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The chemokine receptors CXCR1 and CXCR2 are important pharmaceutical targets due to their key roles in inflammatory diseases and cancer progression. We have previously identified 2-[5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanylmethyl]-phenylboronic acid (SX-517) and 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide (SX-576) as potent non-competitive boronic acid-containing CXCR1/2 antagonists. Herein we report the synthesis and evaluation of aminopyridine and aminopyrimidine analogs of SX-517 and SX-576, identifying (2-{(benzyl)[(5-boronic acid-2-pyridyl)methyl]amino}-5-pyrimidinyl)(4-fluorophenylamino)formaldehyde as a potent chemokine antagonist with improved aqueous solubility and oral bioavailability., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Blockade of undesired neutrophil migration to sites of inflammation remains an area of substantial pharmaceutical interest. To effect this blockade, a validated therapeutic target is antagonism of the chemokine receptor CXCR2. Herein we report the discovery of 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide 6, an antagonist with activity at both CXCR1 and CXCR2 receptors (IC50 values 31 and 21 nM, respectively). Compound 6 exhibited potent inhibition of neutrophil influx in a rat model of pulmonary inflammation, and is hypothesized to interact with a unique intracellular binding site on CXCR2. Compound 6 (SX-576) is undergoing further investigation as a potential therapy for pulmonary inflammation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The G protein-coupled chemokine receptors CXCR1 and CXCR2 play key roles in inflammatory diseases and carcinogenesis. In inflammation, they activate and recruit polymorphonuclear cells (PMNs) through binding of the chemokines CXCL1 (CXCR1) and CXCL8 (CXCR1 and CXCR2). Structure-activity studies that examined the effect of a novel series of S-substituted 6-mercapto-N-phenyl-nicotinamides on CXCL1-stimulated Ca(2+) flux in whole human PMNs led to the discovery of 2-[5-(4-fluorophenylcarbamoyl)pyridin-2-ylsulfanylmethyl]phenylboronic acid (SX-517), a potent noncompetitive boronic acid CXCR1/2 antagonist. SX-517 inhibited CXCL1-induced Ca(2+) flux (IC50 = 38 nM) in human PMNs but had no effect on the Ca(2+) flux induced by C5a, fMLF, or PAF. In recombinant HEK293 cells that stably expressed CXCR2, SX-517 antagonized CXCL8-induced [(35)S]GTPγS binding (IC50 = 60 nM) and ERK1/2 phosphorylation. Inhibition was noncompetitive, with SX-517 unable to compete the binding of [(125)I]-CXCL8 to CXCR2 membranes. SX-517 (0.2 mg/kg iv) significantly inhibited inflammation in an in vivo murine model. SX-517 is the first reported boronic acid chemokine antagonist and represents a novel pharmacophore for CXCR1/2 antagonism.

An acute decrease in cardiac performance can result from a reduced free triiodothyronine (FT3) level following (i) brain death (euthyroid sick syndrome), (ii) a period of cardiopulmonary bypass, and possibly (iii) regional or global myocardial ischemia. The two major pathophysiologic effects of brain death are (i) vascular injury associated with the hemodynamic consequences of the autonomic 'storm', and (ii) a generalized inhibition of mitochondrial function, which results in diminished organ function from the loss of energy stores from a rapid loss of circulating FT3. Deterioration of donor organ function can be reversed by hormonal replacement therapy, in which T3 plays a critical role. This results in (i) an increased number of organs being functionally acceptable, and (ii) increased early and intermediate graft survival. Cardiopulmonary bypass is associated with a reduction in the circulating level of FT3, and this can be associated with deterioration in cardiac function. The administration of T3 at the time of discontinuation of cardiopulmonary bypass reverses this state. In patients undergoing heart transplantation, T3 therapy to both donor and recipient is beneficial.

Although cardioplegia is limited to 4 hours of ice storage, University of Wisconsin solution has successfully extended this period to approximately 12 hours. In this study we have substituted polyethylene glycol for hydroxyethyl starch in a simplified University of Wisconsin solution (Cardiosol). Rabbit hearts were ice stored for 24 hours at 0 degrees C in either University of Wisconsin solution or Cardiosol (containing either 5% or 10% polyethylene glycol). Fresh control hearts were tested immediately after cardiectomy. Function was evaluated in an in vitro working heart model for 1 hour with aortic afterload at 100 cm H2O. Total cardiac output or the proportion of hearts reaching 100 cm H2O were compared. Hearts stored in University of Wisconsin solution for 24 hours functioned at 6% of control levels at 15 minutes of observation. None reached 100 cm H2O or deteriorated further with time (p < 0.05). By contrast, hearts stored in 5% Cardiosol showed progressive recovery during the 1-hour observation. Of the 13 hearts, 11 reached 100 cm H2O with a mean cardiac output of 51% of the control value. Increasing the concentration of polyethylene glycol to 10% improved cardiac output at all observation times, reaching 80% of control heart performance at 1 hour (control > 10% > 5% > University of Wisconsin solution [p < 0.05]). We concluded that 10% polyethylene glycol significantly improved 24-hour ice storage and, hence, viability to a functional level that matched our previously reported microperfusion results.

The use of polyethylene glycol (PEG) in preservation solutions has been associated with a decreased incidence of rejection in clinical and experimental organ transplantation. In this study, we examined the effect of PEG with different molecular configurations on rejection of small bowel allografts in the rat. Male ACI and LEW rats were used as donors and recipients, respectively. Orthotopic small bowel transplantation was performed using the following preservation solutions: lactated Ringer's solution (n = 7), University of Wisconsin solution (n = 7), University of Wisconsin solution without hydroxyethyl starch (sUW; n = 7), sUW with PEG20M (n = 9), sUW with PEG8000 (n = 6), and sUW with PEG20L (n = 7). No immunosuppression was given. In orthotopic small bowel transplantation, only groups with a high molecular weight PEG, PEG20M and PEG20L, demonstrated longer survival (P < 0.01 and P < 0.001, respectively) and delayed onset of unkempt appearance (P < 0.05 and P < 0.001, respectively). In heterotopic small bowel transplantation, sUW was compared with sUW with PEG20L. Rejection occurred later and its progression was slower in the sUW with PEG20L than in the sUW alone. Our observations suggest that the onset and progression of rejection after small bowel transplantation were influenced by the molecular weight and configuration of the PEG molecule. The mechanism is unclear, but high molecular weight PEG appears to reduce or change the immunogenicity of the small bowel allograft.

This investigation was designed to examine the role of glutathione and other reduced sulfhydryl amines during reperfusion of the ischemic rabbit heart. To identify the biochemical features of reduced sulfhydryl amines contributing toward improved myocardial function, we investigated several chemical agents sharing a common property with glutathione (L-leucine, L-glycine, ascorbate, oxidized glutathione, L-cysteine). After a period of 24-hour hypothermic storage of the rabbit heart in a modified University of Wisconsin solution containing polyethylene glycol, the hearts were functionally evaluated on a Langendorff working heart model. The agents were then injected as a bolus (60 mumol/L) during reperfusion, and coronary flow and aortic output were measured. Control hearts were untreated. Reduced sulfhydryl amines (glutathione, L-cysteine) significantly improved coronary flow (p < 0.005) and cardiac output (p < 0.005). Ascorbate, L-leucine, L-glycine, and oxidized glutathione all failed to influence cardiac function.

A terminal rinse (TR) is standard practice in liver preservation with University of Wisconsin solution (UW) to avoid a potassium load. The fact that sodium lactobionate sucrose solution (SLS) is an effective organ preservation solution with a low potassium provided an opportunity to evaluate rat liver preservation without the TR step. Its importance was investigated in 122 rat liver preservation experiments. In study 1, UW and a hydroxyethyl starch-free, modified UW (UWm) were used for 20-hr liver preservation followed by either no TR or Ringer's lactate TR. The 1-week survival was: UW-TR, 2/14; UW-no TR, 1/6; UWm-TR, 0/6; UWm-no TR, 5/5 (P < 0.01). In study 2, livers were stored for 30 hr in SLS, UW, UWm, and UWm + chlorpromazine 5 mg/L, all without a TR. Nine of 11 rats survived 7 days after SLS, but there were no survivors in the other groups (P < 0.05). Study 3 compared no TR with TR with SLS, Ringer's lactate (RL), or a modified Carolina rinse (CRm) after 30-hr SLS preservation. Survival, serum aspartate aminotransferase and alanine aminotransferase, and histology were assessed. One-week survival of 9/11 rats in no TR was significantly better than in the other groups (3/14 in TR-SLS, 0/8 in TR-RL, and 0/14 in TR-CRm, P < 0.01). The values of aspartate aminotransferase (mean +/- SE) 3 hr after transplantation were 1862 +/- 439 U/L, 3334 +/- 817 U/L, 6591 +/- 1944 U/L, and 7028 +/- 1704 U/L, respectively, in no TR, TR-SLS, TR-RL, and TR-CRm. There were significant differences both in aspartate aminotransferase and alanine aminotransferase between no-TR and each of TR-RL and TR-CRm (P < 0.05). Liver specimens from rats killed 3 hr after OLT showed only mild injury in the no TR group and severe injury in the remaining groups. We conclude that a terminal rinse is harmful in rat liver preservation.

In 1969 we described a method for kidney preservation that used a brief flush with a new "intracellular" solution followed by ice storage. This paper stimulated research into optimizing solution composition culminating in the UW solution which is now the accepted standard. Further developments in the design of solutions for hypothermic organ preservation have proceeded along several paths, including: (1) modification and simplification of UW solution, (2) investigation of organ specific requirements, (3) addition of pharmacologic agents particularly calcium antagonists to flush solutions, (4) the concept of "microperfusion" for control of acidosis, (5) the use of solutions containing polyethylene glycol, and (6) the use of a terminal rinse solution. Broadly speaking, the results of these studies have shown that it is possible to improve upon the UW solution by simplification, eliminating several of the components, and that sodium variants, and pharmacological additives, such as chlorpromazine, may yield better results in experimental and clinical trials. It has also been found that there are special requirements for individual organs, rendering the concept of a universal solution unlikely. Of the promising new ideas, microperfusion and polyethylene glycol have been found to be very effective for heart preservation yielding for the first time virtually perfect 24-hour preservation. The concept of a terminal rinse to diminish reperfusion injury has strong experimental support and awaits clinical evaluation.

The effects of the calcium antagonists, chlorpromazine (CPZ), nisoldipine (NIS), trifluoperazine (TFP), and nicardipine (NIC) were compared in rat livers following either 20- or 30-hr ice storage in sodium lactobionate sucrose solution (SLS). Survivals beyond 7 days after orthotopic liver transplantation following 20-hr cold storage were 1/14 in the University of Wisconsin solution, 4/14 in SLS, 4/8 in UW+CPZ, 7/8 in SLS+CPZ. Survivals beyond 7 days after OLT following 30-hr cold storage were 3/8 in SLS+CPZ, 3/8 in SLS+NIS, 2/8 in SLS+TFP, 0/8 in SLS+NIC, and 0/8 in SLS alone. Survival rates were significantly (P less than 0.05) better in both SLS+CPZ and SLS+NIS than in UW and SLS alone. The effluent lactate dehydrogenase (LDH) levels and pH changes were measured at the time of OLT. After 20 hr, LDH levels were 525 +/- 78 IU/L (mean +/- SEM) in UW, 492 +/- 44 in SLS, 322 +/- 35 in UW+CPZ, and 290 +/- 39 in SLS+CPZ. After 30 hr, LDH values were 416 +/- 40 in SLS+CPZ, 450 +/- 25 in SLS+NIS, 448 +/- 21 in SLS+TFP, 573 +/- 18 in SLS+NIC, and 614 +/- 68 in SLS. The LDH levels for SLS+CPZ and SLS+NIS were significantly lower than those of SLS and UW (P less than 0.01). The pH changes in the effluent were significantly less in both the CPZ and NIS groups (P less than 0.01). This study demonstrated improved liver preservation by the use of a simplified colloid-free lactobionate solution containing sodium as the principal cation. The addition of CPZ or NIS to the solution demonstrated the same potency for significant improvement in efficacy of this solution, while NIC was ineffective.

We investigated the effect of the addition of chlorpromazine to a new, simplified organ preservation solution, sodium lactobionate sucrose (SLS), for 20-hour hypothermic rat liver preservation. Survival beyond 7 days after orthotopic transplantation of the stored liver was eight of eight rats in control groups (immediate transplantation, less than 1-hour preservation), one of 14 rats with the University of Wisconsin (UW) solution, four of 14 rats with SLS, seven of eight rats with SLS + chlorpromazine, 1 mg/L, and seven of eight rats with SLS + chlorpromazine, 10 mg/L. The differences is survival between UW and SLS and between SLS and SLS + chlorpromazine were significant (p less than 0.05). Lactic dehydrogenase levels in the effluent after reflushing through the portal vein at the time of transplantation were 145 +/- 20 IU/L (mean +/- SEM) in the controls, 525 +/- 78 IU/L in UW, 492 +/- 44 IU/L in SLS, 290 +/- 39 IU/L in SLS + chlorpromazine, 1 mg/L, 290 +/- 11 IU/L in SLS + chlorpromazine, 10 mg/L. The values for the SLS + chlorpromazine were significantly lower than for SLS and UW (p less than 0.05). The pH of the effluent was 7.10 +/- 0.10 in controls, 6.42 +/- 0.12 in UW, 6.64 +/- 0.18 in SLS, and 7.07 +/- 0.02 in SLS + chlorpromazine, 1 mg/L and 10 mg/L. The pH drop was significantly greater in the groups without chlorpromazine (p less than 0.01). This study shows that superior rat liver preservation was achieved with a simplified lactobionate solution containing sodium as the principal cation, sucrose in place of raffinose, and omitting the colloid and several of the other UW components. The addition of low concentrations of chlorpromazine further enhanced the effectiveness of this solution, without the need for donor pretreatment.

Recent work has shown that UW may be better than standard cardioplegic solutions for short-term heart preservation. In this study we have used a rabbit heart model to evaluate a simplified UW solution in which penicillin, dexamethasone, insulin, allopurinol, and adenosine were omitted and 5% polyethylene glycol (PEG20M) was substituted for hydroxyethyl starch. The test systems consisted of 4-hr cardioplegic storage at 15 degrees C with repeated flushing every 30 min for 2 hr and 24-hr hypoxic low-flow microperfusion (3 ml/g/24 hr) at 0 degrees C. Control groups were arrested with a 15-25 ml flush in iced saline and immediately tested. Cardiac output (CO)* after preservation was measured in a working heart model using an acellular perfusate at 37 degrees C at an aortic pressure of 100 cm H2O. The CO (ml/g heart wt/min) were as follows--Controls: St. Thomas II 20.5 +/- 8.3 (5), UW 34.7 +/- 11.7 (16), PEG20M 41.8 +/- 4.4 (14); 4-hr cardioplegia: St. Thomas II 17.4 +/- 0.9 (4), Bretschneider HTK 14.9 +/- 7.0 (4), UW 25.2 +/- 11.5 (9), PEG20M 41.1 +/- 7.8 (8); 24-hr microperfusion: UW 25.4 +/- 11.1 (18), PEG20M 37.1 +/- 8.2 (18). Following cardioplegic or microperfusion preservation, PEG20M hearts functioned at control levels (P greater than 0.05) and were significantly superior to all other solutions, with approximately double the CO (P less than 0.05, all other groups). We conclude that for heart preservation, 5 components can be eliminated from UW and substitution of PEG20M for HES appears to have improved its performance.

Transplantation of the heart and both lungs is the only therapy that can be offered to certain patients with end-stage pulmonary vascular disease. Our experimental experience with the baboon is presented. Fourteen allotransplants were performed, 12 recipients (inadequately immunosuppressed with cyclosporin A and azathioprine) surviving between 4 and 29 days. In 11 cases death resulted from acute rejection which predominantly involved the lungs, the heart being spared in 10 cases; the remaining death was from bronchopneumonia. Two autotransplanted baboons survived until sacrificed at 6 months. Indications for the operation, selection of both the recipient and the donor, and recent results at other centres are briefly reviewed. It would seem that this operation is recommended in selected patients with idiopathic pulmonary hypertension or Eisenmenger's syndrome whose condition is deteriorating and in whom no other form of therapy is applicable.

Deterioration of function in brain dead baboons is associated with depletion of both myocardial energy stores and certain circulating hormones, notably thyroxine, cortisol, and insulin. We have therefore investigated the effect of the administration of these three hormones to the brain dead pig; their value has been assessed on both the freshly excised and stored donor heart. Brain death was induced by ligation of the two arteries to the upper part of the body which arise from the aortic arch. Storage of selected hearts was by continuous hypothermic perfusion for 20 to 24 hr. Hearts were biopsied for estimation of adenosine triphosphate, creatine phosphate, lactate, and glycogen, and were subsequently functionally tested. Six groups of pigs were studied. Hearts were tested from control pigs which had not undergone brain death (A1), from brain dead pigs which had received intravenous fluid and inotropic support for 4 hr (B1), and from brain dead pigs which had in addition received 2 hr of hormonal therapy (thyroxine 2 micrograms cortisol 100 mg, and insulin 5-10 IU hourly) (C1). A further 3 groups (A2-C2) underwent management identical to A1-C1, but in addition the hearts were stored for 24 hr. Brain death in pigs was followed by a consumption of myocardial energy stores, despite anaerobic glycolysis; this was associated with reduced myocardial function. The administration of hormones to the brain dead pig led to some replenishment of myocardial energy and glycogen reserves and reduction in lactate, with associated improvement in hemodynamic function. A period of hypothermic perfusion storage appeared to reverse the anaerobic metabolism occurring in the heart in the nonhormonally treated brain dead animal, though not in the hormonally treated animal, and led to replenishment of glycogen reserves in nontreated animals. The observation that both better function and an increase in myocardial energy stores occurred in hormonally treated, stored hearts, even though perfusate lactate dehydrogenase rose to significantly higher levels during hypothermic perfusion storage, and tissue lactate levels remained high, suggests that thyroxine promotes both aerobic and anaerobic metabolism in brain dead animals.

Experiments were conducted using rabbit kidneys stored on ice for 48 hr to elucidate the mode of action of "intracellular" flush solutions. Measurements were made of renal function on a shunt and they were correlated with blood flow and the efficiency of the mechanical expulsion of red cells. By comparison with unflushed, ice-stored kidneys, near complete mechanical expulsion of blood by 30 to 60 min of continuous perfusion with hypertonic Ringer's albumin resulted in significantly higher blood flow with little gain in function. Similarly, increasing the content of nonelectrolyte in Ringer's albumin improved blood flow but not function. A simple flush with a low ionic strength sodium solution (LIE), containing impermeant anions and glucose was superior to that with Ringer's albumin. A high-potassium version of the low ionic strength solution (LIC) was in turn significantly better than LIE for kidney preservation by simple flushing and ice storage. These results were interpreted to mean that whereas mechanical flushing is a relatively minor component of the action of flush solutions, the major benefit results from a reduced sodium and elevated potassium content in the presence of impermeant anions. The primary importance of prevention of cell swelling by the inclusion of nonelectrolytes in "extracellular" flush solutions, is questioned.

An easily "replaceable" cardiac valve prosthesis has been designed. It consists of two parts: (1) a sewing ring incorporating a circlip and (2) a functioning valve (either mechanical or tissue). The circlip is encased in a sewing ring, which is sutured into the natural valve annulus, and grips the functional part of the prosthesis, thereby preventing dislodgment. A simple instrument has been designed to open the circlip a few millimeters to allow easy removal or insertion of the functional element. This sewing ring/circlip with the functional element of a Björk-Shiley prosthesis was used in 10 baboons undergoing mitral valve replacement. Removal and replacement of the functional element was carried out at a second operation between 1 and 12 weeks later. There were no operative deaths. Baboons were electively killed one day to twelve months after the second operation. There were no complications related to the prosthesis; cardiac catheterization showed normal hemodynamics before and after the second operative procedure.

The effects of the agonal period and subsequent donor management on renal slice function, using the K+ - Na+ ratio, have been studied in the pig. Brain ischaemia or death resulted in a reduction in renal slice function, whether the pig was maintained normovolemic or hypovolemic by i.v. fluid and dobutamine therapy. This deterioration in function was, however, reversed or prevented by a period of therapy with thyroxine (T3), insulin, and cortisol. A period of 24 hr storage of the kidney slice in a low ionic strength solution in ice resulted in a further deterioration in slice function in all groups studied.

This report describes observations concerning the influence of the addition of 0.3 M cryoprotective agents (propylene glycol or glycerol) to Ringer's albumin solution used for rabbit kidney perfusion for periods of less than 4 and 24 hr. Endogenous creatinine clearance during short-term parabiotic perfusion on a shunt was used to evaluate function after perfusion. The findings were (A) Perfusion for less than 4 hr resulted in a significant loss of function by comparison with 1 hr ice-stored controls. (B) Continuing the perfusion for 24 hr resulted in a further significant fall in function. Much of the early perfusional injury seen in these two groups could be avoided by including 0.3 M cryoprotective agents in the perfusate.

A new technique for 24-hr cardiac preservation is described utilizing very low flow perfusion (microperfusion) with a cold flush solution. Rabbit hearts were arrested with UW solution and then perfused with the same solution through the aortic root at 0 degrees C at a rate of 3-6 ml/gm heart weight/24 hr. When tested on an ex vivo working heart model, the cardiac output (CO) was 28.72 +/- 7.69 ml/g/min compared with fresh UW flushed controls of 26.48 +/- 2.25 ml/g/min. Both oxygenated highflow perfusion with a more conventional perfusate and 24-hr ice storage with UW led to inferior results. Omission of the colloid, hydroxyethyl starch (HES), from the UW solution or prolonged shelf storage were also significantly detrimental. When a previously untested colloid, polyethylene glycol 20,000, was substituted for HES for microperfusion, excellent cardiac function was obtained. In fact, the mean CO of this group, 31.91 +/- 5.70, was significantly above that of fresh HES-UW unstored controls. The suggestion that the UW solution might be improved by this substitution warrants further study.

Vascular resistance changes were measured in response to alteration in perfusate osmolarity in isolated rabbit kidneys perfused at 10 degrees C. The data obtained were found to fit a simple mathematical model of the vascular resistance of the microcirculation in which it was assumed that variation in this parameter depended solely upon osmotic alterations in the size of the cells within and around the blood vessel walls. The model predicts that the volumetric changes due to the different osmolarities are produced in a tissue layer whose thickness is 30% relative to a fixed outer radius. This result is compatible with the hypothesis that the effects are predominantly due to changes of endothelial cell volume and other perivascular capillary cells. The analysis illustrates the significance of perfusate osmolarity as a determinant of vascular resistance, can be used to investigate the hemodynamic effects that occur during the introduction and removal of cryoprotective agents, and is relevant to the interpretation of results obtained with hypertonic solutions in blood volume restoration after hypovolemic shock.