the usefulness of such methods and compositions. Another avenue previously attempted is the immobilisation of an enzyme on polymeric substrates to prevent mobility and self-reactivity. Examples of immobilised enzyme are PEG-papain and chitosan-papain. Whereas the stability of proteolytic activity may be improved by such methods, the enzyme may be chemically altered in an irreversible way and accordingly may not exhibit sufficient activity on specific substrates, especially when applied to complex substrates such as skin and cellular proteins. Moreover, the altered chemical structure of such enzymes could result in deleterious reactions for the end user such as allergies and intolerances. In order to address these problems, studies underpinning the present invention investigated the loss of proteolytic activity of proteases extracted from various sources, such as the Carica papaya (papaya) plant (including papain), and the Ananas comosus (pineapple) plant (including bromelain). Based on these studies, methods were developed for the enhancement and stabilisation of the proteolytic activity of proteases, particularly in pharmaceutical and cosmetic/cosmeceutical compositions containing proteases derived from the papaya and pineapple plants. Surprisingly and unexpectedly, it was found that several of the methods that enhanced the stability of proteolytic activity also significantly enhanced proteolytic activity itself. Summary of the Invention The present inventions is defined in accordance w