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Background and Purpose: Galectin-3 (Gal-3) is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. GB1107 is a novel orally active Gal-3 small molecule inhibitor that has high affinity for Gal-3 >1000-fold selectively over other galectins. The aim of this study was to characterise GB1107 and galectin-3 in vitro and in vivo in the context of fibrosis signalling and liver disease., Experimental Approach: Liver fibrosis was induced by administration of CCl 4 twice weekly by intraperitoneal injection in mice for 8 weeks. GB1107 was orally administered once daily (10 mg/kg) for the last 4 weeks of CCl 4 treatment. Fibrosis was assessed by picrosirius red staining of FFPE sections. Liver enzymes, Gal-3 and downstream biomarkers were assessed in liver and plasma. Paired-end sequencing was performed on the Nextseq 2000 platform. Pathway enrichment analysis was performed to determine enrichment of differentially expressed genes (DEGs) within Reactome pathways and Gene Ontology (GO) terms., Key Results: GB1107 significantly reduced plasma transaminases and liver Gal-3 and reduced liver fibrosis. RNAseq analysis of whole liver showed that 1659 DEGs were identified with CCl 4 treatment compared to control. Pathways enriched in up-regulated genes in the CCl 4 group included those related to the extracellular matrix, collagen biosynthesis and assembly, cell cycle and the immune system. Comparing GB1107 treatment with CCl 4 control 1147 DEGs were identified. GB1107 effectively reversed the majority of the CCl 4 induced gene changes., Conclusions and Implications: GB1107 attenuated liver fibrosis and highlights Gal-3 as a therapeutic target for hepatic fibrosis., Competing Interests: Declaration of competing interest Conflict of interest disclosure: DH, JR, IH, JM, RJS, ACM, AP, FZ, HS: employees, personal fees – Galecto Biotech AB, UJN, HL: shareholders, personal fees – Galecto Biotech AB., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: Overexpression of galectin-3, a β-galactoside-binding lectin, is associated with fibrotic diseases and cancer. Selvigaltin is an oral galectin-3 inhibitor, previously administered as a 50 mg capsule. This study aimed to evaluate the relative bioavailability and food effect of selvigaltin as a 100 mg tablet in healthy volunteers., Methods: In this single-dose, randomized, three-period, crossover study (GALBA-1; NCT05747573), participants received selvigaltin as a 100 mg tablet (under fasted and fed conditions) or as two 50 mg capsules (under fasted conditions). Primary endpoints included plasma and urine pharmacokinetic (PK) parameters. Secondary endpoints were safety and tolerability., Results: Of the 13 enrolled participants, 12 completed the study. Under fasted conditions, geometric mean maximum observed plasma concentration (C max ) and systemic exposure (AUC 0─inf ) of selvigaltin were 161.0% and 84.0% higher, respectively, after administration of a tablet vs. capsules. Under fed vs. fasted conditions, geometric mean C max of the selvigaltin tablet was 20.0% lower, whereas AUC 0─inf was unaffected. Geometric mean percentage of total dose of selvigaltin excreted in urine over 0─96 h was 30.3% and 35.9% for the tablet under fasted and fed conditions, respectively, and 14.5% for the capsules. No treatment-emergent severe or serious adverse events or study discontinuations due to a treatment-emergent adverse event were reported., Conclusion: The tablet formulation of selvigaltin displayed higher bioavailability vs. the capsule formulation, with minimal effect of food on PK. Selvigaltin was well-tolerated during all treatments. These findings warrant further clinical development of the tablet formulation of selvigaltin without specific food restrictions., Clinical Trial Registration: NCT05747573; February 28, 2023., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Objectives: Selvigaltin (GB1211), an orally available small molecule galectin-3 inhibitor developed as a treatment for liver fibrosis and cirrhosis, was evaluated to assess the effect of hepatic impairment on its pharmacokinetics and safety to address regulatory requirements., Methods: GULLIVER-2 was a Phase Ib/IIa three-part study. Parts 1 and 3 had single-dose, open-label designs assessing pharmacokinetics (plasma [total and unbound] and urine), safety, and tolerability of 100 mg oral selvigaltin in participants with moderate (Child-Pugh B, Part 1) or severe (Child-Pugh C, Part 3) hepatic impairment, compared with healthy-matched participants (n = 6 each)., Results: All participants received selvigaltin and completed the study. No adverse events were reported. The median time to reach maximum total plasma concentration following drug administration was of 3.49 and 4.00 h post-dose for Child-Pugh B and C participants, respectively; comparable with controls. Total plasma exposure was higher for participants with hepatic impairment compared with controls. Whilst maximum plasma concentration (C max ) was unaffected in Child-Pugh B participants, area under the plasma concentration-time curve from time zero to infinity (AUC ∞ ) increased by ~ 1.7-fold compared with controls, and half-life was prolonged (geometric mean 28.15 vs 16.38 h). In Child-Pugh C participants, C max increased by ~ 1.3-fold, AUC ∞ increased by ~ 1.5-fold, and half-life was prolonged (21.05 vs 16.14 h). No trend was observed in plasma unbound fractions or urinary excretion of unchanged selvigaltin in either group., Conclusion: Hepatic impairment increased selvigaltin exposure without safety concerns. These data can inform dose recommendations for future clinical programmes., Trial Registration: Clinicaltrials.gov NCT05009680., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Purpose: Galectin-3, a β-galactoside-binding lectin, plays a key role in several cellular pathways involved in chronic inflammation, heart disease and cancer. GB1211 is an orally bioavailable galectin-3 inhibitor, developed to be systemically active. We report safety and pharmacokinetics (PK) of GB1211 in healthy participants., Methods: This phase 1, double-blind, placebo-controlled, first-in-human study (NCT03809052) included a single ascending-dose phase (with a food-effect cohort) where participants across seven sequential cohorts were randomized 3:1 to receive oral GB1211 (5, 20, 50, 100, 200 or 400 mg) or placebo. In the multiple ascending-dose phase, participants received 50 or 100 mg GB1211 or placebo twice daily for 10 days. All doses were administered in the fasted state except in the food-effect cohort where doses were given 30 min after a high-fat meal., Results: All 78 participants received at least one GB1211 dose (n = 58) or placebo (n = 20) and completed the study. No safety concerns were identified. Following single and multiple oral doses under fasted conditions, maximum GB1211 plasma concentrations were reached at 1.75-4 h (median) post-dose; mean half-life was 11-16 h. There was a ~ twofold GB1211 accumulation in plasma with multiple dosing, with steady-state reached within 3 days; 30% of the administered dose was excreted in urine as unchanged drug. Absorption in the fed state was delayed by 2 h but systemic exposure was unaffected., Conclusion: GB1211 was well tolerated, rapidly absorbed, and displayed favorable PK, indicating a potential to treat multiple disease types. These findings support further clinical development of GB1211., Clinical Trial Registration: The study was registered with ClinicalTrials.gov (identifier: NCT03809052)., (© 2023. The Author(s).)

Rationale: High circulating galectin-3 is associated with poor outcomes in patients with coronavirus disease (COVID-19). We hypothesized that GB0139, a potent inhaled thiodigalactoside galectin-3 inhibitor with antiinflammatory and antifibrotic actions, would be safely and effectively delivered in COVID-19 pneumonitis. Objectives: Primary outcomes were safety and tolerability of inhaled GB0139 as an add-on therapy for patients hospitalized with COVID-19 pneumonitis. Methods: We present the findings of two arms of a phase Ib/IIa randomized controlled platform trial in hospitalized patients with confirmed COVID-19 pneumonitis. Patients received standard of care (SoC) or SoC plus 10 mg inhaled GB0139 twice daily for 48 hours, then once daily for up to 14 days or discharge. Measurements and Main Results: Data are reported from 41 patients, 20 of which were assigned randomly to receive GB0139. Primary outcomes: the GB0139 group experienced no treatment-related serious adverse events. Incidences of adverse events were similar between treatment arms (40 with GB0139 + SoC vs. 35 with SoC). Secondary outcomes: plasma GB0139 was measurable in all patients after inhaled exposure and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc analysis of covariance [ANCOVA] over days 2-7; P  = 0.0099 vs. SoC). Plasma biomarkers associated with inflammation, fibrosis, coagulopathy, and major organ function were evaluated. Conclusions: In COVID-19 pneumonitis, inhaled GB0139 was well-tolerated and achieved clinically relevant plasma concentrations with target engagement. The data support larger clinical trials to determine clinical efficacy. Clinical trial registered with ClinicalTrials.gov (NCT04473053) and EudraCT (2020-002230-32).

Galectin-3 is a carbohydrate-binding protein central to regulating mechanisms of diseases such as fibrosis, cancer, metabolic, inflammatory, and heart disease. We recently found a high affinity (nM) thiodigalactoside GB0139 which currently is in clinical development (PhIIb) as an inhaled treatment of idiopathic pulmonary fibrosis. To enable treatment of systemically galectin-3 driven disease, we here present the first series of selective galectin-3 inhibitors combining high affinity (nM) with oral bioavailability. This was achieved by optimizing galectin-3 specificity and physical chemical parameters for a series of disubstituted monogalactosides. Further characterization showed that this class of compounds reduced profibrotic gene expression in liver myofibroblasts and displayed antifibrotic activity in CCl 4 -induced liver fibrosis and bleomycin-induced lung fibrosis mouse models. On the basis of the overall pharmacokinetic, pharmacodynamic, and safety profile, GB1211 was selected as the clinical candidate and is currently in phase IIa clinical trials as a potential therapy for liver cirrhosis and cancer.

Galectin (Gal)-3 is a profibrotic β-galactoside-binding lectin that plays a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and IPF exacerbations. TD139 is a novel and potent small-molecule inhibitor of Gal-3.A randomised, double-blind, multicentre, placebo-controlled, phase 1/2a study was conducted to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of inhaled TD139 in 36 healthy subjects and 24 patients with IPF. Six dose cohorts of six healthy subjects were evaluated (4:2 TD139:placebo ratio) with single doses of TD139 (0.15-50 mg) and three dose cohorts of eight patients with IPF (5:3 TD139:placebo ratio) with once-daily doses of TD139 (0.3-10 mg) for 14 days.Inhaled TD139 was well tolerated with no significant treatment-related side-effects. TD139 was rapidly absorbed, with mean time taken to reach maximum plasma concentration ( C ) values ranging from 0.6 to 3 h and a plasma half-life ( max ) of 8 h. The concentration of TD139 in the lung was >567-fold higher than in the blood, with systemic exposure predicting exposure in the target compartment. Gal-3 expression on alveolar macrophages was reduced in the 3 and 10 mg dose groups compared with placebo, with a concentration-dependent inhibition demonstrated. Inhibition of Gal-3 expression in the lung was associated with reductions in plasma biomarkers centrally relevant to IPF pathobiology (platelet-derived growth factor-BB, plasminogen activator inhibitor-1, Gal-3, CCL18 and YKL-40).TD139 is safe and well tolerated in healthy subjects and IPF patients. It was shown to suppress Gal-3 expression on bronchoalveolar lavage macrophages and, in a concerted fashion, decrease plasma biomarkers associated with IPF progression.T 1/2 ) of 8 h. The concentration of TD139 in the lung was >567-fold higher than in the blood, with systemic exposure predicting exposure in the target compartment. Gal-3 expression on alveolar macrophages was reduced in the 3 and 10 mg dose groups compared with placebo, with a concentration-dependent inhibition demonstrated. Inhibition of Gal-3 expression in the lung was associated with reductions in plasma biomarkers centrally relevant to IPF pathobiology (platelet-derived growth factor-BB, plasminogen activator inhibitor-1, Gal-3, CCL18 and YKL-40).TD139 is safe and well tolerated in healthy subjects and IPF patients. It was shown to suppress Gal-3 expression on bronchoalveolar lavage macrophages and, in a concerted fashion, decrease plasma biomarkers associated with IPF progression., Competing Interests: Conflict of interest: N. Hirani reports grants from Galecto Biotech, during the conduct of the study. Conflict of interest: A.C. MacKinnon reports personal fees from Galecto Biotech, outside the submitted work; and has a patent CA2,794,066 issued, a patent US13/832,672 issued and a patent WO/2014/067986 pending (all patents are fully owned by Galecto Biotech). Conflict of interest: L. Nicol reports grants from Galecto Biotech, during the conduct of the study; personal fees for lectures from Boehringer Ingelheim, outside the submitted work. Conflict of interest: P. Ford reports personal fees and nonfinancial support from Galecto, during the conduct of the study; and has a patent TD139 issued. Conflict of interest: H. Schambye reports personal fees from Galecto Inc, outside the submitted work; and has a patent WO/2016/180483 pending (fully owned by Galecto Biotech). Conflict of interest: A. Pedersen reports personal fees from Galecto Biotech, outside the submitted work. Conflict of interest: U.J. Nilsson has a patent CA2,794,066 issued, a patent US13/832,672 issued, a patent WO/2014/067986 pending, a patent WO/2005/113569 pending and a patent WO/2009/139719 pending (all patents are fully owned by Galecto Biotech). Conflict of interest: H. Leffler has a patent CA2,794,066 issued, a patent US13/832,672 issued, a patent WO/2014/067986 pending and a patent WO/2005/113569 pending (all patents are fully owned by Galecto Biotech). Conflict of interest: T. Sethi reports personal fees from Galecto Biotech, outside the submitted work; and has a patent CA2,794,066 issued, a patent US13/832,672 issued and a patent WO/2014/067986 pending (patents are fully owned by Galecto Biotech). Conflict of interest: S. Tantawi reports personal fees from Galecto Biotech, outside the submitted work. Conflict of interest: L. Gravelle reports personal fees from Galecto Biotech, outside the submitted work; and has a patent WO/2017/103109 pending (fully owned by Galecto Biotech). Conflict of interest: R.J. Slack reports personal fees from Galecto Biotech, outside the submitted work. Conflict of interest: R. Mills has nothing to disclose. Conflict of interest: U. Karmakar has nothing to disclose. Conflict of interest: D. Humphries has nothing to disclose. Conflict of interest: F. Zetterberg reports personal fees from Galecto Biotech, outside the submitted work. Conflict of interest: L. Keeling has nothing to disclose. Conflict of interest: L. Paul has nothing to disclose. Conflict of interest: P.L. Molyneaux has, via his institution, received industry-academic funding from AstraZeneca and has received speaker and consultancy fees from Boehringer Ingelheim and Hoffman-La Roche, outside the submitted work. Conflict of interest: F. Li has nothing to disclose. Conflict of interest: W. Funston has nothing to disclose. Conflict of interest: I.A. Forrest reports personal fees for consultancy and meeting attendance from Boehringer Ingelheim, personal fees for lectures and meeting attendance from Roche Ltd, outside the submitted work. Conflict of interest: A.J. Simpson has nothing to disclose. Conflict of interest: M.A. Gibbons has nothing to disclose. Conflict of interest: T.M. Maher has, via his institution, received industry-academic funding from AstraZeneca and GlaxoSmithKline R&D, and has received consultancy or speaker fees from AstraZeneca, Bayer, Blade Therapeutics, Boehringer Ingelheim, Bristol-Myers Squibb, Galapagos, GlaxoSmithKline R&D, Indalo, Novartis, Pliant, Respivant, Roche and Samumed., (Copyright ©ERS 2021.)

A combination therapy approach is required to improve tumor immune infiltration and patient response to immune checkpoint inhibitors that target negative regulatory receptors. Galectin-3 is a β-galactoside-binding lectin that is highly expressed within the tumor microenvironment of aggressive cancers and whose expression correlates with poor survival particularly in patients with non-small cell lung cancer (NSCLC). To examine the role of galectin-3 inhibition in NSCLC, we tested the effects of galectin-3 depletion using genetic and pharmacologic approaches on syngeneic mouse lung adenocarcinoma and human lung adenocarcinoma xenografts. Galectin-3 -/- mice developed significantly smaller and fewer tumors and metastases than syngeneic C57/Bl6 wild-type mice. Macrophage ablation retarded tumor growth, whereas reconstitution with galectin-3-positive bone marrow restored tumor growth in galectin-3 -/- mice, indicating that macrophages were a major driver of the antitumor response. Oral administration of a novel small molecule galectin-3 inhibitor GB1107 reduced human and mouse lung adenocarcinoma growth and blocked metastasis in the syngeneic model. Treatment with GB1107 increased tumor M1 macrophage polarization and CD8 + T-cell infiltration. Moreover, GB1107 potentiated the effects of a PD-L1 immune checkpoint inhibitor to increase expression of cytotoxic (IFNγ, granzyme B, perforin-1, Fas ligand) and apoptotic (cleaved caspase-3) effector molecules. In summary, galectin-3 is an important regulator of lung adenocarcinoma progression. The novel galectin-3 inhibitor presented could provide an effective, nontoxic monotherapy or be used in combination with immune checkpoint inhibitors to boost immune infiltration and responses in lung adenocarcinoma and potentially other aggressive cancers. SIGNIFICANCE: A novel and orally active galectin-3 antagonist inhibits lung adenocarcinoma growth and metastasis and augments response to PD-L1 blockade. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1480/F1.large.jpg., (©2019 American Association for Cancer Research.)

Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra- and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3.

Discovery of glycan-competitive galectin-3-binding compounds that attenuate lung fibrosis in a murine model and that block intracellular galectin-3 accumulation at damaged vesicles, hence revealing galectin-3-glycan interactions involved in fibrosis progression and in intracellular galectin-3 activities, is reported. 3,3'-Bis-(4-aryltriazol-1-yl)thiodigalactosides were synthesized and evaluated as antagonists of galectin-1, -2, -3, and -4 N-terminal, -4 C-terminal, -7 and -8 N-terminal, -9 N-terminal, and -9 C-terminal domains. Compounds displaying low-nanomolar affinities for galectins-1 and -3 were identified in a competitive fluorescence anisotropy assay. X-ray structural analysis of selected compounds in complex with galectin-3, together with galectin-3 mutant binding experiments, revealed that both the aryltriazolyl moieties and fluoro substituents on the compounds are involved in key interactions responsible for exceptional affinities towards galectin-3. The most potent galectin-3 antagonist was demonstrated to act in an assay monitoring galectin-3 accumulation upon amitriptyline-induced vesicle damage, visualizing a biochemically/medically relevant intracellular lectin-carbohydrate binding event and that it can be blocked by a small molecule. The same antagonist administered intratracheally attenuated bleomycin-induced pulmonary fibrosis in a mouse model with a dose/response profile comparing favorably with that of oral administration of the marketed antifibrotic compound pirfenidone., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Synthesis of doubly 3-O-coumarylmethyl-substituted thiodigalactosides from bis-3-O-propargyl-thiodigalactoside resulted in highly selective and high affinity galectin-3 inhibitors. Mutant studies, structural analysis, and molecular modeling revealed that the coumaryl substituents stack onto arginine side chains. One inhibitor displayed efficacy in a murine model of bleomycin-induced lung fibrosis similar to that of a known nonselective galectin-1/galectin-3 inhibitor, which strongly suggests that blocking galectin-3 glycan recognition is an important antifibrotic drug target.

Introduction: Gentamicin is an important aminoglycoside antibiotic used in clinics to combat infections from especially gram negative bacteria. A frequent side-effect of aminoglycoside antibiotics is a kidney injury consisting of necrosis of proximal tubular cells. It is important both in clinics and in research directed to eliminate or ameliorate this side-effect that a method is available for detection of injury at an early stage., Methods: We have therefore compared the sensitivity of four methods currently used in animal models to assess kidney injury induced by aminoglycoside antibiotics by applying them to the analysis of urine from male rats treated with low doses of gentamicin., Results: Excretion of phospholipids was significantly increased in contrast to excretion of N-acetyl-beta-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL) and protein. Assessment of phospholipiduria thus is the more sensitive noninvasive way to monitor initial renal injury induced by aminoglycoside antibiotics. A protocol is given for the serial analysis of urinary phospholipids allowing a considerable number of determinations to be carried out in the course of 2-3 days., Discussion: It is well known that all four considered methods do detect kidney damage induced in rats by high gentamicin doses far above doses used in clinics. The present investigation shows that only the analysis of the urinary phospholipids will detect damage induced by low doses of gentamicin. The method is relevant for animal model studies but will require considerable and innovative development for use in clinics.

FSH is crucial for oocyte maturation and fertility and is the main component in infertility treatment in assisted reproduction. The granulosa cells expressing the FSH receptor interact with the oocyte and provide nourishing substrates controlling the oocyte maturation. Thus, transcriptome analysis of granulosa cells stimulated by FSH is of major importance in understanding the communication between oocytes and granulosa cells. In this study, gene expression profiles were assessed in human granulosa cells from normal cycling in vitro maturation (IVM) patients using oligonucleotide gene chips. Granulosa cells were stimulated for 2 h with either FSH or a previously generated glycosylated FSH variant (FSH1208) that exhibited increased in vivo activity because of prolonged half-life. The analysis identified 74 significantly FSH/FSH1208 regulated genes. Amongst these were well known FSH regulated genes as well as genes not previously described to be important in the FSH signalling pathway. These novel FSH regulated genes include transcription factors [cAMP responsive element modulator (CREM)/inducible cAMP early repressors (ICER), GATA 6, ZFN 361, Bcl11a, CITED1 and TCF 8] and other regulatory proteins and enzymes (IGF-BP3, syntaxin and PCK1) possibly important for oocyte/granulosa cell interaction and function. Array data were validated for 13 genes by northern blots or RT-PCR. Furthermore, no significant differences in gene regulation were detected between the two FSH analogs. This work uncovers novel data important for understanding the folliculogenesis. Furthermore, the results suggest that FSH1208 has a gene expression profile like FSH and thus, in the light of known prolonged in vivo activity, might be a candidate for improved infertility treatment.

Two nonpeptide ligands that differ chemically by only a single methyl group but have agonistic (L-162,782) and antagonistic (L-162,389) properties in vivo were characterized on the cloned angiotensin AT1 receptor. Both compounds bound with high affinity (K(I) = 8 and 28 nM, respectively) to the AT1 receptor expressed transiently in COS-7 cells as determined in radioligand competition assays. L-162,782 acted as a powerful partial agonist, stimulating phosphatidylinositol turnover with a bell-shaped dose-response curve to 64% of the maximal level reached in response to angiotensin II. Surprisingly, L-162,389 also stimulated phosphatidylinositol turnover, albeit only to a small percentage of the angiotensin response. The prototype nonpeptide AT1 agonist L-162,313 gave a response of approximately 50%. The apparent EC50 values for all three compounds in stimulating phosphatidylinositol turnover were similar, approximately 30 nM, corresponding to their binding affinity. Each of the three compounds also acted as angiotensin antagonists, yet in this capacity the compounds differed markedly, with IC50 values ranging from 1.05 x 10(-7) M for L-162,389 to 6.5 x 10(-6) for L-162,782. A series of point mutations in the transmembrane segments (TMs) of the AT1 receptor had only minor effect on the binding affinity of the nonpeptide compounds, with the exception of A104V at the top of TM III, which selectively impaired the binding of L-162,782 and L-162,389. Substitutions in the middle of TM III, VI, or VII, which did not affect the binding affinity of the compounds, impaired or eliminated the agonistic efficacy of the nonpeptides but with only minor or no effect on the angiotensin potency or efficacy. Thus, in the N295D rat AT1 construct, L-162,782, L-162,313, and L-162,389 all antagonized the angiotensin-induced phosphatidylinositol turnover with surprisingly similar IC50 values (90-180 nM), and they all bound with unaltered, high affinity (22-36 nM). However, L-162,313 and L-162,782 could stimulate phosphatidylinositol turnover to only 20% of that of angiotensin. It is concluded that minor chemical modifications of either the compound or the receptor can dramatically alter the agonistic efficacy of biphenyl imidazole compounds on the AT1 receptor without affecting their affinity, as determined in binding assays, and that a number of substitutions in the middle of the TM segments affect the efficacy of nonpeptide agonists as opposed to angiotensin.

Commercially available solutions for continuous ambulatory peritoneal dialysis (CAPD) affect the viability and function of the cells in the peritoneal cavity. The low biocompatibility of the solutions may be caused by a low pH, hyperosmolality, high glucose content, and lack of potassium, glutamine, and other components essential for normal cellular functions. The nature of the buffer employed is also important for the cytotoxicity of the solutions. Lactate, the most frequently used buffer, has been shown to inhibit cellular functions important for the peritoneal defense system including phagocytosis, bacterial killing, and secretion of cytokines. It is generally believed that the cytotoxicity of lactate is caused by lowering of intracellular pH and impairment of metabolism due to changed redox potentials. However, the cytotoxicity of lactate is highly dependent upon the pH of the solutions, indicating that passive or active diffusion across the cell membrane is determining the effects of lactate. Bicarbonate has been heavily advocated as an alternative buffer because it is the most important naturally occurring buffer in plasma and it enables a pH of approximately 7.4 in the solutions. However, due to sedimentation of calcium carbonate (CaCO3) and production of toxic glucose metabolites it is difficult to prepare and store bicarbonate-based solutions. Moreover, investigations have revealed that even bicarbonate-based solutions are not optimal regarding biocompatibility, presumably due to a paradoxical intracellular acidification caused by influx of carbon dioxide (CO2). More recently, the effect of other buffers such as pyruvate and histidine have been examined. Especially pyruvate is a promising new buffer candidate.

Chimeric constructs between the human and the Xenopus laevis AT1 receptor have demonstrated, that the binding of non-peptide angiotensin antagonists is dependent on non-conserved residues located deep in transmembrane segment VII of the AT1 receptor. Here we have studied four pairs of closely related antagonists each consisting of a competitive and an insurmountable compound differentiated by one out of three different types of minor chemical modifications. None of the antagonists bound to the Xenopus receptor and the binding of all of the compounds to the human receptor was severely impaired by the introduction of non-conserved residues from transmembrane segment VII of the Xenopus receptor. In all four pairs of antagonists the competitive compound was affected more by these substitutions than the corresponding insurmountable one (209 vs. 22, 281 vs. 29, 290 vs. 29 and 992 vs. 325-fold increase in Ki values). A similar pattern was observed in response to substitution of a single non-conserved residue in transmembrane segment VII, Asn295 to Ser. These results indicate that a common molecular mechanism distinguishes the interaction of insurmountable and competitive antagonists with the AT1 receptor.

The renin-angiotensin system is the most important hormone system in the control of blood pressure and electrolyte homeostasis. Pharmacological blockade of the system by means of beta-blockers or ACE-inhibitors is a major tool in the treatment of hypertension and congestive heart failure. Inhibition of the binding of angiotensin to its receptor is, however, theoretically a more direct and selective blocking method. Recently, a series of potent non-peptide antagonists have been developed, which are active when given orally and appear to be promising drug candidates. The clinical and theoretical implications of this discovery are reviewed based upon the present knowledge of the renin-angiotensin system and the available methods for therapeutic intervention in the system.

Five different bicarbonate-based continuous ambulatory peritoneal dialysis (CAPD) solutions (pH: 7.0-7.4; bicarbonate: 10-27 mM; lactate: 20.8-6.7 mM) were produced in order to examine the cytotoxic effects of the different compositions. The migratory capacity of normal human polymorphonuclear (PMN) granulocytes after exposure to the solutions was used as a cytotoxicity assay. All the tested solutions reduced cellular function compared to a standard cell culture medium, but considerable differences between the solutions were observed. The optimal conditions for the PMN migration were at a pH of 7.0 and at bicarbonate and lactate concentrations of 20 mM and 12.5 mM, respectively. Bicarbonate concentrations of more than 25 mM were associated with reduced cellular function as were lactate concentrations of more than 15 mM. The most advantageous CAPD solution regarding cytotoxicity towards normal human PMN's is a combination of a lactate and bicarbonate-based solution, which has a bicarbonate concentration of approximately 20 mM, a lactate concentration of 12.5 mM, and a pH of approximately 7.2.

Unlabelled: Human polymorphonuclear granulocytes (PMN) were tested for migration and phagocytosis after exposure to CAPD solutions and effluents sampled during the first hour of dialysis from patients treated with lactate or bicarbonate based CAPD-solutions. The effluents from the lactate based solutions (Dianeal and Lockolys) reduced the migration and enhanced the phagocytosis compared to values obtained in a standard cell culture medium. Both cell functions increased during the dialysis period. In contrast, the cell-function only changed slightly when 87b, a bicarbonate based CAPD-solution (pH = 7.4, [HCO3-) = 29mM), was employed. During the first 30 minutes, the cells performed at a higher level when exposed to the 87b effluent than when exposed to the lactate effluents. The observations further indicated that optimal conditions for PMNs are at a bicarbonate concentration of less than 20 mM and a lactate concentration of less than 15mM., In Conclusion: PMN migration is reduced by both lactate and bicarbonate based CAPD solutions and effluents collected during the first hour of dialysis. The bio-compatibility of CAPD solutions may be improved by combining the lactate and bicarbonate buffering systems in a solution with a concentration of less than 20 mM of bicarbonate and less than 15 mM of lactate.

Previous in vitro biocompatibility studies have shown bicarbonate-based continuous ambulatory peritoneal dialysis (CAPD) fluids to be superior to those based upon lactate/acetate. To evaluate these findings in vivo, 41 rabbits were subjected to CAPD for four weeks in a randomized prospective study using either Dianeal, a commercially available dialysis fluid containing lactate, or 87b, a bicarbonate-based CAPD fluid. Ten rabbits with CAPD catheters, which were flushed with a heparin solution every 36 hours, served as controls. None of the control rabbits showed clinical or histopathological signs of peritonitis, while 8 of 20 in the Dianeal group and 6 of 21 in the 87b group contracted peritonitis. Four rabbits in the Dianeal group had to be sacrificed early due to severe peritonitis. Post mortem examinations, including scanning and light microscopy, did not reveal any macroscopic or microscopic differences among the three groups of noninfected animals. No significant distinctions between the groups could be made for body temperature, weight gain, dialysate volume, dialysate differential leukocyte count, dialysate protein content, and food intake during the course of the study. In conclusion, the present animal model did not reveal any major difference in the biocompatibility between the lactate- and the bicarbonate-based CAPD fluids.