Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme-antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies on this unique approach to ADEPT.

Adenocarcinoma↗

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A stress-radiographic method to evaluate dogs for passive hip laxity.

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Animals↗

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Pullout properties of 3.5-mm AO/ASIF self-tapping and cortex screws in a uniform synthetic material and in canine bone.

OBJECTIVES--To compare the pullout properties of 3.5-mm AO/ASIF self-tapping screws (STS) to corresponding standard cortex screws (CS) in a uniform synthetic test material and in canine femoral bone. The influence of screw-insertion technique, test material, and test-material thickness were also assessed. STUDY DESIGN--In vitro experimental study. SAMPLE POPULATION--Two independent studies: a uniform synthetic test material and paired femurs from mature dogs. METHODS-Mechanical testing was performed in accordance with standards established by the American Society for Testing and Materials for determination of axial pullout strength of medical bone screws. Completely inserted STS, completely inserted CS, and incompletely inserted STS were tested in 3 groups of 10 test specimens each in 4.96-mm and 6.8-mm thick sheets of synthetic material. In the bone study, group 1 consisted of 24 completely inserted STS compared with 24 completely inserted CS, and group 2 consisted of 24 incompletely inserted STS versus 24 completely inserted CS. Comparisons were made between paired femurs at corresponding insertion sites. Pullout data were normalized, thereby eliminating the effect of test-material thickness on pullout properties. Mean values were compared using 2-way ANOVA. Statistical significance was set at P <.05. RESULTS--In both the 4.96-mm and 6.8-mm synthetic material, pullout testing of the completely inserted STS demonstrated significantly greater yield strength and ultimate strength than completely inserted CS. There was no significant difference between incompletely inserted STS and completely inserted STS. The 6.8-mm test material significantly increased yield strength and ultimate strength for all test groups compared with the 4.96-mm test material. In canine bone, there was no significant difference in yield strength of completely inserted STS and completely inserted CS. Yield strength of completely inserted STS and completely inserted CS were significantly greater than incompletely inserted STS. CONCLUSIONS--Pullout properties of completely inserted STS were significantly greater than corresponding CS in a uniform test material. In canine bone, the pullout strength of STS and CS were not different. Incomplete STS insertion resulted in an 18% reduction in holding power as compared with completely inserted CS and STS in canine bone. CLINICAL RELEVANCE--The length of STS used in canine bone should be such that the cutting flutes extend beyond the trans cortex to maximize pullout strength.

Animals↗

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Biomechanical stability of four cranial cruciate ligament repair techniques in the dog.

Four commonly used cranial cruciate ligament (CCL) repair techniques were compared biomechanically in canine cadaver stifles by assessment of immediate postoperative laxity (cranial drawer) and stiffness. Each joint was tested nondestructively before repair, with the CCL intact and with the CCL excised. The cranio-caudal laxity after each repair was expressed as a percentage of the laxity in the same stifle after CCL excision. The stiffness of a repair was expressed as a percentage of the same intact joint's stiffness at an equivalent displacement. Repaired stifles were tested three consecutive times to the same maximum load (180 N) and the comparative laxity and stiffness in each test were determined. Fibular head transposition resulted in significantly increased stiffness, less laxity, and less decay per test than the other repairs. Lateral imbrication resulted in significantly less laxity on all tests, and significantly greater stiffness than the fascial strip or patellar tendon on the second and third tests to maximum load. The fascial strip resulted in significantly less laxity on all tests than the patellar tendon, but it was not significantly greater in stiffness. These data suggest that, of the reconstructive methods tested, fibular head transposition has biomechanically superior properties in the immediate postoperative period.

Animals↗

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Unilateral and bilateral stifle arthrodesis in eight dogs.

Nine stifle arthrodeses in eight dogs were reviewed retrospectively to evaluate use of the limb, each dog's comfort, complications, and factors that may have influenced the final outcome. Ability to use the limb after unilateral fusion was good (limb used at all times) in three dogs, fair (limb used at all gaits except a gallop) in three dogs, and poor (limb used only when running) in one dog. Factors that appeared to affect the outcome included angle at which the stifle was fused and lesions in the ipsilateral coxofemoral joint. One dog with bilateral arthrodesis had a good outcome with minor limitations. The only potentially devastating complications occurred in one dog in which infection and premature implant loosening jeopardized the fusion. None of the dogs exhibited signs of pain and all owners were satisfied with the results.

Animals↗

PubMedDate not supplied

Perspective missing in fibular head transposition study.

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Animals↗

PubMedDate not supplied

Clinical determination of preoperative and postoperative intra-abdominal pressures in dogs.

Intra-abdominal pressure (IAP) was indirectly measured, using a transurethral catheterization technique, in 20 client-owned dogs before and after elective ovariohysterectomy. Mean preoperative IAP was 4.50 +/- 0.44 cm H2O. Elective abdominal surgery caused significant elevations in mean postoperative IAP (mean 7.50 +/- 0.45 cm H2O, range 0 to 15 cm H2O) that persisted for at least 24 hours. However, the increase in IAP caused no clinically evident complications; thus, after elective abdominal surgery an elevation in IAP up to 15 cm H2O is to be expected. Intra-abdominal pressure was also measured in 20 consecutive clinical cases with gross abdominal distension, before or after laparotomy, or both. Included in this group were dogs with gastric dilation and volvulus, closed pyometra, hemoperitoneum, acute ascites, and diaphragmatic hernias. All dogs with gross abdominal distension had an elevated IAP (> or = 16 cm H2O) either before or after surgery. Severe elevations of IAP were associated with anuria in two dogs, necessitating surgical decompression; one with hemoperitoneum (47 cm H2O) and one after repair of a chronic diaphragmatic hernia (30 cm H2O).

Abdomen↗

PubMedDate not supplied

Postoperative analgesia for stifle surgery: a comparison of intra-articular bupivacaine, morphine, or saline.

A prospective study was undertaken to compare the analgesic effect of intra-articular bupivacaine, morphine, or saline in the 24-hour period following cranial cruciate ligament repair in dogs. Thirty-six clinical patients with ruptured cranial cruciate ligaments were randomly assigned to one of three groups. After surgical stabilization, and before skin closure, an intra-articular injection was given; group one (n = 12) received 0.5% bupivacaine HCl at 0.5 mL/kg, group two (n = 12) received morphine at 0.1 mg/kg diluted with saline to a volume of 0.5 mL/kg, and group three (n = 12) received saline at 0.5 mL/kg. Heart rate, respiratory rate, mean arterial blood pressure, cumulative pain score, visual analog pain score, and pain threshold test on both stifles were recorded preoperatively and at 0 to 6 and 24 hours postoperatively. Surgeons and pain scoring investigators were unaware of the intra-articular medication given. Supplemental analgesia, if needed, was provided in the postoperative period according to subjective assessment of patient discomfort. Postoperative pain scores were lowest in the bupivacaine group and highest in the saline group. Pain threshold, measured by applying calibrated loads to the knee, was higher postoperatively in the bupivacaine group than in the saline group. Dogs in the morphine and bupivacaine groups required less supplemental analgesia than dogs in the saline group. The local provision of analgesia reduces the need for systemic drugs with potential side effects. Both intra-articular morphine and intra-articular bupivacaine provided better postoperative analgesia than intra-articular saline, with intra-articular bupivacaine showing the greatest effect.

Analgesia↗

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The effects of active peripheral skin warming on perioperative hypothermia in dogs.

OBJECTIVE: This study evaluates the efficacy of three perioperative warming protocols to improve control of body temperature in anesthetized dogs. STUDY DESIGN: A randomized controlled clinical trial. ANIMALS OR SAMPLE POPULATION: Thirty-two client-owned dogs. METHODS: We prospectively studied dogs entering the University of Pennsylvania Veterinary Teaching Hospital for orthopedic or dental procedures and assigned them to one of three perianesthetic warming protocols. Group 1 (n = 10) had a single circulating warm water mattress applied over the trunk (single-trunk warming). Group 2 (n = 12) had two circulating warm water mattresses, one placed over and one under the trunk (double-trunk warming). Group 3 (n = 10) had warm circulating mattresses applied only around the feet and legs of all available limbs (peripheral warming). The warm water mattresses were prewarmed and maintained at 40 degrees C (104 degrees F) and applied immediately after induction of general anesthesia. All dogs had a layer of thick terry cloth toweling beneath and above the trunk. Body temperature measurements were recorded every 15 minutes for the first 2.5 hours of anesthesia. RESULTS: The lowest mean temperature for dogs in group 3 was 37.4 +/- 0.2 degrees C (99.5 degrees F), compared with 36.4 +/- 0.2 degrees C (97.4 degrees F) and 36.7 +/- 0.2 degrees C (98.0 degrees F) in groups 1 and 2, respectively. CONCLUSIONS: Dogs in the peripheral warming group maintained significantly higher core body temperatures than dogs in either trunk warming groups throughout the 2.5-hour study period. CLINICAL RELEVANCE: To maintain body heat in dogs during anesthesia, it is more effective to warm the feet and legs than to warm the trunk.

Animals↗