Murray D. Bain

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 …

Diagnosis and management of trimethylaminuria (FMO3 deficiency) in children

SummaryPersistent trimethylaminuria in children is caused by autosomal recessively inherited impairment of hepatic trimethylamine (TMA) oxidation due to deficiency of flavin monooxygenase 3 (FMO3) secondary to mutations in the FMO3 gene. Trimethylaminuria or ‘fish odour syndrome’ is due to excessive excretion into body fluids and breath of TMA derived from the enterobacterial metabolism of dietary precursors. The disorder is present from birth but becomes apparent as foods containing high amounts of choline or of trimethylamine N-oxide (TMAO) from marine (sea or saltwater) fish are introduced into the diet. In our experience, trimethylaminuria (FMO3 deficiency) in children is rare. We have compared the dynamics and diagnostic efficacy of choline loading with marine fish meals in six children with trimethylaminuria. Loading with a marine fish meal provides a simple and acceptable method for confirmation of diagnosis of suspected trimethylaminuria in children, with the effects being cleared more quickly than with a choline load test. However, oral loading with choline bitartrate allows estimation of residual oxidative capacity in vivo and is a useful adjunct to molecular studies. Patients homozygous for the ‘common’ P153L mutation in the FMO3 gene showed virtual complete lack of residual TMA N-oxidative capacity, consistent with a nonfunctional or absent FMO3 enzyme, whereas a patient with the M82T mutation showed some residual oxidative capacity. A patient compound heterozygous for two novel mutations, G193E and R483T, showed considerable residual N-oxidative capacity. A further patient, heterozygous for two novel sequence variations in the FMO3 gene, consistently showed malodour and elevated urinary TMA/TMAO ratios under basal conditions but a negative response to both choline and marine fish meal loading. Comparison of the effects of administration of antibiotics (metronidazole, amoxicillin, neomycin) on gut bacterial production of trimethylamine from choline showed they all reduced TMA production to a limited extent, with neomycin being most effective. ‘Best-practice’ diagnostic and treatment guidelines are summarized.

In vitro and in vivo studies with human carrier erythrocytes loaded with polyethylene glycol-conjugated and native adenosine deaminase

Polyethylene glycol‐conjugated adenosine deaminase (pegademase) is used for enzyme replacement therapy for patients with severe combined immunodeficiency caused by adenosine deaminase deficiency. The entrapment of pegademase within human energy‐replete carrier erythrocytes using a hypo‐osmotic dialysis procedure was investigated with the objective of prolonging the in vivo circulatory half‐life of the enzyme and maintaining therapeutic blood levels. Native unmodified adenosine deaminase (ADA) was similarly studied. The efficiency of pegademase entrapment was low (9%) whereas the entrapment of native unmodified ADA was substantial (50%), suggesting that the polyethylene glycol side‐chains were impeding intracellular entrapment. The biochemical characteristics and the osmotic fragility of these carrier erythrocytes were not adversely affected by the entrapment of either pegademase or native ADA. In vivo survival studies of pegademase‐loaded 51Cr‐labelled carrier erythrocytes in an ADA‐deficient adult patient showed a mean cell half‐life of 16 d. Carrier erythrocyte‐entrapped pegademase and native ADA had in vivo half‐lives of 20 and 12·5 d, respectively, demonstrating that entrapment prolongs the half‐life over that of plasma pegademase, which has a circulating half‐life of 3–6 d. These results provide the basis for a more extensive clinical evaluation of carrier erythrocyte‐entrapped native adenosine deaminase therapy.

Survival of human carrier erythrocytes in vivo

Erythrocytes offer the exciting opportunity of being used as carriers of therapeutic agents. Encapsulation within erythrocytes will give the therapeutic agent a clearance equivalent to the normal life of the erythrocyte therefore maintaining therapeutic blood levels over prolonged periods and also giving a sustained delivery to the monocyte-macrophage system (reticulo-endothelial system). Both the dose and frequency of therapeutic interventions could thus be reduced. Ensuring a near-physiological survival time of carrier erythrocytes is essential to their successful use as a sustained drug delivery system, and this has not been demonstrated in man. In this study we assessed the survival in vivo of autologous unloaded energy-replete carrier erythrocytes in nine volunteers, using a standard 51Cr erythrocyte-labelling technique. Within 144 h after infusion there was a 3 to 49% fall in circulating labelled cells, followed thereafter by an almost complete return to initial circulating levels; surface counting demonstrated an initial sequestration of erythrocytes by the spleen and subsequent release. Mean cell life and cell half-life of the carrier erythrocytes were within the normal range of 89 to 131 days and 19 to 29 days respectively. These results demonstrate the viability of carrier erythrocytes as a sustained drug delivery system.

Secondary analysis of economic data: a review of cost-benefit studies of neonatal screening for phenylketonuria.

STUDY OBJECTIVE: To estimate the net financial benefit of neonatal screening for phenylketonuria (PKU): by a simple pooling of cost data from the literature; and by a more complex modelling approach. DESIGN: A systematic literature review was conducted to identify papers containing data on the monetary costs and benefits of neonatal screening for PKU. The methodological quality of the studies was appraised, and data were extracted on resource use and expenditure. Monetary data were converted to common currency units, and standardised to UK incidence rates. Net benefits were calculated for median, best case and worst case scenarios, and the effect of excluding poor quality studies and data was tested. The net benefit was also estimated from a model based on data from the literature and assumptions appropriate for the current UK situation. Extensive sensitivity analysis was conducted. MAIN RESULTS: The direct net benefit of screening based on the median costs and benefits from the 13 studies identified was 143,400 Pounds per case detected and treated (39,000 Pounds and 241,800 Pounds for worst case and best case scenarios respectively). The direct net benefit obtained by the modelling approach was lower at 93,400 Pounds per case detected and treated. Screening remained cost saving under sensitivity analysis, except with low residential care costs (less than 12,300 Pounds per annum), or very low incidence rates (less than 1 in 27,000). CONCLUSIONS: The economic literature on PKU screening is of variable quality. The two methods of secondary analysis lead to the same conclusion: that neonatal PKU screening is worthwhile in financial terms alone in the UK, and that it justifies the infrastructure for collecting and testing neonatal blood samples. This result cannot necessarily be extrapolated to other countries.

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.

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

Haemorheology in Gaucher disease

Abstract:  In Gaucher disease, a deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within the lysosomes of the monocyte–macrophage system. Prior to the availability of enzyme replacement therapy (ERT), splenectomy was often indicated for hypersplenism. Haemorheological abnormalities could be expected in view of the anaemia and abnormal lipid metabolism in these patients and the role of the spleen in controlling erythrocyte quality. Objectives: To investigate the effect of Gaucher disease on blood and plasma viscosity, erythrocyte aggregation and erythrocyte deformability, and to determine whether observed rheological differences could be attributed to splenectomy. Methods: Haematological and haemorheological measurements were made on blood collected from 26 spleen‐intact patients with Gaucher disease, 16 splenectomised patients with Gaucher disease, 6 otherwise healthy asplenic non‐Gaucher disease subjects and 15 healthy controls. Results: No haemorheological differences could be demonstrated between spleen‐intact patients with Gaucher disease and the control group. Compared to controls, both asplenic Gaucher disease and asplenic non‐Gaucher disease study groups had a reduced MCHC (P = 0.003 and 0.005, respectively) and increased whole blood viscosity at 45% haematocrit (Hct), relative viscosity and red cell aggregation index – all measured at low shear (P < 0.05 for all). Additionally, asplenic patients with Gaucher disease alone showed an increased MCV (P = 0.006), an increased whole blood viscosity at 45% Hct measured at high shear (P = 0.019), and a reduced relative filtration rate (P = 0.0001), compared to controls. Conclusion: These observations demonstrate a direct and measurable haemorheological abnormality in Gaucher disease only revealed when there is no functioning spleen to control erythrocyte quality.

Unexplained gastrointestinal symptoms: Think mitochondrial disease

Defects in mitochondrial function are increasingly recognised as central to the pathogenesis of many diseases, both inherited and acquired. Many of these mitochondrial defects arise from abnormalities in mitochondrial DNA and can result in multisystem disease, with gastrointestinal involvement common. Moreover, mitochondrial disease may present with a range of non-specific symptoms, and thus can be easily misdiagnosed, or even considered to be non-organic. We describe the clinical, histopathological and genetic findings of six patients from three families with gastrointestinal manifestations of mitochondrial disease. In two of the patients, anorexia nervosa was considered as an initial diagnosis. These cases illustrate the challenges of both diagnosing and managing mitochondrial disease and highlight two important but poorly understood aspects, the clinical and the genetic. The pathophysiology of gastrointestinal involvement in mitochondrial disease is discussed and emerging treatments are described. Finally, we provide a checklist of investigations for the gastroenterologist when mitochondrial disease is suspected.

In Vivo Survival of Human Energy-Replete Carrier Erythrocytes

Erythrocytes have been proposed as carriers of encapsulated therapeutic agents. Encapsulation of therapeutic agents within erythrocytes with a normal mean cell life range of 89 to 131 days (normal half-life of 19 to 29 days) should limit the vascular clearance of the administered drug thus reducing the dosage and frequency of therapeutic interventions.