Solomon Epstein

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

Ibandronate: A Clinical Pharmacological and Pharmacokinetic Update

Ibandronate is a potent nitrogen‐containing bisphosphonate. It has a strong affinity for bone mineral and potently inhibits osteoclast‐mediated bone resorption. Ibandronate is effective for the treatment of hypercalcemia of malignancy, metastatic bone disease, postmenopausal osteoporosis, corticosteroid‐induced osteoporosis, and Pagets disease. Oral ibandronate is rapidly absorbed (tmax < 1 hour), with a low bioavailability (0.63%) that is further reduced (by up to 90%) in the presence of food. Ibandronate has a wide therapeutic index and is not metabolized and, therefore, has a low potential for drug interactions. Given its metabolic stability, ibandronate is eliminated from the blood by partitioning into bone (40%‐50%) and through renal clearance (CLR ∼60 mL/min). The CLR of ibandronate is linearly related to creatinine clearance. The sequestration of ibandronate in bone (VD > 90 L) results in a multiphasic elimination (t1/2 range ∼10–60 hours), characterized by the slow release of ibandronate from the bone compartment. The potency of ibandronate and its sequestration into bone allow ibandronate to be developed as oral and intravenous injection formulations that can be administered with convenient extended between‐dose intervals.

Osteoporosis and Diabetes Mellitus

Osteoporosis is defined as microarchitectural deterioration of bone with increased susceptibility to fragility fractures [1]. Between fifteen and twenty million persons in the United States currently have osteoporosis [2]. The presence of a generalized osteoporosis related to diabetes mellitus (DM) is less acknowledged and controversial, although localized bone lesions of the foot are well-recognized complications of DM. Due to the different pathogenetic mechanisms in Type 1 DM and Type 2 DM, the association with osteoporosis is even is less clear. Factors associated with diabetes-osteoporosis, which may account for the pathogenesis of diabetic bone loss have been studied. These include vascular and possibly neuropathic mechanisms, poor glycemic control, abnormalities of calcium and vitamin D metabolism, and hypercalciuria with secondary increase in parathyroid hormone (PTH) secretion. Vitamin D levels have been found to be normal or low. Histomorphometry has revealed decreased bone turnover. Other factors include a negative association with sex hormone binding globulin (SHBG), higher free testosterone, and free estrogen levels. This article will review the relevant literature relating to diabetes and osteoporosis including cellular and animal models. The role of insulin, insulin-like growth factor-1 (IGF-1), glycosylated end products, poor glycemic control, acidosis, ketosis, vitamin D metabolism, BMD, and bone markers will be investigated. Duration of diabetes as well as microvascular complications will also be addressed. The data on cellular mechanisms and experimental models is extensive but despite this, the relevance to the clinical situation is unclear. The presence of Type 1 DM imparts several deleterious consequences for skeletal health, including diminished peak height velocity during the pubertal growth spurt, decreased adult bone density, an increased risk for osteoporosis and fracture, poor bone healing and diabetes-induced skeletal embryopathy, processes which rely on de novo bone formation. Clinical studies in Type 1 DM appears to be concentrated on bone density and bone growth in adolescents, the importance of insulin control, nutrition, celiac disease, and bone markers. The fracture data in this cohort is either absent, or poorly documented. In Type 2 DM, the influence of body weight, fracture incidence, BMD and bone turnover appear to be the prime factors and will be addressed. General recommendations for screening and treatment will be briefly discussed, but once again, the evidence in terms of effective therapy specifically tailored to the diabetic patient is again lacking.

The Roles of Bone Mineral Density, Bone Turnover, and Other Properties in Reducing Fracture Risk During Antiresorptive Therapy

Osteoporosis is a skeletal disorder characterized by compromised bone strength and increased risk of fracture. Properties related to bone strength include rate of bone turnover, bone mineral density, geometry, microarchitecture, and mean degree of mineralization. These properties (with or without bone density) are sometimes collectively referred to as bone quality. Antiresorptive agents may reduce fracture risk by several separate but interrelated effects on these individual properties. For example, antiresorptive agents have been reported to reduce bone turnover, stabilize or increase bone density, preserve or improve microarchitecture, reduce the number or size of resorption sites, and improve mineralization. Although changes in bone architecture and mineralization are not currently measurable in clinical practice, bone turnover is assessed easily in vivo and affects the other bone properties. Moreover, antiresorptive therapies that produce larger decreases in bone turnover markers together with larger increases in bone mineral density are associated with greater reductions in fracture risk, especially at sites primarily composed of cortical bone such as the hip. Reductions in fracture risk are the most convincing evidence of good bone quality. Data from well-designed randomized clinical trials with up to 10 years of continuous antiresorptive therapy have shown that certain antiresorptive agents effectively reduce fracture risk and (together with extensive preclinical data) suggest no deleterious effects on bone quality.

A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+ signaling via CD38/ADP-ribosyl cyclase, a putative intracellular NAD+ sensor

CD38 is an ectocyclase that converts NAD+ to the Ca2+‐releasing second messenger cyclic ADP‐ribose (cADPr). Here we report that in addition to CD38 ecto‐catalysis, intracellularly expressed CD38 may catalyze NAD+→cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3‐E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km ~5.13 μM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38‐EGFP fusion protein. The Δ‐49‐CD38‐EGFP mutant with a deleted amino‐terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+‐induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr‐Δ‐49‐CD38‐EGFP or Δ‐49‐CD38‐EGFP‐Fan mutants, both directed to the plasma membrane but in an opposite topology to the full‐length CD38‐EGFP. Finally, ryanodine inhibited Ca2+ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD+ production to cytosolic Ca2+ signaling.—Sun, L., Adebanjo, O. A., Koval, A., Anandatheerthavarada, H. K., Iqbal, J., Wu, X. Y., Moonga, B. S., Wu, X. B., Biswas, G., Bevis, P. J. R., Kumegawa, M., Epstein, S., Huang, C. L.‐H., Avadhani, N. G., Abe, E., Zaidi, M. A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+ signaling via CD38/ADP‐ribosyl cyclase, a putative intracellular NAD+ sensor. FASEB J. 16, 302–314 (2002)

Blocking FSH induces thermogenic adipose tissue and reduces body fat

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the β-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the β-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.

Disintegration/dissolution profiles of copies of Fosamax (alendronate)

SUMMARY Objective: Poor quality has been reported for some generics and other copies of original products. We performed a pilot study to compare the disintegration/dissolution profiles of FOSAMAX (alendronate) 70 mg tablets with those of copies of FOSAMAX that were manufactured outside the United States. Research design and methods: We used the standard United States Pharmacopeia (USP) disintegration method to evaluate FOSAMAX 70mg tablets and 13 copies. At least 12 (n = 12) dosage units were tested for each product (except Fosmin, n = 10). The dissolution profiles of FOSAMAX and one representative copy were also compared. Results: Nine copies (Osteomax, Defixal, Fosmin, Endronax, Osteomix, Genalmen, Fixopan, Osteoplus, and Fosval) disintegrated two- to tenfold faster than FOSAMAX. Three other copies (Neobon, Regenesis, and Ostenan) disintegrated at least five-fold slower than FOSAMAX. Neobon is a softgel capsule, so special consideration was given to this different dosage form. One copy (Arendal) did not fall into either category but exhibited potentially large inter- and intra-lot variability. Dissolution of alendronate from Regenesis lagged behind that from FOSAMAX. Conclusion: Slower disintegration may reduce efficacy because bisphosphonates must be taken in the fasting state and contact with food or even certain beverages severely reduces bioavailability. Faster disintegration (or the use of gel-caps or other alterations to the drug formulation) could increase the risk of esophagitis, an adverse event associated with prolonged contact of the esophagus with bisphosphonates. These disintegration and dissolution results suggest that important differences may exist between FOSAMAX and its copies with regard to bioavailability, pharmacokinetics, and clinical efficacy and safety profiles. Additional testing is warranted to evaluate the pharmacokinetics and clinical safety of these copies.

Repurposing of bisphosphonates for the prevention and therapy of nonsmall cell lung and breast cancer

Significance Small molecules to target oncogenic signaling cascades in cancer have achieved success in molecularly defined patient subsets. The path to approval is often protracted and plagued with failures. Repositioning Food and Drug Administration-approved drugs with known side effects has become a major focus. Bisphosphonates are a commonly prescribed therapy for osteoporosis and skeletal metastases. The drugs have also been associated with reduced tumor burden in some patients, but the mechanism is unknown. Here we provide evidence that bisphosphonates inhibit the human EGFR (HER) receptor tyrosine kinase, including the commonly mutated forms that drive nonsmall cell lung cancer, as well as a resistance mutation. This new mechanism lays the basis for the future use of bisphosphonates for the prevention and therapy of HER family-driven cancers. A variety of human cancers, including nonsmall cell lung (NSCLC), breast, and colon cancers, are driven by the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. Having shown that bisphosphonates, a class of drugs used widely for the therapy of osteoporosis and metastatic bone disease, reduce cancer cell viability by targeting HER1, we explored their potential utility in the prevention and therapy of HER-driven cancers. We show that bisphosphonates inhibit colony formation by HER1ΔE746-A750-driven HCC827 NSCLCs and HER1wt-expressing MB231 triple negative breast cancers, but not by HERlow-SW620 colon cancers. In parallel, oral gavage with bisphosphonates of mice xenografted with HCC827 or MB231 cells led to a significant reduction in tumor volume in both treatment and prevention protocols. This result was not seen with mice harboring HERlow SW620 xenografts. We next explored whether bisphosphonates can serve as adjunctive therapies to tyrosine kinase inhibitors (TKIs), namely gefitinib and erlotinib, and whether the drugs can target TKI-resistant NSCLCs. In silico docking, together with molecular dynamics and anisotropic network modeling, showed that bisphosphonates bind to TKIs within the HER1 kinase domain. As predicted from this combinatorial binding, bisphosphonates enhanced the effects of TKIs in reducing cell viability and driving tumor regression in mice. Impressively, the drugs also overcame erlotinib resistance acquired through the gatekeeper mutation T790M, thus offering an option for TKI-resistant NSCLCs. We suggest that bisphosphonates can potentially be repurposed for the prevention and adjunctive therapy of HER1-driven cancers.

Nonsteroid Immune Modulators and Bone Disease

Abstract:  Glucocorticoids have been the main agents for preventing organ rejection, but unfortunately they possess serious side effects. Newer immunosuppressive agents have therefore been introduced to overcome these effects and have had a dramatic impact on reducing the incidence of organ rejection, enhancing donor organ acceptance, and hence patient survival posttransplantation. However, calcineurin inhibitors (CIs), such as cyclosporine and tacrolimus, also have serious effects causing rapid and severe bone loss in animal models and humans. The mechanism accounting for this action is unclear at present, but the role of T lymphocyte action via RANKL seems to be of essence in triggering bone loss. The mechanism is complex and in vitro studies often produce results that are opposite to those seen in vivo. In addition to acute, rapid, and severe bone loss (ARSBL), the clinical picture shows an extremely high incidence of fractures at all sites, and depends upon the organ transplanted, preexisting bone disease, interval before transplantation, and the dose and duration of multiple immunosuppressive drugs. Other immune‐modifying drugs, such as azathioprine, mycophenolate mofetil, and sirolimus, which are used in conjunction with glucocorticoids and CIs have not been shown to promote bone loss experimentally or clinically. With the exception of glucocorticoids, all of the agents discussed here demand further investigation with regard to their effects on bone health in the clinical setting.

Bisphosphonates inactivate human EGFRs to exert antitumor actions

Significance For over three decades, bisphosphonates have been used for the therapy of osteoporosis and skeletal metastasis. Here we show that this class of drugs reduces the viability of tumor cells that are driven by the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. We also show that bisphosphonates directly bind to and inhibit HER kinases. Because bisphosphonates are inexpensive and readily available worldwide, our findings may have important healthcare implications by offering an affordable and multiuse alternative or adjunct to current therapies for HER-driven malignancy. Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers.