Bridget E. Bax

Stimulation of osteoclastic bone resorption by hydrogen peroxide

The molecular mechanisms underlying the pathophysiology of bone destruction still remain poorly understood. We have found that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a potent stimulator of osteoclastic bone resorption and cell motility. A marked enhancement of bone resorption was noted when rat osteoclasts, cultured on devitalised bovine cortical bone, were exposed to 10 nM [H2O2]. Apart from exposing osteoclasts to a low extracellular pH, which is known to enhance osteoclastic bone resorption, we provide first evidence for a molecule that stimulates osteoclastic bone resorption in osteoclast cultures that do not respond to parathyroid hormone and 1, 25 dihydroxyvitamin D3. We envisage that both basic biological and practical clinical implications may eventually follow from these studies.

CARRIER ERYTHROCYTE ENTRAPPED THYMIDINE PHOSPHORYLASE THERAPY FOR MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive condition caused by mutations in the nuclear gene ECGF1 coding for thymidine phosphorylase (TP).1,2 Clinical features include gastrointestinal dysmotility, peripheral sensorimotor polyneuropathy, progressive external ophthalmoplegia, and hepatopathy. In vitro evidence and the improvement following stem cell transplantation (alloSCT) in one patient suggest the pathogenesis centers on elevated systemic levels of the TP substrates, thymidine (Thd) and deoxyuridine (dUrd).3,4 ### Case report. In September 2005, a 21-year-old woman presented after a 4-week history of progressive bilateral distal lower limb numbness and foot drop. She had bouts of unexplained gastrointestinal symptoms and weight loss since age 6 years and at 19 developed an acute abdomen leading to a laparotomy that revealed gross small bowel distension (thought to be caused by an elevated ligament of Treitz) and hepatosplenomegaly. A gastrojejunostomy was performed. On neurologic examination, the positive findings were subtle pigmentary retinopathy, partial ptosis, slight reduction of eye abduction, markedly slow horizontal saccades, severe weakness of ankle movements, absent muscle stretch reflexes and plantar reflexes, and stocking diminution for light touch and pain (all findings bilateral and symmetric). Of note on systemic examination: underweight, gross hepatosplenomegaly, tachycardia, and hyperdynamic cardiac apex. Her parents were not related. She left higher education in autumn 2005 due to her illness. After the laparotomy, investigations including hepatitis serology and serum copper were normal; a liver biopsy revealed steatohepatitis. A brain MRI was normal except equivocal diffuse high T2 signal in the centrum semiovale bilaterally. Following the initial presentation, abnormal results included normochromic normocytic anemia; serum lactate 3.72 mmol/L; CSF protein 1.95 g/L; CSF lactate 5.6 mmol/L; plasma Thd 13 μmol/L and …

International seminar on the red blood cells as vehicles for drugs.

The first human transfusion was performed by the pioneer Dr Jean-Baptiste Denis in France in 1667 and now, three centuries later, around 50 millions blood units are transfused every year, saving millions of lives. Today, there is a new application for red blood cells (RBCs) in cellular therapy: the effective use of erythrocytes as vehicles for chemical or biological drugs. Using this approach, the therapeutic index of RBC-entrapped molecules can be significantly improved with increased efficacy and reduced side effects. This cell-based medicinal product can be manufactured at an industrial scale and is now used in the clinic for different therapeutic applications. A seminar dedicated to this field of research, debating on this inventive formulation for drugs, was held in Lyon (France) on 28 January 2011. Drs KC Gunter and Y Godfrin co-chaired the meeting and international experts working on the encapsulation of drugs within erythrocytes met to exchange knowledge on the topic ‘The Red Blood Cells as Vehicles for Drugs’. The meeting was composed of oral presentations providing the latest knowledge and experience on the preclinical and clinical applications of this technology. This Meeting Highlights article presents the most relevant messages given by the speakers and is a joint effort by international experts who share an interest in studying erythrocyte as a drug delivery vehicle. The aim is to provide an overview of the applications, particularly for clinical use, of this innovative formulation. Indeed, due to the intrinsic properties of erythrocytes, their use as a drug carrier is one of the most promising drug delivery systems investigated in recent decades. Of the different methods developed to encapsulate therapeutic agents into RBCs [1,2,] the most widely used method is the lysis of the RBCs under tightly controlled hypotonic conditions in the presence of the drug to be encapsulated, followed by resealing and annealing under normotonic conditions (Figure 1). This results in uniform encapsulation of the material into the cells and a final product with good stability, reproducibility and viability. This process, which has now been developed to an industrial scale, is the technique chosen by the majority of the experts presenting their work in this seminar (by R Franco). Figure 1 The process of reversible hypotonic lysis of RBCs to entrap molecules Keywords: carriers, drug delivery, erythrocytes, red blood cells, targeting 1. Therapeutic enzyme-loaded RBCs Therapy using RBC encapsulated enzymes has the advantage of prolonging the half-life of the enzyme and maintaining therapeutic blood levels, reducing the dosage and frequency of therapeutic interventions, and preventing the need for expensive chemical modification [3]. The therapeutic index can be strongly improved, especially by reducing immunogenic reactions, which are often observed in enzyme replacement (Figure 2). Figure 2 The red blood cell as a bioreactor: the substrate (yellow) contained in the plasma permeates the erythrocyte membrane, with the entrapped enzyme (green) catalyzing the metabolism of the substrate to its normal product inside the red cell 1.1 L-asparaginase-loaded RBCs for ASNS-deficient tumor (by E Dufour) L-asparaginase has been used in the treatment of acute lymphoblastic leukemia for > 40 years. This enzyme converts plasmatic L-asparagine (L-Asn) into L-aspartate plus ammonia. Its use is motivated by the fact that malignant cells (especially leukemic) are deficient in asparagine synthetase (ASNS). Because these cells are unable to synthesize L-Asn to meet metabolic demands, L-Asn deprivation, due to L-asparaginase activity, kills the cancerous cells. However, L-asparaginase can also be responsible for adverse events such as hypersensitivity reactions or blood coagulations disorders, in addition to L-Asn depletion. An approach to decreasing side effects of free L-asparaginase in vivo is to entrap the enzyme in RBCs. Reversible hypotonic dialysis remains the most controlled and reproducible method. Indeed, with this process, human RBCs can be loaded with 116 ± 15 IU of L-asparaginase per milliliter of red cells. The resulting product acts as a bioreactor allowing transport of L-Asn through the RBC membrane where L-asparaginase hydrolyzes it. Due to the RBC membrane, the enzyme is protected from rapid catabolism as well as from potential neutralizing antibodies, resulting in an increased half-life and a reduction in hypersensitivity reactions. A Phase I–II trial testing GRASPA® (ERYTECH Pharma, France) on 24 patients in relapsed acute lymphoblastic leukemia showed a strong reduction in hypersensitive reactions, coagulation disorders and hepatic dysfunctions [4]. The L-asparaginase half-life is enhanced (40 days vs 1 day with the free form) and the mean duration of L-Asn depletion is 18.57 days at a dose of 150 IU/kg in a single injection that corresponds to eight injections of Escherichia coli native L-asparaginase. This improvement in tolerance allows the introduction of L-asparaginase treatment to other hematological malignancies, such as acute myeloid leukemia, and also in solid tumors. Indeed, the level of expression of L-ASNS, the enzyme responsible for the synthesis of L-Asn in mammalian cells, provides a rationale for testing L-asparaginase in several cancers. For example, about 30 and 40% of pancreatic ductal adenocarcinoma patients (85 – 90% of all pancreatic cancer subjects) have no or low level of expression of ASNS, respectively. A Phase I clinical study is ongoing with pancreatic adenocarcinoma patients.

Seeking Optimal Relation Between Oxygen Saturation and Hemoglobin Concentration in Adults With Cyanosis from Congenital Heart Disease

In patients with cyanosis from congenital heart disease, erythropoiesis is governed by many factors that can alter the expected relation between the oxygen saturation (O(2sat)) and hemoglobin concentration. We sought to define the relation between the O(2sat) and hemoglobin in such patients and to predict an ideal hemoglobin concentration for a given O(2sat). Adults with congenital heart defects and cyanosis were studied prospectively using blood tests and exercise testing. Nonoptimal hemoglobin was defined as any evidence of inadequate erythropoiesis (i.e., iron, folate, or vitamin B(12) deficiency, increased erythropoietin, reticulocytosis, or a right-shifted oxygen-hemoglobin curve). For patients without these factors, a linear regression equation of hemoglobin versus O(2sat) was used to predict the optimal hemoglobin for all patients. Of the 65 patients studied, 21 met all the prestudy criteria for an optimal hemoglobin. For all patients, no correlation was found between O(2sat) and hemoglobin (r = -0.22). However, a strong linear correlation was found for those meeting the criteria for optimal hemoglobin (r = -0.865, p <0.001). The optimal hemoglobin regression equation was as follows: predicted hemoglobin = 57.5 - (0.444 × O(2sat)). A negative correlation was found between the hemoglobin difference (predicted minus measured) and exercise duration on cardiopulmonary exercise testing (r = -0.396, p = 0.005) and the 6-minute walk distance (r = -0.468, p <0.001). In conclusion, a strong relation between O(2sat) and hemoglobin concentration can be shown in stable cyanotic patients and used to predict an optimal hemoglobin. This relation might be useful in defining functional anemia in this group.

A 9-yr evaluation of carrier erythrocyte encapsulated adenosine deaminase (ADA) therapy in a patient with adult-type ADA deficiency

Adenosine deaminase (ADA) deficiency is an inherited disorder which leads to elevated cellular levels of deoxyadenosine triphosphate (dATP) and systemic accumulation of its precursor, 2‐deoxyadenosine. These metabolites impair lymphocyte function, and inactivate S‐adenosylhomocysteine hydrolase (SAHH) respectively, leading to severe immunodeficiency. Enzyme replacement therapy with polyethylene glycol‐conjugated ADA is available, but its efficacy is reduced by anti‐ADA neutralising antibody formation. We report here carrier erythrocyte encapsulated native ADA therapy in an adult‐type ADA deficient patient. Encapsulated enzyme is protected from antigenic responses and therapeutic activities are sustained. ADA‐loaded autologous carrier erythrocytes were prepared using a hypo‐osmotic dialysis procedure. Over a 9‐yr period 225 treatment cycles were administered at 2–3 weekly intervals. Therapeutic efficacy was determined by monitoring immunological and metabolic parameters. After 9 yr of therapy, erythrocyte dATP concentration ranged between 24 and 44 μmol/L (diagnosis, 234) and SAHH activity between 1.69 and 2.29 nmol/h/mg haemoglobin (diagnosis, 0.34). Erythrocyte ADA activities were above the reference range of 40–100 nmol/h/mg haemoglobin (0 at diagnosis). Initial increases in absolute lymphocyte counts were not sustained; however, despite subnormal circulating CD20+ cell numbers, serum immunoglobulin levels were normal. The patient tolerated the treatment well. The frequency of respiratory problems was reduced and the decline in the forced expiratory volume in 1 s and vital capacity reduced compared with the 4 yr preceding carrier erythrocyte therapy. Carrier erythrocyte‐ADA therapy in an adult patient with ADA deficiency was shown to be metabolically and clinically effective.

Energy metabolism and glycolysis in human placental trophoblast cells during differentiation.

Energy metabolism and glycolysis of normal human term placental trophoblast in two-sided culture was investigated during differentiation from cytotrophoblast to syncytiotrophoblast, because glycogen metabolism is abnormal in several trophoblast related pregnancy diseases, including pre-eclampsia. After initial recovery of energy and cytoplasmic NADH/NAD+ redox by 24 h of culture, measures of cellular energy state, [ATP], [ADP], [ATP]/[ADP] ratio, ([ATP] + [ADP] + [AMP]), [ATP]/([ATP] + [ADP] + [AMP]) and energy charge remained essentially constant until 72 h, despite periods of increased energy turnover. At 24 h there was a burst of glycogenolysis, and glycolysis indicated by increased lactate production, which coincided with formation of syncytium. Subsequently, there was no resynthesis nor further breakdown of glycogen. At 48 h, oxygen consumption temporarily increased substantially, without increased glycolysis, during functional differentiation of the syncytiotrophoblast. Glucose uptake was constant and largely from the basal (in vivo fetal facing) side. Lactate output into the basal fetal medium was twice as fast as that into the microvillous (maternal) medium, and oxygen uptake was also asymmetrical. The results show that before and after differentiation substantial relatively constant aerobic glycolysis occurs, but that during increased energy demand cytotrophoblast depends on both glycolytic and aerobic energy production whereas syncytiotrophoblast relies on aerobic metabolism.

Clinical and biochemical improvements in a patient with MNGIE following enzyme replacement.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive metabolic disorder caused by a deficiency of thymidine phosphorylase (TP, EC2.4.2.4) due to mutations in the nuclear gene TYMP. TP deficiency leads to plasma and tissue accumulations of thymidine and deoxyuridine which generate imbalances within the mitochondrial nucleotide pools, ultimately leading to mitochondrial dysfunction.1 MNGIE is characterized clinically by leukoencephalopathy, external ophthalmoplegia, peripheral polyneuropathy, cachexia, and enteric neuromyopathy manifesting as gastrointestinal dysmotility. The condition is relentlessly progressive, with patients usually dying from a combination of nutritional and neuromuscular failure at an average age of 37 years.2 Allogeneic hematopoietic stem cell transplantation (AHSCT) offers a permanent cure. Clinical and biochemical improvements following AHSCT have been reported but it carries a high mortality risk and is limited by matched donor availability.3 A consensus proposal for standardizing AHSCT recommends treatment of patients without irreversible end-stage disease and with an optimally matched donor; a majority of patients are ineligible and thus there is a critical requirement for an alternative treatment.4

Drug-loaded erythrocytes: on the road toward marketing approval

Erythrocyte drug encapsulation is one of the most promising therapeutic alternative approaches for the administration of toxic or rapidly cleared drugs. Drug-loaded erythrocytes can operate through one of the three main mechanisms of action: extension of circulation half-life (bioreactor), slow drug release, or specific organ targeting. Although the clinical development of erythrocyte carriers is confronted with regulatory and development process challenges, industrial development is expanding. The manufacture of this type of product can be either centralized or bedside based, and different procedures are employed for the encapsulation of therapeutic agents. The major challenges for successful industrialization include production scalability, process validation, and quality control of the released therapeutic agents. Advantages and drawbacks of the different manufacturing processes as well as success key points of clinical development are discussed. Several entrapment technologies based on osmotic methods have been industrialized. Companies have already achieved many of the critical clinical stages, thus providing the opportunity in the future to cover a wide range of diseases for which effective therapies are not currently available.

In vitro studies of amikacin-loaded human carrier erythrocytes

Erythrocyte-encapsulated antibiotics have the potential to provide an effective therapy against intracellular pathogens. The advantages over the administration of free antibiotics include a lower systemic dose, decreased toxicity, a sustained delivery of the antibiotic at higher concentrations to the intracellular site of pathogen replication, and increased efficacy. In this study, the encapsulation of amikacin by human carrier erythrocytes prepared using a hypo-osmotic dialysis was investigated. The effects of the initial amikacin dialysis concentration and hypo-osmotic dialysis time on the encapsulation efficiency of amikacin were determined, and the osmotic fragility and hematologic parameters of amikacin-loaded carrier erythrocytes were measured. The efficiency of amikacin entrapment by carrier erythrocytes was dependent on the initial dialysis concentration of the drug. Statistically significant differences in the osmotic fragility profiles between control and carrier erythrocytes were observed, which were dependent on the hypo-osmotic dialysis time and on the dialysis concentration of amikacin. Mean hematologic parameters were evaluated and compared with unloaded, native erythrocytes; the mean corpuscular volume (MCV) of amikacin-loaded carrier erythrocytes was statistically significant smaller. Amikacin demonstrated a sustained release from loaded erythrocytes over a 48-h period, which suggests a potential use of the erythrocyte as a slow systemic-release system for antibiotics.

Preclinical Toxicity Evaluation of Erythrocyte-Encapsulated Thymidine Phosphorylase in BALB/c Mice and Beagle Dogs: An Enzyme-Replacement Therapy for Mitochondrial Neurogastrointestinal Encephalomyopathy

Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is currently under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder caused by a deficiency of thymidine phosphorylase. The rationale for the development of EE-TP is based on the pathologically elevated metabolites (thymidine and deoxyuridine) being able to freely diffuse across the erythrocyte membrane where the encapsulated enzyme catalyses their metabolism to the normal products. The systemic toxic potential of EE-TP was assessed when administered intermittently by iv bolus injection to BALB/c mice and Beagle dogs for 4 weeks. The studies consisted of one control group receiving sham-loaded erythrocytes twice weekly and two treated groups, one dosed once every 2 weeks and the other dosed twice per week. The administration of EE-TP to BALB/c mice resulted in thrombi/emboli in the lungs and spleen enlargement. These findings were also seen in the control group, and there was no relationship to the number of doses administered. In the dog, transient clinical signs were associated with EE-TP administration, suggestive of an immune-based reaction. Specific antithymidine phosphorylase antibodies were detected in two dogs and in a greater proportion of mice treated once every 2 weeks. Nonspecific antibodies were detected in all EE-TP-treated animals. In conclusion, these studies do not reveal serious toxicities that would preclude a clinical trial of EE-TP in patients with MNGIE, but caution should be taken for infusion-related reactions that may be related to the production of nonspecific antibodies or a cell-based immune response.