CA2148942C - M-edta pharmaceutical preparations and uses thereof - Google Patents

Abstract

Disclosed are pharmaceutical compositions of a mixture of minocycline and EDTA (M-EDTA) and methods of using the compositions in maintaining the patency of a catheter port. Methods for inhibiting the formation of polysaccharide-rich glycocalyx (such as the glycocalyx of staphylococcal organisms) are also provided using an M-EDTA
solution. A solution including minocycline, EDTA
or both, may also be used to pretreat a medical device to prevent adherence of infectious organisms, such as S. epidermidis and S. aureus. The compositions destroy and prevent the formation of polysaccharide-rich glycocalyx. Methods for treating infections of S. epidermidis and S. aureus where glycocalyx formation are provided with an M-EDTA
solution. The minocycline and EDTA solutions are included together within a pharmacologically acceptable carrier solution, such as saline.

Description

W~ 94f1(1838 . ~ ~ ~ ~rcrfv~93f10893 DESCRIPTION
N-EDTA PFiARMACETJTICAI. PREPARATIONS AND TISES TZ3EREOF
EACRGRO'UND OF THE INVENTION
l a '.C IELD ~~ T11E ~NTI~N
The present invention relates to indwelling medical articles, such as catheters, which may also be flushed or coated with a microbial-inhibiting pharmaceutical preparation. The in.~rention also relates to pharmaceutical preparations useful in maintaining catheter patency and preventing infection. Methods of using the pharmaceutical preparation of the invention in the management and ma~.ntenance of a vascular catheter are also related to the present disclosure.
~ a ~ACI~t"rR~I1ND OF °~E REIi~TED ART
Indwelling medical devices including vascular catheters hare become essential in tie management of hospitalized or chronically ill patients . UnfortunatelyT, vascular catheters have become the major source for ~~ hospital-aac~uired sepsis. ~3er_ce, the benefit derived from indvaelling medical devices such as vascu3.ar catheters is often upset by infectious complications.
Thrombotic occlusions of the lumen of central venous G~theters t~C);is ~noth~~ complication that, will often 3p lead to the removal of catheters.
The current standard care of catheters includes flushing the lumen of the catheter with heparin.
However; heparin'has no antimicrobial ac~iv~ay. Thus, 35 infections, as well as thrombotic occlusion, continue ~o occur frequently despite the prophylactic use of heparin flushes. Knowledge of the pathogenesis and micrabiology t., ,. . . .. ::.: .: , : -;,.. ,;_-:: , . -,.: . .. ,. ,. . ,.. , , ;:.. . ., , . ..;: . ;~ . , - - ..;: . . ,; ;~ ,;
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i~V~ 94/1038 PCf/US93>10893 ~-c~f~central venous catheter-related infections is essential in order to provide effective prevention for this problem. Three essential factors must be considered in controlling for catheter colonization by infectious microbes. The first is controlling the availability of microorganisms tha'~ adhere to the inert catheter surface.
Such microorganisms typically include staphylococci and candida. The second is controlling the produ;coon of a polysaccharide by adherent known as organisms fibrous ~.0 glycocalyx. Production of the glycocalyx is~~essential for the adherence and integrity cf these organisms. The third is control of the formation of a thrombin sheath by the host, which acts to engulf the catheter. The thrombin sheath pro~rides the microorganisms a sticky substrate for enhanced ariherence to the catheter, and thus; continued colonizatgon and infection at the catheter site. The present invent~r~ herein disclose an Nt-EDTA solution unique in its ability to inhibit all three of these essential conditions, and thus provide effective methods far controlling catheter-related ;refection and onset thereof:
Staphy.3ococcus egac~ermidis and S. aureus account for 75a of CVC related infections. Candida species account for another la% to 15% of such infections. The use of antistaphyloco~cal antibiotics to prevent these 3~afectiQns has been found to reduce CVC related bacterial infections, but only at the expense of the occurrence of higher ,rated o~ fua~g~l. (~andida~ infections. The fibrous glyc~ocalyx material produced by staphylococci and Canc3ida helps these organisms adhere and stick to catheter urfaces, thus exacerbating the problem of eliminating these types of infections after they have become established: ~'hese microbial biofilm layers are made of a fibxous glycocalyx material primarily polysaccharide in nature. The protective sheath provided by the glycocalyx at the infected site effectively prevents the elimination 1 1 fVO 9A/10838 .. ~ ~ ~ ~ ~ PCf/US93/1~893 and treatment of these infections. Preparations effective for destroying such a glycocalyx would, therefore, provide a solution far treating established catheter infections, particularly where a glycocalyx is already formed.
Compositionally distinct glycocalyx material is produced by a variety of different organisms. For example, a mostly protein glycocalyx produced by .
~.0 Hymenolepis diminuta (tapeworm? is reportedly eliminated ;
upon treatment with 0.02 i~I-EDTA or 3 ICI F~C112. This material, however, is compositionally distinct from the material of the glycocalyx formed by organisms that typically colona.ze.and cause catheter infection. For IS example, the glycocalyx of several staphylococcus species comprise primarily polysaccharides with only low to nondeductible levels of proteinl3 (Tojo ef aI. at pg.
7~.6,'Table 1). Glycocalyx of microorganisms common to catheter infecta.on are thus compositionally distinct from 20 the glycocal~rx of such organisms as the tapeworm, I~iymenolepis dim3nuta. A pharmaceutical preparation effecta.ve for reducing or eliminating glycocalyx of infectious microorganisms typically associated with catheter colonization axed infection has yet to be 25 identified. ' Infectious microorganisms will typically embed themselves in the protective layer of the glycocalyx, thus ~ ~providing ~ shield, or hiding place, that , protects , 30 staphylococci and fungi from the activity of the host's phagocytic cells: '13n agent or composition that would dissolve or prevent the glycocalyx (biofilm) formation of these clinically important pathogens would thus provide a major br~akthro~xgh in the prevention of typical catheter 35 related Staphyloc~cca.~ and Candida infections.
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w~ 9a"°314 ~ ~ 4 ~ PCT>iJS93/10893 There has also been observed to be a correlation between thrombogenesis and infection. Indwelling vascular catheters are typically engulfed by a fibrin sheath that subsequently acts to cover the internal and external surfaces of a catheter. This fibrin sheath provides such organisms as Staphylococea and Candida, ~f with an enhanced adherence capacity to the~.catheter surface. Unlike these particular microbes', gram-negative bacilli do not adhere well to fabrin and fibronectin. A composition that halts fibrin formation would thus be particularly useful in halting the colonization of these microbes at indwelling catheter sites.
Intraluminal colonization through a catheter hub also constitutes a prelude to catheter-related infections and septicemzas in long-term CVC. The inventors study presented herein of Iong~term C'VC patients (studied by quantitative catheter culture) demonstrates that CVC's with positive cultures as well as matched negative controls evidenced colonization.(as quantitated by EM) ahd biofilm formation of the internal surface at least twice greater"than that of external surface with catheters that stayed longer than 10 days in place. This data is from nontunneled, noncuffed percutaneous CVC.
For tunneled CVCs (Hickman/Eroviac) and ports, internal coloh.izatiox~ was even more prominent. The development of an anticoagulant pharmaceutical preparation effective against,stap~ylQcocci;,, fungi, and polysacchax°ide-rich glycocalyx formation, such as that observed during microorganism intraluminal colonization of a catheter hub, would provide a solution to the treatment and elimination of thrombogenesis and the septicemia associated with lor_g-term CVC.
EDT.A is an anticoagulant used in blood collection, and is also described as having an antibacterial and ,, ~ 2.4842 1~V~ 94/10838 PC'f/US93/10893 -S-antistaphylococcal effect (alone or in combination)1-3.
Ft.oot~ described the efficacy of EDTA in vascular catheters as an antibacterial agent as compared to heparin alone and as compared to a vancomycin-heparin preparation in vitro. These investigators found EDTA to have some bacteridcidal activity. However, no remedy or suggestion for treatment of how a microbial glycocalyx, such as that observed during an viva catheter-related infection, was observed ox described.
zo Glycopeptide antibiotics (vancomycin and teicoplanin) are active against staphylococci in va tra and in tissue. Vancomycin is currently the standard antibiotic used in the treatment of Staphylococcus epidermadis and resistant Staphylococcus aureus. .
Hovaever, th.es~ antibiotics are not active against adherent stapnylocoaci embedded in a biofilm layer, such as glycoe~lyx. In additionp while catheter flushing with such agents may destroy initial strains of staphylococci, such is typically not effective against tolerant and i;esistant strains of the organism that continue the infection. Flushing with an antibiotic preparation; such as ~r~comycin, would therefore- be of only limited therapeutic value agaa.nst catheter infection.
The ideal prophylactic agent or treatment for catheter maintenance would inhibit or eliminate the formation of polysaccharide-rich glycocalyx of microorganisms ~nd~ v~ould also a~nhibit staphylococci ,and fungi irzf ec tious growth.
It is an object of the invention to provide both an anti-staphylococcal and antifungal (anti-Candida) active agerat effective against free-floating as well as adherent 35 organisms embedded in biofilm, as well as to provide an anticoagulant agent and/or method that would prevent or inhibit the formation of polysaccharide-rich fibrous VVO 941I0~38 ~ 1 ~ $ g ~ 2 PC'T/LJS93/10893~ .
.. _6_ glycocalyx biofilm layer. Such a pharmaceutical agent would optimally prevent thrombotic occlusion of the catheter lumen and prevent thrombin formation.
Additional objects of the invention include providing an agent that could be given intraluminally without a toxicity concern t'o humans and to provide a method that would kill adherent Staphylacocca and Candida. Such methods would preferably not include th~v use of pharmaceutical agents typically used t'cs~treat non-~-~Y;, catheter related infections (such asw-.Vancomycin, Ampho B, '.. r.T
Or A~Oles).
The present invention demonstrates that a mixture of minocycline/disodium EDTA (referred to as M-EDTA) does fulfill all of the listed objects.
SR'Y' OF' 'f~iE INVENTION
The present invention provides a unique and effective pharmaceutical preparation that includes mixxocycline and EDTA. These mixtures are shown to be .
effective for maintaining the patency of a medical device, such as a catheter; in vi.vo. As used in the description of the gresent invention, 'tpatency~~ is defined as the state of being freely open or patulous, particularly a catheter opening that is unob~cured by the formation of a microorganismal polysaccharide-rich fibrous glyeocalyx. In a preferred embodiment, minocycline ,and ED'~A ; are included in the disclosed , preparations in pharmacologically effective amounts together in a pharmacologically acceptable carrier s~,lution.
The EDTA of the preparation provides potent 3~ glycocalyx inhibiting potential, while the minocycline at high concentrations has a fungicidal effect and a unique ability to penetrate a polysaccharide-rich glycocalyx ~IVV~ 94!10838 _ ~ ~ 4 ~ 9 ~ 2 PGTlUS93/10893 biofilm layer. The combination of minocycline-EDTA thus provides a uniquely effective anticoagulant, anti-microbial, glycocalyx inhibiting, antibacterial and antifungal agent for the prevention of thrombogenesis, microbial adherence and device-related infections.
The inventors demonstrate that minocycline has the surprising activity of penetrating a polysaccharide-rich microbial glycocalyx biofilm layer at least 6-fold more ~.0 effectively than vancamycin. The inventors also demonstrate that EDTA effectively prevents and dissolves polysaccharide-rich microbial glycocalyx formation at implanted catheter sites in vivo.
Tn still another aspect of the invention, methods for using minocycline as an antimicrabial agent are provided. Minocycline is demonstrated to kill adherent staphylococci (embedded in glycocalyx - Example 4?, and to be superior to vancomycin as an antibacterial agent (Table 3} ~.n actual 3n vivo txials.
For use in maintairain~ catheter patency, the phas:maceutical preparation of the invention may be afficaciousl~r used in conjunction with virtually any device in wh~:ch a clear path for the flaw of biological fluids is necessary. For example, such devices include a central venous catheter, a peripheral intervenous catheter, an arterial catheter, a Swan-Ganz catheter, a hemodaa.lysi~,, ~at~ete,r,; : an umbilical catheter, ., a percutaneous n,ontunneled silicone catheter, a cuffed tunneled central venous catheter as well as a s~,~utaneous central venaus port.
In a preferred embodiment, a solution of the M-EDTA
~a~, ~~ prepared containing a concentration of between about 10 to about 100 mg/mI EDTA (preferably between about 2O to about 60 mg/ml~ and between about 0.00. to _g_ about 100 mg/ml minocycline (preferably between about 2 and about 9 mg/ml). Most preferably, the preparation as a device or catheter flushing solution includes about 30 mg/ml EDTA and about 3 mg/ml minocycline. The minocycline may preferably be prepared as a reconstituted minocycline preparation, such as from a l00 mg vial .of minocycline commercially available (MinocinT"" Intravenous, Lederle (Carolina, Puerto Rico). The carrier solution, by way of example, may comprise saline, preferably sterile saline, available from commercial sources.
In another aspect of the invention, a catheter flushing solution is provided. Most preferably, the catheter flushing solution comprises a glycocalyx inhibiting concentration of EDTA and minocycline in a pharmaceutically acceptable carrier solution. More specifically, the concentration of EDTA in the flushing preparation is between about 1 to about 100 mg/ml with the concentration of minocycline being between about 0.001 to about 100 mg/ml in the preparation. Most preferably, the catheter flushing solution includes about mg/ml EDTA and about 3 mg/ml minocycline.
By way of example, the carrier solution of the 25 flushing preparation is saline, preferably sterile saline. The catheter flushing preparation of the present invention may advantageously be used to inhibit the formation of polysaccharide-rich glycocalyx. In this manner, infections characterized by the presence of a 30 polysaccharide-rich glycocalyx, such as a glycocalyx which is at least 50% polysaccharide by composition, may be effectively treated and/or eliminated.
A "glycocalyx inhibiting concentration" is defined for purposes of describing the present invention as a concentration of minocyclii~e, EDTA or a combination thereof effective to degrade, dissolve, or otherwise '\ ~'1~'~ 94110~3~ ~ ~ ~ g ~ 2 PC'~'fUS93/10$93 _9_ inhibit polysaccharide-rich glycocalyx formation. By way of example, a polysaccharide-rich glycocalyx is characteristic of established staphylococcal infections of S. aureus and S. ep.idermidis.
Another aspect of the present invention provides a method of preparing a biofilm-resistant device, as well as bio-film resistant devices that are coated with at least one of minocycline or EDTA. Most preferably the device is to be coated with a mixture of minocycline and EDTA. The method in one embodiment comprises coating a device with a coating preparation minocycline and EDTA.
While the method may be used to coat virtually any surface where glycocalyx formation is to be desirably a:nhibited, use of the method in coating a catheter device ~;s particularly envisioned. It is anticipated that the method will provide a device resistant to polysaccharide-rich (at least about 50% polysaccharide) glycocalyx formation, such as that characteristic of Staphylococci.
~y of a ~rar~.ety of catheters may be treated or coated according to the described method employing coating ..
techniques well known to th~se of skill in the art. By ~ay'of example; catheters that may be prepared arid treaded according to the invention include a central venous catheter and a triple lumen catheter. ' Tn a most preferred embodiment, the method for .
preparing a biofilm-resistant medical device using a phar~a~eutica~- prep~.r~tion of ;minocycline and,, EDTA, 3Ov Gom rises re arin a solution of at least one of P p p g minocycline or sodium EDTF~; most preferably in a biocompatible adherent coating carrier solution, and treating the surface of the medical device of interest with the solution for a period of time sufficient to 35 allow the formation of a film of the minocyeline, EDTA, or both to the urface of the device. Most preferably, ..:.;
~'C) 94/ ~ 0838 ~ ~ PCT/LJS93/ I 0893 -~.0-the method is to be used in preparing a biofilm-resistant coating on the surface of a catheter.
Alternatively, a coating of the M-EDTA preparation may be provided at the surface of a device by first treating the surface of the device with a;:surfactant.
Such surfactants include, by way of example, tridodecyl-methyl ammonium chloride (TDMACC). Commercially available catheters with such a surfactant already included on the surface are the Eioguard Cook catheters.
Accordingly, a device including a surface surfactant may be immersed in a solution of minocycline, EDTA or M-EDTA
for a period of time sufficient to allow the solution to permeate the device surface: By way of example, the present inventors have found that immersion of a device ih a solution containing about 60 mg minocycline and/or about 60 mg EDTA/m1 for about 15 minutes at room temperature is suff~.cient to provide a coatang of the so7.ution to the surface of the device . The device is 2d preferably air dried and gas sterilized prior to use.
As used in the description of the px'e~ent invention, a "biofilm-resistant" device or surface is defined as a surface or device that will prevent the adherence or ~5 g~.owth of o~°ganisms that produce polysaccharide-rich (about SO~s or greater polysaccharide) formations, such as those formations generally refexred to es a glycocalyx.
Organisms that produce such a glycocalyx include the S,~aphylococcal organisms, particularly the S. au~eus and 30 S. epidexmidis species.However, any organism that produces a polysaccharide-rich material would be equally in&~a.bited by the herein described devices, surface, pharmaceutical preparations and methods.
35 The present methods are particularly eff3c~cious for inhibiting polysaccharide-rich gZ.ycocalyx formation at a cathetex port. The method in one embodiment comprises ~1~~942 i3'~ 94!10838 ,~ PCI'1U~93110893 -11_ flushing the catheter periodically with a preparation including a glycocalyx-inhibiting concentration of at least one of EDTA and minocycline, pharmacologically acceptable carrier solution. In one aspect of the method, the glycoca~.yx°inhibiting concentration of EDTA
in the flushing solution is between about 0.01 mg/ml and about 100 mg/ml. The most preferred concentration of EDTA is about 30 rng/m1. According to one aspect of the described method, the catheter may be described as a tunneled catheter or an untunneled catheter. As part of a catheter maintenance regimen, the catheter most preferably is to be flushed with the aforedescribed preparation at least once every about 24 to about 48 ;.
hours.
In still another aspect of the method, the pharmaceutical preparation includes minocycline. Where included, the glycocalyx-inhibiting concentration of minocycline constitutes a c~ncentration of between about 0:001 mg/ml axed about 100 mg/m1. The most preferred concentration of minocycline is about 3 mg/ml.
particularly statedr the method for eliminating microbial glyeocalyx formation, particularly polysaccharide-rich (Sbaphyl'ococcal) glycocalyx formation, at a catheter lumen comprises preparing a solution comprising minocycline and EDTA in a carrier solution to provide an M-EDTA preparation, and flushing the catheter.;wi~th a, th~raPeutic~lly ,effective amount of thelM-EDTA preparation sufficient to reduce a pol)rsaccharide glycocalyx. In one embodiment, the M-EDTA
preparation includes a concentration of minocycline of .
between about 0.001 to about 1.00 mg/ml (preferably between about 2 and about 9 mg/ml) and between about 10 to about 100 mg/ml (preferably between about 20 to about 60. mg/ml) EDTA. The therapeutically effective amount of the afoxedescribed M-EDTA preparation would, therefore, VN~ 9411083 ~ ~ , PCT/U~93110893 1 2~ ~~9 -7.2-constitute between about 1 to about 10 ml (preferably about 2 to about 3 ml) of the aforedescribed solution.
According to the method, a volume of about 3 ml of the aforedescribed M-EDTA preparation containing about 30 mg/ml EDTA and about 3 mg/ml minocyc~ine is used per flush of ~ catheter or device, dep~riding on the voume of the catheter or device being monitored. For example, a ..
standard size tunneled CVC catheter (Hickman/Brovia? is 0 to be flushed periodically at least once every about 24 hours to about 48 hours with between about 2 to about 3 ml of the M-EDTA preparation. In a preferred aspect of the method, the catheter is to be flushed more frequently, such as.at least once every about 4 hours 1.S with the herein described preparations of M-EDTA.
The M-EDTA preparation will remain therapeutically effective for use as a catheter-flushing agent stored at a refrigerated temperature for at least 1 month after 20 formulation. In use, the M-EDTA solution should be brought to room temperature before use on an animal or patient.
The aforedescribed preparations have been found 25 effective in preventing the adherence and colonization of datheter surfaces by S. aureus, S. epidermid~,s, and fungi, a~ well as effective in both treating and .
eliminating already formed glyeoealyx formations of these infectious ,or~ana.sm~ . . , The anti-ma.crobial resistant medical devices of the gresent invention are further described as a medical device that is processed to include a coating of minocycline, of EDTA or of a combination of M-EDTA. Such 35 a coating would be particularly efficacious for medical devices that include at lest one opening or portal, as the coating is shown by the present inventors to be . :," ... _.;, :..,-. ,:-,. v:. . : .: . .: ;. ~: ~: , :. - , ; . .;;. ~'v: , ~ .
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2~4~942 ~~ W~ 9~/1~838 ' PC'f/~JS93/10893 effective for maintaining the patency of such medical device openings as that characteristic of an indwelling catheter irz ~rivo. The most preferred embodiments of the devices include a coating of M-EDTA. Coating solutions of the M-EDTA include about 60 mg minocycline and about 60 mg EDTA per ml.
Th9 following abbreviations are used in the -;.
descripticin of the present invention:
~. 0 , - y .
= Central Venous Catheters MRD = Modified Bobbins Device M-EDTA = minocycline-EDTA mixture D1Q/W = 10% Dextrose and Water BRIEF DEuCRI~TIf~DI' O~° TFiB DRP~SooTINGS
Figruse 1 - Scanning electron microscopy picture showing staphylococci and biofilm from a control catheter 20 segment that was exp~sed to slime producing S.
e,pic~e~-midis and later immersed in Dextrinase for 24 hours. (Example 6 results.) Figure 2 - Scanning electron microscopy picture 25 showing a layer of biofilm from a catheter segment that exposed to slims producing Ba epidermid.~s and later immersed i.n Urokinase for 24 hours. (Example 6 results.) Fa.gur~ ,3 , Sc~.nz~ing electron microscopy Picture 30 showing a representative ~~.ear surface from a catheter segxn~nt exposed to slime producing S. epiderrnidis and later immersed in EDTA for 24 hours. The wh~.t~ particles are dust partic3.es. (Example 6 results.) ~5 Figure ~ - A scanning electron~micrograph at high magnification showing coccal forms in a biofilm layer from a catheter segment coated with control solution ~V~ 9~/1U838 PCTILJS93/10893 -s~-(sa7s.~ e) and later exposed to slime producing S.
epiderxnidxs. (Example 7 results.) gic,~ar~ 5 - Lawer magnification from a different area S of the catheter of Figure 4 showing coccal forms and biofilm. (Example 7 results . ) ' Gv... 6.
!.'~
F3g~are 6 - Micrograph of a catheter surface pretreated with dextrinase. .A scanning electron 20 mierograph picture showing a ca'Cheter surface pretreated or coated with dextrina~e, upon exposure to staphylococci. The micrograph shows a thick biofilm layer with many coccal formations. These coccal formations are indicative of staphylococcal colonization.
g~gure 7 - Electron microgr~ph demonstrates formation of fibrous glyce~calyx on the surface of a control (saline-treated) catheter segment - before flushing with saline for 4 h~urs.
1~3.g~r~ 8 Electronynicragra~h demonstrates presence of~ deranged fibrous glyco~alyx on the surface of a mgnocy~line cawed catheter segment - before flushing kith saltine for 4 hour:
~~ .:
Fs~r~ g _ Electcron micrograph demonstrates fibrous glycacalyx on the surface of ~ control (saline-treated?
catheter segment - afte~° flushing with saline for 4 hours. ,.,, i Figure 10 - Electron micrograph demonstrating f fibrous gl~rcocalyx on the surface of a minocycline-cowed catheter segment - after flushing with saline for 4 hours.
35 .
Fxg~~e 11 EDTA Stability (~1) and Minocycline Stability (B), _214~9~~
~~ 94/1083 PCT/US93/10893 -15_ , DETATLED DESCRIPTIOIJ OF THE PRE~'ERP,ED EIrISGDIMEISTS
The present invention provides pharmaceutically effective formulations of minocycline, EDTA and combinations thereof. These formulations have been found by the present inventors to be particularly useful in preventing the formation of the "biofilm" or polysaccharide-ra:ch glycocalyx that typically accompanies microbial surface colonization. In particular, the formulations are most effective in breaking down polysaccharide-rich glycocalyx formations and in inhibiting their formation. This feature makes the preparations of the present invention potentially useful in the treatment of staphylococcal infections where a polysaccharide-rich glycocalyac has formed or may potentially be formed,~as well as in the prevention and treatment of ~Staph~lococcal and Carac3ida infection.
The present invention also provides M-EDTA-treated or coated medical devices, such as catheters, that prevent staphylococcal or fungal colonization.
The mi.nocycline (M) used in the studies described in the present disclosure was obtained from Lederle (Mincacin~ (yntravenous, 100 ~ng, Carolina, Fuerto Rico).
Tkxe disodium-EDTA'used in the studies described in the p~.esent disclosure was obtained from Abbott Co. (Endrate°
(Intravenous 150 mg/ml) Chicago, IL). A Modified Robbi,n's:Dev~.ce~ a screening toal customarily, used end accepted as predictive of~ catheter use 'in humansZS'' ~~ , was .
used in the present study of the M-EDTA pharmaceutical preparations described.. The model was c~nstructed at ~eD. ~derson Cancer Center in Houston, Texas.
The following agents were used in the studies disclosed herein:

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Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the present disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.
. The following examples are presented to demonstrate preferred embodiments of the invention, but should not be construed as limiting the claims thereof.
It should be appreciated by those of skill in the .
art that the technique disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and .
thus can be considered to constitute preferred modes for its practice. ~-Iowever, those of skill in the art should, in light of the.pxesent disclosure, appreciate that many changes can be made in the specific embodiment which are disclosed and still obtain like or similar results without departing; again, from the spirit and scope of the present invention.
EXa~iPhE 1 - PREPARATION OF P2-EDTA
pCEUT~CAi~ PREPARATION
~;
~The'present example provides a detailed description of how the M-EDTA pharmaceutical preparation was prepared for'use as a catheter or medical device flushing solution. EDTA was obtained from Sigma. Minocycline was obtained from Lederle.

3~ The M-ED'TA solution was prepared as follows so as to achieve a concentration of about 3 mg/ml minocycline and ~~ 94/10838 ~ PCT/YJS93/10893 2.~ 4~g ~2 _1g_ about 30 mg/ml EDTA in a sterile saline solution.
Separate solutions of EDTA (60 mg/ml) and minocycline (3 mg/ml) were prepared in saline. The EDTA was reconstituted from 200 mg/ml Edetate Calcium Disodium (Versenste~, 3M Riker, Northridge,~~~...CA) or reconstituted from Edetate Disodium (150 mg/ml>>,parenteral concentrate (Endtratem, Abbott, Chicago, IL~,y~or Disotate°, Forest, Maryland Heights, MO). Alternatively, the 60 mg/ml of EDTA could be reconstituted from EDTA powder (Sigma 1.0 Chemical Co., St. Louis, MO). Minocycline was obtained from Lederle and combined with a volume of saline sufficient to constitute about 3 mg/ml minocycline.
The 6 mg/ml minocycline and 60 mg/ml EDTA solutions were mixed in equal volumes to constitute a 3 mg minocycline and 30 mg EDTA/ml solution. The solution was then brought to a physiologically acceptable pH of about 7.4. The solution was stoxed in a stsrile container.
Once fornnulated; ' the M-~DTh may be stored refrigerated at ~°C until use. It is contemplated that 'so f~rmulated, the solution will remain chemically stable and pharmacologically actave for at least ~. month at 4°C.
The preparation is also very stable at room temperature ( 3 °? ° C) for at leas. 72 hours ( Table 1 and Figure ~.1.
) The- ~rreparats.on should be at room temperature before administration to a patient or animal.
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,,r, PC°f/US93/1089's i i~'~ 94/1083~~ ~ 4 _~0_ THE NIODIF'IED ROBBINS DEVICE MODEL
The present example is provided~~to describe the study model employed for illustrating the antimicrobial and therapeutic utility of the minocycline and EDTA

preparations of the present invention.

~.n in vitro model, the Modified Bobbin's Device (MRD), was used to study the formation of.biofilm snd colonisation of catheter segments of S, epa.dermidis.

This is a well established model that is described, in Nickel et al.ZS and Evans and Holmesl6, and provides a study model recognized by those of skill in the art as predictive of an vavo effects ~t a catheter surface:

The NdRD is constructed ~f an acrylic block, 42 cm long, with a lumen of 2x10 mm: The MRD is made of twenty-five evenly spaced specimen plugs each connected to:a catheter latex segment whose anterior surface (0.3 dm~) comes in contact witlx the flushed in~usate coming from a connected tubing end infusion bag. Several studies were conducted using this model, wha.ch are outlined irk the following examples ELE 3 INFIBTTION OF S . EPIDE.~3~Z~IS

i~ i.i ~ 'I1., l!. I ..I.i : .V :.j i~
I The present example ~~s provided t~ demonstrate the X30 utility of the present invention for inhibiting 5.

epidermadis in ah.c3 on a medical device; uch as a catheter. The model described in Example 2 was used in the present study: These results demonstrate the utility .

of the invent~:on for treating and maintaining catheter 35 patency in vivo, grad more specifically for inhibiting S:
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2~.4~942 ,Vd~D 9~t/10838 ~~G°f11JS93110893 epadexm~dis adhesion and glycocalyx formation at a catheter surface.
Catheter segments were placed in the specimen plugs of the Modified Bobbins Device described in Example 2.
After placing the catheter segments in the specimen plugs, the entire apparatus was sterilized with ethylene oxide. A 500 ml loo dextrose/water bag was infected with ~x~.08 CFU/ml of S. epiderrnadis (to produce 8x.05 CFU per ml of D1~/W). The infected infusate was flushed through the NIRD for 3 hours at a 50 ml/hr (using a peristaltic pump). Tn order to remove all free floating and loosely adherent staphylococci, the infected bag was removed and a new sterile bag (of D~p/W) was used to flush the NIRD.
1.5 The I~iRD was flushed with sterile D1~/W for 2~ hours at 40 mls/hr. Following this; catheter segments of equal size were treated with different agents by placing them in tubes containing one of the following so2utions:
Urokinase (5000 units/ml):
2: Heparin (1000 unit/ml):
3: EDTA (50 mg/'ml): and ~. Trypsin (20,000 units/ml).
25 H~presentative catheter segments caere then removed ( in a sterile manner) at 4 and 24 hours arid ~antitatively cultured using the scrape-sonicataon technique described by FChoury:.et a1. (1991) z~ to isolate ~rganisms adherent to catheter surfaces. The Khoizry ~t 30 al. reference is specifically incorporated herein by re~~rence for this purpose. ~.'he experiment was done at 3?°C.
The res~zlts from this study are presented at 35 Table 2. The results demonstrate that treatment of catheter surfaces with EDTA was effective in preventing e~~ ~a/ ~ ~~3~ ~ ~ ~ g 9 ~ 2 TAHLE

NO. OF
ADHERENT
S. EPIDER~IDIS
COLONIES

OBTAINED
FROI~d Q .
3 C~I2 CATHETER
SZTRFACES

Agent Used After ~4 hrs. After 24 hrs.

of treatment of treatment Urakinase . 310 40 I~eparin 54 5 2 0 15 Trypsin 1~0 ~~E ~

!b!-EDTA
AND
THE

PFtEiTENTION
OF BTOFILr~I
FOItMATTO~T

The present example is provided to demonstrate the utility of the M-EDTA
preparation in preventing glycacalyx-rich biofilm formation at the surface of a ~edi~al device, as well as to demonstrate the anti-~5 staphyl~coacal activity ~f minocycline and EDTA

preparations.
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~. ~

~
, The method of Ea~ampl~
3 was used with the following modif ieat~.ons 1: A
more'intense exposure to staphylococci ZS.

epidermir~is and S. aureus) was achieved by flushing the MRD for 6 hours (instead of 3 hours in Example 3) with 3x106 CFU
of ~taphylococci/ml of D5/W;
and adherent S. epidezmidis colonies on a catheter surface after only 4 hours of treatment. In contrast, urokinase, heparin and trypsin treatment of the catheter segments was significantly less effective at~'Liihibiting adherent ;,:.
S. epidermidis colony formation an~~~'adherence after 4 v.,, hours of treatment. . .

214~94~
Pt:T/US93/ 10893 ' CVO 9~/ ~ 0838 2. The growth of adherent staphylococci to the eter segments was Promoted and achieved by exposing cath the catheter segments at '37°C to a 10% broth solution adding 1 ml of trypticase soy broth to 9 ml (prepared by of sterile H2O) of EDTA (30 mg/ml of 10% broth solution) 100 unit~/ml of ~.0% broth) ~ uro3cinase (5.000 heparin ml o~ 10% broth), minocycline (3 mg/ml of 10%
units/
broth). mino/EDTA (3 mg/30 mg per m1 of 10% broth),, 3 mgjml of l0% broth), vancomycin/heparin (3 vancomycin ( com cin plus l00 units heparin/ml of 10% broth) or mg van y D~/10% broth t50 m3/ml o~ l0° broth solution).
results from these studies are demonstrated at The Tab? a 3 (S. epa.dermidis) a~.d Table 4 (S. aureus) -~C.~~~ 3 d~W 0~~..~~. ~ v ~P~~~~~~ ~~~O~~S
~~~~~FROM ~W ~~ ~~ ~~~~~5 A ent Used After 4 hrs. after 24 hrs.
of tgeatment ~f treatment g >5x103 >5x103 ~0 Urokinas~
>5x103 >5x103 geparin 800 a~
EDTA
Minocyeline 10 0 Minocycline/EDTA ~ 0 25 Vancomycin 40 Varicomy~in/H~Par'~.n; ,;4~5 , .
~5x10~ >5x103 D /10% broth s demonstrated in Table 3, the urokinase, heparin extrose solutions alone were equally ineffective in and d eventing and exadicating S. epiderm'idis adherence after Pr s of catheter treatment. The minocycline and ~ or 24 hour minocycla.ne/EDTA provided effective prevention and ~.. ,... .,,. . . . - . ..,; ..
-: , .: .. . ,.. .. , . . :. ., ~:: ;.: . : ; , .:. . , , WO 94/10838 ~ ~ ~ ~ ~ ~ ~ , 1 PCT/U593/10893 Agent Used After 4 hrs. After 24 hrs:
of treatment of treatment 15 U rokinase >5x103 >5x103 Heparin 256 >5x10~

Minocycline p 0 Minocycline/EDTA 0 0 20 Vancomycin 605 230 Vancomycin/Heparin 140 185 D /7.0% broth >5x103 >5x103 * A~.z staphylococcus isalat~s were bloodstream slime-25 producing isplates obtained from human patient cases with catheter-related bacteremia.
. , , ,, , ~, , , Table 4 demonstrates that minocycline and 30 minocycline/EDTA solutions were the most effective ~;nhibitors of 5. aur~us adhesion, with 0 adherent colonies being observed after 4 hours of treatment. EDTA
alone, vancomycin alone and vancomycin/heparin were significantly less effective for preventing adherent S.
irradication of S. elaidermid~.s adhesion after only 4 hours of treatment. Minocycline/EDTA was slightly more effective than minocycline alane at 4 hours. EDTA alone and vancomycin/heparin provided minimal prevention at 4 hours but were more effective after 24 hours. Vancomycin alone provided equal partial prevention at 4 and 24 hours. M-EDTA was superior towall preparations examined, including vancomycin, vancomycin/heparin, minocycline or EDTA alone.

130 . OF ADHERENT S . AUREIIS COLONIES
OBTAINED FROM 0.3 CI~l~ CAT1HETER SURFACES

~~VCD 94/10838 21 ~ ~ g ~ ~ PCT/~.1593/10893 aureus. These later three preparations had some partial anti-adherent activity, particularly after 24 hours of treatment.
These data (Table 3 and Table 4) demonstrate that minocycla~ne alone~or in combination with EDTA was effective for inhibiting ~. epidermidis and S. aureus adherence and colonization of a catheter surface.
EXA~iE~IaE 5 M-EDTA AND T'hiE
INI~iIB~~'I6IwT OF C. ~Zbicans A1?IiESIa~T
1;.;

V~~ 94/0838 P~'/LIS9311089'a B~TO. OF ADHEREI~IT C. A~BICa~JB
OHTAII~7EI' FROM 0.3 Cl~i2 CATZ3ETER Sv'RFACES
Agent 'Used After 4 ~~.hrs After 24 hrs .
. of treatment of treatment Urokinase 5x103 >5x10~

Heparin >5x103 >5x10~

Minocycline 190 535 Minocycline/EDTA 0 0 D /10% broth >5x10~ >5x103 Vancomycin/heparin >5x103 >5x103 vai~comycin 4 70 >5x103 :15 This example demonstrates minocycline/EDTA as a unique anti~taphy7.ococcal and antifungal agent.

Vancomycin (a standard antistaphylococcal agent) when used alone or with heparin fai~.ed to have any anti-C.

albacans activity and was not different from dextrose, 2 0 urokinase ar hepaxin solutions against C. alba. earns .

EDTA alone had some az~ta.-C. albicans activity after 24 hours and minocycline alone had some activity at 4 and probably 24 hours . Tlae combination of minocyclia~e a~xd ~5 EDTA (M-EDTA) pravided a synergistic enhancement of anti-C. albicans activity, demonstrating an essentially total i~hi~itoz~r ef'feet ''against ' fungal adherence after .4 ~nd~~ 24 hours exposure. Therefore, M-EDTA is unique in presenting staph~loaoccal and Candida adherence to 3p catheter surfaces (Staphylococci and Candida contributing to 95% to 1:00s of the pathogenic microbiology of catheter-related infect~.on~?. ;

These results demonstrate that the solutions of a 35 mixf.ure of minocycline and EDTA grovide a more effective ,,. .. , '... ; .__ ;. .. '. _ ; ;
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and rapidly-acting preparation for the prevention of S.
epidexrnidis, S. au.z~eus, and C. albicans adhesion to a catheter surface than any other thrombolytic (urokinase), anticoagulant (heparin, EDTA), or antistaphylococcal preparation (minocycline, vancomycin, vancomycin/heparin).
Epr~E s s enidex~xd~ts s~o~i~rz FoxrsAT~oN

~D ~~~r=~a ~~~z~rASE Arrn D~x~=NAME ~~.EA~

The present example is provided to examine the relative S. epiderm:idis glycocalyx biofilm-destroying activity of heparin, urokinase and dextranase as assessed by scanning electron microscopy of an S. epidermid.as-colonized catheter surface.

Scanning electron r~icrosaopy-was done on various y segnnents o~ a catheter exposed to 5. ep.iderrnzd.zs and then later exgosed to heparin, urokinase or dext~anase for 24 houxs. .A reduction in biofilm (glyc~calyx) was noted on colonized catheter surfaces exposed to EDT for 2~ hours, compared to colonized surfaces later exposed to heparin, urokinase, or dextranase for 24 h~urs (Figure 1 Dextranase; Figure 2 _ Urokinase; Figure 3 = EDTA; Figure 4 - Saline) .

E3~AI~2PLE '1 P~tETREA~F ~A'T~3ETER StTIZFACES W~TI~ ~'.DT,A, Dext anas~ OR SADaI~ : 5. a 'de,~id.ia EZ~F~D~I F'OFtt~iA~'~oN
: ~ r ,.

The-present example is provided to demonstrate the effect of chemically pretreating a catheter surface with EDTA or dextranasa; compared to a saline control; on the forTnation of polysaccharide-rich biofilm formation, such as that characteristic of S. epade~rnidis, and the adhesion of these organisms to a catheter surface:

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. , !~V~ 9~/~0838 ~ ~ 9 ~ '~, PGT/US93/10893 .

Catheter surfaces were coated with EDTA, dextranase ar control (saline), at the concentrations described in Example 5, and then exposed to slime producing S. y ,, epadermidas. . ' No biofilm or organisms~~were observed on EDTA coated surfaces. However, biofilm formation was observed on catheter surfaces pretreated with dextranase (Figure 4 and 5 = saline; Figure 3 - EDTA; Figure 6 = Dextranase pretreated).
E~CAMPLE 6 M~NOCYCLTCOATTI~G OF
A CP.THETER MTCItOEL~IL COLOhi'TZAT~OlIT
l5 The present example is provided to demonstrate the anti--microba.al colonization effect of the use of minocycline at a catheter surface.
Catheter surfaces were coated with minocycline, vanee~myein or control cement with HZO: Catheter surfaces were then exposed to clinical staphylococci isolates.
The Modified Robb~.n's Device was empl~yed in this study (See Example 2. The Modified Bobbin's Device simulates a ~rascular catheter; and therefore provides a model predictive of in ~rivo effects.
One gram of methylr~ethacrylate (cement) was mixed ~~,t~ p,.5 ml; of sterile F3~0 and one o,f the following>
3 p 1. ~ 0 mg c~f minocycline ~: 60 mg of vancomycin 3. 'control (cement + H20 alane) Equal amounts of C~men~ ahne or with minocycline or 35 ~ancomycin were put a.n the lumen of catheter latex segments in a specimen plug of the Modified Bobbin's ~1~9~~
'~0 94/1083 - P(:TlUS93/10~93 De~rice. Twenty-four hours later, a one-liter infusate bag made of 5o dextrose in water was infected with 5 m1 of 105 to 10g colony forming units (Cr~U) per ml of slime producing Staphy~.ococcus epidermidis strains obtained from the blooc3.stream of patients with catheter related bacteremia. Using a peristaltic pump, the infested infusate was run for 2 hours at a rate of &0 ml/hr through the ca~.heter segments of the Modified Bobbin's De~rice .
Eaah catheter segment was made of 30 mm2 silicone with a lumen filled with cement. At the end of 2 hours, some catheter segments (control and antibiotics coated) were taken out from specimen plugs and the cement in the lumen was removed, then the surface that was exposed to the infected fluid was cultured semiquantitatively using the roll-plate technique. Other segments were left behind and flushed with sa7.ine solution for 4 hours, then cultured by roll-plate..
Electron microscopy wras used to document the adherence of staphylococci. and the formation of biofilm layer on the 'surface of control uncoated catheter aegcnents . Leaching of ax~tibiotics from the cement was ~5 dem~nstrated to occur for at least one week by cleter~nining the inhibitian around disc-shaped pieces of Cement placed on blood agar plates that had been i~;oc~.lated yith bacteria . Coming of the catheter i, ~ ;; . ~ i : . . i segments with ahtibiotics was demonstrated by the'~ane~' of 30 inhibitson than.continued to form for at least ~ne week around the disc-shaped catheter segments lwithout cement);
. placed on agar p~.ates that had been inoculated with bacteria. The results from this study are presented in Table 6.
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, 'VV~ 94!10838 . . . p~'f/~S93/1089~ .
-3a-~r~.szE s I~~ . Cala~~?IES OF° S . EP2DERI~iTDIS
g°ROM 3 0 ~1MZ CATFIETER S'URFA.CE
Coating Eefore Fl~xsh. After Flush Control 336n .: 128 Vancomycin 1?'~ 111 Minocycline 48 15 Catheter segments boated with minocycline had a significantly lower number of adherent Staphylococcus t epidermidis colonies, compared to control and vancomycin coated catheter segments (see Table 6). However,'fibrous glycocalyx was not inhibited on the minocycline~coated catheter surfaces. The scanning electron microscopic figures also evidenced these findings. See Figure 7 -spaows fibrous glycocalyx on the surface of a control catheter segment before flush; Figure 8 - shows some deranged (fibrous glycoca7.yx on the surface: of minocycline coated catheter segment before flush; Figure 9 - shows fibrous glycoca:l~x on the surface of ~n~ther control (saline) catheter segment after flush; and Figure 1.0 shows fibrous glycocalyac on the surface of minocycline Coated catheter segment after flush.

~'hese'data demonstrate that the coating of catheters with minocyclia~e alone sigr~:ificantly reduced at~phyloc~c~al~ ~dliex'eriCe a ~;i~~ous 'glycocalyx foxtnati:on is not inhibited with minocycline-coated surfaces.

~~E ~ ~~TL=~Y aF r~=~oc~t~~,~~aE

~D~~ p ' cE~TC~L ~~~~~.~~T~~~

Tkie present studies will be conducted to 35 characterizE the stability of the pharmaceutical M-EDTA

flushing solutions of the present invention. _The M-EDTA

,:. .. . :: .. .. ... ::: . . :.. .. .. . : .: ,: . , . __ . . . ,;. :.,, :
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: .. :.>,....:....,. ~ .-,: . . ,.: . .. ~:... . ,: ,.,. , 2.48942 ~V~ 94/1083 - PCf/~JS93/10~93 solutions are expected to retain their potency for relatively long periods of time when stored refrigerated at about 4°C, i.e., for at least 1 month.

5' The solutions~of the present invention have been examined for retained potency at room temperature (3?°C).
The M-EDT solutions have been observed to retain relativea.y full anti-microbial and anti-fungal potency for at least 72 hours at room temperature. It is 2a therefore expected that the formulation has a shelf life that renders it suitable for routine hospital use (Table 1 and Figure lI ) .
ELE ~.0 15 GOMP,lAFd.l~I.TT~TE GL~IeT~C~ TRTAh OF ~I-EDT~i AID
REP.~R~i~ FOR THE PRE~ENTIOhT OF C~TFiETER-RET~P.TED II~'EGTIONS
The gresent example outlines a study wherein the relative effectiveness of~an M-EDT catheter flushing 2a solution will be cs~mpared to a heparin flushing solution (a.currently used standard preparation) for the prevention of catheter-related infections and occlusions in humans>
25, The objective of these studies is to further document the utility of the M-EDTA fl~xsh solution as compared ~o a heparin flush solution in preventing t. ,, a.nfection and/or occlusion in,central venous catheters i (CVG)~'.
Eligibility for enrollment into these studies v~ilJ.
be based on th.e following inclusion/exclusion criteria:

WO 94/10838 P~T/US93/10893 Inclusion Criteria 1. Patients must have a new (s7 days old) functioning central venous catheter, utilized for infusion of chemotherapy, blood products, or other intermittent infusions.
2. Patients must be able to return to the outpatient clinic for evaluation in case of CVC occlusion or occurrence of fever.
3. Patients must have life expectancy for the planned duration of the study and must have catheter in place for study duration (study duration for a single patient is 6 months).
4. Catheters in the percutaneous/tunneled group will be limited to Hickman/Broviac.
Exclusion Criteria 1. Patients with an occluded central venous catheter.
2. Patients with any existing local or systemic catheter infection.
3. Patients with triple lumen catheters.
4. Patients with polyurethGne or teflonT""catheters.
5. Patients currently taking warfarin.

6. Patients requiring previous catheter removal due to venous thrombosis .

~%~ 94/10~3~ ~ 214 ~ 9 4 ~ P~d'/U593/10~93 _33_ 7. It must be expected that the dwell time of 4 hours will not interfere with routine treatment of the underlying disease.

8. patients ~Nith Groshong catheters.
~t'reat.~aent Pl.aa: Patients will be randomly assigned and in double blind manner to have their CVC flushed with either M-EDTA or heparan according to the following:
1, Tunneled CVC (~Iickman/Broviac) will receive either (a) two mls of I~I~.EDTA (containing 3 mg of minocycline and 30 mg EDTA/ml) q daily (b) two mls of Heparin (100U/ml) q daily, Infusion ports will receive either (a) two mls of M-EDT.A (3 mg minocycline and 30 mg EDTA/ml) q 3 weeks (b) two mls of I~eparin (100U/ml) q 3 weeps Endpoints and Treatment Evaluation: All patients wily be followed up for 6 months and will be evaluated for 2 eradpointd : catheter infecti~n/coloni~ation and occlusion: Catheter irafect~.on will include local CVC-25 related infection or systemic catheter-related septicemia. Catheter colonization v~rill indlude positive quan~.itative catheter culture (flush technigue) ox ,, posit,ive quantitative blood culture through the CVC ire the absence of ~ positive peripheral blood culture Ar 30 clinical manifestations of sep~aa (fever; chills or hypetension). Patients in. the study wh~ develop fever will be evaluated; and sa.multaneous quantcitativ~ blood ctaltures through CVC and peripheral vein conducted.

Catheter ocel~asion ~iJ.l be categorized as complete or ~5 partial depending on whether one cannot withdraw blood, ;.., . ' ,, ... ,. . ._ . . ~. . ;. ::.:. :. :: , .. : v . .:.: :, . ..... . _ .
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infuse fluids through the CVC, or both. This subgroup of infected catheters will ba analyzed separately.
Statistical Considerations:~'~.Based on a recent surveillance study conducted by't~.e inventors (see Table i'.
°7), the rate of CVC-related sepsis in pediatric oncology patients ranges from 25% - ~0.5d flee Table 6). These same ranges will be employed in further establishing the clinical utility of the treatment in adult patients.

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_36_ The results fram this study will be employed in the development of a clinical protocol for the treatment and infection-free maintenance of indwelling catheters in humans. w EXAMPLE l~. PItEPARATIOPt OF' M-EDTA-COATED DEVIOES FOI2 TBiT YTiTQ tTSE
The present example is provided to demonstrate the utility of the M-EDTA solution as a coating material for medical devices, most particularly catheters.
i Any of a trariety of coating techniques may be used for imparting a protective covering of the M-EDTA
~.5 solution to a device. By way of example, such methods include emersion of a medical device, such as a catheter, unto a solution of minoeycline and EDTA. This example provides a description oaf how these particular M-EDTA
coated catheters were prepared.
Bioguard~Cook Catheters with, TDMACC surfactant were v; immersed in antibiotic solutions containing the following:
~.. 60 mg of Min~cycla,ne plus 60 mg of EDTA/m1 2: 60 mg ~f Minocycline/ml 3. 60 mg of EDTA/m1 ~, ", , ,t ~~
Catheterslwere immersed in each of the threw solutions Listed ab~ve for about 15 minutes. Bioguard hook catheters not treated with any of the 3 solutions listed above (which arm not coated by antimicrobials) were used as negative controls. Arrow Card catheters coated with chlorhexidine and silver sulfadiazine were used as positive control devices. The Arrow Gard catheter is coated with antimicrobials. This catheter is i~~ 94/1838 _ PCTIUS93/I~D893 described by Maki et a1. in a clinical study, and reportedly decreased the rate of catheter-related bloodstream infection by five-fold as compared to a standard polyurethane triple-lumen CVC without a chlorhexidine and silver sulfadiazine coating.
Other Coatir~g Methoels for Medical Devices As noted, preparations of the present invention may be advantageously used as a coating preparation for treating the surfaces of a medical device. The medical devices which are amendable to coatings with the subject M-EDTA preparations generally have surfaces composed of thermoplastic or polymeric materials such as polyethylene, Macron, nylon, polyesters, polyt~trafluoroethylene , polyurethane, latex, silicone elastomers and the like. Devices with metallic surfaces axe also amenable to coatings with the disclosed combinations. Such devices, for example indwelling 0 catheters of types listed herein, can be coated by cement mixture containing the subject antibiotic compounds.

Alternatively, de~rices that include a surfactant, such as the Bioguard Cook catheters; may be used whereby the surfactant at the surface of the device allows for the coating of the material onto the device. Particular devices especially suited for application of the M-EDTA

pregarati~n include intravascular, peritoneal, pleural and urological .catheters; heart valves; cardiac ,, ;, i a ~ ~ , ;
. palceinakers; vascular shuzits; and orthopedic, intraocular, or penile prothesis.

'ctarious methods can be employed to coat the surfaces of medical devices with the M-EDTA coating preparation described herein (60 mg EDTA/60 mg minocycline). For example, one of the simplest methods would be to flush the surfaces of the dev~.ce wi h the M-EDTA preparation.

W~ 94/10~3~ PCl'/U593/108g.~

Generally, coating the surfaces by a simple flushing technique would require convenient access to the implantable device. For example, catheters, are generally amenable to flushing w,~.th a solution of EDTA
, ting solutions, the and minocycline. For use as.,coa effective concentration of miriocycline would range from about 0.001 to 100 mg/ml, preferably about 60 mg/ml; and about 1 to x.00 mg/ml EDTA, preferably about 60 mg/m1 EDTA. The coating solution would normally be further composed of a sterile water or a sterile normal saline solutions.

A preferred method of coating the devices would be to first apply or adsorb to the surface of the medical ~.5 device a layer of tridodecylmethyl ammonium chloride (TDMAC) surfactant. followed by a coating layer of the M-EDT~ preparation. This method for coating a device with M-EDTA provides for (first absorbing to the catheter surface a cationic surfactant, such as TDMAC, to medical devices having a polymeric surface, such as polyethylene, silastic elastomers, polytetrafluoroethylene or Darcon, by soaking in a 5% by weight solution of TDMAC far 30 minutes at room temperature: The device should then be air dried, and rinsed ~.n water to remove excess TDMAC.

25 Alternatively, TDMAC pre-coated catheters are commercially available; far,example, arterial catheters coated with TDMAC are available from C~ok Critical Care, Bloomington, Indiana: The device carrying the adsorbed I
I

n~ i tYaen be incubated iii ~a , TDMAC surfactant dating can 30 solution of the M-EDTA combination for between about 15 ~,inutes and an hour, washed in sterile water to remove .

unbound M-EDTA and stored in a sterile package until implantation. xn general; tie soaking solution of M-EDTA

inclu3es between about 10 mg/ml - 100 mg/ml EDTA

35 (preferably about 60 mg/ml) and between about 10 mg/ml -_. , ..: .. .. : ,;,. ,_. :. , .._ . .. ;,_ , ..: :. -. . . -.. , .., ;. .. , ;
, . ~; ,;, _. _ , ._, ,. . ;. , .., . ;;. ., .., . .... .:. .. ..: < :.. ~ .. . . .. . . , , : .. , . . . ...

100 mg/ml minocyclines ;pre~erably about 60 mg/ml) in an aqueous pH 7.4-7.6 buffered solution or sterile water.
Alternative processes and reagents for bonding an agent contained in a solution to a surfactant-coated implantable medical device are provided in U.S. Patent Nos. 4,442,133, 4,67b,660 and 4,749,585, A further method useful to coat the surface of a medical device with an M-EDTA preparation involves first coating the selected surfaces with benzalkonium chloride followed by ionic bonding of the M-EDTA. See, e.g., Solomon, D.D. and Sherertz, R.J.
(1987)17 and U.S. Patent No. 4,442,13318 Other methods of coating surfaces of medical devices with antibiotics are taught in U.S. Patent No. 4,895,566 (a medical device substrate carrying a negatively charged group having a plta of less than 6 and a cationic antibiotic bound to the negatively charged group); U.S.
Patent No. 4,917,686 (antibiotics are dissolved in a swelling agent which is adsorbed into the matrix of the surface material of the medical device); U.S. Patent No.
4,107,121 (constructing the medical device with ionogenic hydrogels, which thereafter absorb or ionically bind antibiotics); U.S. Patent No. 5,013,306 (laminating an antibiotic to a polymeric surface layer of a medical device); and U.S. Patent No. 4,952,419 (applying a film of silicone oil to the surface of an implant and then contacting the silicone film bearing surface with 3C~ an~ibiotic powders).
These and many other methods of coating a-solution to a solid surface appear in numerous patents and medical journal articles. As is evident, one of ordinary skill :.awing benefit of this disclosure would be apprised of pc-rfus~~flos9~
~~ 94f 10838 -~o-several different methods of caating a medical device surface with the subject inventive minacycline and EDTA
coatings.
Medical devices, parti~:ciilarly catheters of the type ' ;., , .
listed in Table 8, may be~coated with the M-EDTA solution and then stored in a sterile packaging material until use.
fi~ZE 8 SHORT-TERM TEMPORARY LONG-TERM INDEFINITE , . ACCESS CATHETER ~IASCULAR ACCESS

Peripheral intravenous ' Peripherally inserted cannulas central venous catheters - winged steel needles (PICC) - peripheral intravenous catheters Arterial catheters Percutaneous nontunneled silicone catheters 20 Central venous catheters Cuffed tunneled central venous catheters (Hickman and Broviac) Swan-Oanz catheters Subcutaneous central ' venous ports (Infusaport; Port-a-cath,. Landmark) Hemodi,alysis catheters Umbilical catheters 25 , , , ~ ;
E~LA~iPLE 12 1~TT30D FOIL I~IN'fAI1~7I11Tt~ C.A~I1~TER PATE~TC3~
~1~TI~ IsiIN~CYCLIl~-EL1~A PFiARMACE~ICAL PRE1~A1~TIO~T
3p The greser~t example demonstrates one embodiment of a method. that may be used in maintaining the patency of an indwelling catheter in.a patient. The regimen described herein is potentially applicable for use.in adult ,, . . . ;.: ~. .:~ : .. , , ., ,.. ; ~:: ~, . . . .. .. , ,. , ;... : .; -: , ::- .. ;.
,. .. . , , d6'~ 94/10838 ~ ~ ~ ~ ~ ~ ~ _PCT/IJ593/10893 -4~-patients, as the dose of M-EDTA in the regimen exposes patients only to relatively low, pharmaceutically acceptable levels of the EDTA and minocycline, and has already been demonstrated by the inventors as effective in pediatric patients.
An indwelling catheter of a patient will be flushed with a solution of minocycline/EDTA. The °'flushing" of the catheter will constitute filling the catheter with a volume of the M-EDTA solution sufficient to provide a concentration of about 9.0 mg minocycline and a concentration of about between 90 mg EDTA in the catheter. For example, assuming a catheter volume of about 2-3 ml., the solution will contain a concentration 1S of EDTA of between about 10 mg/ml - 30 mg/ml, and a concentration of minocycline of between about 1-3 mg/mI.
"Flushing" the catheter with about 3 ml of the M-EDTA
solution will thereby provide a dose of between 3-9 mg minocycline and about 30 - 90 mg EDTA. The solution of M-EDTA will be prepared as outlined in Example 1.
The ''flushing" of the catheter is achieved by adding between 2-3 m1 of the M-EDTA solution to the catheter: The solution is then allowed to diffuse through the catheter to the patient in which it is implanted. The concentration of the EDTA and minocycline in the solution is such that the patient will be exposed only to concentrations of the agents well below pharmacologi;~al;ly tolerable levels., The flushing o~ the catheter is to be repeated at ,~ periodic intervals of at least between every 24 to 72 hours (preferably, every 24 hours? to assure that infectious or~ani~ms are not allowed~an opportunity to colonize the surface or initiate biofilm formation on the catheter surface.
';.~

EFFICACY OF ANTIBIOTIC COATED CATHETERS
AFTER GAS STERILIZATION
The present example is provided to demonstrate the stability of the coated devices to sterilization processes. In order to test the effect of gas sterilization on catheters coated with Minocycline, EDTA
and the combination of drugs, the following studies were performed.
M-EDTA-coated catheters were prepared as described in Example 11. The catheters were then divided into three representative groups. The catheter antibiotic activity was determined in vitro by a modified Kirby-Bauer technique described in Sherertz et al. ((1989) Ar timi crob . Agen is Chemo tier . , 3 = :1174 -117 8 )* The first set of catheters were tESted immediately after immersion without gas sterilization. The second set was tested 24 hours later without gas sterilization. The third set was tested 24 hours after gas sterilization.
The modified Kirby-Bauer technique consisted of growing a strain of slime producing catheter-related bacteremic isolate of Staphylococcus epidermidis for 18 hours in trypticase soy broth, then diluting the solution to 10 CFU ml in phosphate-buffered saline. A cotton swab way placed in the staphylococcal suspension and then rubbed across the surface of a trypticase soy agar plate.
C' Individual catheters were cut into 20 mm lengths pressed into agar overlaid with S. epidermidis and incubated c~~ernight at 37°C. Zone sizes were assessed by measuring the diameter perpendicular to the long axis of the catheter. The data in Table 9 demonstrates that W~ 94/1083 ~ ~_ 4 ~ 9 ~ 2 PCf/US931~0~93 x Catheters treated with the M-EDTA preparations maintained v the greatest post-sterilization zone of inhibition ) -(0 hours =40; 24 hours = 34).
E
a For the in ~rivo'studies, the catheters were cut into 2 am segments and then emersed in 60 mg/ml EDTA, 60 mg/ml minocycline solution each and then immersed in a solution oz a mixture containing 60 mg/ml mina and 60 mg/ml EDTA
for 15 minutes. A11 catheters were allowed to dry for one hour and they were gas sterilized.
Table 9 provides the results achieved with these studies. This data demonstrates the superior anti-S.
epidezanis activity of the M-EDTA coating as compared to non-treated as well as the Arrow Gard catheters.
T~I,E 9 S. E'P.fDE,RP~TDIS
~~NE ~F IIJHI~~T~OIe1 - D~~lETEFt (ate) Tire Sterilization Past Sterilization 0 I-~rs 24 Iirs 24 Hrs Minocycline 36 33 18 EDTA 5 10 1~

Control 0 0 Arrow Gard* 13 15 ~

EFFICACY OF ANTIBIOTIC COATED CATHETERS
AFTER GAS STERILIZATION TO S. AtTRE~S
The present example is provided to further demonstrate the stability of the described M-EDTA
coatings to sterilization processes, such as gas sterilization, particularly as measured through the retained anti-microbial activity of the device.
The protocol of Example 13 was used to prepare the catheters used in this study (using a catheter-related bacteremic strain of S. aureus). The 0 hour catheters, and catheters coated with minocycline or EDTA alone were not included in the study.
TABhE 10 S . AURE~S
ZONE OF INHIBITION - DIAMETER (mm) Pre Post Sterilization Sterilization 24 Hrs 24 Hrs I

Control 0 0 Arrow GardT""*13 13 .

*Coated with chlorhexidine gluconate and silver sulfadiazine The present example demonstrates that gas sterilization did not affect the antibiotic activity of minocycline-EDTA coated catheters and that these catheters were at least two times more active at inhibiting Staphylococcus aureus than the Arrow septic catheters.

_ 2.1 ~~94~
'W~ 94!10838 ' PCT/US93110893 _,~5-EXAMPLE 1.5 COMPARATIVE EFFICACY OF M-EDTA COATED CATHETERS
AGAINST CATHETER RELATED MICR~oRGANISMS
~'~ . 5 The present example demonstrates the broad spectrum activity of M-EDTA catheters. The same zone of inhibition measure described in examples 13 and 14 (with the modified Kirby-Bauer technique) were employed using M-EDTA coated catheters and Arrow Gard catheters (commercia7.ly available) to examine the effectiveness of the various coatings against different catheter-related organisms such as Staphylococcus epiderm.id.zs, Staphylococcus auxeus, Candida albicans and gram negative . bacilli (Pseudomonas aeruginosa, Xanthomonas mat tophalia, and acinetobacter species.) Approximately SO% of catheter infections are caused by S. epidez~midis, 10% by ~. aureus; 1a% by C. a~hicans, 20% by gram negative bacilli (mostly P'. aeruginosa, X. maltophalia, and acinetobaGter species:) These studies show that catheters coated with M-~DTA have a broad spectrum activity agaihst differea~t species of bacteria and fungi as well as different strains of the same species. For purposes of the present studies, a zone of inhibition of z 15 mm is a predictor of excellent efficacy in veins. A

zone of 10 - 15 mm is a predictor of moderate efficacy and a zone of inhibition of s 10 mrn is a predictor of poor efficacy.

y~.l~' ~~~.~. m'.y wr ~.~. . ~ . -~' .~~~ . ~..;~:.y . . . ... :. ~ ~...
....'.: . ~.'':. ' : ,,..,. ...
1~'~ 94/0838 FCT/US93I108y:5 ~
~1.4~942 TABLE 1I - ZONES off' INHIBITION (x~art) (STA~'H~'LOCOCCUS EPIDEE~lIDIS (SE) STRAI2JS
Strain No. Arrow Gard ino/EDTA
M --SE 4392 .. 31 SE 3996 16 2g SE 4345 ~.? 3g SE 4023 1.5 29 SE 93 ~5 3~

Mean (SD) 15.4 (~l.l) 31.8 04.14) p _ 0.QO1 Staphyls~coccus ar~reus T~Ze 12 sets forth data obtained empJ:oying five la ~ifferena strains of Stapk~y-lococeus aureus (SA) in the aforedescribed Zane of inhibition assays. The data ~emor~strate than tlae M-EDTp: coated catheters provided a significantly greater zone of inhibition compared to the non-M-EDTA eath~ters (Arrow Oard).
~ABTaE 12 ZO1~TES OIa° IN$IBIT301C~ - DIT~R fit) Strain No:' Arrow Gard M_ino/EDTA

SA 1445 13 23 , 5~ 1432 z5 28 2 r SA 7.414 12 2 3 SA~ ~ 4 g ; ~;4,:, , 23 :.
i -SA 1411 12 __ , Mean (517) 13 .2 (~1:3 25 : 2 f t4 ) : 9) p ' Q,pp5 C~dada a~.bic~.tas Table 13 sets fox~~h data obtained emp~.oying the organism Caradida albicans;
in the zone of inhibition 21~~942 ' ~W~ 94!10838 PCT/US93/90893 -4?-study protocol, comparing the inhibitory action of the M-EDTA and non-M-EDTA coated catheters especially.
"Ampho B" stands for a broad spectrum antibiotic (xpRS, PDEASE St7ppT,Y) . The data in Table 13 demonstrates that the M-EDTA treated catheters had superior anti-Candida albicans inhibitory activity as compared to contra! and the three other catheter types (coatings) tested.
~.o ~.~~~ i~ - z~rrES o~ xr~~sxTxo~r - nxTER 4) cand~da ~2bic~s ~~

~,~...
Tray Trial #2 Tri_ al #3 M-EDTA
_ ~inocycline ~ 0 Control 0 0 0 Axrow hard 10 ~pho B ~ 18 ~ 8 Table 14 sets f~rth tudies with data obtained i~.
s 2p five strains of Car~c~:ida demonstrates albicans;
and again the broad range anti-microbial the M-EDTA
activity ~f coated catheters.
n i ~ , ; , , ~VC3 94/1083 PCT/(JS93/10~93 TAELE 14 - ~OI3ES OF INFiIEITION - DIAMETER (gin) ( ,TRAII~1S OF C,~I~TDIDA' 1~BICAI~TS (CA) _ ~ ~_~._ _ Strain No. Arrow G_a_rd Mino/EDTA

CA 596 1~ ~-2 cA 27s to to cA a67 to i3 Mean (SD) 7.4 (t4.3) 13.803.2) la p _ 1 0.03 Ac~.xae~obactex (.~c~~n) Ta~rle described ~.S
demons9e~ates the efficacy of the M-EDTA
coatings for inhibiting Acinetobacter.

TAELE

-ZONES
aP' IN~iI~~TIQ~T
-DIAE~iETE~t (gin) ~~
~
~

'_ ~ TMP SMX CFTZ ARROW
Strain No: ~ GARD
M EDTA

2p ACIN639 30_ a0 0~ ao ACIN38B 23 3a 15 t35 ~CI1~T63~ 24 10 00 00 ~CII~T633 10 16 00 09 ~~; ACIriT~:77T '' ' X43'1.4 ;,00 . ;12; ,;

Mean* ~~ 14 3.0 5:2 STDEV 5.3 6.7 M =
Mi.nocyclane;
CFTZ
_ Ceftazidime;

~p,ggv~
_ Tr~.methoprin-Sulfamethoxazole The M-EDTA
efficacy was of the catheter coated with sigrsificantly~ (~ _ 0.01.6) :: :. ,. -., higher . . , ' , . . . - <; than . .,, the , ..
Arrow Gard _.
, .., .
,.:
, :::
.,:., .
..
,.
,.
: ::
.
.
:
.
.
..
--::
:
..;
..
.
:,:

)..1~~, n ~. . : ' ~ . . ' : - . . ' : " - .. ~ . . . ., n -2~.~~9~2 W~ 941~0~38 PCT/US93/10~93 f -49-P. ~l.exugimmosa ns (PS~1) Strai Tab~.e ~.6 s the efficacy M-EDTA
demonstrate of the coated catheters P. aeruginosa trains.
against (PSA) s ';.:.. 5 ~'ABIvE 16 zr~s~~axTZOrr - z~rr~~ o~ - DIAMETER
(~t) Strain No. M EDTA TMP SMX CFTZ AR~20W
GARD

PSA2455 1~ 05 20 00 Means x.2.2 1.0 22 3.0 STDEV 2.17 2.24 6.60 3.0 * The effsaacy catheter coated M-EDTA
of the with was significantly Arrow (p = 0.009) higher than Gard the ;;~ , ' ~ ~. , , , ~Yz.

.;
'!~'~ 9.4/10838 P~f/U~93/10893 ~,~ 50-r X. ,~2altophalia () Stz°alras f Table demonstrates the efficacy of the M-EDTA

catheters against X.
maltoph.zlia strains.

,,: 5 TABLE

ZONES
aF
INF~IBI'TI~N
-DIAMETER
(amp) Strain No. M EDTA TMP SMX CFTZ ARROW CARD
-i XMAL2496 34 22 29 00 i XMAh8929 37 40 20 00 Mean* 29 28.8 23.3 3.0 , STDEV 9.3 10.38 7.05 5.22 z5 *
T
a eff~.cacy of t a catheter coated with M-EDTA
was significantly higher than the Arrow Gard (p =

0.0016) The aforegoing results demanstr~ate that M-EDTA

coated catheters have a broad spectrum activity against various-microbial agents that can cause CVC
related infections.
This activity in the M-EDTA
coated catheters is superior and provides a broader spectrum anti-microbial activity than that provided using Arrow Gard catheters:

~ , ~ ; , Ex~LE s. s Sg;ELF I,~TFE AND STABILITX
, pF THE COATED P5-EDTA (IN SERUM) w Catheters coated wzth M-EDTA, as well as control and Arrow catheters were tested at baseline .(day 1 - Dl) against 5. epidermidi~ using a zone of inhibition measure for relative anti-microbial activity. Then the same '..'~I~:~i !~.r::~h~.
r,.. .: ~:: -.. ...,;, ~ -: ;,, ...;:. ;: . . . :: :: , . ..:::,. ,. . .:.: '.
" .', ... ~. ,;.; ~::

~~ ,Wt's 94/10838 - PC.'T/1JS93/10893 catheters were placed in serum at 37°C and tested at days 3, 7, ~.5 and 30 to determine inhibitory efficacy over time. (Table 17). In addition, segments of the same catheters were kept for.30 and 60 days at 25°C then tested to determine anti-microbial activity as a measure of the shelf life of cetheters with such coatings (Table 1. 8 ) .
STAHII~I~.'St BF ~i-EDTA CDA~'ED CATHETERS
ao ~AB~E s:7 ~c~~S of rr~a~I~zo~a - ~zArfETER (~) M-EDTA 31. 21 16 14 10 Control 0 0 ~ 0 0' Arrow Gard 14 07 07 ~5 03 7~A~LE 18 - ~QIJES OF T1~I~ITTON - DIA'~3ETER (tea) DA'S~' DAY 6 0 . 37oC 25C ~5C

M-EDTA 10 34 3z Control 0 0 0 .row Gard 3 13 1~

25 The foregoing studies demonstrate that M-EDTA
coated catheters tmaint~in excellent anti-microbial (particularly anti.-S epidermidi~~; ;, efficacy two weeks ;,or at in , least C. Whereas.
serum at 37C and at 25 last two months at the efficacy o~ the decreases Arrow Gard catheters ,30 rapidly within .
three days in serum .,.,: ,_ (37oC) .:. :, , :.,.: :;,;
,, r.-.. .. --.. .
-._ . r... . ~: :-:.

IN VIVD EFFICACY OF M-EDTA
COATED CATHETERS IN A RAHHIT MODEL
Animal studies with polyurethane catheters coated with minocycline plus EDTA are provided. The results are consistent with the extensive in vitro data described herein. Currently, the only antibiotic-coated catheters on the market are those produced by Arrow. These catheters are coated with chlorhexidine gluconate and .
silver sulfadiazine. They have been described in a clinical study done as capable of reducing the rate of catheter-related sepsis five fold.
An established in vivo rabbit model was used in the present example (Sherertz et a1. Journal of Infectious Diseases 167:98-106, 1993). Catheters were inserted percutaneously in the rabbit under aseptic conditions.
Tmmediately after insertion, the catheter insertion site was inoculated with 0.1 ml of 105 colony forming units (CFU) of Staphylococcus aureus from the bloodstream of a patient with catheter-related S. aureus bacteremia.
Seven days after insertion, the catheters were removed and the subcutaneous (SQ) as well as the tip of the catheters were cultured by quantitative catheter cultures.
PREPARATION OF M-EDTA COATED CATHETERS
Catheters were first pretreated with a cationic surfactant tridodecylmeylammonium chloride (TDMAC).
(Example 11). Other surfactants, such as benzalkonium chloride, may also be used. Treatment of a catheter with a surfactant will enable subsequent bonding of anionic substances, such as the antibiotic mir_ocycline (mino) and EDTA to the surface. For the present studies, polyethlylene catheters already coated with TDMAC were immersed yn a 60 mg/ml solution of EDTA and 60 mg/ml minocycline, (Bio-Guard T"" AB coating, Cook Critical Care, Bloomington, Ind.). These polyethylene catheters were manufactured by Cook Critical Care (Bloomington, Ind.), 5. 0 Fr (18 ga) , 15 cm (5 7/8 in) ) . For these in vivo studies, the catheters were cut into 6 cm segments before treating with the M-EDTA solution.
Solution A. 3 vials of minocycline (100 mg each) diluted with 0.8 ml of sterile water for injection, USP
to obtain 2.4 ml of 120 mg/ml minocycline;
Solution B. 1.6 ml of 150 mg/ml EDTA was added to.
0.4 ml sterile water to obtain 2 ml of 120 mg/ml EDTA.
Finally, 2 ml of solution A was added to 2 ml solution B, resulting in a 4 ml solution of 60 mg/ml minocycline and 60 mg/ml EDTA.
All catheters used were of the polyurethane type made by Arrow or Cook. The following results were obtain in the experiment where catheters were cultured by either the standard semiquantitative roll-plate culture technique (Maki et al., 1977, N. Engl. J. Med., 296:1305-1309) or the sonication (Sherertz et al., 1990, J. Clin.
Microbiol., 28:76-82) technique.
The data in Tables 19 and 20 demonstrated the consistently inhibitory activity of the M-EDTA coated catheters against S. aureus colonization.

1 v _: r ~f~ 94/~08~8 PCf/US93/~OBys . 1. ~~9 ~~° .

'ABLE ~. ~ - NtTMBER OF' S . AUREIlS
COLONIES C'ULT~RRED BY ROLL-PLACE
Catheter 'I'ye Cathet'~r Tits Catheter SO*

Control ;1000 >1.000 Control >1000 >~.000 _~row Gardt 2 20 Arrow Gard 0 0 Arrow Gard 8 ZO

Arrow Gard ~.0 M-EDTA 0 0 M-ED'T.~3 0 0 Coated w1t11 Chl0r.t1eX1C11ne glLiCOrla~e aaau :mivcs sulf adiazine * Subcutaneous catheter segment TABLE ~ 0 - N~ER Off' ~ . A~
COLONIES ~C~ThTT~RED BY ROLL-PhATE

TABLE 21 - NIJI~ER OF S . AURTUS
COLONIES CULTURED BY SONICATION
Catheter Tvpe Catheter Tip Catheter SO*

Control 40 60 Control 380 >1000 Arrow Gardt 180 >1000 Arrow Gard 0 20 These studies demonstrate the complete prevention of colonization upon using M-EDTA coated catheters compared to the partial prevention achieved using the Arrow Gard catheters.
~CAMPLE 18 IN VIVO EFFICACY OF M-EDTA CATHETERS
The present example further demonstrates the anti-microbial activity of the coated M-EDTA devices in vivo.
The M-EDTA-coated catheters were prepared as described in Example 17. 104 colony forming units of S. aureus were inoculated at the insertion site of a catheter coated with M-EDTA (as described in Example 13), with chlorhexidine gluconate and silver sulfadiazine (the Arrow Gard catheter) or with TDMAC alone with no added antibiotic.
in the present study, New Zealand White rabbits between 2-3 months old and weighing 2-3 ka were useu. The data in Table 2~ demonstrates the super-~or anti-staphylococcal and total inhibitory activity of MEDTA as __ 1 'VVo~ 94/10838 ~ PC.'T/~JS9311a8ys -5s-compared to the partial a.zzti-S. aureus activity achieved with the Arrow Gard catheter.
TABLE 2 2 - ' HER OF S . AffRETI S
S C~L~I~~ES CUL~'L3REI) BY ROLL-PLATE
Catheter Catheter Catheter Catheter Catheter No . Type TiP ~~2 Site Purulence Control > 1000 > 1000 Yes 2 Control > 1000 > 10a0 Yes 3 Control > 1000 > 1000 Yes 4 Control > 100a > 100 Yes 5 Arrow 3 10 No Gard 6 Arrow 15 10 No Gard % g~.row I5 15 No Gard ~5 g 'Arrow 20 15 No Gaxd 9 M-EDTA 0 0 No ~, p M-EDTA 0 0 No 11 M~EDTA 0 ~ No 12 M-EDTA 0 0 No ~~ 13-.. : M_EDT~ (1 4 No 14 M-EDTA p p No 15 M-EDTA 0 0 No ~6:~, ~~ , .~.:~DT~3a~ a ~No .~ _~I4~942 t3~~ 94/10838 PC'f/US93/10893 Catheter Type Catheter ~'ip Catheter SO
_ Control 6n0 5~0 20 Control 400 480 Contral 600 N/A

Control 640 N/A

Gard ~0 80 Arrp w Arrow Gard 120 80 ' 2 5 ~r~ow Gard ~ 0 16 0 ~rbw Gard 80 160 ~_~DT~A

M..E~T~ 0 0 gvrj _ EDTA

30 M~EDTA 0 0 M:EDTA 0 0 M-gDTA 0 0 M_EDTA 0 0 Sheretz et a1. (1990). The Sheretz et a1. article is specifically incorporated herein by reference for this purpose. The sonication of technique is a quantitative culture technique, and is described in Raad et al. (1992) (Diagn. Microbiol. Infect. Dis., 15:13-20). The M-EDTA
catheters were coated as described in Example 11. The chlorhexidine/silver sulfadiazine coated catheters are commercially available fram Arrow International, Inc.
(300 Bernville Road, Reading, PA 19605) (Arrow Gold catheters). the data in Table 23 again demonstrates the consistently effective anti-microbial, particularly the anti-S. aureus, activity of the M-EDTA coated devices of the invention. As well as the superior anti-microbial activity of the M-EDTA devices as compared to the Arrow Gard catheter.
TABLE 2 3 - HER Ol~ S : A~R~'DS
COhOI~ES C~T~D B'X S~i~T~CATION

WO 94/10838 PCT/Ug93/10893 ACTIVITY OF M-EDTA COATED CATHETERS
This study was performed in the rabbit model as described in Example 18. 104 colony forming units (CFU) of S. aureus (a PI strain) were used to infect the catheter insertion site. The catheters were cultured by sonication. The Arrow Gard and Cook catheters coated with EDTA (Example 17) were employed in the study. The control catheters used were again the Cook catheters that have a coating of TIDMAC, but are without antibiotic or EDTA. Table 24 provides the date collected in this study. The results in Table 24 again demonstrate the in vivo effectiveness of the M-EDTA coated catheters for inhibiting infection by S.
aureus in vivo.
Catheters were inserted into the subcutaneous space of New Zealand white rabbits~2-3 months old and weighing 2-3 kg. A 0.1 ml of 10 colonies of a virulent S. aureus strain (PI strain) was injected at the insertion site.
The rabbits were sacrified on day 7. Catheters were aseptically removed and the 2 cm tip cultured by the sonication technique. The results are shown in Table 24.
These results again demonstrate the in vivo effectiveness of M-EDTA against S. aureus infection. These results confirm previous experiments whereby M-EDTA coated catheters prevented staphylococcal colonization and catheter infection compared to partial prevention by the Arrow catheters.

2~.~~94~
9~'~ 94/10838 PCfiUS93l10893 _S9_ TAELE 24 - ND~ER OF S. ALTR.EtTS
COLO~TTES CL~'LTU~ED 8Y 80R~TICAx~0~1 Catheter Catheter Catheter Catheter Site No. Type ~ Tip Purulence Control > 105 Yes 2 Control > 105 Yes 3 Control > 105 Yes 4 Control > 105 Yes 5 Arrow Gird > 1n3 No 1Q 6 Arrow Gard 16 No Arrow Gard 0 No g Arrow Gard 0 No 9 Arrow Gard 4 N

la Arrow Gard o No 15 11 Arrow Gard Q No 12 Arrow Gard 0 No 13 Arrow Gard 0 No ~.4 M-EDTla 0 No 15 M-EDTA o No 2 0 ~: 5 M-EDTA 0 No 1~ M-EDTA 0 No ~g M-EDTA 0 No 19 M-EDTA p No 20 ~-EFTA ~ N

25 21. M-EDTA 0 No ;~
22 M-EDTA 0 No PCTlU593/108y3 HIHLIOGRAPFiY
The following references are specifically incorporated herein by reference for the purposes indicated:
1 . Harper tnTE and Epis '~JA (1987) , Effect of chlorhexidine/EDTT~/Tris against bacterial. isolates from clinical specimens . , Microbios. , S1: x.07-1.12 .
3. 0 2. Said AA et a,~. (1987), Expression of H1 outer-membra.ne protein of Pseudomonas aeruginosa in relation to sensitivity to EDTA and polymyxin B. , J. Med. l~icrobiol . , 24:267-274.

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j ,.. . ....,: ,, -.:. . ._,........,..;.. . .,,......._,. . ....,.., ;........ _, q , ..,r ~.;-... ....,.:~..-..,.., . . .,-:. ... , ,.,, .; : ..... .
...,.....-, _.'.. . :. , ,. .. a ; ....;.,.._.. , , .. .. ... .
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Claims (44)

Claims:

1. An implantable medical device resistant to microbial growth and coated with EDTA and minocycline.

2. A catheter resistant to glycocalyx formation, saiid catheter being coated with EDTA and minocycline.

3. The medical device of claim 1 or catheter of claim 2 defined further as prepared by a process of:
obtaining a medical device or catheter; and treating the device or catheter surface with an anti-microbially effective amount of a preparation comprising minocycline and EDTA.

4. The medical device or catheter of claim 3 wherein the preparation comprises between about 10 to about 100 mg/ml minocycline and between about 10 to about 100 mg/ml EDTA.

5. The medical device or catheter of claim 4 wherein the preparation comprises about 60 mg/ml minocycline and about 60 mg/ml EDTA.

6. The medical device or catheter of claim 3 wherein the device or catheter is further coated with a surfactant.

7. The medical device or catheter of claim 6 wherein the surfactant is tridodecylmethyl ammonium chloride.

8. The medical device or catheter of claim 6 wherein the surfactant is benzalkonium chloride.

9. The medical device of claim 1 or 3 or catheter of claim 2 or 3 further defined as a central venous catheter, a peripheral intravenous catheter, an arterial catheter, a Swan-Ganz catheter, a hemodialysis catheter, an umbilical catheter, a percutaneous nontunneled silicon catheter, a cuffed tunneled central venous catheter or a subcutaneous central venous port.

10. The medical device of claim 1 or 3 or catheter of claim 2 or 3 further defined as a urinary catheter or a peritoneal catheter.

11. A glycocalyx inhibiting pharmaceutical preparation carrying a pharmacologically effective amount of minocycline and at least about 10 mg/ml EDTA.

12. The pharmaceutical preparation of claim 11 comprising between about mg/ml and about 100 mg/ml EDTA.

13. The pharmaceutical preparation of claim 11 comprising between about 0.001 mg/ml and 100 mg/ml minocycline.

14. The pharmaceutical preparation of claim 13 comprising about 30 mg/ml EDTA and about 30 mg/ml minocycline.

15. The pharmaceutical preparation of claim 13 comprising about 60 mg/ml EDTA and about 60 mg/ml minocycline.

16. The pharmaceutical preparation of claim 11 comprising about 3 mg/ml minocycline and about 30 mg/ml EDTA in a saline carrier solution.

17. A minocycline solution for use in preventing glycocalyx formation on a catheter containing between about 0.001 to about 100 mg/ml minocycline and an antimicrobially effective amount of EDTA.

18. The minocycline solution of claim 17 comprising about 3 mg/ml minocycline.

19. An EDTA and minocycline solution for use in preventing glycocalyx formation on a catheter and polysaccharide glycocalyx formation comprising between about 0.001 and 100 mg/ml minocycline and between about 10 and 100 mg/ml EDTA.

20. The EDTA and minocycline solution of claim 19 comprising about 30 mg/ml EDTA and about 3 mg/ml minocycline.

21. A method of preparing a glycocalyx-resistant medical device or catheter comprising coating the device or catheter with the mixture of claim and allowing the coating to dry on the surface of the device or catheter.

22. The method of claim 21 wherein the device or catheter is an indwelling catheter.

23. The method of claim 21 wherein the device or catheter is resistant to formation of polysaccharide-rich glycocalyx.

24. The method of claim 23 wherein the device or catheter is resistant to formation of a staphylococcal glycocalyx.

25. The method of claim 21 wherein the device or catheter is a central venous catheter or a triple lumen catheter.

26. A method for preparing a biofilm-resistant medical device using a pharmaceutical preparation of minocycline and EDTA, comprising:
obtaining a medical device;
preparing a preparation comprising at least about 10 mg/ml minocycline and at least about 10 mg/ml EDTA; and treating the device surface with the pharmaceutical preparation to allow the formation of a film of the preparation on the device surface.

27. The method of claim 26 wherein the medical device is a catheter.

28. The method of claim 26 wherein the biofilm is a microbial glycocalyx.

29. The method of claim 26 wherein the biofilm is a polysaccharide-rich glycocalyx.

30. The method of claim 26 wherein the biofilm is a staphylococcal glycocalyx.

31. The method of claim 26 wherein the pharmaceutical preparation comprises between about 10 to about 100 mg/ml minocycline and between about 10 to about 100 mg/ml EDTA.

32. The method of claim 26 wherein the pharmaceutical preparation comprises about 60 mg/ml minocycline and about 60 mg/ml EDTA.

33. A method for inhibiting polysaccharide glycocalyx formation on a catheter comprising flushing the catheter periodically with a preparation comprising at least about 10 mg/ml EDTA and minocycline in a pharmacologically acceptable carrier solution.

34. The method of claim 33 wherein the preparation comprises between about 10 mg/ml and about 100 mg/ml EDTA.

35. The method of claim 33 wherein the preparation comprises between about 0.001 mg/ml and about 100 mg/ml minocycline.

36. The method of claim 33 wherein the preparation comprises about 30 mg/ml EDTA and about 3 mg/ml minocycline.

37. The method of claim 33 wherein the catheter is a tunneled catheter or an untunneled catheter.

38. The method of claim 33 wherein the catheter is flushed once every 4-24 hours.

39. A method for eliminating microbial glycocalyx formation at a catheter comprising:
preparing a solution of minocycline and EDTA in a carrier solution to provide an M-EDTA preparation; and flushing the catheter periodically with the M-EDTA preparation.

40. The method of claim 39 wherein the solution comprises about 0.001-100 mg/ml minocycline and about 10-100 mg/ml EDTA.

41. The method of claim 39 wherein the solution comprises about 3 mg/ml minocycline and about 30 mg/ml EDTA.

42. The method of claim 39 wherein the microbial glycocalyx is a Staphylococcal glycocalyx.

43. The method of claim 39 wherein the microbial glycocalyx is an S.
epidermis glycocalyx or an S. aureus glycocalyx.

44. The method of claim 39 wherein the catheter is flushed once every about 24 hours with between about 1-10 ml of the solution.