HK1164701B - Hylauronic acid containing compositions for treatment of wounds, scars, post-surgical adhesion formation - Google Patents

Description

Composition containing hyaluronic acid for treating wounds, scars, post-surgical adhesion formation

Technical Field

The present invention relates to pharmaceutical compositions for enhancing the therapeutic effect of biologically active peptides, in particular peptides derived from human lactoferrin. The compositions are effective for treating and/or preventing wounds, scars, and post surgical adhesions.

Background

Peritoneal adhesions are fibrous tissue connections between abdominal structures following surgical trauma or other types of injury. General abdominal surgery, vascular surgery, gynecological surgery, urinary surgery and orthopedic surgery can lead to Adhesion formation in up to 95% of patients (Ellis et al, 1999. advanced-relative adhesions after surgery and physiological surgery: ecological surgery (re-hospitalization for adhesions after abdominal and pelvic surgery: retrospective cohort study).Lancet (Lancet)353, 1476-1480). Postoperative adhesions are believed to be the leading cause of Small bowel obstruction (Menzies et al, 2001.Small bowel organization due to systemic administration: treatment patterns and associated costs for 110hospital admissions).Ann R Coll Surg Engl (annual report of Imperial surgeons college England)83, 40-46.), small bowel obstruction is a well-known cause of secondary infertility in women (Marana et al, 1995. correction between the American Society classification of epithelial and systemic classification, salpinosclerology, and productive outer tubal supply surgery (Correlation between reproductive outcomes in surgical procedures on accessory adhesions and distal tubal occlusion, salpingoscopy, and fallopian tube surgery).Fertil Steril (fertility) He sterile)64, 924-: a proactive nonrandomized long term follow-up study (laparoscopy of chronic abdominal pain: prospective nonrandomized long term follow-up study).J Clin Gastroenterol (J of clinical gastroenterology)39, 110-114). More than 30% of individuals undergoing Lower abdominal surgery are readmitted for some period of their life due to a condition directly or indirectly associated with adhesion formation (Lower et al, 2000.The impact of adhesives on topic readmission over tenears after 8849 open gynaecologanic properties: an assessment from the Surgical and Clinical Adhesives research study (8849 impact of Adhesions 10 years after open gynecological surgery on hospital readmission: evaluation from Surgical and Clinical Adhesions research studies).Bjog (English obstetrics and gynecology journal)107，855-862.)。

For decades, attempts have been made to reduce post-surgical Adhesions by reducing surgical trauma (avoiding dryness, gentle tissue handling, careful hemostasis) and contamination of the abdominal cavity with foreign bodies (using starch-free gloves, lint-free gauze and absorbable sutures) (Holmdahl et al, 1997: adhesives: pathogenesis and prevention-panel discussion and summary).Eur J Surg supply (European journal of surgery),56-62.). Importantly, laparoscopic techniques are not sufficient to overcome the problem of post-operative adhesion formation (Duron et al, 2000. Presence and surgery of small bowel obstruction after laparoscopic abdominal surgery Prevalence and mechanism: retrospective multicenter research).Arch Surg (surgical archive)135, 208-212). Thus, intraperitoneal adhesions remain a significant clinical problem, and it is now believed that future improvements may only be marginally affected by superior surgical techniques. Instead, the focus is to develop a specific product for preventing adhesion formation, which is administered in combination with surgical intervention.

Most therapeutic strategies tested in the prevention of adhesions are medical device products. Different types of physical barriers have been evaluated, where a biodegradable membrane applied during an interventional procedure is used to separate the injured abdominal surface during the critical phase of peritoneal healing. The two most widely used adhesion-reducing barriers are interced (Johnson)&Johnson Medical Inc. (Johnson Medical Inc.), Arlington (Arlington), TX), and Seprafilm^TM(Genzyme, Cambridge, MA, USA). Prepared from sodium hyaluronate and carboxymethyl cellulose(CMC) structured Seprafilm^TMA sticky gel was formed approximately 24-48h after placement, which was slowly absorbed within 1 week (Diamond, 1996.Reduction of the introduction of the tissue mytomy by Sepharose membrane (HAL-F): a blocked, progressive, randomised, multicenter clinical Study (Reduction of adhesions by Sepharose membrane (HAL-F) after hysteromyomectomy): blinding, prospective, randomized, multicenter clinical Study).Fertil Steril (fertility and sterility)66, 904, 910; beck, 1997, The role of seprafilmbioreabsorbable membrane in adhesion prevention.Eur J Surg supply (European journal of surgery),49-55). Seprafilm has been shown^TMReduction of post-surgical adhesions in clinical manifestations (Vrijland et al, 2002. Power intraepithelial adhesions with use of hyaluronic-carboxymethylated cellulose membrane: a random clinical trial (less intraperitoneal adhesions using hyaluronic acid-carboxymethylcellulose membrane: randomized clinical trial).Ann Surg (annual newspaper of surgery)235, 193-; beck et al, 2003.a proactive, randomised, multicenter, controlled study of the safety of the Seprafilm adhesion barrier in small bowel abdominopelvic surgery, and the barrier of the insertion.Dis Colon Rectum (knot) Intestinal and rectal diseases)46, 1310-1319; tang et al, 2003.BioResorbable addition barriers early closure of the depletion ILEssomy after exposure: aproactive, randomised tertiary (bioresorbable adhesion barrier promoting early closure of a secondary ileostomy after rectomy).Dis Colon Rectum (Colon and rectal disease)46, 1200-1207), however, this device is difficult to apply because it adheres to gloves and organs and is fragile (Decerney and DiZerega, 1997.Clinical protocol of endoscopic surgery introduction and diagnosis of Clinical diagnosisbarriers (clinical problem of adhesion formation and use of adhesion prevention barriers in intra-abdominal surgery after general surgery).Surg Clin North Am (North American surgery clinic)77, 671-688). In addition, Seprafilm^TMIncreases the sequelae associated with anastomotic fistulas and is not suitable for laparoscopic procedures (diZerega et al, 2002. A. random, controlled pilot study of the safety and efficacy of 4% icodextrin dissolution in the reduction of adhesions after laparoscopic gynecological surgery (randomized, controlled pilot study of 4% icodextrin solution in reducing adhesions after laparoscopic gynecological surgery).Hum Reprod (human reproduction)17, 1031-1038). Interced, consisting of oxidized regenerated cellulose, is converted into a gel mass covering the damaged peritoneum and has shown efficacy in preventing adhesions in several clinical studies (Mais et al, 1995.Prevention of Prevention of de-novo administration of infected extracellular muscle tissue: an administered third to an affected the infected of oxidized regenerated cellulose absorbent barrier (randomized trial to prevent the formation of adhesions after laparoscopic hysterectomy: evaluate the effectiveness of the oxidized regenerated cellulose barrier to be absorbed)Hum Reprod (human reproduction)10, 3133-3135; mais et al, 1995 Reduction of additive recovery after enzymology and elastometry surgery: a randomizedtral with an oxidized regenerated cellulose absorbable barrier (reduction of adhesion re-formation after laparoscopic endometriosis surgery: randomization test using an oxidized regenerated cellulose absorbable barrier) Obstet Gynecol (gynaecology) 86, 512-515; wallwoener et al, 1998, Adhesion Format of the partial and visual period: an expansion for the control on the use of autogous and alloplastic barriers? (adhesion formation of parietal and visceral peritoneum: explanation of disputes between autologous and allogenic barriersFertil Steril (fertility and sterility)69, 132-137). However, the use of Interceed requires complete hemostasis, since even small intraperitoneal bleeds eliminate any beneficial effect of this barrier (Decerney and DiZerega)1997, supra). A general limitation of using physical barriers is the site specificity of the product, requiring the surgeon to predict where adhesions will occur and where they will most likely cause clinical problems. As an alternative to barriers, different liquids for intra-abdominal perfusion, such as icodextrin (Adept, Baxter Healthcare Corporation, IL, usa) or lactated Ringer's solution, have been administered after surgery in an amount sufficient to allow the abdominal structures to float, thereby preventing impaired surface access to each other (Yaacobi et al, 1991.Effect of Ringer's lactate interference on the formation of a stored adhesive attachment).J Invest Surg (J. Sci. research)4, 31-36; cavallari et al, 2000. opacity of University of Wisconsin solution to reduce pulmonary peritoneal adhesions in rats.Eur J Surg (European journal of surgery)166, 650-; diZerega et al, supra). However, gravity causes problems by preventing even distribution of the liquid in the abdomen. In addition, the solution is absorbed from the peritoneal cavity at a faster rate than the time required for the peritoneal membrane to heal.

A limited number of pharmacologically active compounds have been tested to prevent post-surgical adhesions. As some examples, the inflammatory component of the wound healing cascade and fibroblast proliferation have been targets for drug therapy using steroid and cytotoxic drugs, respectively. However, these agents have shown uncertain efficacy and potentially serious side effects (LeGrand et al, 1995. synthetic efficacy of non-steroidal anti-inflammatory drugs and anti-thrombotic agents in the arabibition adherence-prevention model (comparison of efficacy of non-steroidal anti-inflammatory and anti-thromboxane agents in rabbit adhesion prevention models).J Invest Surg (J. Sci. research)8,187-; li et al, 2004.Synthesis and biological evaluation of a cross-linked hyaluronan-mitomycin C hydrogel (synthetic and biological evaluation of cross-linked hyaluronan-mitomycin C hydrogels).Biomacromolecules (biomacromolecule)5，895-902)。

Due to the limited efficacy and difficulty of the tested therapies, most surgical interventions performed in the Abdominal cavity today do not apply any product to prevent adhesion formation, and post-operative adhesions continue to cause pain to the patient and bring significant costs to society (Ray et al, 1998.Abdominal adhesives: academic Care and adjuvant in the United States of medicine 1994 (Abdominal adhesions lysis: Admission Care and costs of 1994).J Am Coll Surg (journal of American society of surgeons)186，1-9.；2005)。

It is an object of the present invention to provide a means which has the ability to prevent post-operative adhesion formation without the undesirable side effects of currently available pharmaceutical compositions, devices and procedures.

Description of the invention

The present inventors describe a novel method for preventing intra-abdominal adhesion formation using biologically active peptides derived from human lactoferrin formulated in a pharmaceutical composition that enhances the therapeutic efficacy of the peptides. The most important hallmarks of the biologically active peptides on scar formation show an inhibitory effect: reducing the risk of infection, inhibiting inflammation and promoting fibrinolysis. The peptides are formulated with the naturally occurring hydrophilic polymer hyaluronic acid, which provides sustained release properties of the drug and promotes the final result through physical barrier effects. Using a lateral wall defect cecal abrasion model (lateral wall defect-cerumabrasion model) in rats, which is generally recognized as a suitable non-clinical predictor of the clinical efficacy of anti-adhesion drugs, it was shown that biologically active peptides derived from human lactoferrin formulated in hyaluronic acid significantly reduced intra-abdominal adhesions after surgery. The improved effect of the peptides when formulated in hyaluronic acid is unexpected and is significantly synergistic compared to the effect of the peptide administered alone and the effect of hyaluronic acid administered alone.

Accordingly, the present invention relates to pharmaceutical compositions for enhancing the therapeutic effect of biologically active peptides, in particular peptides derived from human lactoferrin.

In one aspect, the present invention provides a pharmaceutical composition for the treatment and/or prevention of wounds, scars and post surgical adhesions, comprising i) one or more biologically active peptides derived from human lactoferrin, and ii) high molecular weight hyaluronic acid.

Another aspect of the present invention provides the use of i) one or more biologically active peptides derived from human lactoferrin, and ii) a high molecular weight hyaluronic acid for the preparation of a pharmaceutical composition for the treatment and/or prevention of wounds, scars, and post surgical adhesions.

In a further aspect, the present invention provides a method for the treatment, prevention and/or prophylaxis of wounds, scars and post surgical adhesions, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising i) one or more biologically active peptides derived from human lactoferrin, and ii) high molecular weight hyaluronic acid.

By "biologically active peptide derived from human lactoferrin" is meant that such biologically active peptide comprises at least one sequence motif derived partially or completely from a human lactoferrin sequence, wherein such sequence motif may comprise one or more amino acid substitutions.

By "biologically active" peptide is meant a peptide having one or more activities such as anti-inflammatory activity, immunomodulatory activity, fibrinolytic activity, anti-angiogenic activity and anti-microbial activity such as antibacterial activity, antiviral activity or antifungal activity.

Biologically active peptides suitable for use according to the present invention are described, for example, in PCT/EP2008/064062, PCT/EP2008/065186, WO 00/01730, corresponding EP 1095061 and US 7253143, which are incorporated herein by reference.

The biologically active peptide may be selected from the group consisting of amino acid sequences

Phe-X1-X2-X3-X4-X5-X6-X7-Lys-Val-Arg (SEQ ID NO: 1),

wherein amino acid X1 is Gln or Ala, amino acid X2 is Trp or Leu, amino acid X3 is Gln, Ala, Orn, Nle or Lys, amino acid X4 is Arg, Ala or Lys, amino acid X5 is Asn, Ala, Orn or Nle, amino acid X6 is Met, Ala or Leu, amino acid X7 is Arg, Ala or Lys.

Preferably said biologically active peptide may be selected from the group consisting of a peptide according to formula (I) and a peptide according to formula (II)

R1-Cys-Phe-X1-X2-X3-X4-X5-X6-X7-Lys-Val-Arg-R2 formula (I)

Wherein R1 is an amino acid-free, Lys or a peptide sequence selected from Gly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys. (SEQ ID NO: 2) and N-terminally truncated fragments thereof, including

Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Ser-Gln-Pro-Glu-Ala-Thr-Lys，

Gln-Pro-Glu-Ala-Thr-Lys，

Pro-Glu-Ala-Thr-Lys，

Glu-Ala-Thr-Lys，

Ala-Thr-Lys，

Thr-Lys-；

And wherein R2 is an amino acid-free, Gly or peptide sequence selected from Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO: 3) and C-terminally truncated fragments thereof, including

Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys，

Gly-Pro-Pro-Val-Ser-Cys-Ile，

Gly-Pro-Pro-Val-Ser-Cys，

Gly-Pro-Pro-Val-Ser，

Gly-Pro-Pro-Val，

Gly-Pro-Pro, and

Gly-Pro。

formula (II)

Wherein amino acid X8 is Gly, Lys, Glu or Asp;

when X8 is Gly, then R3 is Ser- (Arg)_n-X9, and bond α is the peptide bond between the carboxyl group of Gly and the amino group of Ser;

when X8 is Lys, then R3 is X9- (Arg)_n-Ser, and bond α is an amide bond between the e-amino group in Lys and the carboxyl group in Ser; and

when X8 is Glu or Asp, then R3 is Ser- (Arg)_n-X9, and bond α is an amide bond between the γ -carboxy group of Glu or the β -carboxy group of Asp and the amino group of Ser;

amino acid X9 is none or Gly;

and n is an integer from 1 to 10, preferably from 2 to 6, preferably from 4 to 6, or even more preferably from 3 to 4;

and wherein R1 is an amino acid-free, Cys or peptide sequence selected from the group consisting of Gly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys (SEQ ID NO: 48) and N-terminally truncated fragments thereof, including

Gly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Gln-Pro-Glu-Ala-Thr-Lys-Cys，

Pro-Glu-Ala-Thr-Lys-Cys，

Glu-Ala-Thr-Lys-Cys，

Ala-Thr-Lys-Cys，

Thr-Lys-Cys，

And

Lys-Cys，

and wherein R2 is an amino acid-free, Pro or peptide sequence selected from the group consisting of Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO: 49) and C-terminally truncated fragments thereof, including

Pro-Pro-Val-Ser-Cys-Ile-Lys，

Pro-Pro-Val-Ser-Cys-Ile，

Pro-Pro-Val-Ser-Cys，

Pro-Pro-Val-Ser，

Pro-Pro-Val, and

Pro-Pro；

even more preferably the biologically active peptide may be selected from the following peptides:

Ac-Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg-NH₂ (SEQ ID NO：4)

Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：5)

Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg(SEQ ID NO：6)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg(SEQ ID NO：7)

Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg(SEQ ID NO：8)

Gln-Pro-Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg(SEQ ID NO：9)

Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：10)

Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：11)

Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：12)

Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：13)

Cys-Phe-Ala-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：14)

Cys-Phe-Gln-Trp-Ala-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：15)

Cys-Phe-Gln-Trp-Gln-Ala-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：16)

Cys-Phe-Gln-Trp-Gln-Arg-Ala-Met-Arg-Lys-Val-Arg (SEQ ID NO：17)

Cys-Phe-Gln-Trp-Gln-Arg-Asn-Ala-Arg-Lys-Val-Arg (SEQ ID NO：18)

Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Ala-Lys-Val-Arg (SEQ ID NO：19)

Cys-Phe-Gln-Leu-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：20)

Cys-Phe-Gln-Trp-Gln-Lys-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：21)

Cys-Phe-Gln-Trp-Gln-Arg-Asn-Leu-Arg-Lys-Val-Arg (SEQ ID NO：22)

Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Arg-Val-Arg (SEQ ID NO：23)

Cys-Phe-Gln-Trp-Orn-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：24)

Cys-Phe-Gln-Trp-Nle-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：25)

Cys-Phe-Gln-Trp-Gln-Arg-Orn-Met-Arg-Lys-Val-Arg (SEQ ID NO：26)

Cys-Phe-Gln-Trp-Gln-Arg-Nle-Met-Arg-Lys-Val-Arg (SEQ ID NO：27)

Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：28)

Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg (SEQ ID NO：29)

Cys-Phe-Ala-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg (SEQ ID NO：30)

Cys-Phe-Ala-Trp-Gln-Arg-Ala-Met-Arg-Lys-Val-Arg (SEQ ID NO：31)

Cys-Phe-Gln-Leu-Gln-Lys-Asn-Met-Lys-Lys-Val-Arg (SEQ ID NO：32)

Cys-Phe-Ala-Leu-Lys-Lys-Ala-Met-Lys-Lys-Val-Arg (SEQ ID NO：33)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：34)

Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：35)

Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：36)

Lys-Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：37)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys (SEQ ID NO：38)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile (SEQ ID NO：39)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys (SEQ ID NO：40)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：41)

Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：42)

Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：43)

Lys-Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg (SEQ ID NO：44)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys (SEQ ID NO：45)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile (SEQ ID NO：46)

Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Lys-Arg-Ala-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys (SEQ ID NO: 47), and

most preferably, the biologically active peptide is selected from the group consisting of

Ac-Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Arg-NH₂ (SEQ ID NO：4)

And

Ac-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Ser-Arg-Arg-Arg-Arg-Gly-NH₂；

(SEQ ID NO：50)。

peptides comprising two cysteine residues may be in the form of cyclic peptide structures in which the two cysteines form a cysteine bridge.

Thus, a preferred biologically active peptide is the peptide

When present, the amino acid Cys may advantageously be replaced by acetamidomethyl-cysteine (denoted CysM) to avoid the peptide forming a disulfide bridge with another cysteine-containing peptide.

According to a preferred aspect of the invention, the carboxy terminus of the peptide has been capped, i.e. the free COOH of the carboxy terminus has been converted to CONH by e.g. amidation₂(is represented by-NH)₂)。

According to another preferred aspect of the invention, the amino terminus of the peptide has been capped, i.e. free NH at the amino terminus₂The radical having been converted into the amide CH by, for example, acetylation₃CONH- (denoted as Ac-).

According to yet another preferred aspect of the invention, both the carboxy-terminal and the amino-terminal of the peptide are capped.

In the case where the peptide according to the invention is described as being capped at the carboxy-terminus and/or the amino-terminus, the corresponding uncapped peptide may also be used according to the invention.

In the case where the peptide according to the invention is described as being capped at the carboxy-terminus and/or the amino-terminus, the corresponding capped peptide may also be used according to the invention.

The advantage of the capped form is that the N and C terminal amino acids of these peptides are neutral and uncharged and therefore have altered electrostatic properties. Assuming that the receptors bind to the corresponding sequences of human lactoferrin where there is no N and C terminal charge, the capped peptides should bind better because they resemble the native protein more than the uncapped peptides in this respect.

Preferably the biologically active peptide is present in the pharmaceutical composition in a concentration of between 0.1mg/ml and 100mg/ml, most preferably between 0.5mg/ml and 25 mg/ml.

The biologically active peptides may be present in the form of pharmaceutically acceptable salts.

Preferably the high molecular weight hyaluronic acid has a molecular weight of above 300,000Da, most preferably above 800,000 Da.

Preferably the high molecular weight hyaluronic acid is present in the pharmaceutical composition in a concentration between 0.1 and 10% (w/w), most preferably between 0.5 and 2.5% (w/w).

The high molecular weight hyaluronic acid may be present in the form of a pharmaceutically acceptable salt.

The pharmaceutical composition according to the present invention can be used for preventing the formation of scars, adhesions, keloids after surgical operations associated with the treatment of various tissues such as skin, muscles, tendons, nervous tissue, blood vessels and in different parts of the body such as eyes, ears, vocal cords, hands, spinal cord, abdominal cavity, thoracic cavity, intracranial cavity, oral cavity, gynecological operations, endometrium (endometris), phimosis (phimosis).

The present invention surprisingly found that the biological effect of a peptide derived from human lactoferrin can be significantly enhanced if the peptide derived from human lactoferrin is administered in a pharmaceutical composition comprising the peptide together with high molecular weight hyaluronic acid.

This enhancement cannot be explained only by the possible effect of hyaluronic acid itself, but is due to an unexpected synergistic effect.

Description of the drawings

Figure 1. behaviour of PXL01 loaded sodium hyaluronate gel at 37 ℃.

PXL 01-loaded sodium hyaluronate gel behavior at 37 ℃. The concentration of PXL01 was 6mg/ml in 1.5% sodium hyaluronate solution. The cumulative drug released is expressed as% drug released at time t. Data are presented as mean SDV of three independent product formulations with the addition of a moving average trend line.

Figure 2 PXL01 prevented adhesion formation in a rat abdominal surgical model.

(A) The incidence of adhesion formation between the abdominal wall and the site of the injury to the cecum was expressed as the percentage of animals in each group that had wall-to-wall (wall to wall) adhesions that had occurred connecting these injuries. (B) The cumulative score scale shows the total number of adhesions found in the abdominal cavity expressed as mean ± SEM. (C) Adhesion scores expressed as mean ± SEM according to Nair scale (scores criteria are listed in the examples). (D) Percentage of animals without any adhesion formation in the abdominal cavity of each group. (E) Weight change during 6 days of survival after surgery, expressed as a percentage of the initial weight, n (control) 20, n (0.5 ml PXL01(6mg/ml) in 1 application dH 2O) 10, n (0.5 ml PXL01(2mg/ml) in 1 application dH2O associated with surgery and 3 applications dH2O 24 and 48 hours after surgery) 18, n (1 application 1.5% sodium hyaluronate) 20, n (1 ml PXL01(1.5mg/ml) in 1.5% sodium hyaluronate 10, n (1.5 ml PXL01(6mg/ml) in 1 application 1.5% sodium hyaluronate) 10). Statistical significance was estimated by Fisher's exact test (a, D) or by non-parametric rank sum test (Mann Whitney test) (B, C).^*，p＜0.05；^**And p < 0.01 represents the statistical difference compared to the surgical control group of animals. Adm, administration; SH, sodium hyaluronate, dH20, distilled water.

Examples

Experiment of

Peptides

The peptide PXL01(SEQ ID NO: 56) was used in the experiments.

Preparation of PXL01 in sodium hyaluronate hydrogel

PXL01 dissolved in sodium chloride solution was added to a 2.5% sodium hyaluronate solution at a volume ratio of 2/5PXL01 solution to 3/5 sodium hyaluronate solution to obtain 1.5 or 6mg/ml PXL01 in 1.5% sodium hyaluronate. The mixture was aspirated several times through a 2.1mm diameter needle to homogenize the solution.

Characterization of the formulated product

The concentration and homogeneity of PXL01 in sodium hyaluronate was determined by high performance liquid chromatography with a UV detector (Agilent model 1100) at 220 nm. The analytical column used was Vydac218TP (C18, 5 μm, 250 x4.6mm). The mobile phase used (0.1% aqueous TFA with 1% acetonitrile (solvent A) and 0.1% TFA/acetonitrile (solvent B)) was run in a gradient at a flow rate of 1.0 ml/min. Diluted PXL01 standard samples were applied to generate calibration curves.

The samples were prepared by adding a hyaluronidase solution (hyaluronidase from Streptomyces hyalurolyticus, Sigma-Aldrich (Sigma Aldrich), St Louis, MO) with an enzymatic activity of 500 units/ml to the sample solution. The mixture was stirred at room temperature for 2h and the sample was diluted with aqueous TFA as required, followed by additional mixing. Samples were centrifuged at 7000rpm for 5min before injection into the column.

Extracorporeal delivery system set-up

0.25ml of the formulated product was placed in wells of a Tissue culture plate (24-Flat Well Tissue culture plate, Techno Plastic Products AG (TPP Co.)) to produce a film of approximately 1.3 mm. The plate was placed in a thermostat (37 ℃) for 1h to allow the product to reach a temperature of 37 ℃. 0.5ml of release medium (PBS, pH 7.4) re-equilibrated at 37 ℃ was carefully layered on the gel surface and the tissue culture plates were transferred to a constant temperature shaker (60rpm, 37 ℃). At predetermined time intervals, 10 microliter aliquots of the aqueous solution were withdrawn from the release medium. The concentration of PXL01 released was monitored at a wavelength of 230nm using spectrophotometric measurements. Since the measurement of absorbance at 230nm enables detection of the peptide in the release medium as well as dissolved sodium hyaluronate, a control release medium was used which had an equivalent amount of sodium hyaluronate with PXL01 without any drug.

Animal model for evaluating post-surgical adhesion prevention

Female Sprague-Dawley rats (200-250g, Charles River Laboratories, Sulzfeldt, Germany) were maintained on a 12 hour light-dark cycle, under laboratory animal protection regulations. The study was conducted after prior approval by the local ethics committee.

Cecal abrasion and abdominal wall resection are performed to induce de novo adhesions as described previously (Harris et al, 1995.Analysis of the kinetics of epithelial adhesion formation in the ratio and evaluation of potential anti-adhesive agents in rats).Surgery (Surgery)117, 663-669). Briefly, rats were administered isoflurane (vet, Shering-Plough Animal Health, Farum, Denmark, was anesthetized and buprenorphine (48 mg/kg, Temgesic, Shering-Plough, Brussels, Belgium) was administered as a postoperative pain relief drug. A 5-cm-long abdominal median incision was made and a rectangular full-thickness lesion (5mmx25mm) was made on the peritoneal wall through both the parietal and myofascial peritoneum. In addition, in the serosal region on both sides of the cecum, approximately 10mmx15mm, light rubs with cotton gauze until petechial bleeding occurs. Rats were randomly assigned to either untreated control or treated groups. Excess blood from the lesion is removed and a syringe is used to apply the test substance over the abraded area. The laparotomy wound was closed with continuous sutures and the skin was closed with a metal clip (apple ULC35W, tyco healthcare Group LP (taike medical devices limited), Norwalk (Norwalk), CT, US). Animals were sacrificed 6 days after surgery with excess sodium Pentobarbital (pentabarbital vet, APL, Stockholm, sweden). The abdomen was opened and adhesions were scored by an evaluator blinded to the treatment. The incidence of adhesions between abdominal incisions and abraded ceca was quantified as the percentage of animals in each group that developed wall-to-wall adhesions that joined these lesions. In addition, to evaluate the overall adhesion formed in the abdominal cavity (including adhesion away from surgical trauma)And) the total number, two different rating schemes are used. Bothin describes a cumulative scoring scale (Bothin et al, 2001.The intestinal flora infilfurations addition formation of agricultural effectors and demographics (intestinal flora influences adhesion formation around surgical anastomoses).Br J Surg (English journal of surgery)88, 143-145) specifies the total number of adhesions present in the abdominal cavity: each observed adhesion was given 1 point, which was added to form a score. According to the adhesion rating scale of Nair (Nair et al, 1974.Role of proteolytic enzyme in the prevention of adhesions in the abdomen after proteolytic enzyme surgery).Arch Surg (surgical archive)108, 849-85) combined both the total number of adhesions and the incidence of adhesions between target organs, the latter given a higher rating (0, no adhesions; 1, individual adherent strips of viscera to target organs; 2, two adherent strips of viscera to target organs; 3, viscera to target organ more than two adhesive strips, 4, viscera attached directly to abdominal wall, regardless of the number and extent of adhesive strips). Finally, the percentage of rats in each group without any abdominal adhesions was evaluated. Any possible signs of peritoneal inflammation (erythema and/or edema) or interrupted wound healing associated with autopsy were recorded. As a general indicator of health, the body weights of animals before and 6 days after surgery were compared.

Rat middle and large intestine anastomosis model

Female Sprague Dawley rats (200-250g, Charles River Laboratories, Sulzfeldt, Germany) were maintained on a 12 hour light-dark cycle, under laboratory animal protection regulations. The study was conducted after prior approval by the local ethics committee. With isoflurane (vet, expering-plus Animal Health, Farum, denmark) induced anesthesia and rats received buprenorphine intramuscularly (48 mg/kg; temgesic, Shering-Plough, Brussels (Brussel)Er)) for postoperative pain relief and was administered subcutaneously prior to surgery to receive Bimotrim (80 mg/kg; bimeda, uk).

The abdominal wall was shaved and an approximately 3cm abdominal median incision was made. The colon was exposed and transected to 2cm of the distal cecum. Endocumentary myofascial anastomosis was performed using an 8-fold interrupted suture using 6/0 monocryl (Y432H, Ethicon Inc. (Irishikang Co., St-Stevens Woluwe, Belgium). Small almond cakes (macaroon) are placed in the colon at the anastomotic orifice during the suturing process as a scaffold. Rats were randomly assigned to groups (n-8) or untreated groups (n-8) receiving PXL01(6mg/ml) in 1.5% sodium hyaluronate covering the anastomoses and surrounding the peritoneum. The abdomen was closed in the muscle layer with continuous sutures (4-0 monocryl, Y3100H, Ethicon Inc. (erichson) and in the skin with a stapler. 2ml of isotonic saline was administered subcutaneously to prevent dehydration.

Animals received an additional dose of buprenorphine (24 mg/kg: Temgesic, Shering-Plough (Prolingpauy), Brussels (Brussels)) twice daily subcutaneously for two days following surgery. Animals were killed 7 days after surgery with an excess of sodium Pentobarbital (pentabarbital vet, APL, Stockholm, sweden). The abdomen was opened and a 4-cm-long small intestine section was excised, with the anastomotic region in the middle. The tube to which the pressure monitor is connected is inserted into one side of the small intestine section, the other side being ligated at the end. The small intestine section was immediately placed under isotonic sodium chloride, and stained saline was perfused into the small intestine section through the tube, and the intra-luminal pressure was monitored using a Grass recorder (Grass instruments co, Quincy, Ohio, usa). The maximum pressure before the anastomotic rupture is recorded as burst pressure. The appearance of stained saline around the stoma indicates the point in time of rupture. The evaluator was unaware of the treatment each animal received.

Results

Release behavior of PXL01 in sodium hyaluronate

PXL01 dissolved in sodium chloride solution was mixed with sodium hyaluronate solutionA homogeneous PXL 01-containing hydrogel was produced. In vitro release experiments showed that PXL01 released suddenly from sodium hyaluronate gel formulation (burst release), with approximately 70% of PXL01 released within 1 hour (fig. 1). The release behaviour characterized by an initial burst has been demonstrated by other soluble compounds formulated in sodium hyaluronate (Sherwood et al, 1992.Controlled antibody delivery systems).Biotechnology (Biotechnology)(N Y)10, 1446-. This has functional utility in providing an initial dose during drug delivery, minimizing any lag phase. Importantly, the release profiles (release profiles) of PXL01 from the formulated products prepared on three separate occasions largely overlap, indicating that the preparation of PXL 01-loaded sodium hyaluronate gels is highly reproducible (fig. 1).

Prevention of peritoneal adhesions by PXL01

The anti-adhesion effect of PXL01 was demonstrated using the lateral wall defect-cecal abrasion model in rats (Arnold et al, supra). Without treatment, this model produced reliable and consistent adhesions between the two injured surfaces, with direct cecal-peritoneal wall adhesions occurring in 85% of the rats in the control group (fig. 2A). No significant reduction in adhesion formation was observed when 3mg of PXL01 aqueous solution was administered as a single dose in association with surgery (fig. 2A-D). However, animals treated with 3 doses of 1mg PXL01 in water showed a significant reduction in adhesion formation compared to the rat control group (fig. 2A, C). These results indicate that the slow release of PXL01 in the surgical field is beneficial compared to a single treatment with an aqueous solution of the peptide.

Sodium hyaluronate was chosen as the carrier to obtain controlled release of PXL 01. PXL01 appears to be readily soluble and sufficiently stable in sodium hyaluronate, and sodium hyaluronate hydrogel containing PXL01 is also bioadhesive and easily applied to the surgical field using a syringe. When PXL01 was applied in a 1.5% high molecular weight sodium hyaluronate formulation, the formation of abdominal adhesions was significantly reduced compared to the control group. There was a 4-fold reduction according to the cumulative adhesion score scale (FIG. 2B) and adhesion according to NairThere was a greater than 3-fold reduction in running score (fig. 2C). Compared to 5% of animals in the control group and 20% of animals in the group treated with sodium hyaluronate, 60% of animals treated with 6mg/ml PXL01 in sodium hyaluronate were completely non-adherent (fig. 2D). Sodium hyaluronate itself has been shown to reduce adhesion formation by several scale scores, presumably due to physical barrier effects (Burns et al, 1995.Prevention of tissue injury and post-surgical adhesions by pre-coating the tissue with a hyaluronic acid solution).J Surg Res (J. research)59，644-652.)。

No adverse effects associated with the treatment were recorded during the studies on wound healing or peritoneal inflammation assessed at necropsy. In addition, the average body weight of the rats in the treated group increased compared to their pre-surgical weight, although the difference from the control group did not reach statistical significance (fig. 2E). Importantly, PXL01 in sodium hyaluronate applied around the stoma of the small intestine did not reduce healing potential as estimated by the stoma burst pressure measured 7 days post-surgical (206.3 ± 14.3mm Hg for the treatment group (n ═ 8) versus 197.4 ± 9.6mm Hg for the sham group (n ═ 8)).

The ability of PXL01 to prevent adhesions was limited in aqueous solutions (fig. 2A-D), possibly due to the fact that peptides were rapidly cleared from the peritoneum. However, the peptides formulated in sodium hyaluronate were highly effective (fig. 2A-D), resulting in a significant reduction of adhesions according to the different rating scales, including adhesions formed between two damaged surfaces as well as adhesions formed in the abdominal region distant from the application site. Sodium hyaluronate is a natural component of the extracellular matrix and is catabolized locally, or brought to the lymph nodes or the general circulation, from where it is cleared by the endothelial cells of the liver (Fraser et al, 1988.Uptake and degradation of hyaluronan in lymphatic tissue).Biochem J (journal of biochemistry)256, 153-158; laurent and Fraser 1992 Hyaluronan (Hyaluronan)).Faseeb J (American Association of the society for laboratory and Biotechnology) Magazine)6,2397-2404.). Sodium hyaluronate may enhance the effect of PXL01 by maintaining local drug concentrations through controlled release. In vitro experiments showed that PXL01 was released from sodium hyaluronate for a relatively short period of time (fig. 1), suggesting that the duration of drug release required for in vivo adhesion prevention may be quite limited. This is consistent with previous evidence that a key event in the formation of adhesions in the abdominal cavity occurred during the first 36h (Harris et al, 1995.Analysis of the kinetics of epithelial adhesion formation in the rat and evaluation of potential anti-adhesive agents).Surgery (Surgery)117, 663-669.). Previously, several microparticle-based carrier systems have been shown to induce adhesions or cause inflammation (Hockel et al, 1987.Prevention of adhesions in the rat with a supramented transdermal therapeutic delivery by a novel therapeutic system dexamethasone delivered continuously intraperitoneally has been shown.Ann Chir Gynaecol76, 306 and 313; kohane et al, 2006, Biodegradable polymeric microspheres and nanospheres for drug delivery in the peritonium.J Biomed Mater Res A (Bio) Journal of medical materials research A)77, 351-361). No significant adverse events such as burnout, peritoneal inflammation or inhibition of wound healing were observed in animals treated with PXL01 at any concentration. All treatment groups maintained or exceeded their pre-surgical weight at sacrifice (fig. 2E). Importantly, PXL01 in sodium hyaluronate applied around small intestinal anastomoses does not interfere with the healing potential of the anastomoses.

In summary, the inventors describe the surprising observation that the biological effect of peptides from lactoferrin on preventing post-surgical adhesion formation can be significantly enhanced if the peptides are administered in a pharmaceutical composition comprising the peptides together with high molecular weight hyaluronic acid. The effect is significantly synergistic compared to the effect of the peptide alone and the effect of hyaluronic acid alone. Previously, several microparticle-based carrier systems have been shown to induce adhesions or cause inflammation (Hockel et al, 1987. supra; Kohane et al, 2006. supra). In addition, the use of physical barriers for adhesion prevention has been shown to lead to side effects such as anastomotic fistulas due to interference with the wound healing process (diZerega et al, 2002, supra). In this study, it was shown that sodium hyaluronate does not increase adhesion, but rather acts synergistically with lactoferrin peptide in the prevention of adhesion. Importantly, the administration of the peptide in sodium hyaluronate is not accompanied by any safety issues related to healing of the anastomotic orifice, so this product demonstrates superior safety compared to the previously described anti-adhesion agents. The peptide-loaded sodium hyaluronate gel is easy to handle and administer and is suitable for laparotomy and laparoscopy. In summary, it is expected that the product provides a comprehensive adhesion prevention treatment regimen, not only preventing the formation of adhesions at the site of the surgical procedure, but also preventing de novo adhesions at sites not directly involved in the surgical procedure, due to unintended tissue damage during the surgical procedure, without causing any adverse reactions to healing.

Claims (7)

1. A pharmaceutical composition comprising

i) A biologically active peptide, the amino acid sequence of which is

And are and

ii) high molecular weight hyaluronic acid or a pharmaceutically acceptable salt of high molecular weight hyaluronic acid, wherein the high molecular weight hyaluronic acid has an average molecular weight of more than 300,000 Da.

2. The pharmaceutical composition according to claim 1, wherein the high molecular weight hyaluronic acid has an average molecular weight higher than 800,000 Da.

3. The pharmaceutical composition according to any one of claims 1-2, wherein the peptide is present in a concentration between 0.1mg/ml and 100 mg/ml.

4. The pharmaceutical composition according to claim 3, wherein the peptide is present in a concentration between 0.5mg/ml and 25 mg/ml.

5. The pharmaceutical composition according to any one of claims 1-2, wherein the hyaluronic acid is present in a concentration between 0.1% w/w and 10% w/w.

6. The pharmaceutical composition according to any one of claims 1-2, wherein the hyaluronic acid is present in a concentration between 0.5% w/w and 2.5% w/w.

7. Use of a pharmaceutical composition according to any one of claims 1 to 6 in the manufacture of a medicament for the prevention of the formation of post-surgical scars, adhesions and keloids associated with surgical procedures.