Lakshminarayan Ranganath

Efficacy and safety of recombinant human parathyroid hormone (1-84) in hypoparathyroidism (REPLACE): a double-blind, placebo-controlled, randomised, phase 3 study

BACKGROUND Hypoparathyroidism results in impaired mineral homoeostasis, including hypocalcaemia and hyperphosphataemia. Treatment with high-dose oral calcium and active vitamin D does not provide adequate or consistent control of biochemical indices and can lead to serious long-term complications. We aimed to test the efficacy, safety, and tolerability of once-daily recombinant human parathyroid hormone 1-84 (rhPTH[1-84]) in adults with hypoparathyroidism. METHODS In this double-blind, placebo-controlled, randomised phase 3 study (REPLACE), we recruited patients with hypoparathyroidism (≥ 18 months duration) aged 18-85 years from 33 sites in eight countries. After an optimisation period, during which calcium and active vitamin D doses were adjusted to achieve consistent albumin-corrected serum calcium, patients were randomly assigned (2:1) via an interactive voice response system to 50 μg per day of rhPTH(1-84) or placebo for 24 weeks. Active vitamin D and calcium were progressively reduced, while rhPTH(1-84) could be titrated up from 50 μg to 75 μg and then 100 μg (weeks 0-5). The primary endpoint was the proportion of patients at week 24 who achieved a 50% or greater reduction from baseline in their daily dose of oral calcium and active vitamin D while maintaining a serum calcium concentration greater than or the same as baseline concentrations and less than or equal to the upper limit of normal, analysed by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00732615. FINDINGS Between June 23, 2009, and Feb 28, 2011, 134 eligible patients were recruited and randomly assigned to rhPTH(1-84) (n=90) or placebo (n=44). Six patients in the rhPTH(1-84) group and seven in the placebo group discontinued before study end. 48 (53%) patients in the rhPTH(1-84) group achieved the primary endpoint compared with one (2%) patient in the placebo group (percentage difference 51.1%, 95% CI 39.9-62.3; p<0.0001). The proportions of patients who had at least one adverse event were similar between groups (84 [93%] patients in the rhPTH[1-84] group vs 44 [100%] patients in the placebo group), with hypocalcaemia, muscle spasm, paraesthesias, headache, and nausea being the most common adverse events. The proportions of patients with serious adverse events were also similar between the rhPTH(1-84) group (ten [11%] patients) and the placebo group (four [9%] patients). INTERPRETATION 50 μg, 75 μg, or 100 μg per day of rhPTH(1-84), administered subcutaneously in the outpatient setting, is efficacious and well tolerated as a PTH replacement therapy for patients with hypoparathyroidism.

Alkaptonuria – a review of surgical and autopsy pathology

Alkaptonuria is a rare, inherited defect of homogentisic acid 1,2‐dioxygenase that leads to the widespread deposition of polymeric homogentisic acid, and clinical symptoms from degeneration of joints and the aortic valve. Pathological descriptions are few and mainly those of late‐stage changes related to joint or valve failure. In this review, the macroscopic and histopathological changes in the tissues in alkaptonuria are illustrated by the detailed autopsy study of a 74‐year‐old female who died from disseminated ovarian carcinoma. The pathology is discussed in the context of the literature and in relation to potential pathogenic mechanisms of tissue damage. This review highlights the heterogeneity of some of the manifestations. In symptomatic patients, degenerative changes in synovial and intervertebral joints are usually well advanced, while early changes include diffuse cartilage pigmentation and chondrocyte necrosis. The initial stage of pigment deposition in the cardiovascular system may be influenced by intravascular pressure and flow disturbances, whereas more intense pigmentation affects fibrolipid components of atheromatous plaques. Pigmentation of the aortic and mitral valve cusps and valve rings is a result of intracellular and extracellular pigment deposition and is associated with calcification and clinically significant aortic stenosis.

Plasma obestatin levels are lower in obese and post- gastrectomy subjects, but do not change in response to a meal

Objective:To investigate a potential role for obestatin in humans by examining response to a fixed energy meal.Context:A new anorectic peptide hormone, obestatin has recently been isolated from rat stomach. The significance of this peptide in humans is unknown.Study design:Case-control study.Setting:Hospital-based study.Patients:Nine healthy controls, nine morbidly obese subjects and eight post-gastrectomy subjects.Intervention:Subjects attended after an overnight fast and were given a fixed energy meal (1550 kJ).Main outcome measure:The response of obestatin to a meal in the different groups.Results:Fasting obestatin was significantly lower in obese subjects as compared to lean subjects (27.8±4 vs 17.2±2 pg/ml, P=0.03). Obestatin was also decreased in gastrectomy subjects but this did not reach statistical significance (27.8±4 vs 21.9±3 pg/ml, P=0.3). Obestatin did not change significantly from baseline in response to the meal. Lean and obese subjects had a similar obestatin/ghrelin ratio (0.04±0.003 vs 0.05±0.009, P=0.32), but this was higher in the gastrectomy group (0.04±0.003 vs 0.1±0.01, P<0.001).Conclusions:Obestatin does not vary significantly with a fixed energy meal, but is significantly lower in morbidly obese subjects as compared to lean subjects supporting a possible role for obestatin in long-term body weight regulation. Obestatin tended to be lower in gastrectomy subjects and their obestatin/ghrelin ratio differed from healthy controls. Hence, the expression of obestatin is altered following gastrectomy, suggesting other sites outside the stomach may also secrete obestatin.

Recent advances in management of alkaptonuria (invited review; best practice article)

Alkaptonuria (AKU) is an autosomal recessive condition arising as a result of a genetic deficiency of the enzyme homogentisate 1,2 dioxygenase and characterised by accumulation of homogentisic acid (HGA). Oxidative conversion of HGA leads to production of a melanin-like polymer in a process termed ochronosis. The binding of ochronotic pigment to the connective tissues of the body leads to multisystem disorder dominated by premature severe spondylo-arthropathy. Other systemic features include stones (renal, prostatic, salivary, gall bladder), renal damage/failure, osteopenia/fractures, ruptures of tendons/muscle/ligaments, respiratory compromise, hearing loss and aortic valve disease. Detection of these features requires systematic investigation. Treatment in AKU patients is palliative and unsatisfactory. Ascorbic acid, low protein diet and physiotherapy have been tried but do not alter the underlying metabolic defect. Regular surveillance to detect and treat complications early is important. Palliative pain management is a crucial issue in AKU. Timely spinal surgery and arthroplasty are the major treatment approaches at present. A potential disease modifying drug, nitisinone, inhibits 4-hydroxy-phenyl-pyruvate-dioxygenase and decreases formation of HGA and could prevent or slow the progression of disease in AKU. If nitisinone therapy is able to complement the biochemical ‘cure’ with improved outcomes, it will completely alter the way we approach the management of this disease. Greater efforts to improve recognition and registration of the disease will be worthwhile. Improved laboratory diagnostics to monitor the tyrosine metabolic pathway that includes plasma metabolites including tyrosine to monitor efficacy, toxicity and safety postnitisinone will also be required.

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment

Background Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. Methods Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. Findings A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. Conclusions In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. Trial registration number EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria

OBJECTIVE Alkaptonuria is a genetic disorder of tyrosine metabolism, resulting in elevated circulating concentrations of homogentisic acid. Homogentisic acid is deposited as a polymer, termed ochronotic pigment, in collagenous tissues, especially cartilages of weight-bearing joints, leading to a severe osteoarthropathy. We undertook this study to investigate the initiation and progression of ochronosis from the earliest detection of pigment through complete joint failure. METHODS Nine joint samples with varying severities of ochronosis were obtained from alkaptonuria patients undergoing surgery and compared to joint samples obtained from osteoarthritis (OA) patients. Samples were analyzed by light and fluorescence microscopy, 3-dimensional scanning electron microscopy (SEM), and the quantitative backscattered electron mode of SEM. Cartilage samples were mechanically tested by compression to determine Youngs modulus of pigmented, nonpigmented, and OA cartilage samples. RESULTS In alkaptonuria samples with the least advanced ochronosis, pigment was observed intracellularly and in the territorial matrix of individual chondrocytes at the boundary of the subchondral bone and calcified cartilage. In more advanced ochronosis, pigmentation was widespread throughout the hyaline cartilage in either granular composition or as blanket pigmentation in which there is complete and homogenous pigmentation of cartilage matrix. Once hyaline cartilage was extensively pigmented, there was aggressive osteoclastic resorption of the subchondral plate. Pigmented cartilage became impacted on less highly mineralized trabeculae and embedded in the marrow space. Pigmented cartilage samples were much stiffer than nonpigmented or OA cartilage as revealed by a significant difference in Youngs modulus. CONCLUSION Using alkaptonuria cartilage specimens with a wide spectrum of pigmentation, we have characterized the progression of ochronosis. Intact cartilage appears to be resistant to pigmentation but becomes susceptible following focal changes in calcified cartilage. Ochronosis spreads throughout the cartilage, altering the mechanical properties. In advanced ochronosis, there is aggressive resorption of the underlying calcified cartilage leading to an extraordinary phenotype in which there is complete loss of the subchondral plate. These findings should contribute to better understanding of cartilage-subchondral interactions in arthropathies.

Ultrastructural examination of tissue in a patient with alkaptonuric arthropathy reveals a distinct pattern of binding of ochronotic pigment

SIR, we report a female with known alkaptonuria (AKU) undergoing routine hip replacement surgery due to alkaptonuric arthropathy. AKU is caused by a deficiency in the enzyme that breaks down homogentisic acid (HGA), resulting in elevated circulation of HGA levels in the body. HGA is deposited as a polymerized pigment in collagenous connective tissues, predominantly in the weight-bearing joints [1]. It has also been shown to affect other non-joint tissues [2, 3]. Ligamentous capsule was processed routinely for histology and electron microscopy. Macroscopically, ochronotic pigment was clearly visible alongside non-pigmented regions. Haematoxylin and eosin (H&E) staining showed the presence of extracellular pigmentation associated with the collagen fibres and also intracellular pigmentation within fibroblasts. This novel intracellular pigmentation appeared as numerous individual granules located within the cytoplasm of single fibroblasts (Fig. 1A). Extracellular pigmentation appeared as two distinct types: a granular deposition, similar to that within the cells, but also more homogeneous pigmentation that completely encrusted the collagen fibres. Ultrastructural analysis revealed that collagen fibres in transverse section had numerous electron-dense granules of ochronotic pigment associated with them. These granules varied in position and distribution among the fibres. Some had single granules of pigment located within the cross-section of …

The entero-insular axis: implications for human metabolism.

Abstract Incretins such as glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are intestinal hormones that are released in response to ingestion of nutrients, especially carbohydrate. They have a number of important biological effects, which include release of insulin, inhibition of glucagon and somatostatin, maintenance of β-cell mass, delay of gastric emptying, and inhibition of feeding. These properties allow them to be potentially suitable agents for the treatment of type 2 diabetes (T2D). Incretin receptors are also present in other parts of the body including the brain, where their effects are beginning to be understood and their relevance to disorders of nutrition and ageing are being explored. There is currently a pandemic of obesity and diabetes, and existing treatments are largely inadequate in regard to efficacy as well as their ability to tackle important factors in the pathogenesis of T2D. There is increasing evidence that current treatments do not address the issue of progressive β-cell failure in T2D. As obesity is the engine that is driving the epidemic of diabetes, it is disappointing that most treatments that succeed in lowering plasma glucose are also associated with weight gain. It is now well established that intensively treated T2D has a better outcome than standard treatment. Consequently, achieving better control of diabetes with lower HbA1c is the goal of optimal treatment. Despite the use of usual therapeutic agents in T2D, often in high doses and as combinations, such as metformin, sulphonylurea, α-glycosidase inhibitors, thiazolidinediones and a number of animal and human insulin preparations, optimal control of glycaemia is not achieved. The use of incretins as therapeutic agents offers a new approach to the treatment of T2D. Incretin metabolism is abnormal in T2D, evidenced by a decreased incretin effect, reduction in nutrient-mediated secretion of GIP and GLP-1 in T2D, and resistance to GIP. GLP-1, on the other hand, when administered intravenously in T2D is able to increase insulin secretion and improve glucose homeostasis. As GLP-1 has a very short half-life, due to rapid degradation by the enzyme dipeptidyl peptidase IV (DPPIV), analogues of GIP and GLP-1 that are resistant to the action of DPPIV have been developed and clinical trials have shown their effectiveness. Another novel agent, naturally resistant to DPPIV that is given by subcutaneous injection is a synthetic peptide called exenatide, has recently been approved for treatment of T2D in the USA. Efforts are underway to develop agents that can be given orally and include a DPPIV inhibitor that has been licensed for the treatment of T2D in the USA, and several other agents are undergoing clinical trials. Strategies to augment the biological actions of GIP and/or GLP-1 in T2D are expected to minimise weight gain, reduce hypoglycaemic episodes and prevent progressive β-cell failure by increasing β-cell mass. The optimal agent(s) that may mimic and replace the endogenous incretin effect is not fully known and awaits the outcome of clinical trials that are still ongoing. The potential therapeutic role in non-diabetic states, including obesity and neurodegenerative disease, is intriguing and depends upon results from ongoing research. Clin Chem Lab Med 2008;46:43–56.

Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone

Background Alkaptonuria (AKU) is a rare metabolic disease caused by deficiency of homogentisate 1,2 dioxygenase, an enzyme involved in tyrosine catabolism, resulting in increased circulating homogentisic acid (HGA). Over time HGA is progressively deposited as a polymer (termed ochronotic pigment) in collagenous tissues, especially the cartilages of weight bearing joints, leading to severe joint disease. Objectives To characterise blood biochemistry and arthropathy in the AKU mouse model (Hgd−/−). To examine the therapeutic effect of long-term treatment with nitisinone, a potent inhibitor of the enzyme that produces HGA. Methods Lifetime levels of plasma HGA from AKU mice were measured by high-performance liquid chromatography (HPLC). Histological sections of the knee joint were examined for pigmentation. The effect of nitisinone treatment in both tissues was examined. Results Mean (±SE) plasma HGA levels were 3- to 4-fold higher (0.148±0.019 mM) than those recorded in human AKU. Chondrocyte pigmentation within the articular cartilage was first observed at 15 weeks, and found to increase steadily with mouse age. Nitisinone treatment reduced plasma HGA in AKU mice throughout their lifetime, and completely prevented pigment deposition. Conclusions The AKU mouse was established as a model of both the plasma biochemistry of AKU and its associated arthropathy. Early-stage treatment of AKU patients with nitisinone could prevent the development of associated joint arthropathies. The cellular pathology of ochronosis in AKU mice is identical to that observed in early human ochronosis and thus is a model in which the early stages of joint pathology can be studied and novel interventions evaluated.

Circulating ghrelin exists in both lipoprotein bound and free forms.

Introduction Ghrelin is a gastric peptide that has been implicated in the development of obesity and cardiovascular disease. It has been reported that ghrelin binds to lipoproteins, although the different binding patterns of acylated ghrelin (AG) and unacylated ghrelin (UAG) are still to be determined. Methods Lipoprotein fractions were generated using a self-generating iodixanol gradient. AG and UAG were measured using specific enzyme immunoassays. Results AG bound to all lipoproteins in approximately equal concentrations (VLDL 26%, LDL 22%, HDL 23%) and was present as a plasma protein (27%). UAG bound more specifically to HDL (49%) and was present as a plasma protein (48%). Conclusions The different binding patterns of AG and UAG may have significant implications for their biological effects, including roles in energy metabolism, the development of obesity and potentially in the modulation of cardiovascular disease.