Parallel synthesis and screening of peptide conjugates.

Peptide conjugates represent an emerging class of therapeutics. However, in contrast to that of small molecules and peptides, the discovery and optimization of peptide conjugates is low in throughput, resource intensive, time-consuming, and based on educated decisions rather than screening. A strategy for the parallel synthesis and screening of peptide conjugates is presented that (1) reduces variability in the conjugation steps; (2) provides a new method to rapidly and quantitatively measure conversion in crude conjugation mixtures; (3) introduces a purification step using an immobilized chemical scavenger that does not rely on protein-specific binding; and (4) is supported by robust analytical methods to characterize the large number of end products. Copper-free click chemistry is used as the chemoselective ligation method for conjugation and purification. The productivity in the generation and screening of peptide conjugates is significantly improved by applying this strategy as is demonstrated by the optimization of the anti-Angiopoietin-2 (Ang2) CovX-body, CVX-060, a peptide-antibody scaffold conjugate that has advanced in clinical trials for oncology indications.

Ambulatory phlebectomy under tumescent local anesthesia in a kidney-transplant patient.

INTRODUCTION: Tumescent local anesthesia (TLA) is widely used for ambulatory surgery. Patients with transplanted or gans are on immunosuppressive therapy and with risk for organ rejection or severe infection. CASE REPORT: Saphe nectomy with phlebectomy on the left leg under TLA was performed in a patient with kidney transplantation per formed four years ago. A combination of 35 mg of 1% prilocaine-hydrochloride, 5 mL of 8.4% sodium bicarbonate and 500 microg of epinephrine in 460 mL of normal saline was used for TLA. Overall 750 mL of the solution was used. The patient had satisfactory postoperative analgesia and was discharged home on the same day. Blood levels of urea, cre atinine, estimated glomerular filtration rate (eGFR) and ta crolimus concentration, measured preoperatively and on the second postoperative day, were in a regular range. Prilocaine blood concentrations determined on the 4th, 10th and 16th postoperative hours, were below toxic levels. CONCLUSION: TLA in a kidney-transplanted patient performed for saphe nectomy with phlebectomy proved to be a safe and reliable anesthesia method.

Development of a long acting human growth hormone analog suitable for once a week dosing.

Human growth hormone was conjugated to a carrier aldolase antibody, using a novel linker by connecting a disulphide bond in growth hormone to a lysine-94 amine located on the Fab arm of the antibody. The resulting CovX body showed reduced affinity towards human growth hormone receptor, reduced cell-based activity, but improved pharmacodynamic properties. We have demonstrated that this CovX-body, given once a week, showed comparable activity as growth hormone given daily in an in vivo hypophysectomized rat model.

Development of novel linkers to conjugate pharmacophores to a carrier antibody.

We have developed modified maleimide novel linkers with improved chemical stability that could potentially be used in conjugating various pharmacophores such as oligo nucleotides, peptides, and proteins to antibodies to afford novel biologics with well-defined therapeutic benefits and improved pharmacokinetic properties. These linkers expand the array of tools available for bioconjugation of pharmacophores to antibodies.

Epidemiology of nosocomial colonization/infection caused by Acinetobacter spp. in patients of six surgical clinics in war and peacetime.

BACKGROUND/AIM: Acinetobacter spp. has emerged as nosocomial pathogen during the past few decades in hospitals all over the world, but it has increasingly been implicated as a serious nosocomial pathogen in military hospitals. The aim of this study was to analyse and compare the surveillance data on Acinetobacter nosocomial colonization/infection (NCI) collected during the wartime with the data collected in peacetime. METHODS: We conducted a prospective study of incidence of Acinetobacter spp. colonization/infection. Also, the two nested case-control studies were conducted. The patients with nosocomial infection (cases) were compared with those with nosocomial colonization (controls) during the two different periods, wartime and peacetime. The patients with NCI by Acinetobacter spp. were identified by the case-based surveillance. The surveillance covered all the patients in 6 surgical clinics. RESULTS: During the study periods a total of 166 patients had cultures that grew Acinetobacter spp. and the pooled rates of Acinetobacter spp. colonization and infection were significantly higher in wartime. When patients with NCI in wartime were compared with those with NCI in peacetime significant differences were observed. In the war year, the patients were more significantly males (p < 0.000). In a period of peace, most of the colonization/infections were reported from patients with certain chronic diseases (p = 0.020) and the survival of patients was more significant (p = 0.049). During the peacetime, proportions of Acinetobacter isolates resistent to ciprofloksacin, imipenem and meropenem were significantly higher (p < 0.001). CONCLUSION: This study provides additional important information about the risk factors of nosocomial Acinetobacter spp. infections in a large cohort of surgical patients. This is also the first study that directly examines epidemiological differences between NCI caused by Acinetobacter spp. during the war and peace period.

Splenic artery pseudoaneurysm as a complication of pancreatic pseudocyst.

INTRODUCTION: Pancreatic pseudocyst presented as pseudoaneurysm of the splenic artery is a potential serious complication in patients with chronic pancreatitis. CASE REPORT: A 42-year-old male patient with a long-standing evolution of chronic pancreatitis and 8-year long evolution of pancreas pseudocyst was referred to the Military Medical Academy, Belgrade due to worsening of the general condition. At admission, the patient was cachectic, febrile, and had the increased values of amylases in urine and sedimentation (SE). After clinical and diagnostic examination: laboratory assessment, esophagogastroduodenoscopy (EGDS), ultrasonography (US), endoscopic ultrasonography (EUS), multislice computed scanner (MSCT) angiography, pseudoaneurysm was found caused by the conversion of pseudocyst on the basis of chronic pancreatitis. The patient was operated on after founding pancreatic pseudocyst, which caused erosion of the splenic artery and their mutual communication. Postoperative course was duly preceded without complications with one year follow-up. CONCLUSION: Angiography is the most reliable and the safest method for diagnosing hemorrhagic pseudocysts when they clinically present as pseudoaneurysms. A potentially dangerous complication in the presented case was treated surgically with excellent postoperative results.

[Impossibility of the treatment of inoperable liver multicystic echinococcosis due to adverse reactions to antihelminitics].

INTRODUCTION: In some cases of multicystic forms of liver echinococcal disease, the advanced method for treatment of cystic echinococcosis faces great problems relating to the final outcome of the treatment. CASE REPORT: In May 2005, a computerized tomography of the abdomen obtained in a 27-year-old female patient with abdominal pain revealed more than 20 echinococcal cysts measuring up to 6.7 cm in both lobes of the liver. Laboratory analyses found the value of eosinophils 6.8%, gamma globulins 29.9%, immunoglobulin E 29 600 IU/mL and the indirect hemagglutination for echinococcosis 1:8,196. The treatment started in December that year with the continuous administration of a daily dose of 800 mg (14.5 mg/kg body weight) of albendazole, but it was terminated two months later due to high serum transaminases values. By the end of 2006, the largest cyst detected in the left lobe of the liver had a diameter of 5.7 cm and the one in the right lobe of the liver measured 4.1 cm. There were lesions of germinative membrane found on both cysts. Six months later, praziquantel at daily dose of 2,500 mg (45.3 mg/kg body waight) was introduced into the therapy, but the treatment was terminated after eight days because of the development of exanthema. The computerized tomography of the abdomen obtained in February 2008 revealed the presence of a large number of echinococcal cysts in the liver. The largest among those cysts measured 3.5 cm while calcifications of the cyst walls were observed on some of them. None of the remaining therapeutic options for further treatmetnt of echinococcal disease could be applied. CONCLUSION: The presented case confirms medical therapy as the only option for the treatment of some forms of cystic echinococcosis. Benzimidazole carbamates (albendazole, mebendazole) and praziquantel are only efficacious antihelminitics currently available, and when they have to be withdrawn due to serious adverse affects, futher treatment of a patient with liver multicystic echinococcosis is impossible. Because of that there is a need to search for new and more efficient drugs for the treatment of ehinococcal disease.

[Mesoatrial shunt in Budd-Chiari syndrome].

BACKGROUND: Budd-Chiari syndrome (BCS) represents partial or total occlusion of the hepatic veins with or without simultaneous obstruction of vena cava inferior (VCI). The symptoms of BCS are abdominal pain, hepatomegaly, ascites, varices of the abdominal wall, sometimes bleeding from the upper part of gastointestinal tract (GIT), lower limbs swelling and jaundice. Primary BSC is a relatively rare condition occuring in one per 100,000 of the population worldwide. CASE REPORT: A male patient, 25-year-old, facing tooth postextraction complications, was presented with acute BCS. On admission, physical examination revealed pale-grayish complexion, more pronounced veins over the thorax and abdomen, ascites, enlarged liver rising 8 cm below the right costal arch and having a minor pleural effusion by the right side. The patient was submitted to Doppler sonography and computed tomography (CT) that verified the right leg deep veins thrombosis, as well as the presence of a thrombus in the intrahepatic portion of the VCI. Multislice computed tomography (MSCT) showed occlusion of hepatic veins (Budd-Chiari syndrome) and thrombosis of the VCI in the retrohepatic part 6 cm long. Also, increased values of transaminases and gamma GT and reduced values of albumines and serum ferrum were registered. Molecular examination revealed Factor V Leiden mutation--heterozygote. After preoperative preparations a mesocaval shunt was made using Gore-Tex ring graft of 12 mm. Intraoperatively, the blue enlarged liver was found with almost black zones of tense capsule. After a graft making, liver congestion decreased followed by the change of colour and volume. Within postoperative course metabolic and synthetic liver functions were obvious. CONCLUSION: In patients with BCS medicamentous treatment does not yield adequate results, but even causes worsening of general condition. Surgical therapy in the presented patient was performed timely regarding the stage of the disease due to which irreversible liver changes were prevented while decompression of the portal system provided time overbridging up to liver transplantation.

5,6-Dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase.

5,6-Dihydro-1H-pyridin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4ad displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; IC(50) (1a)<25nM, EC(50) (1b)=16nM), good in vitro DMPK properties, as well as moderate oral bioavailability in monkeys (F=24%).

[Adverse effects of longterm, continual administration of high doses of albendazole in the treatment of echinococcal disease].

BACKGROUND/AIM: Modern treatment of cystic echinococcosis, except for surgical treatment and percutaneous drainage of cyst considers also administration of albendazole as a type of individual therapy. However, clinicians fear of the serious adverse effects of high doses of albendazole, first of all the elevation of serum transaminases activity, very frequently results in subdosing of albendazole and wrong conclusions its efficacy and safety. The aim of this study was to investigate adverse effects of a longterm, continual administration of high doses of albendazole in the treatment of patients with echinococcal disease. METHODS: A total of 42 patients (mean age 40.4 +/- 18.3 years) with echinococcal disease were included in the study. They were treated with continual administration of high doses of albendazole within the period of 4 to 6 months. The subgroups of 27 and 15 patients were treated with 15-20 mg/kg/day and with 21-25 mg/kg/day albendazole, respectively. The patients in the control group (18 with surgical treatment, 6 with percutaneous drainage of cyst) were treated with 800 mg albendazole per day (< 15 mg/kg body weight) in the cycles of 28 days (1-3 cycles) and a two-week pause between them. RESULTS: In the study group adverse effects of albendazole were registered in 20 (47.6%), whereas in the control group in 6 (30.0%) of the patients. In both subgroups elevated activity of serum transaminases were found more frequently in the study group compared to the control one (35.7% vs 25%, p < 0.05), especially in the patients who were treated with higher doses of albendazole. The patients in the study group, compared to the patients in the control group had significantly higher mean activity of serum alanin aminotransferase in the course of the second and third month of the therapy (p < 0.05). Administration of albendazole due to adverse effects was stopped in 3 (7.1%) of the patients in the study group. Two (4.8%) of them had a very high activity of serum transaminases and one had a muscle pains and high activity of serum creatine kinase. After the interruption of the therapy we documented a nonnalization of serum enzyme levels in all the patients. CONCLUSION: Longterm, continual administration of high doses of albendazole in the patients with echinococcal disease results in significant elevation of serum transaminases activity, compared to the patients treated with albendazole in the cycles, but in the majority of the patients serum transaminases activity was normalizated by the end of a 6-month period.

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 3: Further optimization of the 2-, 6-, and 7'-substituents and initial pharmacokinetic assessments.

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)<10nM; IC(50) (1a)=22 nM; EC(50) (1b)=5nM], good stability toward human liver microsomes (HLM t(1/2)>60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda6-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones: Part 4. Optimization of DMPK properties.

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as potent inhibitors of genotype 1 HCV NS5B polymerase focusing on the optimization of their drug metabolism and pharmacokinetics (DMPK) profiles. This investigation led to the discovery of potent inhibitors with improved DMPK properties.

Effects of acclimation on cognitive performance in soldiers during exertional heat stress.

This study investigates the effects of exertional heat stress and acclimation status on physiological and cognitive performance. Forty male soldiers performed an exertional heat stress test (EHST) either in a cool (20 degrees C, 16 degrees C wet bulb globe temperature), or in a hot environment (40 degrees C, 29 degrees C wet bulb globe temperature), unacclimatized, or after 10 days of passive or active acclimation. Mean skin and tympanic (Tty) temperatures and heart rates (HR) measured physiological strain. A cognitive test (the computerized Cambridge Neuropsychological Test Automated Batteries attention battery) is administered before and immediately after EHST. EHST in hot conditions induced physiological heat stress (increase in Tty and HR), which caused mild deficits in attention in U group (decreased number of correct responses, and prolonged movement time). Acclimated (passive and active) soldiers suffered no detrimental effects of exertional heat stress, despite almost the same degree of heat strain, measured by Tty and HR.

Immune cytokine response in combat casualties: blast or explosive trauma with or without secondary sepsis.

The aim of this study was to assess the prognostic value of tumor necrosis factor (TNF) alpha, interleukin (IL)-8, IL-4, and IL-10 in combat casualties. Fifty-six casualties with severe trauma (blast and explosive) who developed sepsis and 20 casualties with the same severity of trauma without sepsis were enrolled in this study. Fifty-five casualties developed multiple organ dysfunction syndrome; 36 died. Blood was drawn on the first day of trauma. Concentrations of IL-8, TNF-alpha, IL-4, and IL-10 were determined in plasma using enzyme-linked immunosorbent assays. Mean values of IL-8 were 230-fold, IL-10 were 42-fold, and TNF-alpha were 17-fold higher in trauma and sepsis group (p < 0.01). Mean values of IL-8 were 60-fold, TNF-alpha were 43.5-fold, and IL-10 were 70-fold higher in the multiple organ dysfunction syndrome group (p < 0.01). Mean values of IL-8 were 2.3-fold and IL-10 were 1.4-fold higher in nonsurvivors and TNF-alpha were 2.2-fold higher in survivors (p < 0.01). IL-4 had no significance as a predictor of severity and outcome.

[Preoperative evaluation of the feasibility of laparoscopic cholecystectomy].

AIM: To determine the possibility of preoperative evaluation of the feasibility of laparoscopic cholecystectomy based on the standard preoperative examinations and findings. METHODS: During 1997, 100 consecutively operated patients with the diagnosis of chronic calculous cholecystitis were followed up. Sex and age, and the results of blood count, sedimentation rate, ultrasonography (US), and intravenous cholangiography (IVC) were monitored. Based on adhesions, fibrosis in Calot's triangle and pericholecystitis, surgical interventions were classified as minor and major. RESULTS: Minor operations were performed in 57, and major in 43 patients. Earlier surgical interventions had been carried out in 8 (18.6%) patients from the major surgery group, while 9 (15.79%) patients had undergone minor surgery. Out of 57 patients with minor surgery, IVC verified the contrast medium filling of the gallbladder in 55 (96.49%) of the patients. Tense gallbladder or wall stratification was not revealed by ultrasonography in any of the patients from this group. Out of 43 patients with major surgery, the gallbladder was not filled with the contrast medium during IVC in 34 (79.07%) patients, while the stratified and tense gallbladder was found by US in 2 (4.65%) patients. The mean sedimentation rate was 14.3 in the patients with minor surgery, and 23.5 in major surgery group. Mean WBC in the patients with minor surgery was 7.4 x 10(9). The patients with major surgery had slightly increased mean value of the white cell count. It was 8.3 x 10(9). CONCLUSION: Statistically significant difference (p < or = 0.05) was found between the variables of the IVC, sedimentation rate, the white blood count, and the earlier operations. No significant difference was found between other analyzed variables.

Liver hydatid disease: morphological changes of protoscoleces after albendazole therapy.

BACKGROUND: Postoperative recurrence of the liver hydatid disease befalls approximately 10-30% of patients. Preoperative or postoperative therapy with albendazole in single therapeutic protocol (800 mg/d, within 28 days) indicated the need to evaluate the hydatid cyst liquid protoscoleces viability. Morphological changes of protoscoleces following the treatment with drugs are not well known. AIM: To estimate the viability of protoscoleces after preoperative or postoperative albendazole therapy, and their ability for cystic metamorphosis. METHODS: A prospective, randomized clinical trial included 30 patients with liver hydatid disease, treated with albendazole and surgically (I group), and 30 patients in the control group treated only surgically (II group). The concentration of albendazole and its active metabolite albendazole sulphoxide in the cysts contents were determined using HPLC. Estimation of protoscoleces viability was based on the established micromorphologic criteria, and compared between the patients treated with albendazole, and the patients treated only surgically. Biological assessment of the viability was performed on protoscoleces with uncertain signs of the disturbed viability (unchanged structure, evaginated, without movements) using intraperitoneal injection of 1 ml of protoscoleces prepared suspension to AO type of rats. RESULTS: The concentration of albendazole in cysts' contents ranged from 0 to 64.9 microg/ml, and of its active metabolite from 0.5 to 40.8 microg/ml. The presence of fully viabile protoscoleces in the albendazol treated patients was significantly lower than in the control group. A significant difference was noticed in the presence of disintegrated protoscoleces without movements in the albendazol treated group, compared to the control group. Biological assessment of the viability showed incapability of these protoscoleces for cystic metamorphoses. CONCLUSION: Low viability of parasites due to medicamentous therapy is very useful and important to surgeons, because the fertility of cysts is lower, and the risk of the disease recurrence is reduced.

[Significance of iron reduction for the therapy of chronic hepatitis C]. / Znacaj uklanjanja gvozda u terapiji hronicnog hepatitisa C.

BACKGROUND: It has been established that many patients with chronic hepatitis C have elevated serum iron, feritin levels and iron deposites in the liver. Therefore, the liver damage due to hepatitis C virus may be aggravated with iron overload. In many studies higher levels of iron in the blood and the liver were connected with the decreased response to interferon-alfa therapy for chronic viral hepatitis C. Recent introduction of pegylated interferons plus ribavirin has improved the therapeutic response, so it is now possible to cure more than 50% of the patients. CASE REPORT: Three patients with chronic hepatitis C and iron overload were presented. Iron reduction therapy using phlebotomy or eritrocytapheresis with plasmapheresis was done at different times in regard to specific antiviral therapy or as a sole therapy. CONCLUSION: It has been shown that iron reduction, sole or combined with antiviral therapy, led to the deacreased aminotransferase serum activity and might have slown down the evolution of chronic hepatitis C viral infection.

War liver injuries.

AIM: To provide a retrospective analysis of our results and experience in primary surgical treatment of subjects with war liver injuries. METHODS: From July 1991 to December 1999, 204 subjects with war liver injuries were treated. A total of 82.8% of the injured were with the liver injuries combined with the injuries of other organs. In 93.7%, the injuries were caused by fragments of explosive devices or bullets of various calibers. In 140 (68.6%) of the injured there were minor lesions (grade I to II), treated with simple repair or drainage. There were complex injuries of the liver (grade III-V) in 64 (31.4%) of the injured Those injuries required complex repair (hepatorrhaphy, hepatotomy, resection debridement, resection, packing alone). The technique ofperihepatic packing and planned reoperation had a crucial and life-saving role when severe bleeding was present. Routine peritoneal drainage was applied in all of the injured. Primary management of 74.0% of the injured was performed in war hospitals. RESULTS: After primary treatment, 72 (35.3%) of the injured were with postoperative complications. Reoperation was done in 66 injured. Total mortality rate in 204 injured was 18.1%. All the deceased had significant combined injuries. Mortality rates due to the liver injury of the grade III, IV and V were 16.6%, 70.0% and 83.3%, respectively. CONCLUSION: Complex liver injuries caused very high mortality rate and the management of the injured was delicate under war circumstances (if the injured reached the hospital alive). Our experience under war circumstances and with war surgeons of limited knowledge of the liver surgery and war surgery, confirmed that it was necessary to apply compressive abdominal packing alone or in combination with other techniques for hemostasis in the treatment of liver injuries grade III-V, resuscitation and rapid transportation to specialized hospitals.