WO1995023511A1

WO1995023511A1 - Wound disinfection and repair

Info

Publication number: WO1995023511A1
Application number: PCT/US1995/002821
Authority: WO
Inventors: Robert D. Kross
Original assignee: Alcide Corporation
Priority date: 1994-03-04
Filing date: 1995-03-01
Publication date: 1995-09-08
Also published as: EP0748161A4; AU2096795A; CA2184813C; EP0748161A1; JPH09509861A; CA2184813A1

Abstract

Methods for preventing and treating microbial wound infections, especially of peritoneal cavity wounds including those having indwelling catheters, and enhancing wound repair while minimizing adhesions and scar formation involve the infusion or irrigation of the wound with a solution containing a pharmacologically acceptable carrier and chlorine dioxide in an amount ranging from about 5 ppm to 1000 ppm, and having a chlorine dioxide to chlorite ratio of at least 5:1. Typical solutions contain chlorine dioxide in isotonic saline and exhibit a pH of from about 5 to about 7.5. Sodium chlorite is employed in preferred embodiments.

Description

WOUND DISINFECTION AND REPAIR

Related Application Data

This application is a continuation-in-part of co- pending U.S. application serial number 08/133,465, filed October 8, 1993, which was a continuation-in-part of application serial number 07/854,286, filed March 20, 1992, which issued on October 12, 1993 as U.S. Pat. No. 5,252,343, the full disclosures of which are hereby fully incorporated herein by reference.

Technical Field

This invention relates generally to the disinfec¬ tion and repair of wounds, especially peritoneal cavity wounds and surgical sites.

Background of the Invention

With the possible exception of teeth, tissues and organs of the body are capable of repairing injuries. An injury may be broadly defined as an interruption in the continuity of tissues, and these are repaired by reestab¬ lishing that continuity. Tissue repair is achieved pri¬ marily by proliferation, migration and differentiation of involved cells. Epithelial tissue heals chiefly by cel¬ lular migration, presumably because epithelium is essen¬ tially two-dimensional. With mesodermal tissues, howev¬ er, the three-dimensional configuration is correlated with a somewhat different mode of repair that takes the form of an aggregate of cells that migrate into the le- sion where they eventually redifferentiate into the tis¬ sue in question. These repair aggregates may take the form of granulation tissue in the case of dermis, frac¬ ture callus in the case of broken bones, or a comparable accumulation of cells between the cut ends of a severed tendon. (For a very brief review, see Cohen, I.K., et al . , eds., Wound Healing, W.B. Saunders Co., Philadel¬ phia, 1992, page 24).

The physiological process of wound healing or tissue repair has been arbitrarily divided into three major phases: the inflammatory phase, the proliferative phase, and the remodeling phase. A complex series of physiological and biochemical events can be correlated with macroscopic and microscopic changes in the wound as it heals.

The first, or inflammatory, phase of wound repair is initiated by a sequence of biochemical and cellular events that begins once the integrity and homeostasis of the tissue membranes are disrupted and involves both humoral and cellular components. A commonly observed feature of inflammation is the release of various eicosa- noids and the appearance of polymophonuclear neutrophils, which migrate into both infected and noninfected wounds. The neutrophils are followed by monocytic macrophages that remove wound debris and contribute soluble mediators including additional eicosanoids that promote wound re¬ pair.

In infected wounds or in wounds with massive tis¬ sue destruction, the process of phagocytosis by both cell types is accompanied by a sequence of biochemical events that sharply increase oxygen uptake, producing a number of oxygen-derived free radicals including superoxide, hydroxyl and singlet oxygen, and derivative products such as hydrogen peroxide. These reactive products generate chemotactic factors for phagocytes and may be used by the phagocyte in destruction of infectious wound con¬ taminants, but phagocyte-derived oxygen reduction prod¬ ucts can also cause tissue damage and mediate ischemic injury (id . , pages 302 to 303). Increased levels of in¬ flammatory mediators, especially the vasoconstrictive ones, have been shown experimentally to enhance bacterial proliferation and microabscess formation. In infected wounds, this sets up a vicious cycle resulting in an im- balance of mediators normally useful in the repair pro¬ cess (id. , pages 292 and 298) .

In man, collagen plays a pivotal structural role in the proliferative and remodelling phases of wound healing and tissue repair (id. , pages 146 to 147) . The collagenous scaffold of the extracellular matrix compris¬ es at least 13 genetically distinct types of collagen (ijbid.), and the role the types play in the pathophysiol- ogy of tissue repair and their interplay with noncollage- nous matrix materials is incompletely understood, espe- cially the more recently described collagen types VI to

XIII. In general, however, it has been observed that connective tissue reaction to injury eventually leads to the appearance of increased numbers of fibroblasts and finally to the accumulation of numerous rather large fibrils derived from type I collagen molecules. Fiber- rich scar tissue that ultimately forms contains fibrils predominantly derived from this type of collagen (ibid . ) . Investigators who have described the fibrillar components involved in wound healing of other tissues have observed type I collagen deposition in fracture healing of bone, both types I and III collagen deposition in the dermis, and type II in cartilage (ibid . ) . Type IV collagen forms a eshlike scaffold in basement membranes and type V collagen appears to be involved in the migration and movement of capillary endothelial cells during angiogene- sis (ibid. ) . In a study of controlled epithelial repair in guinea pigs, it was reported that collagen deposition in artificially infected incisions was inhibited by treat¬ ment with an acidified chlorite solution (Kenyon, A.J. , et al . , Am. J. Vet Res . 47 : 96-101 (1986)). Treated wounds consistently epithelialized rapidly and seldom gaped or exhibited desquamated wound edges, but in the course of healing exhibited less wound breaking strength than control wounds. In the same study, treatment had an antimicrobial effect against Staphylococcus aureus exper¬ imentally introduced into the wounds.

The overall union of the opposing surfaces of a wound results in an adhering or uniting process referred to as an adhesion. The adhesion may involve tissue for- mation without differentiation of new elements, resulting in scarring and/or unnatural tissue associations. In surgery, foreign body reactions to lint and starch, sero- sal damage due to handling, ischemia and tension imposed by suturing and handling, and impaired fibrinolysis can contribute to adhesion formation. Fibrovascular adhe¬ sions complicate gynecological, intestinal, tendon, and cardiac surgery (Cohen, et al . , cited above, page 576), and may result in ischemia. Adhesion prevention after surgical procedures has been attempted by reducing fibrin deposition using heparin and fibrinolytic agents, inhib¬ iting fibroblast proliferation and collagen deposition using antihistamines or steroids, using careful surgical techniques and separating organs using various tech¬ niques, including separation with resorbable fabric. These methods have met with limited success due to the multiple and poorly understood etiology of adhesion for¬ mation (ibid. ) .

In past years, the management of wound treatment and repair was directed to wound healing after surgery or physical trauma. However, certain procedures currently employed in medicine such as those involving indwelling catheters for cleansing or administration of drugs neces¬ sitate the routine maintenance of what amount to infec¬ tion-free wounds. For example, in the treatment of irre- versible renal failure (briefly reviewed in Wyngaarden, J.B., et al . , Cecil 's Textbook of Medicine , 19th ed. , Harcourt Brace Jovanovich, Philadelphia, 1992, pages 541 to 545) , continuous ambulatory or cycling peritoneal dialysis (respectively called C.A.P.D. and C.C.P.D.) are now employed as an alternative to, or an adjunct with, hemodialysis or kidney transplantation. Each year, ap¬ proximately 1.3 in 10,000 people in the United States develop end-stage renal disease, which is characterized by the accumulation of solutes in the body that can be removed by dialysis, diffusion across a semipermeable membrane down a chemical concentration. Peritoneal dial¬ ysis allows for the clearance of larger, and sometimes more toxic, substances than hemodialysis because of the greater permeability of the peritoneal membrane to larger molecules and the longer duration of treatment. Perito¬ neal dialysis is the dialysis treatment of choice for diabetics and patients with peripheral vascular disease or congestive heart failure because the method provides less cardiovascular stress than hemodialysis, and for children, because they have relatively good peritoneal clearance. Peritoneal dialysis also allows for greater freedom and schedule flexibility and is thus often pre¬ ferred by working or disabled patients.

In peritoneal dialysis, the dialyzing solution is introduced into and removed from the peritoneal cavity through an abdominal incision. C.A.P.D. makes use of the fact that small molecular weight solutes reach complete equilibration with peritoneal fluid in 4 to 6 hours. Typically, a patient on C.A.P.D. exchanges 1.5 to 3.0 liters of sterile dialysate containing hypertonic glucose and physiologic electrolytes three to five times a day, introduced and removed through a peritoneal dialysis catheter. C.C.P.D. is becoming increasingly popular because the number of daily connects is reduced from four to two by employing the cycler during sleep and a single prolonged, C.A.P.D.-type daytime exchange. Many patients have been managed successfully with peritoneal dialysis for 5 to 10 years, but long-term technique failure rates remain higher than for chronic hemodialysis, mainly be¬ cause of problems with the peritoneal catheter or recur- rent peritonitis (ijbid.).

One method almost universally employed for treat¬ ing wounds of all types, including those surrounding indwelling catheters, is simple cleansing. Surgeons commonly employ mechanical forces to rid wounds of bacte- ria and other particulate matter retained on wound sur¬ faces. Irrigation can rid wounds of large foreign bod¬ ies, but high pressure irrigation is necessary to remove smaller ones, and these can damage tissue and imbed for¬ eign bodies in exposed tissue. Antibiotics added to surgical irrigation solutions appear to provide no addi¬ tional benefit over saline alone (Hau, T., and Nishikawa, R. , Surg. Gyn. & Obstet . 156 : 25-30 (1983)). Disinfec¬ tants added to the solutions are potentially toxic; many disinfectants that can be employed on unbroken skin cause tissue damage and damage to tissue defenses when employed on wounds, especially surgical wounds (Cohen, et al . , cited above, pages 584-586) . Exposure of blood to either Hibiclens^® or Betadine^® surgical scrub solutions, for example, damage its cellular components. Some surgical irrigants employ a surfactant such as Poloxamer 188 which does not produce discernible toxic effects or allergic reactions in tissues, but this has the disadvantage of exhibiting no antibacterial activity. For wounds that are prone to infection, systemic antibiotic treatment must be employed (ibid. ) . It would be advantageous to have a quick-acting, broad-spectrum antimicrobial for use as a surgical irri- gant or catheter and wound cleanser that rapidly degrades and does not irritate tissues. It would be especially advantageous to have an antimicrobial that not only does not interfere with wound healing, but, through interac¬ tion with the complex processes involved, actually pro¬ motes wound healing through a more beneficial route by minimizing scar and adhesion formation.

Summary of the Invention

It is an object of the invention to provide a method for treating and disinfecting wounds, particularly for the therapeutic and prophylactic treatment of wounds to the peritoneal cavity.

It is another object of the invention to provide a method for disinfecting indwelling catheters and dialy- zates of patients undergoing continuous ambulatory or cycling peritoneal dialysis.

These and other objects are accomplished by the present invention, which provides methods for using wound irrigants especially suitable for the peritoneal cavity which may be employed surgically or post-surgically or after physical trauma. The irrigants exhibit broad-spec¬ trum antimicrobial activity, counter inflammation, and minimize scar and adhesion formation during the wound healing process.

The method involves irrigation or infusion of a wound, or catheter cleansing, with a solution containing dissolved chlorine dioxide. The solutions typically com- prise from about 0.0005% (5 parts per million, ppm) to about 0.1000% (1000 ppm) of chlorine dioxide (C10₂) . One embodiment efficacious in peritoneal dialysis solutions employs from about 5 to about 75 ppm C10₂; another effi¬ cacious as a post-surgical irrigant, from about 5 to about 250 ppm C10₂; and a third especially suitable as a surgical irrigant, from about 40 ppm to about 600 ppm C10₂. The chlorine dioxide solutions used in the inven¬ tion have a relative molar ratio of chlorine dioxide to residual chlorite of at least 5:1, typically at least 7.5:1, and preferably at least 10:1.

The chlorine dioxide solution may be provided in a number of ways. For example, it may be formed immedi¬ ately prior to infusion or irrigation by combination of a chlorine dioxide liberating compound (such as water-solu¬ ble alkali or alkaline earth metal chlorites or mixtures thereof) with a mineral acid such as sulfuric acid, hy- drochloric acid, and/or phosphoric acid, followed by adjustment of the pH to about 5 to 7.5. Sodium chlorite is employed in some preferred embodiments.

Alternatively, chlorine dioxide can be formed by reacting a chlorine dioxide liberating compound such as the chlorites mentioned above with an organic acid having a pK of from about 2.8 to about 4.2, such as malic acid, lactic acid, citric acid, mandelic acid, tartaric acid, and mixtures thereof. Lactic acid is employed in one embodiment; organic acids other than lactic acid are employed in other embodiments. Chloride ion can option¬ ally be used in these formulations, as can carbohydrate triggering substances that accelerate the formation of chlorine dioxide.

Alternatively, chlorine dioxide can be formed by reacting a chlorine dioxide liberating compound, such as the chlorites mentioned above, with a saccharide which has been heat-activated by heating the saccharide to a temperature of from about 50^βc to about 150°c for at least about 1 minute, typically from about 5 to 240 min- utes and preferably from about 20 to 120 minutes, in a solution in the presence of an organic acid having a pK of about 2.8 to about 4.2 and at a pH below about 5.5. Typical saccharides used in these formulations include glucose, galactose, mannose, ribose, rhamnose, lactose, sucrose, maltose, and mixtures thereof.

The volume of chlorine dioxide-containing solution used as a wound irrigant according to the method of the invention varies with the size of the wound and the mam- mal and the scope of the infection or potential infec¬ tion, and can range between about 10 milliliters to about 5 liters.

Solutions employed for wound irrigation according to the method of the invention typically contain a phar- maceutically acceptable carrier. Pharmaceutically ac¬ ceptable carriers include any that do not irritate the wound or cavity, including dialysis solutions and iso¬ tonic solutions containing saline and other inorganic (e.g., phosphates and sulfates) and organic salts. Typi- cal solutions have a pH of from about 5 to 7.5. The solutions may optionally contain suitable wetting agents and emollients.

Brief Description of the Figures

Figure 1 is a graphical plot of data obtained in lactate dehydrogenase assays of isolated polymorphonu- clear leukocytes in the presence of chlorine dioxide solutions used according to the method of the invention and antinflammatory ibuprofen controls. The figure plots lactate dehydrogenase activity, expressed as % of a posi- tive control, versus dilutions of chlorine dioxide solu¬ tions (•) or ibuprofen (I) , and shows the effect of chlo¬ rine dioxide or ibuprofen on cell integrity with (solid lines) or without (dashed lines) incubating with 0.05% bovine serum albumin (BSA) prior to the assay. A nega¬ tive control is denoted by 0.

Figure 2 is a graphical plot of data collected using chlorine dioxide and ibuprofen at various dilution levels run concurrently in a chemotaxis assay employing 10% bovine serum albumin with polymorphonuclear leukocyte cells. In the figure, •—• and •—• show the chlorine dioxide results, ■—■ and ■—■ show ibuprofen results, and 0 is a negative control.

Figure 3 shows the results of a polymorphonuclear leukocyte chemotaxis assay that employs chlorine dioxide (•) and ibuprofen (■) at various dilution levels and bovine serum albumin levels of 0.05%. In the figure, the solid lines represent data obtained after incubation of the solutions with bovine serum albumin prior to addition of cells, and the dashed lines represent incubation of the cells with the solutions prior to addition of bovine serum albumin. A negative control is shown as 0.

Detailed Description of the Invention

This invention is based upon the finding that a solution containing chlorine dioxide having defined chlo¬ rine dioxide-to-chlorite molar ratios that limit tissue irritation are efficacious as wound irrigants both pro- phylactically and therapeutically.

In one embodiment, the present invention is di¬ rected to the use of effective amounts of chlorine diox¬ ide as a peritoneal cavity irrigant. The enteric organ¬ isms which enter the peritoneal cavity through contamina¬ tion by gastrointestinal contact, whether by physical trauma or during surgery, are all susceptible to the cidal effects of chlorine dioxide, which is a broad-spec¬ trum antimicrobial. Chlorine dioxide has demonstrated activity against all relevant organisms, both aerobic and anaerobic, including lactobacilli, streptococci, con¬ forms, Klebsiella species, Enterobacter species, Bacte- roideε species, clostridia and eubacteria. Irrigation of the peritoneal cavity with saline, in concert with sys¬ temic antibiotic therapy, has been found to be beneficial in the treatment of peritonitis. However, as mentioned in the Introducticn, addition of antibiotics to the irri¬ gating solution has not provided any additional benefit (Hau and Nishikawa, cited above) , and it is presumed that the peritoneal irrigation derives from its mechanical effect. The ineffectivenss of local antibiotic solutions has been surprising to researchers, since the antibiotics that have been used were capable of in vitro inhibition of the bacterial species present in the peritoneal cavi¬ ty. It may be that the lack of effect was attributable to the brevity of contact of the antibiotic with the organisms, since usual flush times approximate five min¬ utes or less in duration, which would be too short for significant cidal action to occur.

This invention provides a quick-acting, broad- spectrum antimicrobial, that is not absorbed systemically because it is rapidly eliminated or degraded. The inven¬ tive compositions have the requisite, extended range of antimicrobial action, rapidity of kill and degradability for this application. For example, 50 ppm C10₂ solutions have been shown to kill 6-logs/ml of a wide variety of organisms within one minute, including Pseudomonas aeru- ginosa , Staphylococcus species, Escherichia coli and other coliforms, Klebsiella pneumoniae , Streptococcus species, and Bacteroides species. C10₂ reacts with oxi- dizable bonds of these organisms and other organic matter and, as a result, is reduced to lower oxidation states: initially chlorite and ultimately chloride. The latter is present at high levels in the body (ca . 0.5% of cellu¬ lar fluids) , so that the small amount formed by C10₂ degradation (in the parts per million range) would disap¬ pear into that pool. Compatability of these disinfecting peritoneal flush solutions is also appropriate, since the C10₂ is generated or dissolved in solutions which have osmotic pressures that are suitably isotonic with cellu¬ lar fluids.

As has been mentioned, the inflammatory process resulting from bacterial infection of wounds in the peri¬ toneal cavity, e . g. , peritonitis, can, upon treatment, either resolve with no residual effects or can lead to the formation of scarring and/or fibrous adhesions. An additional feature of C10₂ and related oxychlorine systems is their capability to inhibit the stimulation of fibro- plasia, the proliferation of fibroblast cells and expres- sion of collagen, which ordinarily ensues following wounding. As shown in the examples that follow, the oxychlorines resulting from acidification of chlorite to form chlorous acid significantly inhibits fibroblast proliferation and collagen formation.

A further study confirming the effect of C10₂ on wound healing set out in greater particularity below combines an investigation of the immediate cellular re¬ sponse (chemotaxis) of the body to injury using isolated polymorphonuclear leukocytes, and another investigation assessing the interaction of C10₂ with free radicals which form during the course of tissue repair collagen synthe¬ sis. In the former case, C10₂ was found to control chemo¬ taxis by affecting the quality and morphology of the associated polymorphonuclear leukocytes. In the latter case, the C10₂, which is a stable free radical containing one unpaired electron, was found to neutralize the super- oxide and related radicals involved in the process of collagen production. In another embodiment, this invention is directed to the use of oxychlorines as a prophylactic or therapeu¬ tic rinse for individuals on peritoneal dialysis. Over 100,000 persons in the United States require dialysis treatments to supplement or replace the activity of fail¬ ing or failed kidneys. The majority of these people rely on hemodialysis, which involves several hour sessions, three times per week, during which time their blood is passed through an external hemodialysis apparatus. As discussed in the Introduction, an alternative treatment is Continuous Ambulatory or Cycling Peritoneal Dialysis (C.A.P.D. and C.C.P.D.), where their peritoneal cavities are filled with phosphate-buffered sugar solution, and the peritoneal membrane acts as a dialyzing filter for waste products that have accumulated in the blood. This technique frees the patient from thrice-weekly immobili¬ zation on a dialysis machine, but has a tendency to pro¬ mote peritoneal infections in the cavity by transfer of skin organisms into the peritoneum when the indwelling catheter that delivers the fluid is inserted. Other transfer of organisms takes place when connecting the daily dialysis fluid supply. Once an organism has reached the cavity, typically Staphylococcus epidermidis , it deposits on the catheter surface and grows a protec- tive glycocalyx structure around its colonies. As a result, the average C.A.P.D. or C.C.P.D. patient has a peritonitis infection at least once a year, which dis¬ suades many people from this otherwise less-encumbering dialysis procedure. This invention, which is efficacious in the destruction of S. epidermidis encased in biofilms as illustrated below, provides a method to reduce this incidence.

The C10₂-containing isotonic solutions of the invention can be used for C.A.P.D. patients ^'in one of two ways. It can be included in the daily volume of the dialyzate infused into the patient, as a prophylactic means of destroying any incidental organisms that might be transferred during the daily exchange of fresh fluid for spent dialyzate. In this situation, a separate sat¬ ellite bag of C10₂ solution can be transferred to the main dialyzate volume prior to introduction into the peritone- urn. The satellite bag itself can be a dual container of components which, when mixed, would release C10₂ at an appropriate level such that its dilution in the dialyzate fluid would approximate the desired final concentration (e. g. , 5 to 75 ppm) .

In another application, the method of the inven¬ tion is employed to treat peritonitis in C.A.P.D. pa¬ tients which arises from penetration of organisms into the peritoneum. While free-floating organisms are sus¬ ceptible to the cidal effects of C10₂, S. epidermidis colonies on indwelling catheters can also be effectively destroyed. These colonies intermittently recontaminate the peritoneal cavity, which may be subsequently treated with an antibiotic without destroying the protected colo¬ ny on the catheter surface. As set out in the examples hereinafter, the cidal effectiveness of C10₂ was demon¬ strated using S. epidermidis biofilms that were grown on flat plates and exposed to a variety of disinfectants and antibiotics.

The method of the invention can also be used post- surgically. Following surgery, particularly abdominal surgery, it is generally necessary to flush the area of the operation with sterile saline solution to remove organic debris, including blood and serous fluids. When the surgery involves the gastrointestinal tract, there is generally the added presence of those organisms which inhabit that system. Even without such involvement, organisms in the surgical environment, although much reduced from normal levels, can deposit on the site, and must be removed. Without such removal, serious infec- tions can result after closure of the incision or wound. It is desirable therefore to incorporate an antimicrobial material in the flush, but broad use of antibiotics is discouraged and use of antiseptics such as hypochlorite or peroxide is discouraged because of tissue incompati¬ bility at levels that might be considered effective.

It is an advantage of the invention that solutions containing fairly significant levels of C10₂ are particu¬ larly well tolerated by tissues. For example, solutions of 350 ppm in buffered saline have been shown to be safe¬ ly infusable into cows' udders without stimulation of any immune response by the animals. C10₂ is capable of being reduced to lower oxidation states, such as chlorite and chloride, upon contact with organic materials, but is less reactive than, for example, hypochlorite, and a volume of C10₂-containing saline is expected to retain significant antimicrobial activity when used to flush out organic debris from an incised area. Appropriate use levels of C10₂ will vary, depending on the amount of organic detritus in the site, but generally concentra¬ tions of about 5 to 250 ppm in approximately isotonic solution can be used with good effect.

It is a further advantage of the invention that C10₂ is a broad-spectrum antimicrobial, capable of rapid destruction of aerobic and non-aerobic, gram-positive and gram-negative bacteria, as well as fungal organisms and viruses. It is particularly suitable for inclusion in a surgical irrigant or flush, since there is a diversity of microorganisms that could contact exposed areas during surgical procedures. The unique benefit of C10₂, in addition to its high cidal capacity, is its ability to interfere with the biochemical processes that lead to augmented collagen production. As such, it can signifi¬ cantly suppress undesirable adhesions that often form between incised tissues during the course of post-surgi¬ cal healing.

The method of the invention employs the broad- spectrum antimicrobial, chlorine dioxide (C10₂) , typically provided from chlorine-dioxide generating compounds, in solution for body cavity, catheter, and wound disinfec¬ tion. Preformed chlorine dioxide is too unstable to be stored in neutral disinfectant solutions for any signifi¬ cant period of time. The methods of the invention can be employed as a treatment for peritonitis, as a prophylac¬ tic or therapeutic rinse for people undergoing peritoneal dialysis, as a surgical irrigant, and as a disinfecting ear flush.

In contrast to antibiotics, systemic absorption of the compositions of the invention is minimized in treat¬ ments using them because of the inorganic nature of chlo¬ rine dioxide, and its reductive degradation to chloride as a result of its interaction with organic matter (in¬ cluding bacteria, fungi, and viruses) . It is important to note that materials may be non-inflammatory (i . e . , not provoke inflammation) but not anti-inflammatory (i.e., counter the effects of inflammation) . Chlorine dioxide has been found to be non-inflammatory, by virtue of being infusible into the peritoneal cavity and other body cavi- ties without evoking the inflammatory response, as well as being anti-inflammatory; see the copending U.S. ap¬ plication serial number 08/133,465, filed October 8, 1993, and parent case U.S. Pat. No. 5,252,343, cited and incorporated by reference above. In order to utilize the germ-killing and non-inflammatory qualities of chlorine dioxide, it is preferable to isolate it from chlorites and its acidic form, chlorous acid (which have detrimen¬ tal cytotic effects) . To minimize the negative effects caused by chlo¬ rite (and chlorous acid in lower pH solutions) , techni¬ ques are employed which preferably either a) deliver the soluble chlorine dioxide gas in a solution relatively free of harmful chlorite, or b) employ a pre-infusion chemical reaction whereby the chlorite species has sub¬ stantially converted to chlorine dioxide leaving rela¬ tively little chlorite remaining. In both cases, the relative molar ratio of chlorine dioxide to residual chlorite is at least 5:1, typically at least 7.5:1, and preferably at least 10:1. The concentration of chlorine dioxide in the infusate or irrigant typically varies from about 5 ppm ( g/liter) to about 1000 ppm in most embodi¬ ments, and is generally at least about 10 ppm. One em- bodiment employs from about 5 to about 75 ppm C10₂; as mentioned above, this is especially efficacious in peri¬ toneal dialysis solutions. Another employs from about 5 to about 250 ppm C10₂; this is especially suitable as a post-surgical irrigant. A third particularly useful as a surgical irrigant employs from about 40 to about 600 ppm C10₂.

As addressed below, the concentration requirement depends, to a significant degree, on the type of use for which the solution is employed, and the total volume used, since it is the total quantity of chlorine dioxide (i.e., concentration times volume) that is critical to the goal of overcoming the neutralizing effects of organ¬ ic matter in the wound in order to achieve the antimicro¬ bial and beneficial healing effects of chlorine dioxide.

Any means may be used for the preparation of a chlorine dioxide composition for use in the method of the invention. In many embodiments, a chlorine dioxide gen¬ erating compound is reacted with an acid in an aqueous solution. Exemplary chlorine dioxide generating com- pounds are water-soluble chlorites such as alkali metal chlorites, alkaline earth metal chlorites, and mixtures of these. Sodium chlorite is employed in preferred em¬ bodiments.

In conventional means of producing chlorine diox- ide solutions, a mineral acid is reacted with a chlorite such as sodium chlorite at such concentrations as to pro¬ vide rapid evolution of chlorine dioxide. Typical miner¬ al acids include, but are not limited to, sulfuric acid, hydrochloric acid, phosphoric acid, and the like. Such admixture, however, results in a very acidic solution that requires neutralization before use. Typical solu¬ tions are neutralized to a pH of from about 5 to about 7.5 prior to use.

Alternatively, a chlorine dioxide generating co - pound such as the chlorites mentioned above can be react¬ ed with a weaker acid, such as an organic acid having a pK of from about 2.8 to about 4.2. Typical acids include lactic acid, citric acid, malic acid, glycolic acid, mandelic acid, tartaric acid, and mixtures thereof. One embodiment employs lactic acid; the lactate so formed may enhance angiogenesis, collagen synthesis and deposi¬ tion, and modulate the response of fibroblasts to growth factors in the wound as summarized by Ninikowski, J., et al . , in Janssen, H. , et al . , eds., Wound Healing, Wright- son Biomedical Publishing Ltd, Petersfield, U.K., 1991, pages 169 to 170. In embodiments where these effects are not desired, organic acids other than lactic acid are employed.

The preparation of these types of formulations is set out in U.S. Pat. No. 4,986,990 to Davidson and Kross, the disclosure of which is hereby incorporated herein in its entirety by reference. In that patent, concentra¬ tions of sodium chlorite and activating acid are both below about 0.01- 0.02%, in isotonic saline. Such solu- tions have been found to be appropriate for use in the uterine infusion treatment of the present invention for chlorine dioxide levels up to about 125 ppm. The solu¬ tions may require pH adjustment, e. g. , to about 5 to about 7.5. The reactions, upon admixture, are virtually complete within several minutes, and can generate chlo¬ rine dioxide solutions in excess of 40 ppm with solution pH's compatible with the peritoneal cavity. When higher levels of chlorine dioxide are required, stronger acids and higher levels of chlorite may be used, with subse¬ quent neutralization prior to infusion. Typical solu¬ tions can contain up to about 1000 ppm chlorine dioxide, but higher concentrations, e. g. , up to about 5000 ppm chlorine dioxide are employed in some embodiments.

The inclusion of small amounts of certain activat¬ ing sugars (e.g., ribose, galactose, mannose) in the formulation, for example, at levels at or below about 1%, can further increase the speed and efficiency of chlorine dioxide formation. It has been found that this reaction, with or without the addition of sugar triggers, can pro¬ vide the requisite chlorine dioxide-to-chlorite molar ratios of at least 5:1 that are necessary to limit tissue irritation.

The composition may contain chloride ion, which is typically in the form of an alkali^' or alkaline earth metal salt. For sodium chloride, for example, concentra¬ tions can range between about 0.5% to 1.5% by weight; use of other salts requires an appropriate weight percent adjustment. In solutions below pH about 7, chloride ion causes chlorite ion to decompose in an accelerated man¬ ner, via the degradation of chlorous acid to form chlo¬ rine dioxide. Preferred embodiments of the invention employ solutions that are approximately isotonic to the peritoneal cavity or peritoneal dialysis solutions. In another embodiment, chlorine dioxide formation in the reaction between chlorite and weaker acids is catalyzed by heat-activated saccharides. Heat-activated saccharides are prepared by heating saccharides to a temperature of from about 50"C to about 150°C for at least about 1 minute, typically from about 5 to 240 min¬ utes. Some heat-activated saccharides are prepared by heating to about 75°C to about 110°C for about 20 to 120 minutes. Exemplary saccharides include, but are not limited to, glucose, galactose, mannose, ribose, rham¬ nose, and disaccharides such as sucrose, lactose, and maltose, and mixtures thereof. Glucose is preferred in one embodiment. Heat-activated saccharides useful in these embodiments of the invention are described in grea- ter detail in U.S. Pat. No. 5,019,402 to Kross and

Scheer, the disclosure of which is hereby incorporated herein in its entirety by reference.

The chlorine dioxide solutions are generally buf¬ fered mixtures that maintain the irrigant at a pH compat- ible with the peritoneal or other cavity. The pH typi¬ cally varies between about 4.5 or 5.0 and about 7.5. The solutions can contain other ingredients typical in washes and irrigants such as, for example, wetting agents (such as nonylphenoxy polyoxyethylene (9)), soothing emol- lients, and the like. Suitable carriers are chosen for their ability to dissolve or disperse chlorine dioxide as well as provide a composition conducive to infusion or irrigation. Many such compositions are known in the art, and can include thickening and emulsifying agents and the like, and such carriers are referred to herein as pharma¬ cologically acceptable carriers.

The solutions may be prepared immediately before infusion or irrigation in one embodiment. In another em¬ bodiment, the chlorine dioxide solution may be prepared and stored below a pH of about 5.5. For more particulars about storage, including storage of components and mixing of these prior to use, reference is made to U.S. Pat. Nos. 4,986,990 and 5,019,402, cited above, and references cited therein. Briefly stated, for the delivery of pre- formed aqueous chlorine dioxide, the following criteria should be met: 1) a storage pH below about 5.5 to mini¬ mize the degradation of chlorine dioxide to chlorite and other species; 2) a concentration of sodium chloride or equivalent material sufficient to render the solution approximately isotonic (e. g. , from about 0.80 to about 1.0% NaCl) ; 3) a package container that is virtually impermeable to, and non-reactive with, chlorine dioxide, such as glass and certain grades of polyacrylonitrile and polyvinylidene chloride. Immediately before the infusion or irrigation, a suitable buffer is typically added to these solutions. Formulations for particular applica¬ tions such as use in peritoneal dialysis and catheter cleansing are discussed more fully below.

The volume of infusates varies with the size of the animal and the degree of infection or potential in¬ fection, and can range between about 10 milliliters to about 5 liters. Example volumes are given hereinafter.

When solutions of the invention are infused into the peritoneal cavity, they are well tolerated, producing no noticeable irritation effects in the animals tested. Infusions with solutions of the invention are especially efficacious in the treatment of peritonitis.

Another important feature associated with the use of chlorine dioxide infusions or irrigations for the con- trol of peritonitis and related disorders is its fairly rapid reduction to chloride ion, which is a common compo¬ nent of body fluids and tissues in general. This reduc¬ tion occurs by interaction with organic matter, including bacteria. Topical agents that are non-toxic to cells without systemic absorption are especially needed for the destruction of potentially resistant isolates and for the treatment of dialysis patients who have recurrent infec¬ tions.

Since a sufficient excess of the chlorine dioxide provides microbiocidal activity at a more rapid rate than the rate of depletion of the molecule's oxidizing power by the organic environment, some antimicrobial efficacy may be achieved from the chlorine dioxide despite its rapid reduction by organic matter. Thus, a chlorine dioxide infusion into the peritoneal cavity which may contain significant amounts of organic material (e.g., mucus, serum, and sloughed cells) can still provide anti¬ microbial activity.

Both reduction of wound organic matter with the chlorine dioxide and longer contact time increase the antimicrobial effectiveness of the chlorine dioxide solu¬ tion. These data suggest that the infusion of a suffi¬ cient volume of a chlorine dioxide solution into a wound such as a peritoneal cavity wound, to overcome residual quantities of materials present during infection, could destroy microorganisms present in the environment. In some embodiments, volumes of 1 to 4 liters are employed in repeated irrigations during surgery. Higher chlorine dioxide concentrations in these volumes would similarly enhance the cidal activity, and are particularly advanta¬ geous in the cleansing of catheters. Since residence times of such infusions can be lengthened, there would be ample time for the antimicrobial to operate before being chemically neutralized.

The ability of the chlorine dioxide solutions to bring about a marked reduction in Staphylococcus epider¬ midis biofilms frequently observed on the surface of C.A.P.D. and C.C.P.D. catheters described in greater particularity below is significant. Also significant are studies illustrating the ability of oxychlorines to mini¬ mize collagen formation (noted by Kenyon, et al . , cited above, in epidermal tissue, who reported a 50% drop in collagen formation) , and scavenge free radicals involved in wound repair as set out below. Thus, irrigation of wounds with combined lactic acid and chlorite solutions significantly provides not only disinfection, but pro¬ motes healing and epithelization by minimizing collage- nous scar formation.

Use of the chlorine dioxide solutions of this invention, with their rapid degradation to chloride salts, further allows for their application as a prophy¬ lactic treatment, which is especially useful in irriga- tion of surgical sites, peritoneal cavity surgery, and in the cleaning of indwelling peritoneal catheters. Infu¬ sion or irrigation with chlorine dioxide solutions could significantly reduce the impact of microbial invasion of the peritoneum.

EXAMPLES

The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard. Unless otherwise indicated, all parts and percentages are by weight, and are given for the particular processing step described.

Example 1

This example compares and contrasts the cidal effectiveness of compositions of the invention with other disinfectants and antibiotics against Staphylococcus epidermidis biofilms, which are frequently observed on the surface of C.A.P.D. peritoneal catheters even in the absence of peritonitis or exit site infections. A strain of S. epidermidis with characteristic abundant slime production, and derived from a human source, is used to prepare the biofilms. The isolate is fully sensitive in the fluid phase to all commonly used antibiotics as assayed using routine test methods. Ali- quots of the bacteria are frozen at -70°C in 10% glycerol broth or propagated on 5% defibrinated horse blood agar. Standardized biofilms are formed on soda glass microscope slides or slides coated with Silastic^® (the basic materi- al of indwelling catheters; see Cecil 's, cited above, on page 542) , placed in Petri dishes containing 20 ml of tryptone soya broth (Oxoid Ltd. , England) , seeded with 10* cfu S. epidermidis and incubated for 18 hours at 37°C. Reproducible uniform and confluent S. epidermidis bio- films of minimal density are formed.

Antibiotic activity against S. epidermidis bio¬ films are determined using a supravital dye which acts as an electron acceptor in coupled oxidative systems in biofilm bacteria. The method allows the bacteria to be examined undisturbed within the biofilm matrix in situ .

Briefly stated, the biofilms are exposed to antibiotic solutions and incubated at 37^βC for varying periods of time. Positive (viable, 1% peptone water) and negative (sterilized, 4% formol-peptone) controls are included in all experiments. After a designated exposure at 22°C for varying periods of time between 15 seconds to 6 hours, the biofilms are rinsed twice in sterile water and al¬ lowed to drain. They are then placed in a flat agar medium incorporating substantive dyes and incubated at 37'C for 4 hours, then 24 hours. Viability is assessed visually by a change in dye color and confirmed after 24 hours by mechanically scraping and subsequently culturing the biofilms. Sterilization is indicated by the absence of color changes and confirmed by culture of both intact and mechanically fragmented biofilms. Inhibition of growth is indicated by a lack of color change after a 4- hour incubation and confirmed by a positive color change by 24 hours.

Using this technique, the following results are obtained:

Test Formulation Exposure Time (22°C) for Film Sterilization

50 ppm C10₂ Solution 5 seconds

Dakin's Solution (0.5% Hypochlorite) 5 seconds

Povidone-Iodine (10%) 5 seconds

Hydrogen Peroxide (3%) 10 minutes

4% Formaldehyde/Saline 3 hours

Rifambin (100 μg/ml) 6 hours

38 Other Antibiotics^* >24 hours

^*None of the following antibiotics showed any activity against the bacteria after a 24 hour exposure to concen¬ trations shown in parenthesis: amikacin (60 μg/ml) , ampicillin (30 μg/ml) , bacitracin (20 μg/ml) , Bactrim^® (25 μg/ml) , cefadroxil (30 μg/ml) , cefamandole (60 μg/ml) , cefazolin (30 μg/ml) , cefoperazone (30 μg/ml) , cefotaxime (30 μg/ml) , cefsulodin (30 μg/ml) , ceftazidime (30 μg/ml) , cefuroxime (30 μg/ml) , cephalexin (30 μg/ml) , chloramphenicol (20 μg/ml), ciprofloxacin (12.5 μg/ml), clindamycin (20 μg/ml) , cloxacillin (20 μg/ml) , erythro- ycin 45 μg/ml) , fusidic acid (30 μg/ml) , gentamicin (30 μg/ml) , imipenem (20 μg/ml) , moxalactam (30 μg/ml) , neo- mycin (30 μg/ml) , norfloxacin (20 μg/ml) , novobiocin (60 μg/ml) , oleandomycin (45 μg/ml) , penicillin G (25 μg/ml) , rifampin and clindamycin or rifampin and gentamicin or rifampin and tetracycline (at concentrations of 20 μg/ml rifampin and 10 μg/ml other antibiotic) , spectinomycin (20 μg/ml) , streptomycin (20 μg/ml) , subactam and ampi- cillin (20 μg/ml each) , tetracycline (60 μg/ml) , tobra- mycin (20 μg/ml) , trimethoprim-sulfamethoxazole, and vancomycin (60 μg/ml) .

The materials in the table which are as effective as the 50 ppm Clθ₂ solution are ones that would be con¬ sidered to be too irritating and/or corrosive for use as an irrigant in a peritoneal cavity. The 50 ppm C10₂ solution, on the other hand, is well tolerated by mamma¬ lian tissue, as, for example, when used as an eye irri- gant, to treat bacterial infections induced in rabbits, to infuse into a cow's udder, or as a uterine douche for mares and cows in the prevention and treatment of endome- tritis.

Moreover, the 0.5% hypochlorite will also be sub- ject to significant loss of activity as a result of its interaction with organic materials in peritoneal fluid. Hydrogen peroxide, at a concentration of 3%, requires a contact time 1200 times longer than the 50 ppm C10₂ to de¬ stroy the organisms in the biofilm, and also is likely to be too corrosive for infusion into the peritoneum. The

4% formaldehyde/saline solution is similarly inappropri¬ ate for such treatment, and would take more than 2000 times longer for sterilization. The best antibiotic of those tested, rifampin (Rimactane^®, obtained from Ciba- Geigy) , took 6 hours to accomplish the same steriliza¬ tion. Thus, the use of an isotonic C10₂ solution for eliminating the focal point of recurring peritoneal in¬ fections in patients undergoing Continuous Ambulatory Peritoneal Dialysis (C.A.P.D.) or Continuous Cycling Peritoneal Dialysis (C.C.P.D.) is particularly appropri¬ ate.

This conclusion is underscored in studies that assess the efficacy of methods employed using composi- - 27 -

tions of the invention in combination with an infusate used in C.A.P.D. and C.C.P.D. practice, Inpersol^®, Abbott Laboratories, Montreal, Canada, which contains 4.25% dextrose and has a pH of 5.2. A 50 ppm C10₂ solution is diluted from 1:2 to 1:10 with normal saline (denoted below as NS) , Inpersol^® peritoneal dialyzate (denoted as PD) , and peptone-saline (a standardized protein digest obtained from Oxoid, denoted as PS) . Four percent formol saline, a known potent environmental disinfectant of considerable toxicity, is used as a reference. The re¬ sults are expressed as the minimal time of exposure at 22°C to effect sterilization of S. epidermidis biofilms:

50 ppm C10₂ Formol Saline

Dilution NS PD PS NS PD PS

1/2 15 sec 5 sec 1 min 8 hr 6 hr 8 hr

1/3 30 sec 1 min 3 min 12 hr 6 hr 12 hr

1/5 5 min 5 min 1 hr 18 hr 24 hr 24 hr

1/7 15 min 7 min 4 hr 24 hr 24 hr >24 hr

1/10 1 hr 10 min 24 hr 24 hr 24 hr >24 hr

It can be seen that though diluting with the peptone solutions has an inhibitory effect on the sterilizing activity of C10₂ solution, the peritoneal dialysis solu¬ tion markedly enhances antimicrobial activity of the C10₂ solution.

Example 2

To test whether methods of the invention can con¬ trol the inflammatory response, isolated polymorphonu¬ clear leukocytes, which are among the first cells to be found at a wound or a site of potential infection, are studied with respect to their response to chlorine diox- ide solutions and compared with a control containing ibuprofen, a known non-steroid anti-inflammatory com¬ pound. The choice of in vitro assay is chemotaxis.

Isolated polymorphonuclear leukocytes are studied with respect to their response to a C10₂ solution varying in concentration from 3 to 300 ppm and an ibuprofen con¬ trol. Cellular response and integrity (chemotaxis) is assayed in vitro after incubating with both compounds by measuring lactate dehydrogenase activity and/or release concurrently with observing morphology using scanning electron microscopy and/or transmission electron micros¬ copy.

The results are graphically illustrated in Figures 1 to 3. The x-axis in the figures show dilutions of a 0.5 mg/ml ibuprofen solution, and chlorine dioxide solu¬ tions prepared by combining lactic acid with sodium chlo¬ rite and diluting to yield chlorine dioxide concentra¬ tions as follows: 1:1 is 300 ppm C10₂, 1:10 is 60 ppm C10₂, 1:20 is 30 ppm C10₂, 1:50 is 12 ppm C10₂, 1:100 is 6 ppm C10₂, 1:150 is 4 ppm C10₂, and 1:200 is 3 ppm C10₂.

Figure 1 shows the data obtained from the lactate dehydrogenase assays. The figure plots lactate dehydro¬ genase activity, expressed as % of a positive control, versus dilutions of chlorine dioxide solutions or ibupro- fen, and shows the effect of chlorine dioxide or ibupro¬ fen on cell integrity with or without incubating with 0.05% bovine serum albumin (BSA). In the figure, •—• shows the results of incubating chlorine dioxide and BSA for 15 minutes, adding cells, centrifuging and then as- saying the supernatant, and •—• plots data obtained by incubating chlorine dioxide and cells for 15 minutes and then adding BSA, centrifuging and assaying the superna¬ tant. For comparison, ■—■ shows ibuprofen incubated with BSA for 15 minutes, followed by adding cells, cen¬ trifuging and assaying, and ■—I, incubation with cells for 15 minutes followed by adding BSA, centrifuging and assaying. A negative control is denoted by 0.

Figure 2 shows chemotaxis when chlorine dioxide and ibuprofen are run concurrently in a chemotaxis assay. The figure shows the effect of 0.10% bovine serum albumin on the cells. The albumin is added to the cells and incubated 15 minutes, and then cells are mixed in equal volumes with dilutions of chlorine dioxide or ibuprofen and incubated; 250 μl of each mixture is then applied to the top well of a modified Boyden chamber. The y-axis plots percent of a positive control. In the figure, t—• and •—• show the chlorine dioxide results, ■—■ and ■—■ show ibuprofen results, and 0 is a negative control.

Figure 3 shows the results of the chemotaxis as¬ say. It demonstrates the effect of the difference in the sequence in which reagents are added and incubated. In one experiment, for each dilution, there is a known vol- ume of chlorine dioxide (•—•) or ibuprofen (■—■) to which bovine serum albumin is added to a final concentra¬ tion of 0.05%, the mixture is incubated for 15 minutes, and equal volumes of cells are added to each to a final concentration of 2.5 x 10⁶ cells/ml. In a second experi- ment, for each dilution, a known volume of cells contain¬ ing approximately 6.0 x 10⁶ cells/ml and an equal volume of chlorine dioxide (•—•) or ibuprofen (■—■) are incu¬ bated for 15 minutes and then bovine serum albumin is added to a final concentration of 0.05%. In each assay, 250 μl is applied to the top well of a modifed Boyden chamber. A negative control is shown as 0.

It can be seen that the C10₂ solution affected both the quality and morphology of polymorphonuclear leuko¬ cytes. Example 3

Since recent studies indicate that the superoxide radical is the active form of oxygen in the prolyl and lysyl hydrolyase reactions during collagen synthesis, this example assesses the effects that compositions of the invention have on free radicals.

Free radicals are generated in vitro by the action of xanthine oxidase (XO) on xanthine. The generation of free radicals is monitored by the reduction of ferricyto- chrome C by the system. The action of chlorine dioxide is compared with the scavenging action of the well known free radical scavengers superoxide dismutase (SOD) and catalase.

C10₂ (75 ppm) is generated by mixing 0.25% chlorite and 1.4% lactic acid. One hundred-fold dilutions are tested within one minute after mixing using electron paramagnetic spectroscopy (ESR) and a LKB luminometer. A strong free radical signal is generated using both meth- ods, and the signal is quenched using the free radical scavengers.

For the ESR assay, the chlorine dioxide solution is frozen in liquid nitrogen to -167°C and electron spin resonance analyzed using a Varian E3 instrument.

The luminometer assay utilizes a modified luminol assay for radical detection. Luminol (LH₂) reacts with free radicals (R- ) as follows:

LH₂ + R- - LH- LH* + 0₂ → LOO^" (endoperoxide) LOO^" - L₂ + AP* (aminophthalate dianion, active state)

AP* - AP (inactive) + hv The pH optimum for the reaction is 10 to 12. The assay is standardized with an enzymatic system in which the generation of free radicals could be quantified by mea¬ suring generation of -0₂" , obtained with the following reaction, hypoxanthine + xanthine oxidase - urate + -0₂ ^" by the reduction of ferricytochro e C.

The experiments are divided into three groups. In the first group, 50 μmoles of xanthine, 0.0217 units/mg XO, 0.1 mM EDTA are mixed in a total volume of 1.0 ml

Hank's Balanced Salt Solution (HBSS) . This generated 64 ± 8 nmoles oxygen. In the second group, 0.3125 units/mg SOD and 1,300 units catalase are added to the reagents employed in group 1; this yields 19.4 nmoles oxygen. Significant inhibition of free radical formation by the SOD and catalase is thus observed. In the third group, chlorine dioxide is substituted for the SOD and catalase in the same system; a chlorine dioxide concentration of about 7.5 ppm inhibits free radical formation by 74% and at a concentration of 15 ppm, by 100%.

It can be seen from the data that solutions of the invention clearly scavenge free radicals.

Example 4

A lactic acid-activated chlorite solution is prepared by mixing 0.25% sodium chlorite with 1.5% lactic acid, which yields 150 to 600 ppm Clθ₂. The solution inhibits fibroblast proliferation in rat fetal lung fi- broblasts.

If the solution is prepared in the presence of a phosphate buffer which immediately neutralizes the lactic acid, the formation of chlorine dioxide is inhibited in the system. The solution has no inhibitory effect with - 32 -

rat fetal lung fibroblasts. Thus, it is not chlorite ion that is operative, but C10₂.

The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Claims

1. A method for the treatment or prevention of microbial infections in a mammal having a peritoneal cavity cathe¬ ter comprising infusing into the cavity or around the cathether an effective amount of a composition comprising a pharmacologically acceptable carrier and chlorine diox¬ ide in an amount ranging from about 5 ppm to about 1000 ppm, the molar ratio of chlorine dioxide to any residual chlorite in the composition is at least 5:1, and the pH of the composition is compatible with the peritoneal cavity of the mammal.

2. A method according to claim 1 wherein the pharmaco¬ logically acceptable carrier is peritoneal dialysis solu¬ tion.

3. A method according to claim 2 wherein the composition contains from about 5 to about 75 ppm chlorine dioxide.

4. A method according to claim 1 wherein the chlorine dioxide is produced by reacting an acid with an alkali metal chlorite.

5. A method according to claim 4 wherein the chlorite is sodium chlorite and the composition has a pH of about 5 to about 7.5.

6. A method according to claim 4 wherein the acid is an organic acid selected from the group consisting of citric acid, malic acid, lactic acid, mandelic acid, tartaric acid, and mixtures thereof.

7. A method according to claim 6 wherein the acid is lactic acid.

8. A method for irrigating a peritoneal cavity wound to disinfect the wound and promote healing, comprising in¬ fusing the wound with a composition comprising about 5 ppm to 1000 ppm chlorine dioxide in a pharmaceutically acceptable carrier, wherein the chlorine dioxide is gen¬ erated by reaction of an acid with a water-soluble chlo¬ rite selected from the group consisting of alkali metal chlorites, alkaline earth metal chlorites, and mixtures thereof, provided that the molar ratio of chlorine diox- ide to chlorite in the composition is at least 5:1.

9. A method according to claim 8 wherein the wound is a surgical wound.

10. A method according to claim 9 wherein the composi¬ tion contains from about 5 to about 250 ppm chlorine dioxide.

11. A method according to claim 9 wherein the composi¬ tion contains from about 40 to about 600 ppm chlorine dioxide.

12. A method according to claim 9 wherein the wound con- tains an indwelling catheter.

13. A method according to claim 12 wherein the composi¬ tion contains from about 5 to about 75 ppm chlorine diox¬ ide.

14. A method according to claim 8 wherein the composi¬ tion is adjusted to a pH of about 5 to about 7.5 after the reaction.

15. A method according to claim 14 wherein the acid is a mineral acid selected from the group consisting of sulfu- ric acid, hydrochloric acid, and phosphoric acid.

16. A method according to claim 14 wherein the acid is an organic acid selected from the group consisting of citric acid, malic acid, lactic acid, mandelic acid, tartaric acid, and mixtures thereof.

17. A method according to claim 14 wherein the composi¬ tion further comprises isotonic chloride ion.

18. A method according to claim 8 wherein the reaction is carried out in the presence of a heat-activated sac¬ charide compound prepared by heating a saccharide select¬ ed from the group consisting of glucose, galactose, an- nose, ribose, rhamnose, lactose, sucrose, maltose, and mixtures thereof, to a temperature of from about 50°C to about 150°C for at least about 1 minute.

19. A method for treating or preventing a microbial in¬ fection in the peritoneal cavity of a mammal comprising infusing into the cavity an effective amount of a compo¬ sition comprising chlorine dioxide in a pharmacologically acceptable carrier, wherein the molar ratio of chlorine dioxide to chlorite in the composition is at least 5:1.

20. A method according to claim 19 wherein the composi¬ tion contains from about 5 ppm to about 1000 ppm chlorine dioxide.

21. A method according to claim 19 wherein the chlorine dioxide in the composition is produced by the reaction of a chlorine dioxide generating compound and an acid.

22. A method according to claim 21 wherein the chlorine dioxide generating compound is a water-soluble chlorite selected from the group consisting of an alkali metal chlorite, an alkaline earth metal chlorite, and mixtures thereof.

23. A method according to claim 22 wherein the chlorine dioxide in the composition is produced by a process se¬ lected from the group consisting of:

(a) reaction of a chlorite with a mineral acid and adjustment of the pH to about 5 to about 7.5;

(b) reaction of a chlorite with an organic acid having a pK of from about 2.8 to about 4.2; and

(c) reaction of a chlorite with a saccharide, which has been heat-activated by heating the saccharide to a temperature of from about 50°C to about 150°C for at least about 1 minute, in a solution in the presence of an organic acid having a pK of from about 2.8 to about 4.2 and a pH below about 5.5.

24. A method according to claim 23 wherein the organic acid is lactic acid.

25. A method according to claim 23 wherein the saccha¬ ride is selected from the group consisting of glucose, galactose, mannose, ribose, rhamnose, lactose, sucrose, maltose, and mixtures thereof.

26. A method according to claim 23 wherein the chlorite is sodium chlorite.