Clinical development innovation in rare diseases: overcoming barriers to successful delivery of a randomised clinical trial in alkaptonuria-a mini-review.

Alkaptonuria is a rare inherited disorder for which there was no disease-modifying treatment. In order to develop a successful approved therapy of AKU multiple barriers had to be overcome. These included activities before the conduct of the study including deciding on the drug therapy, the dose of the drug to be used, clarify the nature of the disease, develop outcome measures likely to yield a positive outcome, have a strategy to ensure appropriate patient participation through identification, build a consortium of investigators, obtain regulatory approval for proposed investigation plan and secure funding. Significant barriers were overcome during the conduct of the multicentre study to ensure harmonisation. Mechanisms were put in place to recruit and retain patients in the study. Barriers to patient access following completion of the study and regulatory approval were resolved.

Metabolic profiles and fingerprints for the investigation of the influence of nitisinone on the metabolism of the yeast Saccharomyces cerevisiae.

Nitisinone (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, NTBC) is considered a potentially effective drug for the treatment of various metabolic diseases associated with disorders of L-tyrosine metabolism however, side-effects impede its widespread use. This work aimed to broaden the knowledge of the influence of NTBC and its metabolites 2-amino-4-(trifluoromethyl)benzoic acid (ATFA), 2-nitro-4-(trifluoromethyl)benzoic acid (NTFA), and cyclohexane-1,3-dione (CHD) on the catabolism of L-tyrosine and other endogenous compounds in Saccharomyces cerevisiae. Based on a targeted analysis performed by LC-ESI-MS/MS, based on multiple reaction monitoring, it was found that the dissipation kinetics of the parent compound and its metabolites are compatible with a first-order reaction mechanism. Moreover, it has been proven that formed NTBC metabolites, such as CHD, cause a decrease in L-tyrosine, L-tryptophan, and L-phenylalanine concentrations by about 34%, 59% and 51%, respectively, compared to the untreated model organism. The overall changes in the metabolism of yeast exposed to NTBC or its derivatives were evaluated by non-targeted analysis via LC-ESI-MS/MS in the ion trap scanning mode. Based on principal components analysis, a statistically significant similarity between metabolic responses of yeast treated with ATFA or NTFA was observed. These findings facilitate further studies investigating the influence of NTBC on the human body and the mechanism of its action.

Determinants of tyrosinaemia during nitisinone therapy in alkaptonuria.

Nitisinone (NIT) produces inevitable but varying degree of tyrosinaemia. However, the understanding of the dynamic adaptive relationships within the tyrosine catabolic pathway has not been investigated fully. The objective of the study was to assess the contribution of protein intake, serum NIT (sNIT) and tyrosine pathway metabolites to nitisinone-induced tyrosinaemia in alkaptonuria (AKU). Samples of serum and 24-h urine collected during SONIA 2 (Suitability Of Nitisinone In Alkaptonuria 2) at months 3 (V2), 12 (V3), 24 (V4), 36 (V5) and 48 (V6) were included in these analyses. Homogentisic acid (HGA), tyrosine (TYR), phenylalanine (PHE), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and sNIT were analysed at all time-points in serum and urine. Total body water (TBW) metabolites were derived using 60% body weight. 24-h urine and TBW metabolites were summed to obtain combined values. All statistical analyses were post-hoc. 307 serum and 24-h urine sampling points were analysed. Serum TYR from V2 to V6, ranging from 478 to 1983 µmol/L were stratified (number of sampling points in brackets) into groups < 701 (47), 701-900 (105), 901-1100 (96) and > 1100 (59) µmol/L. The majority of sampling points had values greater than 900 µmol/L. sPHE increased with increasing sTYR (p < 0.001). Tyrosine, HPPA and HPLA in serum and TBW all increased with rising sTYR (p < 0.001), while HPLA/TYR ratio decreased (p < 0.0001). During NIT therapy, adaptive response to minimise TYR formation was demonstrated. Decreased conversion of HPPA to HPLA, relative to TYR, seems to be most influential in determining the degree of tyrosinaemia.

Application of Multiple Strategies to Improve the Production of the Potential Cancer Drug 4-Acetylantroquinonol B (4-AAQB) by the Rare Fungus Antrodia cinnamomea.

4-Acetylantroquinonol B (4-AAQB) was identified in the rare fungus Antrodia cinnamomea and has been proven to be a potential therapeutic agent for cancer treatment. But the extraction of 4-AAQB from the fruit body led to a low yield and limited its further application in the pharmaceutical field. In this work, 4-AAQB production was enhanced in the submerged fermentation by the combination of exogenous additives, surfactants with the in situ extractive fermentation. 4-Methylbenzoic acid was proven to be an efficient additive for the accumulation of 4-AAQB by Antrodia cinnamomea, while 2% (w/v) Tween-80 added on the first day as surfactant and 30% (w/v) oleic acid added on the sixteenth day as extractant were the most available couples for 4-AAQB production in the in situ extractive fermentation. The combination of these multiple strategies resulted in the yield of 4-AAQB to 17.27 mg/g dry cell weight with a titer of 140 mg/L, which was the highest titer of 4-AAQB reported so far. It showed that the combination of these strategies had a significant promotion on 4-AAQB production by A. cinnamomea, which laid a good foundation for its large-scale production and also provided a viable method for the cultivation of other rare fungi.

Nitisinone treatment during two pregnancies and breastfeeding in a woman with tyrosinemia type 1 - a case report.

OBJECTIVES: Tyrosinaemia type 1, an inherited disorder of tyrosine metabolism, is usually treated with a tyrosine-defined diet and since 2000 with nitisinone. So far, data about effects of nitisone during pregnancy and breastfeeding are rare. This is the first report of two pregnancies in a patient with tyrosinaemia type 1 while under treatment with nitisinone. CASE PRESENTATION: We here present a 20-year-old female patient with tyrisonemia type 1 receiving treatment with nitisinone and a tyrosine-defined diet since she was diagnosed with tyrosinaemia type 1 at the age of 18 months. During two pregnancies blood concentrations of tyrosine, succinylacetone and nitisinone were measured regularly. Neither infant has tyrosinaemia type 1 and both showed an initial increase in concentrations of tyrosine, succinylacetone and nitisinone. All three metabolites dropped within two weeks after birth. Both were exclusively breastfed for about two weeks. Both children show age-appropriate physical and mental development. CONCLUSIONS: Nitisinone therapy during pregnancy and the short breastfeeding period did not result in adverse events in our patient or her children. Regular assessments of tyrosine, succinylacetone and nitisinone should be made during pregnancy and the breastfeeding period in both the mother and the infant. For better understanding, in principle, all cases of pregnancy and breastfeeding with tyrosinemia type 1 should be assessed and followed to further evaluate the implications of tyrosinaemia type 1 and its treatment during pregnancy. Additionally, even though experience with breastfeeding is limited, medication with nitisinone is safe and there is no reason to consider breastfeeding unsafe or to not recommend it.

Preventive use of nitisinone in alkaptonuria.

Alkaptonuria (AKU, OMIM 203500) is a rare congenital disorder caused by a deficiency of the enzyme homogentisate-1,2,-dioxygenase. The long-term consequences of AKU are joint problems, cardiac valve abnormalities and renal problems. Landmark intervention studies with nitisinone 10 mg daily, suppressing an upstream enzyme activity, demonstrated its beneficial effects in AKU patients with established complications, which usually start to develop in the fourth decade. Lower dose of nitisinone in the range of 0.2-2 mg daily will already reduce urinary homogentisic acid (uHGA) excretion by > 90%, which may prevent AKU-related complications earlier in the course of the disease while limiting the possibility of side-effects related to the increase of plasma tyrosine levels caused by nitisinone. Future preventive studies should establish the lowest possible dose for an individual patient, the best age to start treatment and also collect evidence to which level uHGA excretion should be reduced to prevent complications.

Treatment adherence in tyrosinemia type 1 patients.

BACKGROUND: While therapeutic advances have significantly improved the prognosis of patients with hereditary tyrosinemia type 1 (HT1), adherence to dietary and pharmacological treatments is essential for an optimal clinical outcome. Poor treatment adherence is well documented among patients with chronic diseases, but data from HT1 patients are scarce. This study evaluated pharmacological and dietary adherence in HT1 patients both directly, by quantifying blood levels nitisinone (NTBC) levels and metabolic biomarkers of HT1 [tyrosine (Tyr), phenylalanine (Phe), and succinylacetone]; and indirectly, by analyzing NTBC prescriptions from hospital pharmacies and via clinical interviews including the Haynes-Sackett (or self-compliance) test and the adapted Battle test of patient knowledge of the disease. RESULTS: This observational study analyzed data collected over 4 years from 69 HT1 patients (7 adults and 62 children; age range, 7 months-35 years) who were treated with NTBC and a low-Tyr, low-Phe diet. Adherence to both pharmacological and, in particular, dietary treatment was poor. Annual data showed that NTBC levels were lower than recommended in more than one third of patients, and that initial Tyr levels were high (> 400 µM) in 54.2-64.4% of patients and exceeded 750 µM in 25.8% of them. Remarkably, annual normalization of NTBC levels was observed in 29.4-57.9% of patients for whom serial NTBC determinations were performed. Poor adherence to dietary treatment was more refractory to positive reinforcement: 36.2% of patients in the group who underwent multiple analyses per year maintained high Tyr levels during the entire study period, and, when considering each of the years individually this percentage ranged from 75 to 100% of them. Indirect methods revealed percentages of non-adherent patients of 7.3 and 15.9% (adapted Battle and Haynes tests, respectively). CONCLUSIONS: Despite initially poor adherence to pharmacological and especially dietary treatment among HT1 patients, positive reinforcement at medical consultations resulted in a marked improvement in NTBC levels, indicating the importance of systematic positive reinforcement at medical visits.

Evaluating the aortic stenosis phenotype before and after the effect of homogentisic acid lowering therapy: Analysis of a large cohort of eighty-one alkaptonuria patients.

AIMS: A large alkaptonuria (AKU) cohort was studied to better characterise the poorly understood phenotype of aortic stenosis of rare disease AKU. METHODS AND RESULTS: Eighty-one patients attended the National Alkaptonuria Centre (NAC) between 2007 and 2020. Nine only attended once. Fifty-one attended more than once and received nitisinone 2 mg daily. Twenty-one attended at least twice without receiving nitisinone. Assessments included questionnaire analysis, standard transthoracic echocardiography, as well as photographs of ochronotic pigment in eyes and ears at baseline when 2 mg nitisinone was commenced, and yearly thereafter. Blood and urine samples were collected for chemical measurement. The prevalence of aortic stenosis and aortic valve replacement were 22.2 and 6.2% in the current group. Aortic maximum velocity (Vmax) was directly related to varying degrees to age (R = 0.58, p < 0.001), systolic blood pressure (R = 0.32, p < 0.05), serum homogentisic acid (sHGA) (R = 0.28, p < 0.05), ochronosis scores (R = 0.72, p < 0.001), and alkaptonuria severity score index (AKUSSI) (R = 0.58, p < 0.001) on linear regression analysis. Age and ochronosis scores were significantly related to Vmax on multiple regression analysis (p < 0.005). Nitisinone decreased sHGA, 24-h urine HGA (uHGA24), ochronosis scores and AKUSSI significantly at all visits post-nitisinone. Nitisinone decreased Vmax change scores at final visit comparison, with a similar pattern at earlier visits. CONCLUSION: Aortic valve disease is highly prevalent in this NAC cohort, and strongly associated with ochronosis and disease severity. Nitisinone decreases ochronosis and had a similar significant effect on Vmax.

Long-term safety and outcomes in hereditary tyrosinaemia type 1 with nitisinone treatment: a 15-year non-interventional, multicentre study.

BACKGROUND: Since the EU approval of nitisinone in 2005, prognosis for patients with hereditary tyrosinaemia type 1 has changed dramatically, with patients living with the disease now reaching adulthood for the first time in history. This study aimed to assess the long-term safety and outcomes of nitisinone treatment in patients with hereditary tyrosinaemia type 1. METHODS: We did a non-interventional, non-comparative, multicentre study in 77 sites across 17 countries in Europe and collected retrospective and prospective longitudinal data in patients with hereditary tyrosinaemia type 1 who were treated with oral nitisinone during the study period (Feb 21, 2005, to Sept 30, 2019). There were no specific exclusion criteria. Patients were followed-up with an investigator at least annually for as long as they were treated, or until the end of the study. The primary endpoints, occurrence of adverse events related to hepatic, renal, ophthalmic, haematological, or cognitive or developmental function, were assessed in the complete set (all patients already receiving treatment at the index date [Feb 21, 2005] or starting treatment thereafter) and the index set (the subset of patients who had their first dose on the index date or later only). FINDINGS: 315 patients were enrolled during the study period (complete set). Additionally, data from 24 patients who had liver transplantation or died during the post-marketing surveillance programme were retrieved (extended analysis set; 339 patients). Median treatment duration was 11·2 years (range 0·7-28·4); cumulative nitisinone exposure was 3172·7 patient-years. Patients who were diagnosed by neonatal screening started nitisinone treatment at median age 0·8 months versus 8·5 months in those who presented clinically. Incidences of hepatic, renal, ophthalmic, haematological, or cognitive or developmental adverse events were low. Occurrence of liver transplantation or death was more frequent the later that treatment was initiated (none of 70 patients who started treatment at age <28 days vs 35 [13%] of 268 patients who started treatment at age ≥28 days). 279 (89%) of 315 patients were assessed as having either very good or good nitisinone treatment compliance. Treatment and diet compliance declined as patients aged. Suboptimal plasma phenylalanine and tyrosine levels were observed. The majority of patients were reported to have good overall clinical condition throughout treatment; 176 (87%) of 203 during the entire study, 98% following 1 year of treatment. INTERPRETATION: Long-term nitisinone treatment was well tolerated and no new safety signals were revealed. Life-limiting hepatic disease appears to have been prevented by early treatment start. Neonatal screening was the most effective way of ensuring early treatment. Standardised monitoring of blood tyrosine, phenylalanine, and nitisinone levels has potential to guide individualised therapy. FUNDING: Swedish Orphan Biovitrum (Sobi).

A Synthetic Curcuminoid Analogue, 2,6-Bis-4-(Hydroxyl-3-Methoxybenzylidine)-Cyclohexanone (BHMC) Ameliorates Acute Airway Inflammation of Allergic Asthma in Ovalbumin-Sensitized Mice.

2,6-Bis-(4-hydroxyl-3-methoxybenzylidine) cyclohexanone (BHMC), a synthetic curcuminoid analogue, has been shown to exhibit anti-inflammatory properties in cellular models of inflammation and improve the survival of mice from lethal sepsis. We further evaluated the therapeutic effect of BHMC on acute airway inflammation in a mouse model of allergic asthma. Mice were sensitized and challenged with ovalbumin (OVA), followed by intraperitoneal administration of 0.1, 1, and 10 mg/kg of BHMC. Bronchoalveolar lavage fluid, blood, and lung samples were collected, and the respiratory function was measured. OVA sensitization and challenge increased airway hyperresponsiveness (AHR) and pulmonary inflammation. All three doses of BHMC (0.1-10 mg/kg) significantly reduced the number of eosinophils, lymphocytes, macrophages, and neutrophils, as well as the levels of Th2 cytokines (IL-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (BALF) as compared to OVA-challenged mice. However, serum level of IgE was not affected. All three doses of BHMC (0.1-10 mg/kg) were effective in suppressing the infiltration of inflammatory cells at the peribronchial and perivascular regions, with the greatest effect observed at 1 mg/kg which was comparable to dexamethasone. Goblet cell hyperplasia was inhibited by 1 and 10 mg/kg of BHMC, while the lowest dose (0.1 mg/kg) had no significant inhibitory effect. These findings demonstrate that BHMC, a synthetic nonsteroidal small molecule, ameliorates acute airway inflammation associated with allergic asthma, primarily by suppressing the release of inflammatory mediators and goblet cell hyperplasia to a lesser extent in acute airway inflammation of allergic asthma.

Mechanistic evaluation of a novel cyclohexenone derivative's functionality against nociception and inflammation: An in-vitro, in-vivo and in-silico approach.

The synthesis of a novel cyclohexanone derivative (CHD; Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-enecarboxylate) was described and the subsequent aim was to perform an in vitro, in vivo and in silico pharmacological evaluation as a putative anti-nociceptive and anti-inflammatory agent in mice. Initial in vitro studies revealed that CHD inhibited both cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes and it also reduced mRNA expression of COX-2 and the pro-inflammatory cytokines TNF-α and IL-1ß. It was then shown that CHD dose dependently inhibited chemically induced tonic nociception in the abdominal constriction assay and also phasic thermal nociception (i.e. anti-nociception) in the hot plate and tail immersion tests in comparison with aspirin and tramadol respectively. The thermal test outcomes indicated a possible moderate centrally mediated anti-nociception which, in the case of the hot plate test, was pentylenetetrazole (PTZ) and naloxone reversible, implicating GABAergic and opioidergic mechanisms. CHD was also effective against both the neurogenic and inflammatory mediator phases induced in the formalin test and it also disclosed anti-inflammatory activity against the phlogistic agents, carrageenan, serotonin, histamine and xylene compared with standard drugs in edema volume tests. In silico studies indicated that CHD possessed preferential affinity for GABAA, opioid and COX-2 target sites and this was supported by molecular dynamic simulations where computation of free energy of binding also favored the formation of stable complexes with these sites. These findings suggest that CHD has prospective anti-nociceptive and anti-inflammatory properties, probably mediated through GABAergic and opioidergic interactions supplemented by COX-2 and 5-LOX enzyme inhibition in addition to reducing pro-inflammatory cytokine expression. CHD may therefore possess potentially beneficial therapeutic effectiveness in the management of inflammation and pain.

4-Acetylantroquinonol B ameliorates nonalcoholic steatohepatitis by suppression of ER stress and NLRP3 inflammasome activation.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is an inflammatory lipotoxic disorder with a prevalence of over 25% worldwide. However, safe and effective therapeutic agents for the management of NAFLD are still lacking. We aimed to investigate the hepatoprotective effect and molecular mechanism of 4-acetylantroquinonol B (4-AAQB), a natural ubiquinone derivative obtained from the mycelia of Antrodia cinnamomea. METHODS: RAW264.7 and J774A.1 cells were treated with 4-AAQB and then stimulated with LPS or tunicamycin (TM) for 24 h. Inflammatory responses, markers of endoplasmic reticulum (ER) stress, and NOD-like receptor protein 3 (NLRP3) inflammasome were analyzed in both cell lines. In the applied in vivo model, male C57BL/6J mice were fed with chow or a methionine/choline-deficient (MCD) diet along with vehicle or 4-AAQB (10 mg/kg, i.p. injected, once a day) for 10 consecutive days. Plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Liver tissues were analyzed using histological techniques; protein levels involved in ER stress, NLRP3 inflammasome, and inflammatory responses were measured. RESULTS: 4-AAQB significantly ameliorated the plasma levels of ALT and AST as well as the NAFLD activity score (NAS) in mice fed the MCD diet. In addition, 4-AAQB suppressed inflammatory responses, ER stress, and NLRP3 inflammasome activation, but increased the nuclear factor erythroid 2-related factor 2 (Nrf2) and Sirtuin 1 (SIRT1) signaling pathways in both in vitro and in vivo models. CONCLUSIONS: We suggest that 4-AAQB treatment might be a tangible therapeutic strategy in the management of NAFLD/NASH.

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Type 1 tyrosinemia in Finland: a nationwide study.

BACKGROUND: Introduction of nitisinone and newborn screening (NBS) have transformed the treatment of type 1 tyrosinemia, but the effects of these changes on the long-term outcomes remain obscure. Also, the predictors for later complications, the significance of drug levels and the normalization of laboratory and imaging findings are poorly known. We investigated these issues in a nationwide study. RESULTS: Type 1 tyrosinemia was diagnosed in 22 children in 1978-2019 in Finland. Incidence was 1/90,102, with a significant enrichment in South Ostrobothnia (1/9990). Median age at diagnosis was 5 (range 0.5-36) months, 55% were girls and 13 had homozygotic Trp262X mutation. Four patients were detected through screening and 18 clinically, their main findings being liver failure (50% vs. 100%, respectively, p = 0.026), ascites (0% vs. 53%, p = 0.104), renal tubulopathy (0% vs. 65%, p = 0.035), rickets (25% vs. 65%, p = 0.272), growth failure (0% vs. 66%, p = 0.029), thrombocytopenia (25% vs. 88%, p = 0.028) and anaemia (0% vs. 47%, p = 0.131). One patient was treated with diet, seven with transplantation and 14 with nitisinone. Three late-diagnosed (6-33 months) nitisinone treated patients needed transplantation later. Kidney dysfunction (86% vs. 7%, p = 0.001), hypertension (57% vs. 7%, p = 0.025) and osteopenia/osteoporosis (71% vs. 14%, p = 0.017) were more frequent in transplanted than nitisinone-treated patients. Blood/serum alpha-fetoprotein decreased rapidly on nitisinone in all but one patient, who later developed intrahepatic hepatocellular carcinoma. Liver values normalized in 31 months and other laboratory values except thrombocytopenia within 18 months. Imaging findings normalized in 3-56 months excluding five patients with liver or splenic abnormalities. Low mean nitisinone concentration was associated with higher risk of severe complications (r = 0.758, p = 0.003) despite undetectable urine succinylacetone. CONCLUSIONS: Prognosis of type 1 tyrosinemia has improved in the era of nitisinone, and NBS seems to provide further benefits. Nevertheless, the long-term risk for complications remains, particularly in the case of late diagnosis and/or insufficient nitisinone levels.

Dehydroxymethylepoxyquinomicin, a novel nuclear factor-κB inhibitor, prevents the development of cyclosporine A nephrotoxicity in a rat model.

BACKGROUND: Cyclosporine A (CsA) is an essential immunosuppressant in organ transplantation. However, its chronic nephrotoxicity is an obstacle to long allograft survival that has not been overcome. Nuclear factor-κB (NF-κB) is activated in the renal tissue in CsA nephropathy. In this study, we aimed to investigate the effect of the specific NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in a rat model of CsA nephrotoxicity. METHODS: We administered CsA (15 mg/kg) daily for 28 days to Sprague-Dawley rats that underwent 5/6 nephrectomy under a low-salt diet. We administered DHMEQ (8 mg/kg) simultaneously with CsA to the treatment group, daily for 28 days and evaluated its effect on CsA nephrotoxicity. RESULTS: DHMEQ significantly inhibited NF-κB activation and nuclear translocation due to CsA treatment. Elevated serum urea nitrogen and creatinine levels due to repeated CsA administration were significantly decreased by DHMEQ treatment (serum urea nitrogen in CsA + DHMEQ vs CsA vs control, 69 ± 6.4 vs 113.5 ± 8.8 vs 43.1 ± 1.1 mg/dL, respectively, p < 0.0001; serum creatinine in CsA + DHMEQ vs CsA vs control, 0.75 ± 0.02 vs 0.91 ± 0.02 vs 0.49 ± 0.02 mg/dL, respectively, p < 0.0001), and creatinine clearance was restored in the treatment group (CsA + DHMEQ vs CsA vs control, 2.57 ± 0.09 vs 1.94 ± 0.12 vs 4.61 ± 0.18 ml/min/kg, respectively, p < 0.0001). However, DHMEQ treatment did not alter the inhibitory effect of CsA on urinary protein secretion. The development of renal fibrosis due to chronic CsA nephrotoxicity was significantly inhibited by DHMEQ treatment (CsA + DHMEQ vs CsA vs control, 13.4 ± 7.1 vs 35.6 ± 18.4 vs 9.4 ± 5.4%, respectively, p < 0.0001), and these results reflected the results of renal functional assessment. DHMEQ treatment also had an inhibitory effect on the increased expression of chemokines, monocyte chemoattractant protein-1, and chemokine (c-c motif) ligand 5 due to repeated CsA administration, which inhibited the infiltration of macrophages and neutrophils into the renal tissue. CONCLUSIONS: These findings suggest that DHMEQ treatment in combination therapy with CsA-based immunosuppression is beneficial to prevent the development of CsA-induced nephrotoxicity.

Development of pH/Glutathione-Responsive Theranostic Agents Activated by Glutathione S-Transferase π for Human Colon Cancer.

Two novel theranostic agents HJTA and HJTB have been designed and synthesized by covalently linking a ß-carboline derivative, with antitumor activities and pH-responsive fluorescence, with a 2-exomethylenecyclohexanone moiety, which can be activated by the tumor-targeting glutathione (GSH)/glutathione S-transferase π (GSTπ). These agents showed pH- and GSH-dual-responsive fluorescence in tumor cells but not in normal cells. Importantly, HJTA selectively illuminated tumor tissue for up to 7 h and generated precise visualization of orthotopic colonic tumors through the blood circulation system in intraoperative mice. Furthermore, HJTA exhibited potent and selective antiproliferative activities and colonic tumor inhibition in mice. Finally, HJTA induced great cancer cell apoptosis and autophagy by regulating the expression of apoptotic and autophagic proteins. Therefore, this pH/GSH-dual-responsive fluorescent probe with cancer-targeting therapeutic activity provides a novel tool for precise diagnosis and tumor treatment, therefore broadening the impact of multifunctional agents as theranostic precision medicines.

First Scandinavian case of successful pregnancy during nitisinone treatment for type 1 tyrosinemia.

Background Type 1 tyrosinemia is a hereditary metabolic disease in which tyrosine metabolites damage the liver and kidneys. Nitisinone medication revolutionized the treatment, but the effects of the drug during human pregnancy are unknown. Case presentation A 17-year-old tyrosinemia patient became pregnant. Nitisinone was continued throughout pregnancy with a varying serum concentration and dose ranging from 0.8 to 1.4 mg/kg/day. Blood tyrosine remained stable until it increased in late pregnancy. α-fetoprotein increased to 284 µg/L without new changes in liver. Urine succinylacetone remained undetectable, but there were signs of possibly reoccurring kidney tubulopathy. Fetal ultrasound monitoring was normal throughout the pregnancy and the newborn healthy. After the delivery, α-fetoprotein normalized, but tyrosine continued to rise for up to 1 year. The child is developing normally. Conclusions Pregnancy during nitisinone was successful, but tailoring of the drug dose and possibly reappearing complications, as also increasing serum tyrosine concentration after delivery warranted intensified surveillance.