IMPLANTABLE MEDICAL DEVICES WITH INCREASED IMMUNE TOLERANCE, AND METHODS FOR MAKING AND IMPLANTING

Abstract

The present invention relates to the contacting of one or more surfaces of an implantable medical device with one or more diketopiperazines (DKPs).

Claims

1-87. (canceled)

88. A medical device coated with DA-DKP and a component selected from the group consisting of N-acetyl tryptophan, caprylate, caprylic acid, and combinations thereof.

89. The medical device of claim 88, wherein the component is N-acetyl tryptophan.

90. The medical device of claim 88, wherein the component is caprylate, caprylic acid, or both caprylate and caprylic acid.

91. The medical device of claim 88, wherein the component is N-acetyl tryptophan and caprylate.

92. The medical device of claim 88, wherein the component is N-acetyl tryptophan and caprylic acid.

93. The medical device of claim 88, wherein the component is N-acetyl tryptophan, caprylate and caprylic acid.

94. The medical device of claim 88, which is implantable.

95. The medical device of claim 88, which is selected from the group consisting of a graft, catheter, stent, prosthetic, breast implant, pump, tube, pin, rod, screw, brace, plate and pacemaker.

96. The medical device of claim 88, which is a tracheal tube.

97. The medical device of claim 96, which is an endotracheal tube.

98. A method of preparing a medical device, comprising contacting the medical device with DA-DKP and a component selected from the group consisting of N-acetyl tryptophan, caprylate, caprylic acid, and combinations thereof.

99. The method of claim 98, wherein the component is N-acetyl tryptophan.

100. The method of claim 98, wherein the component is caprylate, caprylic acid, or both caprylate and caprylic acid.

101. The method of claim 98, wherein the component is N-acetyl tryptophan and caprylate or N-acetyl tryptophan and caprylic acid.

102. The method of claim 98, wherein the device is implantable.

103. The method of claim 98, wherein the device is selected from the group consisting of a graft, catheter, stent, prosthetic, breast implant, pump, tube, pin, rod, screw, brace, plate and pacemaker.

104. The method of claim 98, wherein the device is a tracheal tube.

105. The method of claim 104, wherein the device is an endotracheal tube.

106. The method of claim 98, wherein the DA-DKP is coated onto the surface of the device.

107. A method for implanting a medical device into a subject, wherein a surface of the device is coated with DA-DKP, N-acetyl tryptophan, caprylate, caprylic acid, or combinations thereof.

Description

EXAMPLES

Example 1

[0094] This example shows the results of an analysis of biofilms on extracted orthopedic devices to determine if the presence of a diketopiperazine is correlated with bacterial colonization. Bacteria use small molecular weight N-acylhomoserine lactones and diketopiperazines to initiate biofilm formation and regulate colony growth. An Aspartate, Alanine-Diketopiperazine (DA-DKP) formed by the cleavage and cyclization of the N-terminal amino acids of human serum albumin has previously been demonstrated to be immunomodulatory for memory but not naive human T lymphocytes.

[0095] Methods: This study was an institutional review board (IRB) approved study. Twenty-two patients undergoing hardware removal were enrolled. The removed orthopedic devices were stripped of surface biofilm using methanol/ammonium formate. The ≤3 kD MW material was collected and diketopiperazine levels analyzed using anion exchange high pressure liquid chromatography coupled to negative electrospray ionization mass spectrometry.

[0096] Results: The thirty-three patients ranged in age from 6 to 91 years, with a mean of 54. There were fifteen males and eighteen females. Ten devices were reported by the clinical laboratory to be culture positive. In five cases the main organism was Staphylococcus. In one of the three cases Bacteroides and Streptococcus species were also isolated. In all thirty-three cases detectable amounts of DA-DKP were identified with a mean level of 120 ng/ml. Higher amounts of DA-DKP (9.75-235 ng/ml) were detected in the culture positive devices versus the culture negative (1.78-34.7 ng/ml). In addition, one device removed from a case with osteomyelitis had a DA-DKP content of 3,063 ng/ml. (see Table 1).

[0097] Conclusion: DA-DKP is an important immune modulator in biofilm formation on orthopedic implants. Its presence in biofilms found on extracted orthopedic devices suggests innate physiologic mechanisms conferring tolerance to the implanted device possibly correlated to the presence of inflammation/rejection reactions.

TABLE-US-00001 TABLE 1 Study Group DA-DKP amounts Sex of subject Concentration Age of F = female Culture of DA-DKP subject M = male Device Removed Results ng/ml 73 F Pin (hip) None 4.63 66 F Rod + Screws (hip) Negative 19.7 89 F Rod + Screws (hip) Staph 87.0 (positive) 74 M Staple (knee) None 3.03 67 F Septic hip prosth. Staph aureus 84.8 44 M Plate (clavicle) None 2.40 45 F Rod + Screws (spine) Negative 30.9 87 F Screws (hip arthritis) None 19.9 33 M Rod + Screws (knee) None 6.03 45 M Rod + Screws (ulna) None 9.05 31 F Rod + Screws (ankle) Bacteroides + 53.7 Strep 52 F Brace + Screws None 9.02 (tibia) 58 F Screws (femur) Staph 28.4 (positive) 74 M Brace (clavicle) Negative 7.02 6 M Plate + Screws None 22.4 60 F Rod (osteomyelitis Negative 3,063 tibia) 58 F Hip Prosth. Negative 23.0 66 F Rod (femoral) Staph + Strep 235 45 M Rod + Screws (tibia) Gram + 12.9 55 M Nails (femoral) Gram + 70.4 54 F Ankle Prosth. Negative 34.7 24 M Screws (tibia) Gram + 9.75 91 F Screws (hip) None 3.91 44 M Plate + Screws None 14.2 (ankle) 46 F Screws (ankle) None 9.32 24 M Screws (femur) Staph aureus 53.1 68 F Knee Prosth. Negative 1.78 56 F Screws (knee) None 3.41 41 F Brace + Screws Yeast 20.4 (ankle) 61 M Screws (femur) Negative 4.21 34 M Plate + Screws Negative 9.78 (radius) 77 M Plate + Rod (hip) None 2.13 28 M Plates + Screw None 12.7 (ankle)

Example 2

[0098] Isolation and characterization of peptides and proteins from endotracheal tubes. The results of this example further demonstrate that DKPs form on implantable medical devices such as endotracheal tubes, when they are implanted within a subject. The presence of the DKPs on these tubes helps the subject to confer tolerance to the tubes. This again demonstrates the unique finding of coating implantable medical devices, such as endotracheal tubes, with DPKs prior to implantation so as to increase the subject's immune tolerance and/or to decrease a subject's inflammatory response to the tube.

[0099] Endotracheal tubes discarded from mechanically ventilated trauma patients are collected into sterile biohazard pouches and transported immediately to the Trauma Research Lab. As controls for the absence of biofilms, discarded endotracheal tubes form surgical patients that were only used for a few hours during surgery were used.

Method

[0100] Biofilm and/or mucus is stripped from the proximal ends of endotracheal tubes by placing in a sterile centrifuge tube containing 1-2 ml of chromatography each analysis buffer consisting of methanol 60% plus 50 mM ammonium formate 40% with extensive washing using a pipette and agitation on a vortexer. After the biofilm is stripped from the endotracheal tube, the sediment is pelleted by centrifugation and frozen for later analysis of bacterial content. The biofilm supernatant is collected for analysis of protein and large molecular width peptides. An aliquot of the biofilm supernatant is placed in an ultrafiltration spin column (Vivaspin 500, 3,000 MWCO, Sartorius, Hannover, Germany) for centrifugation at 15,000×g. The filtrate is collected for analysis of <3 kD molecular weight peptides.

[0101] Supernatants containing higher molecular weight material are analyzed by high performance liquid chromatography (HPLC, Waters, Milford, Mass., USA) coupled to positive electrospray ionization time of flight mass spectrometry (+ESI-TOF MS, Micromass, UK). Each supernatant is diluted 1:10 with dH.sub.2O. 10 μL of each sample is injected onto a YMC-Pack Protein-PR, 150 mm×4.6 mm, 5 u, HPLC column heated at 50° C. (Waters, Milford, Mass., USA) using a 20-minute linear gradient method used water/0.1% trifluoroacetic acid (A) and acetonitrile/0.1% TFE (B). The output of the HPLC is split 1:20 (v:v) and injected into the mass spectrometer with a scan range of 500 to 3500 m.z, cone voltage of 30 eV, source temperature of 100° C., and gas temperature of 250° C. Albumin (a molecular standard) elutes at 8.15 minutes and is visualized as a charge envelope from 1100 to 2500 m/z representing +44 to +26 charges. The spectrum is the deconvolved to the uncharged parent mass using MaxEnt 1 (Micromass, UK). The parent mass spectrum is then integrated and relative proportions of each species were calculated.

[0102] 50 μl of each of the <3000-Da filtrate fractions of biofilm supernatant is injected into high performance liquid chromatography (HPLC, 2795 system, Waters, Mass.) coupled to a mass spectrometer (LCT-TOF, Micromass, UK), and quantified using a storage anion exchange column (Supelcosil, SAX1 250 mm×4.6 mm, Supelco) and a 70:30 v/v methanol/water with 25 mM ammonium acetate (Sigma Aldrich, St. Lois, Mo.) as the mobile phase in an isocratic mode at 1 ml/min. The output of the HPLC is split 1:20 (v/v) and injected into the mass spectrometer using negative electrospray ionization (−ESI MS) with a scan ranges of 80-1000 m/z, cone voltage of 30 eV, source temperature of 100° C. and a gas temperature of 250° C. DA-DKP, as a molecular standard, is measure by monitoring the mass 185 in time which corresponds to DA-DKP minus a single proton (−H+). DA-DKP elutes at 5.8 mins and is quantified by integrating the area under the curve. The area was compared with a standard curve derived from synthetic DA-DKP standard (DMI Synthesis, Newport, Wales) of known concentrations (5000 ng/ml, 1000 ng/ml, 200 ng/ml, 40 ng/ml, 8 ng/ml). The calibration curve was found to be very linear in this range within R2 of 0.99998.

[0103] The concentration of DKP on over 100 endotracheal tubes as detected by the method described above is presented in Table 2. The DKP concentration ([DKP]) provided on Table 2 has already been adjusted per volume added to dissolve biofilm. The following are indicated on Table 2:

[0104] ID #: Subject identification number

[0105] Sex: Subject's sex either male (M) or female (F)

[0106] Age: Age in years of the subject

[0107] [DKP] ng/ml: The DKP concentration in ng/ml already adjusted per volume added to dissolve biofilm

[0108] Bacteria identified: Type of bacteria detected on endotracheal tube

[0109] Vent Days: The number of days the endotracheal tube was implanted with the subject

[0110] Protein identified: The proteins that were determined on the endotracheal tube

[0111] AIS: Abbreviated injury score/scale, with a score of 1 being a minor injury, 2=moderate, 3=serious, 4=severe, 5=critical, 6=maximum, 9=not further specified.

[0112] ISS: Injury severity score, assesses trauma severity and correlates with mortality, morbidity and hospitalization time after trauma.

[0113] GCS: Glascow coma score/scale-neurological scale to help assess the status of the central nervous system and used acutely to grade the severity of a subject's trauma and mental function.

[0114] GOS: Glascow outcome score/scale (R=rehabilitation; L=long term acute care; 1=dead; 5=good recovery)—a 5-point score given to victims of traumatic brain injury at some point in their recovery.

TABLE-US-00002 TABLE 2 DKP Concentration on Endotracheal Tubes ID [DKP] Vent Proteins # Sex Age ng/ml Bacteria Days ID AIS ISS GCS GOS  18 M 25 150.06 8 PRP1, 2 38 15 5 HIG2, AT5G1  24 M 47 607.73 S.pneumon 11 Defensin 3 20 15 R MRSA 1, 2, 3 LL-37, Lysozyme C  25 M 90 <20 3 Defensin 3 14 6 1 1, 2, 3 LL-37  28 F 22 <20 1 Defensin 3 21 7 R 1, 2, 3 LL-37  30 F 55 1,071.55 Enterob. 4 2 17 3 R Sakazakii    30-2 F 55 <20 Enterob. 4 2 17 3 R Sakazakii  31 M 60 1,402.85 9 S10A8 3 10 15 5  34 M 20 <20 H. 30 5 45 3 R influenza  35 F 71 116.29 7 4 30 6 L  36 M 46 1,580.08 Pneumo- 11 3 34 9 R thorax 36-2 M 46 <20 Pneumo- 11 3 34 9 R thorax  39 M 58 <20 5 4 29 15 L  41 M 35 72.53 K. oxytoca 20 3 43 3 L S. aureus  44 M 35 <20 8 4 25 4 5  45 M 17 34.09 S. 3 1 21 3 5 marcesens  50 M 76 362.29 1 3 22  52 M 34 159.56 <1 2 17 12 R  53 M 21 156.85 <1 3 14 6 1  58 M 25 <20 36 5 29 15 L  60 M 15 124.82 1 1 41 5  70 M 28 104.63 4 3 26 3 R  79 M 73 33.48 K. oxytoca 8 4 L  87 M 49 79.92  90 F 56 103.79  96 F 49 637.7  97 M 30 1,752.89  98 F 24 952.71 102 M 32 1,311.48 106 F 80 1,083.15 108 F 59 2,648.23 109 M 20 629.67 114 M 76 379.51 121 F 84 2,325.85 123 M 15 8,009.14 129 M 27 500.95 131 F 65 16,194.74 132 M 32 282.84 133 F 48 480.59 138 M 49 137.37 140 F 81 567.2 143 M 28 426.93 145 M 68 1,655.73 149 M 21 58.68   149-2 M 21 749.03 150 M 31 111.68 151 M 50 389.23 152 M 25 134.8 153 F 51 272.45 156 M 29 25.45 157 F 18 634.13 158 M 51 2,183.93 161 M 77 386.28   161-2 M 77 691.98 162 M 18 364.63 163 M 63 107.4 164 F 15 69.15 166 F 18 856.74 169 F 30 <20 170 M 73 368.9 178 M 53 48.55 179 M 25 <20 182 M 67 1059.7   182-2 M 67 255.73 183 M 36 279.76 184 M 37 <20 190 M 34 624.44 191 M 29 2,207.58   191-2 M 29 798.37 192 M 66 <20 195 M 28 2,608.52 196 F 23 <20 198 M 35 404.87 200 M 33 816.38 202 M 67 <20 203 M 50 112.64 204 1,290.96 205 2,108.70 206 656.8 208 <20 209 276.96 210 76.19 211 76.98 212 <20   212-2 505.63 213 218.41 215 503.16   215-2 322.54 216 163.25 219 12,019.74 221 177.19 222 155.14 225 247.88 228 190.86 229 92.4 231 243.68 233 72.03 234 22.96 235 356.11   235-2 68.11 237 509.7 239 1,620.48 Surg 2,648.55 <1 001 Surg 500.22 <1 002 Surg 56.74 <1 003 Surg 505.67 <1 004 Surg <20 <1 005 Surg F 83 89.23 92 Surg F 83 67.87 92-2 R 2235.63 111 R 1216.05 113

[0115] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following exemplary claims.