calcitonin and Breast-Neoplasms

This study aimed to investigate calcitonin as an effective therapy for osteoporosis in patients with bone pain during the anastrozole treatment of breast cancer. Ninety-one patients, who were on anastrozole treatment for breast cancer and also suffered anastrozole-induced bone pain, were randomly divided into two groups: the calcitonin group received salmon calcitonin and Caltrate D, and the control group received Caltrate D. All patients were evaluated by the visual analogue scale (VAS) and underwent the dual energy x-ray absorptiometry test for bone mineral density (BMD), and serum osteocalcin (BGP), alkaline phosphatase (ALP), calcium (Ca), and phosphorus (P) were measured at three months before and after the treatment. Significant differences in serum Ca, P, BGP, and ALP were found in each group between before and after treatment (P < 0.05), while no differences between the calcitonin and control groups were found. No difference was observed in femur BMD between the two groups, or between before and after treatment in each group. There was a significant difference in spine BMD between before and after treatment in the control group (P < 0.05) but not in the calcitonin group, while no difference was found between the calcitonin and control groups. Futhermore, VAS score significantly declined in each group after treatment (P < 0.05), but much more in the calcitonin group than the control group (P < 0.05). Our finding suggests that calcitonin may alleviate bone pain during the anastrozole treatment of breast cancer but has no effect on bone loss during cancer treatment.

Topics: Absorptiometry, Photon; Aged; Alkaline Phosphatase; Anastrozole; Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Bone Density; Bone Density Conservation Agents; Breast Neoplasms; Calcitonin; Calcium; Female; Femur; Humans; Middle Aged; Nitriles; Osteocalcin; Osteoporosis; Pain; Phosphorus; Spine; Triazoles

Salmon calcitonin 100 MRCU/day or a saline placebo were given in daily injections for at least three months to 49 patients with bone metastases from breast cancer in a randomized double-blind trial. All patients were normocalcemic, and most patients had stable or regressing disease at start of trial. No improvement in general performance or bone pain was detected as measured by a visual analogue scale, the daily duration of pain or consumption of analgetic drugs. Calcitonin had no effect on disease progression as judged by bone scans and radiographs. Calcitonin therapy did not affect serum calcium, alkaline phosphatase, bone gla-protein, or the urinary excretion of calcium and hydroxyproline. Serum phosphate and magnesium decreased significantly during calcitonin treatment (p = 0.01, and 0.00005, respectively). It was concluded that salmon calcitonin in this dosage has no discernible effect on skeletal pain, general performance, bone metabolism or disease progression in patients with breast cancer metastatic to bone. A significant decrease in serum phosphate and magnesium probably indicated an effect of calcitonin on the renal excretion of these ions.

Topics: Bone Neoplasms; Breast Neoplasms; Calcitonin; Clinical Trials as Topic; Double-Blind Method; Electrolytes; Female; Humans; Pain; Random Allocation

Salmon calcitonin (sCT) is approved for the short-term treatment of Paget's disease and hypercalcemia. As pulmonary delivery might improve the drug's efficacy, a variety of liposomal sCT formulations for inhalation were prepared and characterized with the intention of developing a controlled release formulation.. The influence of pH of the loading buffer, charge of the vesicular surface, and membrane rigidity on particle size, ζ-potential, and sCT encapsulation efficiency of formulations was studied. The most promising systems were investigated for their ability to withstand nebulization stresses using an Aeroneb(®) vibrating mesh device. In vitro studies were carried out to determine sCT release from the vesicles and the bioactivity of the peptide post nebulization. Lastly, pharmacokinetics of sCT liposomes following intratracheal aerosolization in an experimental rat model were investigated and compared with intravenous injection.. Liposomes prepared with acidic loading buffer and comprising rigid lipid membranes showed an optimal compromise between small particle size, high encapsulation efficiency, and sCT stability. Polyethylene glycol (PEG) liposomes showed the highest encapsulation efficiency overall, regardless of the ζ-potential of the vesicles. Positive surface charge, however, yielded higher entrapment in non-PEGylated liposomes. All liposomes tested were stable during nebulization. The bioactivity of sCT after formulation into liposomes was 52-55%. Intratracheal nebulization in rats revealed that the bioavailability and other pharmacokinetic parameters were not enhanced by liposomes, when compared with sCT solution. Following intravenous administration, however, liposomes showed significantly higher bioavailability and AUCinf (area under the curve to the infinity time point) than controls.. The developed liposomal formulations were not optimal carriers for pulmonary delivery of sCT. Due to the low amounts of peptide released from the vesicles, enzymatic digestion by peptidases in the airspace reduced the bioavailability significantly. Liposomal encapsulation of sCT, nevertheless, resulted in improved pharmacokinetics following injection.

Topics: Administration, Inhalation; Animals; Area Under Curve; Biological Availability; Bone Density Conservation Agents; Breast Neoplasms; Buffers; Calcitonin; Cell Line, Tumor; Chemistry, Pharmaceutical; Delayed-Action Preparations; Drug Carriers; Female; Hydrogen-Ion Concentration; Injections, Intravenous; Liposomes; Lung; Male; Nebulizers and Vaporizers; Particle Size; Polyethylene Glycols; Rats; Rats, Wistar; Stress, Mechanical; Surface Properties

Synthetic salmon Calcitonin (sCT) is currently used to treat and manage conditions associated with low bone mass, and elicits its antiresorptive effect by acting upon Calcitonin receptors (CTRs) located on bone-resorbing osteoclast cells. However, CTRs are also widely distributed in many non-skeletal tissues (such as kidney, brain, and lung), and the competitive uptake of available sCT amongst such CTRs likely reduces sCT availability for bone resident osteoclast cells, particularly if the drug is administered systemically and not specifically targeted to bone. Hence, the objective of this study was to synthesize and characterize a bisphosphonate (BP)-mediated bone targeting delivery system for sCT and to determine whether the bioactivity of sCT was retained after BP conjugation. BP-sCT conjugates were synthesized by initially reacting sCT with sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) in dimethyl formamide in the presence of triethylamine (TEA) at room temperature. Thiolated (Thiol)-BP was then reacted with the sCT-sulfo-SMCC conjugates to generate sCT-BP conjugates, which were purified by dialysis and assayed using the micro-BCA protein assay. Non-BP containing control sCT-Cysteine conjugates were also synthesized using the same procedure. Reactions were monitored and characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF) analysis and Tris-Tricine SDS-PAGE. Conjugates were evaluated for in vitro bone mineral affinity using a hydroxyapatite binding test, for bone mineral specificity using different calcium salt binding affinity assays, and for continued sCT bioactivity after conjugation using an intracellular cAMP stimulation in human T47D breast cancer cells. Our results confirmed that BP-conjugated sCT exhibited significantly greater affinity and specificity for bone mineral over unmodified sCT, and that sCT-BP conjugates retained strong CT bioactivity after conjugation. Our conjugation strategy holds the promise of facilitating the delivery of sCT preferentially to skeletal bony tissues, thereby increasing its local concentration to bone surfaces. This peptide hormone-bisphosphonate drug system represents a new class of antiresorptive drug that has not previously been attempted, nor has a bone targeting formulation of sCT been reported.

Topics: Bone and Bones; Bone Density; Bone Density Conservation Agents; Breast Neoplasms; Calcitonin; Cell Line, Tumor; Dimethylformamide; Drug Delivery Systems; Electrophoresis, Polyacrylamide Gel; Ethylamines; Humans; Maleimides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Calcitonin is currently used to treat hypercalcemia of many clinical types. However, we encountered a woman who suffered severe hypercalcemia and status epilepticus, both of which developed 8 days after the administration of salmon calcitonin for the treatment of breast cancer. When the patient first presented her serum calcium level was 15.5mg/dl, intact parathyroid hormone level 118 pg/ml, calcitonin <2 pg/ml, magnesium 1.2mg/dl, and phosphate 1mg/dl. Her serum calcium level returned to the reference range within 48 h after correction. At follow-up no hypercalcemia had developed, although the patient had received no further treatment for her breast cancer and multiple metastases were subsequently detected. Her hypercalcemia is ascribed to exogenous calcitonin supplementation. These conflicting events may be due to functionally heterogeneous calcitonin receptors or to activation of 1 alpha-hydroxylase by exogenous calcitonin.

Topics: Bone Neoplasms; Breast Neoplasms; Calcitonin; Female; Humans; Hypercalcemia; Middle Aged; Osteoporosis; Status Epilepticus

Human adrenomedullin (hADM), human calcitonin gene-related peptide (hCGRP), and salmon calcitonin (sCT)-activated adenylyl cyclase with EC50 values of 132, 764, and 0.5 nM, respectively, in human breast cancer cell line, T 47D. Treatment of T 47D cell membranes with near maximal concentrations of sCT, hADM and hCGRP had no additive effect on adenylyl cyclase activity. Salmon calcitonin (8-32)[sCT (8-32)], selective antagonist of calcitonin receptor, inhibited the activation of adenylyl cyclase by these three peptides. On the other hand, the putative ADM receptor antagonist, ADM (22-52), and CGRP receptor antagonist, CGRP (8-37), failed to inhibit ADM-, CGRP- or sCT-activated adenylyl cyclase. These results suggest that in T47D cells, both ADM and CGRP activated adenylyl cyclase through sCT receptors.

Topics: Adenylyl Cyclases; Adrenomedullin; Breast Neoplasms; Calcitonin; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Cell Membrane; Drug Interactions; Enzyme Activation; Female; Humans; Membrane Proteins; Peptide Fragments; Peptides; Receptors, Adrenomedullin; Receptors, Calcitonin; Receptors, Peptide; Tumor Cells, Cultured

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Calcitonin; Carcinoma, Lobular; Female; Humans; Hypercalcemia; Middle Aged; Palliative Care; Paraneoplastic Syndromes; Radiotherapy Dosage

Stable transfectants expressing a recombinant human calcitonin receptor respond to calcitonin via increased cyclic adenosine 3',5' monophosphate (cAMP, EC50 = 0.06 nM salmon calcitonin [sCT]) and a transient mobilization of intracellular calcium ([Ca2+]i) coincident with turnover of inositol phosphate (IP; EC50 = 6 nM sCT). Millimolar increases in extracellular calcium ([Ca2+]o, EC50 = 8 mM) cause a rapid elevation in [Ca2+]i after a calcitonin dose-dependent pretreatment of cells (pretreatment EC50 = 0.2 nM sCT). Cells exhibit persistent sensitivity to increased [Ca2+]o up to 3 h after hormone exposure and even after multiple cycles of increased [Ca2+]o followed by wash. Calcitonin pretreatment of cells also allows apparent influx of elevated extracellular strontium and manganese, but little or no effect is observed on addition of barium, cadmium, or lanthanum. Human amylin (100 nM) causes a rapid and transient increase in [Ca2+]i comparable to that of calcitonin; however, no significant response to increased [Ca2+]o is observed after amylin addition. Human calcitonin gene-related product (hCGRP) (300 nM) and forskolin do not increase [Ca2+]i or activate a sensitivity to increased [Ca2+]o. Nevertheless, human amylin and human calcitonin gene-related product (hCGRP) activate adenylate cyclase with EC50s of 0.7 nM and 8 nM, respectively. The calcium-channel drugs verapamil, BAY K 8644, diltiazem, and nifedipine have little effect on [Ca2+]i increases. The calcitonin-induced transient mobilization of calcium is inhibited by treatment of cells with cholera toxin or 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8); whereas, the response to subsequent increased [Ca2+]o is inhibited by lanthanum chloride (200 microM) and lower pH (6.0).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Amyloid; Animals; Breast Neoplasms; Calcitonin; Calcitonin Gene-Related Peptide; Calcium; Calcium Channel Blockers; Carcinoma; Cations, Divalent; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Inositol Phosphates; Islet Amyloid Polypeptide; Receptors, Calcitonin; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Amyloid; Animals; Base Sequence; Binding, Competitive; Breast Neoplasms; Calcitonin; Calcitonin Gene-Related Peptide; Carcinoma; Cyclic AMP; DNA, Complementary; DNA, Neoplasm; Enzyme Activation; Female; Gene Expression Regulation; Genes, Reporter; Humans; Islet Amyloid Polypeptide; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Neoplasm Proteins; Protein Conformation; Receptors, Calcitonin; Second Messenger Systems; Tumor Cells, Cultured

Calcitonin (CT) down-regulates its receptor in several cancer cell lines, including T47D human breast cancer cells. Removal of CT results in the recovery of CT receptor (CTR) binding. However, homologous regulation of the CTR in osteoclasts is not well understood. To elucidate these phenomena in cells of the osteoclast lineage, mouse osteoblasts and bone marrow cells were cocultured on type 1 collagen gels. For the experiments, osteoclast-like cell (OCL)-enriched populations were subcultured from the collagen gels into multiwell dishes on days 7-8, and CT regulation of the CTR was determined and compared with that in T47D cells. When cells of either type were treated with CT for 1 h and then washed, binding capacity for [125I] salmon CT ([125I]sCT) was decreased dependent upon the preincubating concentration of CT. After removal of CT, the binding capacity in OCLs recovered toward the control level over 12 h. However, in contrast to that in T47D cells, recovery was transient, so that 24 h after removal of CT, the binding capacity in preincubated cells was strikingly reduced compared with that in control cells. This occurred even when the preincubating concentration of CT was too low to cause down-regulation of binding in 1 h. Scatchard analysis showed a decrease in receptor number in CT-treated compared with control OCLs 24 h after CT removal, with unchanged receptor affinity. By autoradiography, decreased CTR density on multinuclear OCLs was indicated. Preexposure of either OCLs or T47D cells to CT caused elevation of intracellular cAMP, which persisted for 6-12 h after removal of CT. In addition, there was desensitization to a rechallenge with CT, which, in T47D cells, recovered by 24-36 h. In contrast, OCLs showed incomplete recovery of desensitization. These data correlated with the results of semiquantitative reverse transcription-polymerase chain reaction studies; the CTR messenger RNA level was increased to about 150% of the control level in sCT-treated T47D cells 18-36 h after sCT removal; the level was markedly decreased to about 20% of the control value in sCT-treated OCLs 12-48 h after sCT removal and remained suppressed. This study suggests that CT-induced homologous down-regulation is a potential cause of the "escape" phenomenon, by producing a population of CT-resistant osteoclasts.

Topics: Animals; Base Sequence; Binding Sites; Breast Neoplasms; Calcitonin; Cyclic AMP; DNA Primers; Down-Regulation; Female; Humans; Kinetics; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Osteoclasts; Receptors, Calcitonin; RNA, Messenger; Tumor Cells, Cultured

Topics: Adult; Antineoplastic Agents; Breast Neoplasms; Calcitonin; Female; Fluid Therapy; Gallium; Humans; Hypercalcemia

The use of calcitonin (CT) is established as a treatment of Paget's disease of bone and postmenopausal osteoporosis (PMO). Salmon calcitonin (sCT), which differs in 14 of the 32 amino acids from human calcitonin, has found a wider distribution world wide, although antibody formation against sCT has been reported in more than 70% of the patients on continuous sCT treatment. The clinical significance of these antibodies has been discussed controversially, because the occurrence of antibodies is not always associated with the development of secondary resistance. Using an in vitro bioassay, based on the CT-mediated increase of the cyclic AMP (cAMP) production of the human breast cancer cell line T 47 D we could identify a neutralizing activity against sCT in the serum of a subset of patients with formation of antibodies against sCT which was related to the development of secondary resistance. Antibody formation against human calcitonin (hCT) has been reported only once before. Binding and neutralizing antibodies were now observed in 1 of 33 patients with PMO treated with hCT. Due to a low neutralizing activity, clinical sequelae were not to be expected in this patient. The formation of neutralizing antibodies against calcitonin is common after treatment with salmon but a rare phenomenon after treatment with human calcitonin. We recommend monitoring of patients with postmenopausal osteoporosis and Paget's disease of bone on long term treatment with sCT or hCT for neutralizing antibody formation in order to evaluate the therapeutic effect of treatment.

Topics: Antibodies; Antibody Formation; Breast Neoplasms; Calcitonin; Cyclic AMP; Drug Resistance; Female; Humans; Iodine Radioisotopes; Osteitis Deformans; Osteoporosis, Postmenopausal; Receptors, Calcitonin; Tumor Cells, Cultured

Exposure of T 47D human breast cancer cells to salmon calcitonin (sCT) resulted in a reduction of binding capacity for [125I]iodo-sCT in washed cells. The reduction was both time and concentration dependent. Recovery of binding capacity in CT-pretreated T 47D cells occurred in the absence of CT, but was prevented by inhibitors of protein synthesis. Studies were carried out to determine the mechanism of CT-induced reduction of binding capacity. When T 47D cells were treated with sCT at 37 C or 4 C and washed with buffer at neutral pH, subsequently measured binding capacity was lost as a function of time of pretreatment. Cells pretreated under the same conditions were washed with isotonic buffer at pH 2.5 to release cell-surface bound sCT and to allow assessment of cell surface receptor concentration. It was found that at 37 C sCT induced a time-dependent loss of cell surface receptors, so that initially the lost binding capacity was largely reclaimable by acid treatment, whereas after longer exposure to sCT, acid treatment was much less effective in regenerating binding capacity. The CT-induced reduction in binding capacity was not observed when cells were pretreated with sCT at 4 C or in the presence of inhibitors of cellular metabolic energy. These results are consistent with the view that initially CT-induced loss of CT receptors in T 47D cells is primarily due to occupancy of cell-surface receptors and later to a reduction in the concentration of cell-surface receptors mediated by an energy requiring internalization process involving the CT-receptor complex; reappearance of receptors requires new protein synthesis.

Topics: Breast Neoplasms; Calcitonin; Cell Line; Cell Membrane; Cold Temperature; Energy Metabolism; Female; Humans; Hydrogen-Ion Concentration; Iodine Radioisotopes; Receptors, Calcitonin; Receptors, Cell Surface