Use of beat-to-beat cardiovascular variability data to determine the validity of sham therapy as the placebo control in osteopathic manipulative medicine research.

Osteopathic manipulative medicine researchers often use sham therapy as the placebo control during clinical trials. Optimally, the sham therapy should be a hands-on procedure that is perceptually indistinguishable from osteopathic manipulative treatment, does not create an effect on its own, and is not a treatment intervention. However, the sham therapy itself may often influence the outcome. The use of cardiovascular variability (eg, beat-to-beat heart rate variability) as a surrogate for the autonomic nervous system is one objective method by which to identify such an effect. By monitoring cardiovascular variability, investigators can assess autonomic nervous system activity as a response to the sham therapy and quickly determine whether or not the selected sham therapy is a true placebo control. The authors provide evidence for assessment of beat-to-beat heart rate variability as one method for assuring objectivity of sham therapy as a placebo control in osteopathic manipulative medicine research.

Metabolism-guided discovery of a potent and orally bioavailable urea-based calcimimetic for the treatment of secondary hyperparathyroidism.

A series of urea based calcimimetics was optimized for potency and oral bioavailability. Crucial to this process was overcoming the poor pharmacokinetic properties of lead thiazole 1. Metabolism-guided modifications, characterized by the use of metabolite identification (ID) and measurement of time dependent inhibition (TDI) of CYP3A4, were essential to finding a compound suitable for oral dosing. Calcimimetic 18 exhibited excellent in vivo potency in a 5/6 nephrectomized rat model and cross-species pharmacokinetics.

Cinacalcet attenuates hypercalcemia observed in mice bearing either Rice H-500 Leydig cell or C26-DCT colon tumors.

Excessive secretion of parathyroid hormone-related protein (PTHrP) by tumors stimulates bone resorption and increases renal tubular reabsorption of calcium, resulting in hypercalcemia of malignancy. We investigated the ability of cinacalcet, an allosteric modulator of the calcium-sensing receptor, to attenuate hypercalcemia by assessing its effects on blood ionized calcium, serum PTHrP, and calcium-sensing receptor mRNA in mice bearing either Rice H-500 Leydig cell or C26-DCT colon tumors. Cinacalcet effectively decreased hypercalcemia in a dose- and enantiomer-dependent manner; furthermore, cinacalcet normalized phosphorus levels, but did not affect serum PTHrP. Ribonuclease protection assay results demonstrated presence of PTHrP receptor, but not calcium-sensing receptor mRNA in C26-DCT tumors. The mechanism by which cinacalcet lowered serum calcium was investigated in parathyroidectomized rats (i.e., without PTH) made hypercalcemic by PTHrP. Cinacalcet attenuated PTHrP-mediated elevations in blood ionized calcium, which were accompanied by increased plasma calcitonin. Taken together these results suggest that the cinacalcet-mediated decrease in serum calcium is not the result of a direct effect on tumor cells, but rather is the result of increased calcitonin release. In summary, cinacalcet effectively reduced tumor-mediated hypercalcemia and corrected hypophosphatemia in mice. Further investigation of cinacalcet for treatment of hypercalcemia of malignancy is warranted.

The effect of conspiracy beliefs and trust on HIV diagnosis, linkage, and retention in young MSM with HIV.

Conspiracy beliefs about HIV may result in delayed diagnosis, medication non-adherence, and low retention in care. The impact of such beliefs is not well described for minority youth. We assessed conspiracy beliefs, trust in physicians, and trust in the health care system in 47 HIV-infected, minority, adolescent men who have sex with men (MSM). We identified correlations of these factors with two intermediate outcomes (general self-efficacy and medication attitudes) and with three clinical outcomes (CD4 cell count at diagnosis, linkage to care, and retention in care). Greater conspiracy beliefs were associated with negative medication attitudes (r=-0.37, p=.01), while trust in physicians was correlated with positive medication attitudes (r=0.42, p=.003). Neither conspiracy beliefs nor trust was correlated with self-efficacy, nor were they correlated with any of the three clinical outcomes. Conspiracy beliefs and lack of trust did not predict delayed diagnosis or poor linkage and retention in this population of young, minority MSM.

Centralization of HIV services in HIV-positive African-American and Hispanic youth improves retention in care.

African-American and Hispanic HIV-infected youth are a high risk group for not remaining in HIV care. We examined differences in retention in care among 174 HIV-infected African-American and Hispanic youth between 13 and 23 years old who presented for HIV primary care between 1 January 2002 and 31 August 2008. Patients were included in three service eras, based on when they entered the clinic: when no youth-specific services were available (the decentralized era), after formation of a youth clinic staffed by adolescent providers and a case-manager (the centralized era), and after educational activities and support groups were added and the social services staff were trained in the use of motivational interviewing (the centralized with supportive services era). Patient and attendance data for the 12-months following entry into care were captured. Retention in HIV care was examined using two different measures: adequate visit constancy (at least three quarters with at least one visit in each quarter) and having a gap in care (two consecutive medical visits ≥180 days apart). Adequate visit constancy improved by service era from 31% in the decentralized era to 57% in the centralized era and 65% in the centralized with supportive services era (p=0.01). The percent of patients with no gap in care remained stable at about 80% in the decentralized and centralized eras, but then increased to 96% in the centralized with supportive services era (p=0.04). Results suggest that centralizing youth-specific care and expanding youth services can improve retention in HIV care. These system changes should be considered when resources allow.

FGF23 neutralization improves chronic kidney disease-associated hyperparathyroidism yet increases mortality.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is associated with secondary hyperparathyroidism (HPT) and serum elevations in the phosphaturic hormone FGF23, which may be maladaptive and lead to increased morbidity and mortality. To determine the role of FGF23 in the pathogenesis of CKD-MBD and development of secondary HPT, we developed a monoclonal FGF23 antibody to evaluate the impact of chronic FGF23 neutralization on CKD-MBD, secondary HPT, and associated comorbidities in a rat model of CKD-MBD. CKD-MBD rats fed a high-phosphate diet were treated with low or high doses of FGF23-Ab or an isotype control antibody. Neutralization of FGF23 led to sustained reductions in secondary HPT, including decreased parathyroid hormone, increased vitamin D, increased serum calcium, and normalization of bone markers such as cancellous bone volume, trabecular number, osteoblast surface, osteoid surface, and bone-formation rate. In addition, we observed dose-dependent increases in serum phosphate and aortic calcification associated with increased risk of mortality in CKD-MBD rats treated with FGF23-Ab. Thus, mineral disturbances caused by neutralization of FGF23 limited the efficacy of FGF23-Ab and likely contributed to the increased mortality observed in this CKD-MBD rat model.

Development of a label-free assay for sodium-dependent phosphate transporter NaPi-IIb.

The most widely used assay format for characterizing plasma membrane transporter activity measures accumulation of radiolabeled substrates in tissues or cells expressing the transporters. This assay format had limitations and disadvantages; therefore, there was an unmet need for development of a homogeneous, nonradioactive assay for membrane transporter proteins. In this report, the authors describe the development of a label-free homogeneous assay for the sodium-dependent phosphate transporter NaPi-IIb using the Epic system. The addition of phosphate stimulated a dynamic mass redistribution (DMR) profile unique to cells expressing NaPi-IIb but not on parental cells. This DMR profile was phosphate specific because sulfate or buffer alone did not elicit the same response. Furthermore, the DMR response observed was phosphate and sodium dependent, with Km values in the micromolar and millimolar range, respectively. A known NaPi-IIb noncompetitive inhibitor was shown to completely inhibit the phosphate-stimulated DMR response, suggesting that this observed DMR response is an NaPi-IIb-mediated cellular event. The results demonstrate that a novel label-free assay was developed for studying transporter-mediated cellular activity, and this DMR assay platform could be applicable to other membrane transporter proteins.

Localization, distribution, and function of the calcium-sensing receptor in sperm.

The intracellular movement of calcium, through calcium channels, plays a major role on sperm cell function. The calcium-sensing receptor (CaSR), a molecular mechanism by which many cells detect changes in extracellular calcium concentration, has not been described in spermatozoa. The aim of this study was to investigate the expression of the CaSR in testicular tissue and sperm cells and the functional consequences of spermatozoid CaSR activation by calcimimetics. CaSR mRNA and protein were identified both in rat testicular tissue and in rat spermatozoa using real-time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. Functionality of CaSR was evaluated by studying the influence of calcimimetic AMG 641 on rat and pig sperm motility. Treatment with AMG 641 100 nM for 1 hour increased rat sperm motility from a score of 1.0 ± 0.1 to 3.8 ± 0.3 (P < .05). AMG 641 also resulted in a modest but significant increase in the pig sperm motility parameters evaluated by computer-assisted sperm analysis. AMG 641 was effective in a wide range of concentrations but resulted in a more marked effect at 50-100 nM. In addition, AMG 641 did not have any negative effect on sperm viability, which was measured by flow cytometry. In conclusion, our results demonstrate the expression of functional CaSR in testicular tissue and sperm, which can be activated by calcimimetic AMG 641.

Cinacalcet HCl prevents development of parathyroid gland hyperplasia and reverses established parathyroid gland hyperplasia in a rodent model of CKD.

BACKGROUND: Secondary hyperparathyroidism (sHPT) represents an adaptive response to progressively impaired control of calcium, phosphorus and vitamin D in chronic kidney disease (CKD). It is characterized by parathyroid hyperplasia and excessive synthesis and secretion of parathyroid hormone (PTH). Parathyroid hyperplasia in uremic rats can be prevented by calcium-sensing receptor (CaSR) activation with the calcimimetic cinacalcet (Sensipar®/Mimpara®); however, it is unknown, how long the effects of cinacalcet persist after withdrawal of treatment or if cinacalcet is efficacious in uremic rats with established sHPT. METHODS: We sought to determine the effect of cinacalcet discontinuation in uremic rats and whether cinacalcet was capable of influencing parathyroid hyperplasia in animals with established sHPT. RESULTS: Discontinuation of cinacalcet resulted in reversal of the beneficial effects on serum PTH and parathyroid hyperplasia. In rats with established sHPT, cinacalcet decreased serum PTH and mediated regression of parathyroid hyperplasia. The cinacalcet-mediated decrease in parathyroid gland size was accompanied by increased expression of the cyclin-dependent kinase inhibitor p21. Prevention of cellular proliferation with cinacalcet occurred despite increased serum phosphorus and decreased serum calcium. CONCLUSIONS: The animal data provided suggest established parathyroid hyperplasia can be reversed by modulating CaSR activity with cinacalcet and that continued treatment may be necessary to maintain reductions in PTH.

Discovery of a calcimimetic with differential effects on parathyroid hormone and calcitonin secretion.

Calcimimetics are positive allosteric modulators to the calcium-sensing receptor (CaSR). Activation of the CaSR inhibits the secretion of parathyroid hormone (PTH), stimulates the secretion of calcitonin, and decreases serum calcium (Ca(2+)). Cinacalcet, a second-generation calcimimetic, is used therapeutically to control PTH in patients with chronic kidney disease who are on dialysis with secondary hyperparathyroidism. A calcimimetic that displays increased separation of PTH versus Ca(2+) lowering in patients would potentially allow the use of calcimimetics to treat patients in earlier stages of renal disease because hypocalcemia can develop in this population. Toward this end, we developed a third-generation calcimimetic, determined the molecular pharmacological properties of it using an operation model of allosteric modulation/agonism, and measured the compound effects on PTH, serum ionized Ca(2+), and calcitonin levels in 5/6 nephrectomized rats. We found the new molecule effectively reduced PTH levels without promoting calcitonin secretion or hypocalcemia. Furthermore, our third-generation molecule was less efficacious at promoting calcitonin secretion from human thyroid carcinoma cells compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic. These data provide evidence that calcimimetics with increased potency can be used to lower PTH without production of significant hypocalcemia because the threshold for inhibition of PTH secretion is much lower than the threshold for calcitonin secretion.

Effect of calcium and the calcimimetic AMG 641 on matrix-Gla protein in vascular smooth muscle cells.

Vascular calcification (VC) is frequently observed in patients with chronic renal failure and appears to be an active process involving transdifferentiation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Reports of VC prevention in uremic rodents by calcimimetics coupled with identification of the calcium-sensing receptor (CaSR) in VSMCs led us to hypothesize that CaSR activation in arterial cells and VSMCs may elicit expression of an endogenous inhibitor of VC. Toward this end, we determined the effects of calcium and the calcimimetic AMG 641 on arterial wall and isolated VSMC expression of matrix-Gla protein (MGP). Bovine VSMCs were incubated with increasing calcium chloride or AMG 641 concentrations, while in vivo experiments were carried out on healthy and uremic rats. Both AMG 641 and hypercalcemia induced MGP expression in the arterial wall in healthy and uremic rats. The results obtained in vitro supported those from in vivo experiments. In conclusion, selective CaSR activation, either by extracellular calcium or AMG 641, increased MGP expression in vivo in the arterial wall and in vitro in bovine VSMCs. This local upregulation of MGP expression provides one potential mechanism by which calcimimetics prevent VC.

The discovery of an orally efficacious positive allosteric modulator of the calcium sensing receptor containing a dibenzylamine core.

The discovery of a series of novel and orally efficacious type II calcimimetics, developed from the lead compound 1, is described herein. Compound 22 suppressed plasma PTH levels relative to vehicle when dosed orally in a rat pharmacodynamic model.

Design and synthesis of 6-oxo-1,6-dihydropyridines as CDK5 inhibitors.

Cyclin-dependent kinase 5 (CDK5) is a serine-threonine protein kinase that plays a significant role in neuronal development. In association with p25, CDK5 abnormally phosphorylates a number of cellular targets involved in neurodegenerative disorders. Using active site homology and previous structure-activity relationships, a new series of potent CDK5 inhibitors was designed. This report describes the optimization of 6-oxo-1,6-dihydropyridines as CDK5 inhibitors.

Discovery and optimization of substituted 1-(1-phenyl-1H-pyrazol-3-yl)methanamines as potent and efficacious type II calcimimetics.

Our efforts to discover potent, orally bioavailable type II calcimimetic agents for the treatment of secondary hyperparathyroidism focused on the development of ring constrained analogues of the known calcimimetic R-568. The structure-activity relationships of various substituted heterocycles and their effects on the human calcium-sensing receptor are discussed. Pyrazole 15 was shown to be efficacious in a rat in vivo pharmacodynamic model.

The calcimimetic AMG 641 abrogates parathyroid hyperplasia, bone and vascular calcification abnormalities in uremic rats.

Calcimimetics and vitamin D sterols reduce serum parathyroid hormone (PTH) in patients with secondary hyperparathyroidism receiving dialysis, a disease state associated with parathyroid hyperplasia, vascular calcification, bone disease, and increased mortality. The aim of this study was to determine the effects of the research calcimimetic AMG 641 (Amgen, Inc., Thousand Oaks, CA) or calcitriol (Sigma Aldrich Corporation, St. Louis, MO) on vascular calcification in a rodent model of progressive uremia with accompanying secondary hyperparathyroidism induced by dietary adenine. Treatment effects on parathyroid gland hyperplasia and bone loss were also investigated. Rats were treated daily with vehicle, calcitriol (10 ng), AMG 641 (3 mg/kg), or no treatment during the 4 week period the animals were fed adenine. The uremia-induced increases in serum PTH levels were significantly attenuated by both AMG 641 (>90%) and calcitriol (approximately 50%). AMG 641 significantly reduced calcium-phosphorus product (CaxP) and significantly attenuated the development of both parathyroid hyperplasia and vascular calcification. In addition, AMG 641 prevented the defects in trabecular bone volume, trabecular number, and bone mineralization, as well as increases in trabecular spacing in this rodent model of secondary hyperparathyroidism. Calcitriol (10 ng/rat) decreased osteoid surface/bone surface, but had no effects on other bone parameters, or parathyroid hyperplasia (likely due to the lower PTH suppressive effect of calcitriol at the dose used in this study). However, this dose of calcitriol significantly exacerbated vascular calcification. These results suggest that calcimimetics can reduce the development of vascular calcification, parathyroid hyperplasia and bone abnormalities associated with secondary hyperparathyroidism.

Calcimimetic inhibits late-stage cyst growth in ADPKD.

In polycystic kidney disease (PKD), genetic mutations in polycystin 1 and 2 lead to defective intracellular trafficking of calcium, thereby decreasing intracellular calcium and altering cAMP signaling to favor proliferation. We hypothesized that calcimimetics, allosteric modulators of the calcium-sensing receptor, would reduce cyst growth by increasing intracellular calcium. We randomly assigned 20-wk-old male rats with a form of autosomal dominant PKD (heterozygote Cy/+) to one of four groups for 14 to 18 wk of treatment: (group 1) no treatment; (group 2) calcimimetic R-568 formulated in the diet; (group 3) R-568 plus calcium-supplemented drinking water (R-568 plus Ca); or (group 4) Ca-supplemented drinking water with a normal diet (Ca). Severity of PKD did not progress in any of the three treatment groups between 34 and 38 wk. Compared with no treatment, cyst growth was unaffected at 34 wk by all treatments, but cyst volume and fibrosis were lower at 38 wk, with both R-568-treated groups demonstrating a greater reduction than calcium alone. Between 34 and 38 wk, the total kidney weight increased by 78% in the control group (P < 0.001) and by 19% in the Ca group (P < 0.01), but did not increase in the R-568 or R-568 plus Ca groups, suggesting inhibition of disease progression despite equivalent suppression of parathyroid hormone. In summary, treatment of hyperparathyroidism halts late-stage progression of rodent cystic kidney disease. The benefit of R-568 alone suggests calcium-sensing receptor modulation may have additional inhibitory effects on late-stage cyst growth resulting from a direct modulation of intracellular calcium.

R-568 reduces ectopic calcification in a rat model of chronic kidney disease-mineral bone disorder (CKD-MBD).

BACKGROUND: Chronic kidney disease-mineral bone disorder (CKD-MBD), a newly defined disorder in patients with CKD, describes the interacting triad of (1) biochemical abnormalities of calcium, phosphorus and parathyroid hormone (PTH), (2) extraskeletal calcification and (3) abnormal bone. METHODS: We studied the effects of the calcimimetic R-568, R-568 with calcium (R-568 + Ca) or calcium (Ca) alone compared with control CKD rats on this triad in the Cy/+ male rat, a model of progressive CKD that spontaneously develops CKD-MBD on a normal phosphorus diet. Animals were treated for either 14 or 18 weeks beginning at 20 weeks of age (34-week and 38-week animals, respectively). RESULTS: The results demonstrated similar efficacy of R-568, R-568 + Ca and Ca in lowering PTH levels. R-568 alone lowered plasma calcium compared to control over time, but increased phosphorus compared to control early in the course of the disease, but not at 38 weeks. Animals treated with Ca alone or R-568 + Ca had lower phosphorus levels; the Ca alone group had elevated Ca levels. Bone volume improved in the calcium-treated groups. In contrast, arterial and cardiac calcification worsened by most assessments in the R-568 + Ca and Ca alone treated animals compared with R-568 alone whereas R-568 alone treatment showed beneficial effects on most sites of extraskeletal calcification. CONCLUSION: Thus, R-568, with or without Ca, improved the biochemical abnormalities of hyperparathyroidism but with higher and lower calcium levels, respectively, compared with controls. However, R-568 + Ca had more dramatic improvement in bone volume, but more extraskeletal calcification than R-568 alone. This complexity demonstrates that treatment of one component of CKD-MBD may lead to undesirable effects on other components.

The calcimimetic AMG 641 accelerates regression of extraosseous calcification in uremic rats.

The purpose of the present study was to test the hypothesis that extraskeletal calcification regresses in uremic rats after reduction in phosphorus intake and treatment with calcimimetics. Extraosseous calcification was induced in five to six nephrectomized rats fed a high-phosphorus (1.2%) diet who received calcitriol (80 ng/kg ip) every other day for a period of 14 days. Next, dietary phosphorus was reduced to 0.6%, and rats were treated with vehicle (n = 20), calcitriol [80 ng/kg ip/48 h (n = 20)], or the calcimimetic AMG 641 [1.5 mg/kg sc/48 h (n = 20)]. Aortic and soft-tissue calcium and phosphorus content was evaluated after 14 and 28 days. At 28 days, reduction of phosphorus intake resulted in a significant decrease in tissue mineral content in vehicle- and AMG 641-treated rats but not in rats receiving calcitriol. Aortic calcium and phosphorus was lower in rats treated with AMG 641 (96.7 +/- 26.4 mg/g) than in rats receiving vehicle (178.3 +/- 38.6 mg/g). An infiltrate of phagocytic cells expressing the calcium-sensing receptor was identified in areas surrounding foci of calcification. Additional studies in parathyroidectomized rats demonstrated that AMG 641 increased the urinary excretion of calcium (6.2 +/- 0.6 vs. 3.1 +/- 0.5 mg/day, vehicle) (P < 0.001). In conclusion, experimentally induced extraosseous calcification in uremic rats can be partially resolved by reducing phosphorus intake; the addition of calcimimetics may accelerate the regression process through mechanisms potentially involving a direct stimulatory effect on mineral phagocytic cells plus an increase in urinary calcium excretion.

Osteopathic manipulative treatment and its relationship to autonomic nervous system activity as demonstrated by heart rate variability: a repeated measures study.

BACKGROUND: The relationship between osteopathic manipulative treatment (OMT) and the autonomic nervous system has long been acknowledged, but is poorly understood. In an effort to define this relationship, cervical myofascial release was used as the OMT technique with heart rate variability (HRV) as a surrogate for autonomic activity. This study quantifies that relationship and demonstrates a cause and effect. METHODS: Seventeen healthy subjects, nine males and eight females aged 19-50 years from the faculty, staff, and students at Oklahoma State University Center for Health Sciences College of Osteopathic Medicine, acted as their own controls and received interventions, administered in separate sessions at least 24 hours apart, of cervical myofascial OMT, touch-only sham OMT, and no-touch control while at a 50-degree head-up tilt. Each group was dichotomized into extremes of autonomic activity using a tilt table. Comparisons were made between measurements taken at tilt and those taken at pre- and post-intervention in the horizontal.The variance of the spectral components of HRV, expressed as frequencies, measured the response to change in position of the subjects. Normalized low frequency (LF) and high frequency (HF) values, including LF/HF ratio, were calculated and used to determine the effect of position change on HRV. RESULTS: Predominantly parasympathetic responses were observed with subjects in the horizontal position, while a 50-degree tilt provided a significantly different measure of maximum sympathetic tone (p < 0.001). Heart rate changed in all subjects with change in position; respirations remained constant. When OMT was performed in a sympathetic environment (tilt), a vagal response was produced that was strong enough to overcome the sympathetic tone. There was no HRV difference between sham and control in either the horizontal or tilt positions. CONCLUSION: The vagal response produced by the myofascial release procedure in the maximally stimulated sympathetic environment could only have come from the application of the OMT. This demonstrates the association between OMT and the autonomic nervous system. The lack of significance between control and sham in all positions indicates that HRV may be a useful method of developing sham controls in future studies of OMT. TRIAL REGISTRATION: clinicaltrials.gov NCT00516984.