Use of substances having oxytocin activity for preparation of medicaments for wound healing

Substances having oxytocin activity can be used for the preparation of a pharmaceutical composition for the curative or prophylactic treatment of wounds, such as chronic wounds. The wound healing effect can be strengthened or prolonged by combining oxytocin with estrogen.

The present invention refers to a new use of oxytocin.
 BACKGROUND OF THE INVENTION
 A lesion or wound can be obtained either by external damage, giving a
 traumatic wound emanating from an accident or a surgical operation, or by
 a pathological process in the tissue. An ulcer, such as peptic and bone
 ulcer, mainly refers to a wound in the skin or membranes. Lesions also
 comprises damages in varying other tissues, such as bone fractures. In
 this description the expression wound is used for all types of damages of
 the human or animal body. Wounds can be classified as an acute or a
 chronic wound.
 The mechanisms involved in wound healing can generally be divided into four
 phases, that is hemostasis, inflammation, proliferation and maturation.
 During the inflammation leucocytes are accumulated in order to combat
 bacteria and the permeability of the walls of the vessels is increased
 leading to swelling. Many of said substances also promotes an improved
 microcirculation leading to redness and heat. If an infection is not
 developed the number of leucocytes will be reduced and replaced by
 monocytes that is macrophages and lymphocytes releasing i.e. growth
 factors (cytokines) as well as a number of chemical substances, such as
 histamine, serotonin, prostaglandins, which are the main regulators of the
 beginning as well as the continuation of the wound healing process. The
 most important regulating cell is the macrophage. In the proliferation
 phase connective tissue is formed, new blood vessels are growing in and
 injured tissue is regenerated. Fibroblasts are the dominating cells after
 about a week, the inflammation decreases and the strength of the wound is
 rapidly increased. During the maturation phase, finally, the tissue
 protein collagen is stabilised and scar tissue is formed. This phase might
 go on for a long time during which the strength is improved and the
 regeneration is continued, for instance of nerve tissue. In order to
 obtain an optimal wound healing the supply of different vitamins and trace
 elements as well as nutrients should be sufficient as well as the oxygen
 supply.
 Uncompromised cellular homeostasis is essential for wound healing and
 repair and in the healing tissue certain conditions are required for the
 development of granulation tissue and epithelial budding. The homeostatic
 conditions include the degree of hydration, sufficient blood perfusion,
 availability of various growth factors, an appropriate partial pressure of
 oxygen, acceptable levels of nonpathogenic microflora and maintenance of
 voltage gradients between the wound and adjacent normal skin. In a
 homeostatic environment cells are bathed in body fluids that allow for
 normal growth and repair processes.
 Chronic wounds or indolent, nonhealing wounds may arise from different
 causes including infection, the presence of foreign bodies or toxic
 irritants, burns, prolonged cutaneously applied pressure and poor blood
 supply owing to impaired circulation. In a chronic wound the tissue
 homeostasis and the wound environment are compromised so that either
 healing fails to occur or healing begins but is subsequently halted.
 Factors involved in said conditions are tissue necrosis, dehydration,
 chronic wound edema, fibrotic induration and small blood vessel disease.
 Leg ulcer is one type of chronic wounds which is found in a substantial
 part of the population, increasing with advancing age. The course of the
 disease can last from a couple of months up to several decades and will
 bring about heavy expenses for the society as well as a great suffering
 for the individual patient. Pressure ulcer, decubitus ulcer, or bedsore is
 another type of chronic wounds which mainly affects elderly people.
 Chronic non-healing ulcers are a serious problem in diabetic patients.
 In order to provide a homeostatic environment the wound can be covered with
 an occlusive dressing designed to keep the wound moist. This will however
 bring about a risk of promoting wound infection.
 One of the oldest therapeutic methods used for treating chronic open wounds
 is hydrotherapy, which today can be performed in a whirlpool bath. By this
 adherent dressings can be soaked off and necrotic tissue debrided.
 In chronic dermal and indolent ulcers cell migration from the wound
 periphery may not occur if necrotic, desiccated tissue is obstructing it.
 The necrotic tissue impedes the formation of granulation tissue and
 prevents epithelial cells from migrating across the wound. Debridement,
 that is the removal of necrotic tissue from a wound, is therefore
 necessary not only to remove dead tissue on which bacteria survive to
 allow the healing processes of granulation development and fibroblastic
 deposition of collagen upon which the epidermal cells can migrate and
 close the wound, but also to remove many of the microorganisms that may be
 present. This debridement can be performed by topical application of
 enzymes, by facilitating autolysis, that is self-digestion of necrotic
 tissue by enzymes that are naturally present in wound fluids, with
 synthetic dressings or by surgical excision. Once debrided a wound will
 normally undergo granulation, contraction and epithelialization.
 There are a number of topical agents which can have a profound effect on
 the rate of wound healing. Topical antimicrobials and cleansers have
 frequently been overused in wound therapy resulting in harmful cytotoxic
 effects and minimal antimicrobial and cleansing effectiveness. As examples
 of such agents can be mentioned iodophor, sodium hypochlorite solution,
 hydrogen peroxide and zinc oxide.
 It has been hypothesized that impaired wound healing may result from lack
 of adequate stimulation by growth factors.
 In fact growth factors have been used in clinical trials to test if they
 can improve wound closure in patients with chronic nonhealing wounds.
 Recently promising animal experiments on wound healing have been performed
 with growth factors, but a local administration on chronic wounds in
 humans has not been as successful. It has been suggested to use several
 different growth factors in combination with a protease inhibitor.
 Another approach has been to transplant epithelial cells alone or in
 combination with fibroblasts and extracellular matrix components. It has
 also been speculated in the use of genes regulating the synthesis of
 healing polypeptides in the transplanted cells or applied directly on the
 wound.
 PRIOR ART
 Growth factors that are mitogenic or chemotactic or that promote the
 differentiation of one or more types of cells that participate in the
 repair of injured skin could, in principle, promote cutaneous healing. The
 fibroblast growth factors (FGFs) are a family of nine known homologous
 mitogens that stimulate cells of mesodermal and ectodermal origin through
 at least four unique membrane-spanning tyrosine kinase receptors and their
 alternatively transcribed variants. Acidic FGF (aFGF or FGF-1) appears to
 be unique among the characterized FGFs in its ability to bind all known
 FGF receptors with high affinity. This broad receptor specificity
 presumably contributes to its multiple cellular targets in vitro and
 diverse therapeutically relevant biologic activities in animal models of
 tissue repair. aFGF is a short polypeptide that is a potent mitogen for
 dermal fibroblasts, vascular endothelial cells, and epidermal
 keratinocytes, the principal cells in skin. In addition, aFGF is
 chemotactic for vascular endothelial cells in vitro and induces new blood
 vessel growth in vivo. In rats and mice, exogenously applied aFGF promotes
 healing of full-thickness dermal wounds and produces a transient increase
 of tensile strength in incisional wounds. Liying Sun et al., Transfection
 with aFGF cDNA improves wound healing, The Journal of Investigative
 Dermatology, Vol. 108, No. 3, 1997. Applications of aFGF have been
 limited, however, because of the necessity to administer relatively large
 amounts of recombinant aFGF that is often blocked from reaching the target
 tissue by eschar. The short half-life of aFGF is another drawback of
 topical application that necessitates frequent doing. Frequent application
 of aFGF with high doses to overcome diffusion problems can be costly.
 Another family of growth factors is transforming growth factor .beta.,
 TGF-.beta., comprising a broad variety of polypeptides with multiple
 biologic activities. The important role of TGF-.beta.1, -.beta.2 and
 -.beta.3 in wound repair has been demonstrated. TGF-.beta.1 is an
 important modulator of skin morphogenesis and cutaneous wound repair, and
 exogenous application to a wound has been shown to enhance the wound
 healing process in animals. In order to gain insight into the mechanisms
 of TGF-.beta.1 action genes regulated by this factor in cultured
 keratinocytes have been identified and cloned, see B. Munz et al.,
 Differential Expression of the Calpactin I Subunits Annexin II and p11 in
 Cultured Keratinocytes and During Wound Repair, The Journal of
 Investigative Dermatology, Vol. 108, No. 3, 1997. In addition said
 substances were also increased by epidermal growth factor, EGF, and
 keratinocyte growth factor, KGF, which both are stimulating the
 keratinocyte proliferation.
 P. Luppi et al., NGF is released into plasma during human pregnancy; an
 oxytocin-mediated response?, Neuroreport, 4(8), 1063-5, 1993, has
 demonstrated a five-fold increase of biologically active nerve growth
 factor, NGF, in the peripheral circulation of women during pregnancy,
 labour and lactation. As intravenous injection of oxytocin in female rats
 has also produced an increase in the hypothalamic levels of NGF it is
 speculated that the increased amounts of circulating NGF might be
 correlated with either the high levels of oxytocin in plasma or the
 activation of the hypothalamo-pituitary-adrenal axis.
 G. Jansen et al., Increased survival of ischaemic musculocutaneous flaps in
 rats after acupuncture, Acta Physiol. Scand., 135, 555-558, 1989,
 investigated the effects of acupuncture on the survival of flaps in rats
 and showed that manual acupuncture and electro-acupuncture significantly
 increased the survival of said flaps compared to untreated controls.
 DESCRIPTION OF THE INVENTION
 It has now surprisingly been found that oxytocin has a wound healing
 effect. This healing effect can at least in part be ascribed to the fact
 that oxytocin activates growth factors of importance for the healing of
 different tissues. In particular it has now been found that growth factors
 of the FGF type and/or their receptors are activated by the administration
 of oxytocin.
 The invention refers to the use of a substance with oxytocin activity for
 the preparation of a pharmaceutical composition for the curative or
 prophylactic treatment of wounds in mammals including man.
 A preferred use is for the treatment of wounds in skin, bone, mucus,
 tendons, muscles, nerves, vessels and connective tissue.
 Another preferred use of the invention is for the treatment of chronic
 wounds.
 Oxytocin was one of the first peptide hormones to be isolated and
 sequenced. It is a nonapeptide with two cysteine residues that form a
 disulfide bridge between positions 1 and 6 and corresponds to the formula
 (SEQ ID No: 9)
 ##STR1##
 For a long time the only effects attributed to oxytocin were its
 stimulating effects on milk ejection and uterine contractions, but in the
 past decades it has been shown that oxytocin exerts a wide spectrum of
 effects within the CNS. It has been suggested that oxytocin participates
 in the control of memory and learning processes and of various types of
 behaviour such as feeding, locomotion, as well as maternal and sexual
 behaviour. Oxytocin is also suggested to participate in the control of
 cardiovascular functions, thermoregulation, pain threshold and fluid
 balance. There is also evidence that oxytocin is involved in the control
 of various immunological processes. It has recently been demonstrated that
 oxytocin injections cause a lowering of blood pressure and increased
 weight gain--long lasting effects after repetitive administration.
 There are different processes described for the synthetical production of
 oxytocin; commercial processes are for instance described in U.S. Pat.
 Nos.2,938,891 and 3,076,797.
 In the human body oxytocin is produced in the paraventricular nucleus, PVN,
 and the supraoptic nucleus, SON, of the hypothalamus. It differs by only
 two amino acids from vasopressin, which is also produced in these nuclei.
 The magnocellular oxytocinergic neurons of the SON and PVN send
 projections into the posterior pituitary gland from which oxytocin or
 vasopressin are secreted into the circulation. Parvocellular neurons that
 originate in the PVN project into multiple areas within the central
 nervous system, CNS. The oxytocin-producing cells are innervated by
 cholinergic, catecholaminergic as well as peptidergic neurons. The
 presence of oxytocin in different tissues outside the brain, such as the
 uterus, ovaries, testis, thymus, adrenal medulla and pancreas has been
 demonstrated and oxytocin is suggested to exert local effects in these
 organs.
 A parallel secretion of oxytocin into the brain regions and into the
 circulation occurs in response to some stimuli such as suckling, but other
 stimuli may cause separate activation of oxytocinergic neurons,
 terminating in the brain or the pituitary.
 In this context oxytocin refers, whenever applicable, in addition to
 oxytocin also to precursors, metabolic derivatives, oxytocin agonists or
 analogues displaying the same properties.
 Substances with oxytocin activity according to the invention can be
 described by the general formula (SEQ ID No: 10)
 ##STR2##
 wherein
 W is selected from the group consisting of Ile, Cha, Val, Hoph and Phe;
 X is selected from the group consisting of Gln, Ser, Thr, Cit, Daba and
 Arg;
 Y is selected from the group consisting of Leu, Ile, Arg, Hos, Daba, Cit
 and Val; where Leu, Ile and Val give oxytocin analogues and Arg, Hos, Daba
 and Cit give vasopressin analogues; and
 Z is selected from the group consisting of Gly and Ala.
 The unnatural amino acids in said substances have the following structures:
 ##STR3##
 Said amino acids are all commercially available, for instance from Bachem
 and Sigma.
 The amino acids in the substances according to the invention can be either
 L- or D-amino acids.
 Substances according to the invention also include nonapeptides having
 sequences with reversed peptide bonds. These sequences are preferably
 inverted sequences; more preferably comprising D-amino acids.
 The nonapeptides of the formula I are believed to present oxytocin activity
 owing to the structural similarity to oxytocin having the formula (SEQ ID
 No: 11):
 ##STR4##
 In the oxytocin structure the positions 1, 2, 5, 6 and 7 have remained
 unchanged, that is the disulfide bridge, and the amino acids believed to
 stabilize said bridge and to be of critical importance for the properties,
 that is Tyr in position 2, Asn in position 5 and Pro in position 7. In
 position 3 the hydrophobic Ile can be exchanged for other hydrophobic
 amino acids and in position 4 the hydrophilic Gln can be exchanged for
 other hydrophilic amino acids. In position 8 oxytocin analogues are
 obtained if Leu is exchanged for the hydrophobic amino acids Ile or Val,
 and vasopressin analogues are obtained if exchanged for the hydrophilic
 amino acids Arg, Hos, Cit or Daba. The nonapeptides of the invention have
 been compiled through methods described by S. Hellberg et al., "Peptide
 Quantitative Structure-Activity Relationships, a multivariate approach",
 J. Med. Chem. 1987, 30, 1126 and J. Jonsson et al., "Multivariate
 parametrization of 55 coded and non-coded amino acids" Quant. Struct.-Act.
 Relat., 8. 204-209 (1989). The peptides can be synthezised according to
 known methods (e.g. Merrifields solid phase synthesis for instance as
 described in Streitwieser and Heathcock, Introduction to Organic Chemistry
 3rd ed, p 949-950.) Sequences with reversed peptide bonds can also be
 prepared through e.g retro-inverso-modification (see e.g. S. Muller et al,
 PNAS vol 94 November 1997, 12545-12550).
 The use of oxytocin constitutes a preferred embodiment of the invention.
 Another preferred embodiment of the invention is the use of the substances
 mesotocin, isotocin, vasopressin or vasotocin having the formulas (SEQ ID
 No: 10):
 Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Ile-Gly-NH.sub.2 (mesotocin) (when in formula I
 W=Ile, X=Gln, Y=Ile and Z=Gly)
 Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly-NH.sub.2 (isotocin) (when in formula I
 W=Ile, X=Ser, Y=Ile and Z=Gly)
 Cys-Tyr-Phe-G ln-Asn-Cys-Pro-Arg-Gly-NH.sub.2 (vasopressin) (when in
 formula I W=Phe, X=Gln, Y=Arg and Z=Gly)
 Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH.sub.2 (vasotocin) (when in formula I
 W=Ile, X=Gln, Y=Arg and Z=Gly)
 Other preferred substances for use according to the invention are:
 Cys-Tyr-Cha-Cit-Asn-Cys-Pro-Arg-Gly-NH.sub.2 (SEQ ID NO: 1),
 Cys-Tyr-Val-Thr-Asn-Cys-Pro-Leu-Gly-NH.sub.2 (SEQ ID NO: 2),
 Cys-Tyr-Hoph-Thr-Asn-Cys-Pro-Val-Gly-NH.sub.2 (SEQ ID NO: 3),
 Cys-Tyr-Phe-Cit-Asn-Cys-Pro-Leu-Gly-NH.sub.2 (SEQ ID NO: 4),
 Cys-Tyr-Cha-Arg-Asn-Cys-Pro-Hos-Ala-NH.sub.2 (SEQ ID NO: 5),
 Cys-Tyr-Val-Daba-Asn-Cys-Pro-Daba-Ala-NH.sub.2 (SEQ ID NO: 6),
 Cys-Tyr-Hoph-Daba-Asn-Cys-Pro-Cit-Ala-NH.sub.2 (SEQ ID NO: 7) and
 Cys-Tyr-Phe-Arg-Asn-Cys-Pro-Val-Ala-NH.sub.2 (SEQ ID NO: 8).
 Experiments have shown that oxytocin by way of a central action increases
 the activity of the central .alpha..sub.2 -receptors in rats. These
 receptors have an inhibitory action and counteracts the activating aspects
 of noradrenalin in the brain which are mainly mediated via .alpha..sub.1
 -receptors, which activate cyclic AMP. When .alpha..sub.2 -receptor
 stimulation dominates over .alpha..sub.1 -receptor stimulation, activity
 is exchanged by relaxation and energy is shunted towards growing and
 healing, i.e. is not used for stress or muscular contraction and activity.
 As a consequence parasympathetic nerve tone dominates over sympathetic
 nervous tone and the musculature is relaxed. It can be presumed that
 oxytocin exerts a similar effect also in humans. During breast feeding--a
 situation characterized by repetitive oxytocin secretion--all the effects
 observed in experimental animals following repeated oxytocin
 administration are seen. It is not known how the effect by oxytocin on
 .alpha..sub.2 -receptors is mediated, but probably not by a classical
 oxytocin receptor mediated effect.
 The effect of oxytocin can be extended or strengthened by administration in
 combination with drugs increasing the release of oxytocin and/or the
 number of receptors, such as estrogen, or drugs having an .alpha..sub.2
 -agonistic effect, such as clonidine.
 The invention also refers to the use of oxytocin in combination with
 estrogen for the preparation of a pharmaceutical composition for a
 curative or prophylactic wound healing. Oxytocin and estrogen are either
 administrated together or estrogen is given first and oxytocin
 subsequently. Estrogen increases release and synthesis of oxytocin, the
 synthesis of oxytocin receptors, and it also upregulates the activity of
 .alpha..sub.2 -receptors.
 The short term effects of oxytocin, such as short term pain relief, short
 term sedation and hormone release, are reversed by oxytocin antagonists
 and can thus be described as direct effects.
 The long term effects, amounting to days-months of relief, which are
 obtained after repeated treatments, for 3-10 days, preferably for 5-8
 days, in intervals ranging from weeks to months, are surprising also in a
 general perspective and have not previously been reported for any other
 drug in patients. When said effect has been obtained it can be maintained
 by for instance once in a week treatment. This long term effect is
 reversed by .alpha..sub.2 -antagonists and potentiated by .alpha..sub.2
 -agonists.
 The pharmaceutical composition containing oxytocin can be formulated for
 topical, iontophoretical, nasal, intra-pulmonary, parenteral, such as
 subcutaneous, intraperitoneal or intravenous, intrathecal or
 intracerebroventricular administration. The preferred ways of
 administration are topically and intranasally. A parenteral composition is
 for instance a solution or emulsion for subcutaneous, intra-muscular or
 intravenous injection. A topical composition can be a lotion, cream,
 ointment or gel, for instance incorporated into a plaster.
 Doses to be given in topical administration can be 1-1000 .mu.g/cm.sup.2,
 preferably 10-100 .mu.g/cm.sup.2 ; in parenteral administration 0.05-1
 mg/kg; and in nasal administration 1-100 IU/d, preferably 25-50 IU/d. 1 IU
 or international unit corresponds to 2 .mu.g. To children with navel colic
 a preferred dose might be 20-50, preferably 25-30 IU/d nasally, preferably
 administered 2-3 times during 1-2 hours. In i.c.v. or i.t. administration
 the doses are generally 1/10 to 1/1000 of the parenteral doses.
 In the market there are today commercial solutions for injection and nasal
 sprays.

BIOLOGICAL TESTS
 An established model of wound healing is to investigate the survival of
 musculocutaneous flaps in rats. In plastic surgery, reconstructive
 problems are often solved by surgical flaps. The main disadvantage of
 surgical flaps has been their tendency to develop ischemia and eventually
 necrosis of the distal part. This complication can in the end ruin what
 postoperatively seemed to be an excellent result. Much research has been
 devoted to explain the mechanisms of surgical skin flap survival and how
 to prevent and reverse established ischemia.
 For studying the effects of cutaneous wound-healing agents on chronic
 non-healing dermal ulcers the mutant C57BL/Ks-db/db diabetic mouse has
 emerged as a potentially relevant healing-impaired model. These homozygous
 genetically diabetic mice develop obesity and hyper-glycemia that is
 resistant to insulin, analogous to human type II diabetes. They also show
 signs of diabetes such as excessive drinking and urination and greatly
 elevated urinary glucose levels. Wounds in these mice exhibit a marked
 delay in cellular infiltration, granulation tissue formation, and wound
 closure. Previous studies have used this model to evaluate formulations of
 aFGF.
 Test 1. Survival of flaps in rats after administration of oxytocin
 The aim of this study was to investigate the effects of oxytocin on the
 survival of musculocutaneous flaps in rats.
 71 female albino rats (Sprague-Dawley, weight 180-220 g) were anaesthetized
 with chloralhydrate (0.4 g/kg), shaved on the back and a dorsal cranially
 based standard flap (2.times.7 cm) was elevated and then sutured back in
 position. The flap was raised from the deep fascia of muscle and included
 the superficial fascia, panniculus carnosus, subcutaneous tissue and skin.
 24 h and 1 h before operation, respectively, as well as 24 h after
 operation the rats were treated with 1 mg/kg oxytocin s.c. (n=20), an
 oxytocin antagonist, that is 1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-oxytocin
 in an amount of 1 mg/kg (n=15), with 1 mg/kg of said oxytocin antagonist
 and then after 30 minutes with 1 mg/kg oxytocin (n=16), or with saline
 (n=20).
 Six days after the operation the survival of the flaps were estimated. The
 oxytocin-treated rats had a significantly higher survival of the flaps
 when compared to the saline-treated controls (p&lt;0.05). When the oxytocin
 antagonist was administrated before the oxytocin injection this effect was
 abolished. The oxytocin antagonist alone decreased the flap survival
 compared to the saline-treated rats (no significance, p=0.13) and the
 oxytocin-treated rats (p&lt;0.05).
 The result of the study is demonstrated in FIG. 1, showing the survival of
 musculocutaneous flaps in rats treated s.c. with saline (NaCl), oxytocin
 (Ox), oxytocin antagonist (OxA) and oxytocin antagonist+oxytocin (OxA+Ox).
 The results are shown as means.+-.SD. Statistical evaluation was performed
 with a one-way ANOVA followed by Student's t-test.
 Test 2. Wound healing in diabetic mice after administration of oxytocin
 Female C57BL/Ks-db/db diabetic mice, 11 weeks old, were obtained from
 Jackson Laboratories (Bar Harbor, Me.). The mice were housed in sterile
 microisolator boxes (four per box) with sterile water and bedding and were
 kept in a semi-barrier quarantine facility. Supplemental heat was provided
 with electric heating pads under half of each box after wounding to
 prevent hypothermia and reduce mortality.
 Mice were anesthetized with intraperitoneal injections of 110 mg ketamine
 and 9 mg xylazine per kg of body weight. The mid-back and thoracic skin
 was shaved and disinfected with a 2% Chlorhexidine surgical scrub,
 followed by 70% ethanol. A template 1.6 cm in diameter was used to mark a
 2.0 cm.sup.2 circle on the mid-dorsal area, and a single full-thickness
 wound was created by blunt excision with sterile curved iris scissors.
 Wound areas typically increased to approximately 2.3 cm.sup.2 soon after
 injury, presumably because of contraction of the dermis along the wound
 perimeter. All surgical and subsequent healing analysis procedures were
 performed in a laminar flow hood using full aseptic techniques. Doses were
 administrated on 5 consecutive days before, and on day 0, 3 and 7 after
 injury. Oxytocin was administrated at different doses, 0.001, 0.01, 0.1,
 1.0, 10.0 and 100.0 .mu.g/cm.sup.2 , respectively, to groups of 16 mice
 per dose. The wounds were covered with a semitransparent Bioclusive
 dressing (Johnson & Johnson Products, New Brunswick, N.J.) for protection,
 maintenance of a moist environment and prevention of crust formation.
 Wound fluid samples were taken at day 10 after injury and cultured for
 aerobic and anaerobic microorganisms. Animals from which cultures
 exhibited more than 50 colonies per wound were excluded from the study
 analysis. Prevention of even mild or subclinical infection resulted in a
 consistent maximal healing impairment, thus providing a large
 wound-healing window for evaluation of treatment effects.
 Wound appearances were recorded photographically and their perimeters were
 traced onto sterile glass slides applied directly to the exposed wound
 surfaces after pre-wetting with a drop of sterile physiologic saline.
 Measurements were made immediately after wounding and twice weekly
 thereafter until the wounds were completely closed. Wound areas and
 perimeters were determined from the glass-slide tracings using
 computerized image analysis. Statistical significances of differences
 between groups were evaluated using an unpaired two-tailed Student t-test.
 Healing expressed as a decrease in percent initial area was converted to
 linear ingrowth from the wound edges by dividing the differences in wound
 areas by the average wound perimeters at sequential time intervals. Total
 ingrowth at a specific time is the sum of the incremental ingrowth
 distances up until that time. This transformation linearized closure as a
 function of time, thereby allowing expression of healing as kinetic rates
 that were constant over time.
 Wounds, examined twice weekly to monitor time to total closure, were
 considered fully healed when moist granulation tissue was no longer
 apparent, indicating a functional epidermal water-permeability barrier.
 Closure was also confirmed histologically. Time-point comparisons of the
 fraction of healed versus unhealed wounds were analyzed by the X.sup.2
 statistic with Yates correction, and group comparisons of absolute days to
 total closure for individual animals were analyzed by an unpaired
 two-tailed Student t-test.
 Full-thickness dermal excisional wounds 1.6 cm in diameter closed
 substantially faster in response to three doses of 10 .mu.g per cm.sup.2
 initial wound area administered during the first week after wounding
 compared with treatment with the corresponding vehicle control.
 Statistically significant enhancements of the healing rate (p.ltoreq.0.01)
 of treated wounds were seen as early as day 7 after injury and continued
 throughout the test period. A 50% maximum difference in percent of initial
 area between oxytocin and vehicle-treated wounds occurred from 14 d after
 injury. This difference corresponded to a 10-fold decrease in wound size
 in treated mice at day 14 compared with those receiving the corresponding
 PBS/heparin vehicle. Although wounds receiving a liquid vehicle healed
 somewhat faster than wounds receiving nothing, treatment with either PBS
 or normal saline resulted in healing rates equivalent to those with the
 heparin-containing PBS vehicle (data not shown).
 Wound closure was nonlinear as a function of time, so the magnitude of the
 effect of oxytocin treatment appeared to change over time. Because these
 full-thickness wounds closed by progressive healing from the wound
 perimeter, we transformed the data from percent initial wound area to
 average linear healing length from the original wound edges. This
 calculation linearized the average healing rates after an initial 3 d lag.
 Treatment with oxytocin significantly (p.ltoreq.0.01) increased the linear
 rate of healing from the wound edges at all time intervals. The slope of
 cumulative linear healing length as a function of time was determined for
 each animal. These slopes, grouped by treatment, showed that 10
 .mu.g/cm.sup.2 oxytocin increased the mean linear healing rate to 0.45
 mm/d, from 0.18 mm/d observed in the vehicle-treated group
 (p.ltoreq.0.01).
 Treatment with this dose of oxytocin also clearly reduced the time to total
 wound closure. Statistically significant (p.ltoreq.0.05) increases in
 total closure were seen by day 14. The median time to complete closure
 decreased from 52 d in vehicle-treated wounds to only 23 d in those
 treated with three doses of oxytocin during the first week after injury
 (p.ltoreq.0.0001).
 Three topical applications of oxygen, ranging from 1 to 100 .mu.g/cm.sup.2
 per dose, applied during the first week after injury resulted in
 significant dose-related increases in healing rates over paired vehicle
 controls. The 0.001 .mu.g/cm.sup.2, 0.01 .mu.g/cm.sup.2 and 0.1
 .mu.g/cm.sup.2 doses were ineffective, thus establishing a "no effect"
 level between 0.001 and 1 .mu.g/cm.sup.2.
 A dose response relation was also seen in the time to complete wound
 closure. Application of from 1 to 100 .mu.g/cm.sup.2 per dose
 significantly reduced the time required for these wounds to close
 completely. In the dose range of 1.0 to 10.0 .mu.g/cm.sup.2, wounds closed
 approximately 10 d sooner than their respective vehicle controls. The
 closure rates, the 0.001-.mu.g/cm.sup.2 (not shown) and 0.01
 .mu.g/cm.sup.2 doses were ineffective in decreasing the time to total
 closure.
 The concentration of FGF was compared in skin biopsies taken from the
 injured area ("ulcer") of ten mice treated with 10 .mu.g/cm.sup.2 oxytocin
 and ten mice treated with PBS 7 days after injury. The results show that
 the oxytocin treated mice synthesized 162% more FGF-1 as compared to the
 PBS vehicle treated mice. This increase in concentration was highly
 significant (p&lt;0.001) and strongly support the hypothesis that oxytocin
 increases wound healing through an increased expression and synthesis of
 FGF-1.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 11
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 1
 Cys Tyr Xaa Xaa Asn Cys Pro Arg Gly
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 2
 Cys Tyr Val Thr Asn Cys Pro Leu Gly
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 3
 Cys Tyr Xaa Thr Asn Cys Pro Val Gly
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 4
 Cys Tyr Phe Xaa Asn Cys Pro Leu Gly
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 5
 Cys Tyr Xaa Arg Asn Cys Pro Xaa Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 6
 Cys Tyr Val Xaa Asn Cys Pro Xaa Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 7
 Cys Tyr Xaa Xaa Asn Cys Pro Xaa Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: MOD_RES
 &lt;222&gt; LOCATION: (9)
 &lt;223&gt; OTHER INFORMATION: AMIDATION
 &lt;400&gt; SEQUENCE: 8
 Cys Tyr Phe Arg Asn Cys Pro Val Ala
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequencePEPTIDE
 &lt;400&gt; SEQUENCE: 9
 Cys Tyr Ile Gln Asn Cys Pro Leu Gly
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 10
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequencePEPTIDE
 &lt;400&gt; SEQUENCE: 10
 Cys Tyr Xaa Xaa Asn Cys Pro Xaa Xaa
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 11
 &lt;211&gt; LENGTH: 9
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial SequencePEPTIDE
 &lt;400&gt; SEQUENCE: 11
 Cys Tyr Ile Gln Asn Cys Pro Leu Gly
 1 5