Efficacy of Alkaline Phosphatase in Critically Ill Patients with COVID-19: A Multicentre Investigator-Initiated Double-Blind Randomised Placebo-Controlled Trial.

BACKGROUND: Efforts to identify therapies to treat hospitalised patients with COVID-19 are being continued. Alkaline phosphatase (AP) dephosphorylates pro-inflammatory adenosine triphosphate (ATP) into anti-inflammatory adenosine. METHODS: In a randomised controlled trial, we investigated the safety and efficacy of AP in patients with SARS-CoV-2 infection admitted to the ICU. AP or a placebo was administered for four days following admission to the ICU. The primary outcome was the duration of mechanical ventilation. Mortality in 28 days, acute kidney injury, need for reintubation, safety, and inflammatory markers relevant to the described high cytokine release associated with SARS-CoV-2 infection were the secondary outcomes. RESULTS: Between December 2020 and March 2022, 97 patients (of the intended 132) were included, of which 51 were randomised to AP. The trial was terminated prematurely based on meeting the threshold for futility. Compared to the placebo, AP did not affect the duration of mechanical ventilation (9.0 days vs. 9.3 days, p = 1.0). No safety issues were observed. After 28 days, mortality was 9 (18%) in the AP group versus 6 (13%) in the placebo group (p = 0.531). Additionally, no statistically significant differences between the AP and the placebo were observed for the other secondary outcomes. CONCLUSIONS: Alkaline phosphatase (AP) therapy in COVID-19 ICU patients showed no significant benefits in this trial.

Alkaline phosphatase to treat ischaemia-reperfusion injury in living-donor kidney transplantation: APhIRI I feasibility pilot study.

AIMS: Ischemia-reperfusion injury (IRI) during kidney transplant procedures is associated with adverse outcome. Alkaline phosphatase (AP) is an enzyme that has the potential to dampen IRI. Prior to this study, it had not been tested in the setting of kidney transplantation. This study aimed to evaluate the safety and feasibility of peri-procedural AP administration in living donor kidney transplantation. METHODS: In this double blind, randomized, placebo-controlled, single-center pilot study, all eligible recipients of living donor kidneys were asked to give informed consent. AP (bRESCAP) or a placebo was administered intravenously over 24 hours after the transplantation procedure. The primary outcome-graft function at 1 year-was represented by iohexol measured glomerular filtration rate (mGFR). Serum and urine biomarkers within seven days after surgery were used as surrogate markers of kidney function and injury. RESULTS: Eleven patients were enrolled of whom five were treated with bRESCAP and six with placebo. After 1 year, mGFR was not different between groups. No specific adverse events were observed in the bRESCAP group. Urine expression of injury biomarkers CCL14, NGAL and Cystatin C was lower in the bRESCAP group at day seven. This was statistically significant. CONCLUSION: This study illustrates that bRESCAP treatment is feasible in kidney transplantation, might have a dampening effect on IRI induced renal inflammation, and raises no safety concerns. Future research will evaluate the effects of bRESCAP treatment in donation after circulatory death kidney transplantation where IRI is more pronounced.

Clinical and genetic analyses of a Dutch cohort of 40 patients with a nephronophthisis-related ciliopathy.

BACKGROUND: Nephronophthisis is an autosomal recessive ciliopathy and important cause of end-stage renal disease (ESRD) in children and young adults. Diagnostic delay is frequent. This study investigates clinical characteristics, initial symptoms, and genetic defects in a cohort with nephronophthisis-related ciliopathy, to improve early detection and genetic counseling. METHODS: Forty patients from 36 families with nephronophthisis-related ciliopathy were recruited at university medical centers and online. Comprehensive clinical and genotypic data were recorded. Patients without molecular diagnosis were offered genetic analysis. RESULTS: Of 40 patients, 45% had isolated nephronophthisis, 48% syndromic diagnosis, and 7% nephronophthisis with extrarenal features not constituting a recognizable syndrome. Patients developed ESRD at median 13 years (range 5-47). Median age of symptom onset was 9 years in both isolated and syndromic forms (range 5-26 vs. 5-33). Common presenting symptoms were fatigue (42%), polydipsia/polyuria (33%), and hypertension (21%). Renal ultrasound showed small-to-normal-sized kidneys, increased echogenicity (65%), cysts (43%), and abnormal corticomedullary differentiation (32%). Renal biopsies in eight patients showed nonspecific signs of chronic kidney disease (CKD). Twenty-three patients (58%) had genetic diagnosis upon inclusion. Thirteen of those without a genetic diagnosis gave consent for genetic testing, and a cause was identified in five (38%). CONCLUSIONS: Nephronophthisis is genetically and phenotypically heterogeneous and should be considered in children and young adults presenting with persistent fatigue and polyuria, and in all patients with unexplained CKD. As symptom onset can occur into adulthood, presymptomatic monitoring of kidney function in syndromic ciliopathy patients should continue until at least age 30.

Relation between duration of the prodromal phase and renal damage in ANCA-associated vasculitis.

BACKGROUND: In ANCA-associated vasculitis the acute phase of the disease is often preceded by prodromal symptoms. The aim of the present study was to analyze the relation between the duration of the prodromal phase and renal damage. METHODS: Patients with ANCA-associated vasculitis and renal involvement from a retrospective single-center cohort were divided into two equal groups based on the duration of the prodromal phase. The prodromal phase was defined as the time between first vasculitis related symptoms and the date of diagnosis. Clinical characteristics at diagnosis and renal items on the vasculitis damage index at 6 months were compared between the two groups. In addition, the relation between a long prodromal phase and 3-year end-stage renal disease and mortality as a composite outcome was studied. RESULTS: A total of 72 patients were included (age 64 ± 12 years; 74% male; 96% Caucasian). At diagnosis, in patients with a prodromal phase ≤22 weeks versus >22 weeks estimated glomerular filtration rate and proteinuria did not differ significantly (35 (interquartile range 50) versus 30 (50) ml/min p = 0.84; 75% versus 87%, p = 0.21 respectively). Furthermore, Birmingham Vasculitis Activity Scores were comparable (7 (3), p = 0.71). At 6 months, a long prodromal phase was associated with proteinuria (odds ratio 5.38, 95% confidence interval (CI) 1.47-19.62), but not with an estimated glomerular filtration rate ≤ 50 ml/min (odds ratio 0.89, 95% CI 0.33-2.37) in multivariable analyses. In addition, a long prodromal phase was associated with end-stage renal disease/mortality (hazard ratio 5.22, 95% CI 1.13-24.20). CONCLUSIONS: A long prodromal phase was associated with proteinuria and 3-year end-stage renal disease/mortality, but not with a reduced renal function at 6 months. These results underline the importance of an early diagnosis in ANCA-associated vasculitis patients in order to improve renal outcomes.

Inactivating PSMB5 mutations and P-glycoprotein (multidrug resistance-associated protein/ATP-binding cassette B1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno)proteasome inhibitors in mononuclear blood cells from patients with rheumatoid arthritis.

Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also been proposed as a potential antirheumatic agent. Its reported side effects, however, make it unappealing for long-term administration, and resistance may also develop. To overcome this, second-generation PIs became available. Here, we investigated whether a novel class of peptide epoxyketone-based PIs, including carfilzomib, N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide (ONX0912), and (S)-3-(4-methoxyphenyl)-N-((S)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanamide (ONX0914), might escape two established BTZ-resistance mechanisms: 1) mutations in the proteasome ß5 subunit (PSMB5) targeted by these PIs, and 2) drug efflux mediated by ATP-binding cassette transporters. THP1 myeloid sublines with acquired resistance to BTZ (54- to 235-fold) caused by mutations in the PSMB5 gene displayed marked cross-resistance but less pronounced cross-resistance to carfilzomib (9- to 32-fold), ONX0912 (39- to 62-fold), and ONX0914 (27- to 97-fold). As for ATP-binding cassette transporter-mediated efflux, lymphoid CEM/VLB cells with P-glycoprotein (Pgp)/multidrug resistance 1 overexpression exhibited substantial resistance to carfilzomib (114-fold), ONX0912 (23-fold), and ONX0914 (162-fold), whereas less resistance to BTZ (4.5-fold) was observed. Consistently, ß5 subunit-associated chymotrypsin-like proteasome activity was significantly less inhibited in these CEM/VLB cells. Ex vivo analysis of peripheral blood mononuclear cells from therapy-naive patients with rheumatoid arthritis revealed that, although basal Pgp levels were low, P-glycoprotein expression compromised the inhibitory effect of carfilzomib and ONX0914. However, the use of P121 (reversin 121), a Pgp transport inhibitor, restored parental cell inhibitory levels in both CEM/VLB cells and peripheral blood mononuclear cells. These results indicate that the pharmacologic activity of these PIs may be hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion via Pgp.

ABC drug transporters and immunity: novel therapeutic targets in autoimmunity and cancer.

ABC transporters were identified originally for their contribution to clinical MDR as a result of their capacity to extrude various unrelated cytotoxic drugs. More recent reports have shown that ABC transporters can play important roles in the development, differentiation, and maturation of immune cells and are involved in migration of immune effector cells to sites of inflammation. Many of the currently identified, endogenous ABC transporter substrates have immunostimulating effects. Increasing the expression of ABC transporters on immune cells and thereby enhancing immune cell development or functionality may be beneficial to immunotherapy in the field of oncology. On the contrary, in the treatment of autoimmune diseases, blockade of these transporters may prove beneficial, as it could dampen disease activity by compromising immune effector cell functions. This review will focus on the expression, regulation, and substrate specificity of ABC transporters in relation to functional activities of immune effector cells and discusses implications for the treatment of cancer on the one hand and autoimmune diseases on the other.

Involvement of breast cancer resistance protein expression on rheumatoid arthritis synovial tissue macrophages in resistance to methotrexate and leflunomide.

OBJECTIVE: To determine whether multidrug-resistance efflux transporters are expressed on immune effector cells in synovial tissue from patients with rheumatoid arthritis (RA) and compromise the efficacy of methotrexate (MTX) and leflunomide (LEF). METHODS: Synovial tissue biopsy samples obtained from RA patients before treatment and 4 months after starting treatment with MTX (n = 17) or LEF (n = 13) were examined by immunohistochemical staining and digital image analysis for the expression of the drug efflux transporters P-glycoprotein, multidrug resistance-associated protein 1 (MRP-1) through MRP-5, MRP-8, MRP-9, and breast cancer resistance protein (BCRP), and the relationship to clinical efficacy of MTX and LEF was assessed. RESULTS: BCRP expression was observed in all RA synovial biopsy samples, both pretreatment and posttreatment, but not in control noninflammatory synovial tissue samples from orthopedic patients. BCRP expression was found both in the intimal lining layer and on macrophages and endothelial cells in the synovial sublining. Total numbers of macrophages in RA patients decreased upon treatment; in biopsy samples with persistently high macrophage counts, 2-fold higher BCRP expression was observed. Furthermore, median BCRP expression was significantly increased (3-fold) in nonresponders to disease-modifying antirheumatic drugs (DMARDs) compared with responders to DMARDs (P = 0.048). Low expression of MRP-1 was found on synovial macrophages, along with moderate expression in T cell areas of synovial biopsy specimens from one-third of the RA patients. CONCLUSION: These findings show that the drug resistance-related proteins BCRP and MRP-1 are expressed on inflammatory cells in RA synovial tissue. Since MTX is a substrate for both BCRP and MRP-1, and LEF is a high-affinity substrate for BCRP, these transporters may contribute to reduced therapeutic efficacy of these DMARDs.

Folate receptor beta as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients.

OBJECTIVE: To determine the expression of folate receptor beta (FRbeta) in synovial biopsy tissues and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients and to identify novel folate antagonists that are more selective in the targeting and internalization of FRbeta than methotrexate (MTX). METHODS: Immunohistochemistry and computer-assisted digital imaging analyses were used for the detection of FRbeta protein expression on immunocompetent cells in synovial biopsy samples from RA patients with active disease and in noninflammatory control synovial tissues. FRbeta messenger RNA (mRNA) levels were determined by reverse transcription-polymerase chain reaction analysis. Binding affinities of FRbeta for folate antagonists were assessed by competition experiments for 3H-folic acid binding on FRbeta-transfected cells. Efficacy of FRbeta-mediated internalization of folate antagonists was evaluated by assessment of antiproliferative effects against FRbeta-transfected cells. RESULTS: Immunohistochemical staining of RA synovial tissue showed high expression of FRbeta on macrophages in the intimal lining layer and synovial sublining, whereas no staining was observed in T cell areas or in control synovial tissue. Consistently, FRbeta mRNA levels were highest in synovial tissue extracts and RA monocyte-derived macrophages, but low in peripheral blood T cells and monocytes. Screening of 10 new-generation folate antagonists revealed 4 compounds for which FRbeta had a high binding affinity (20-77-fold higher than for MTX). One of these, the thymidylate synthase inhibitor BCG 945, displayed selective targeting against FRbeta-transfected cells. CONCLUSION: Abundant FRbeta expression on activated macrophages in synovial tissue from RA patients deserves further exploration for selective therapeutic interventions with high-affinity-binding folate antagonists, of which BCG 945 may be a prototypical representative.

Noninvasive imaging of macrophages in rheumatoid synovitis using 11C-(R)-PK11195 and positron emission tomography.

OBJECTIVE: Noninvasive imaging by positron emission tomography (PET) of macrophages in inflamed joints of patients with rheumatoid arthritis (RA) may allow early detection of disease activity. We undertook this study to investigate whether rheumatoid synovitis can be visualized by PET using the tracer 11C-(R)-PK11195, which binds to peripheral benzodiazepine receptors (PBRs) on macrophages. METHODS: Knee joints of 11 RA patients with active arthritis of at least 1 knee joint were imaged with 11C-(R)-PK11195 PET. Tissue uptake of 11C-(R)-PK11195 was quantified. PET was followed by arthroscopy of the most inflamed knee joint of each RA patient. Synovial tissue samples were subjected to immunohistochemical staining. RESULTS: 11C-(R)-PK11195 uptake on the PET scans was significantly higher in severely inflamed joints than in joints with moderate or mild signs of inflammation. In addition, tracer uptake in contralateral uninflamed knee joints of RA patients was significantly higher than in uninflamed joints of control patients without inflammatory joint disease, suggesting the presence of subclinical disease activity. PET tracer uptake in joints correlated significantly with PBR staining in the sublining of synovial tissue. PBR staining correlated significantly with CD68 staining of macrophages. CONCLUSION: 11C-(R)-PK11195 PET imaging allows noninvasive in vivo imaging of macrophages in rheumatoid synovitis and possibly even in subclinical synovitis. Noninvasive visualization of macrophages may be useful both for detecting early synovitis and for monitoring synovitis activity during treatment.

Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein.

The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45- to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome beta5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to beta5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) restoration of bortezomib sensitivity in bortezomib-resistant cells by siRNA-mediated silencing of PSMB5 gene expression. Collectively, these findings establish a novel mechanism of bortezomib resistance associated with the selective overexpression of a mutant PSMB5 protein.

Drug Insight: resistance to methotrexate and other disease-modifying antirheumatic drugs--from bench to bedside.

The chronic nature of rheumatoid arthritis (RA) means that patients require drug therapy for many years. Many RA patients, however, have to discontinue treatment because of drug-related toxic effects, loss of efficacy, or both. The underlying molecular cause for loss of efficacy of antirheumatic drugs is not fully understood, but it might be mediated, at least in part, by mechanisms shared with resistance to anticancer drugs. This Review outlines molecular mechanisms that could be involved in the onset of resistance to, or the loss of efficacy of, disease-modifying antirheumatic drugs in RA patients, including methotrexate, sulfasalazine, chloroquine, hydroxychloroquine, azathioprine, and leflunomide. The mechanisms suggested are based on findings from experimental laboratory studies of specific drug-uptake and drug-efflux transporters belonging to the superfamily of multidrug-resistance transporters, alterations in intracellular drug metabolism, and genetic polymorphisms of drug transporters and metabolic enzymes. We also discuss strategies to overcome resistance and the current clinical studies aiming to predict response and risk of toxic effects. More in-depth knowledge of the mechanisms behind these features could help facilitate a more efficient use of disease-modifying antirheumatic drugs.