CV706, a prostate cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity.

Radiation is an effective means of treating localized prostate cancer. However, up to 40% of men with certain risk factors will develop biochemical failure 5 years after radiotherapy. CV706, a prostate cell-specific adenovirus variant, is currently in clinical trials for the treatment of recurrent organ-confined prostate cancer. We demonstrated previously that a single administration of CV706 at 5 x 10(8) particles/mm3 of tumor eliminated established tumors within 6 weeks in nude mouse xenografts (Rodriguez et al., Cancer Res. 57: 2559-2563, 1997). We now demonstrate that CV706-mediated cytotoxicity is synergistic with radiation. In vitro, addition of radiation to CV706 resulted in a synergistic increase of cytotoxicity toward the human prostate cancer cell line LNCaP and a significant increase of virus burst size, with no reduction in specificity of CV706-based cytopathogenicity for prostate cancer cells. In vivo, prostate-specific antigen (+) LNCaP xenografts of human prostate cancer were treated with CV706 (1 x 10(7) particles/mm3 of tumor), 10 Gy of single fraction local tumor radiation, or both. Tumor volumes of the group treated with CV706 or radiation was 97% or 120% of baseline 6 weeks after treatment. However, when the same dose of CV706 was followed 24 h later with the same dose of radiation, the tumor volume dropped to 4% of baseline at this time point and produced antitumor activity that was 6.7-fold greater than a predicted additive effect of CV706 and radiation. Histological analyses of tumors revealed that, compared with CV706 or radiation alone, combination treatment with two agents increased necrosis by 180% and 690%, apoptosis by 330% and 880%, and decreased blood vessel number by 1290% and 600%, respectively. Importantly, no increase in toxicity was observed after combined treatment when compared with CV706 or radiation alone. These data demonstrate that CV706 enhances the in vivo radioresponse of prostate tumors and support the clinical development of CV706 as a neoadjuvant agent with radiation for localized prostate cancer.

The effects of alendronate on stature and the spine deformity index.

Several diagnostic tools are now available to assess the presence and severity of vertebral deformity in patients with osteoporosis. In addition to categorical x-ray confirmation of fracture, a spine deformity index (SDI) has been developed to quantify vertebral deformity. Sequential measurements of stature are also valuable, and both methodologies have been used in Phase III clinical trials to assess the efficacy of alendronate to treat postmenopausal osteoporosis. In these clinical trials, the SDI increased (worsened) in 41% of placebo-treated patients and 33% of alendronate-treated patients (P = 0.028). Alendronate reduced mean stature loss by 35% (P = 0.005). These beneficial effects were consistent in older and younger women, and in those with and without baseline vertebral fractures. Spine deformity index and stature results corresponded with a 48% reduction in categorical vertebral fractures (assessed by means of digitized x-ray analysis) in these trials. Taken together, these data show that treatment with alendronate progressively reduced the risk for vertebral deformity height loss and vertebral fracture over 3 years of therapy.

Early changes in biochemical markers of bone turnover predict the long-term response to alendronate therapy in representative elderly women: a randomized clinical trial.

Although the antiresorptive agent alendronate has been shown to increase bone mineral density (BMD) at the hip and spine and decrease the incidence of osteoporotic fractures in older women, few data are available regarding early prediction of long-term response to therapy, particularly with regard to increases in hip BMD. Examining short-term changes in biochemical markers incorporates physiologic response with therapeutic compliance and should provide useful prognostic information for patients. The objective of this study was to examine whether early changes in biochemical markers of bone turnover predict long-term changes in hip BMD in elderly women. The study was a double-blind, placebo-controlled, randomized clinical trial which took place in a community-based academic hospital. One hundred and twenty community-dwelling, ambulatory women 65 years of age and older participated in the study. Intervention consisted of alendronate versus placebo for 2.5 years. All patients received appropriate calcium and vitamin D supplementation. The principal outcome measures included BMD of the hip (total hip, femoral neck, trochanter, and intertrochanter), spine (posteroanterior [PA] and lateral), total body, and radius. Biochemical markers of bone resorption included urinary N-telopeptide cross-linked collagen type I and free deoxypyridinoline; markers of bone formation included serum osteocalcin and bone-specific alkaline phosphatase. Long-term alendronate therapy was associated with increased BMD at the total hip (4.0%), femoral neck (3.1%), trochanter (5.5%), intertrochanter (3.8%), PA spine (7.8%), lateral spine (10.6%), total body (2.2%), and one-third distal radius (1.3%) in elderly women (all p < 0.01). In the placebo group, bone density increased 1.9-2.1% at the spine (p < 0.05) and remained stable at all other sites. At 6 months, there were significant decreases in all markers of bone turnover (-10% to -53%, p < 0.01) in women on alendronate. The changes in urinary cross-linked collagen at 6 months correlated with long-term bone density changes at the hip (r = -0.35, p < 0.01), trochanter (r = -0.36, p < 0.01), PA spine (r = -0.41, p < 0.01), and total body (r = -0.34, p < 0.05). At 6 months, patients with the greatest drop in urinary cross-linked collagen (65% or more) demonstrated the greatest gains in total hip, trochanteric, and vertebral bone density (all p < 0.05). A 30% decrease in urinary cross-linked collagen at 6 months predicted a bone density increase of 2.8-4.1% for the hip regions and 5.8-6.9% for the spine views at the 2.5-year time point (p < 0.05). There were no substantive associations between changes in biochemical markers and bone density in the placebo group. Alendronate therapy was associated with significant long-term gains in BMD at all clinically relevant sites, including the hip, in elderly women. Moreover, these improvements were associated with early decreases in biochemical markers of bone turnover. Early dynamic decreases in urinary cross-linked collagen can be used to monitor and predict long-term response to bisphosphonate therapy in elderly women. Future studies are needed to determine if early assessment improves long-term patient compliance or uncovers poor compliance, thereby aiding the physician in maximizing the benefits of therapy.

Prevention of nonvertebral fractures by alendronate. A meta-analysis. Alendronate Osteoporosis Treatment Study Groups.

OBJECTIVE: To evaluate the effect of treatment with alendronate sodium, a potent aminobisphosphonate, on the incidence of nonvertebral fractures in postmenopausal women with osteoporosis. DATA SOURCES: Published data and data on file at Merck Research Laboratories. STUDY SELECTION: All completed prospective, randomized, placebo-controlled alendronate trials of at least 2 years' duration (5 studies). DATA EXTRACTION: All subjects were women with osteoporosis between the ages of 42 and 85 years, postmenopausal at least 4 years, with lumbar spine bone mineral density (measured using dual-energy x-ray absorptiometry) at least 2.0 SD below the mean for young adult women. All women randomized to treatment with placebo or alendronate at a dose higher than 1 mg per day for at least 2 years were included. DATA SYNTHESIS: In the placebo group (n=590), 60 women reported nonvertebral fractures during 1347 patient-years at risk (overall rate, 4.45 women with fractures per 100 patient-years at risk). In the alendronate group (n = 1012), 73 women reported nonvertebral fractures during 2240 patient-years-at risk (overall rate, 3.26 women with fractures per 100 patient-years at risk). The estimated cumulative incidence of nonvertebral fractures after 3 years was 12.6% in the placebo group and 9.0% in alendronate group. The relative risk for nonvertebral fracture estimated using the Cox proportional hazards model was 0.71 (95% confidence interval,0.502-0.997) (P=.048). A reduction in risk was consistent across each of the studies and at each major site of osteoporotic fracture, including the hip and wrist. CONCLUSION: In postmenopausal women with osteoporosis, treatment with alendronate reduces the risk of nonvertebral fractures over at least 3 years.

Treatment with alendronate prevents fractures in women at highest risk: results from the Fracture Intervention Trial.

BACKGROUND: The efficacy of antiresorptive therapy in preventing fractures in women at highest fracture risk, such as very elderly women or those with severe osteoporosis, is uncertain. PARTICIPANTS AND METHODS: Using data from a double-blind, randomized, placebo-controlled clinical trial that enrolled 2027 postmenopausal women aged 55 to 81 years with low femoral neck bone mineral density (BMD) and existing vertebral fractures, we examined the consistency of the effect of treatment with alendronate sodium in preventing fractures within a priori-specified risk subgroups defined at baseline by age, bone density, number of preexisting vertebral fractures, and history of postmenopausal fracture. The women were randomized to oral administration of alendronate or placebo and followed up for an average of 2.9 years. The initial dose of alendronate sodium was 5 mg/d; the dosage was increased from 5 to 10 mg/d at 24 months. New vertebral fractures, the primary end point of this arm of the trial, were defined by morphometry as a decrease of 20% and at least 4 mm in any vertebral height between baseline and a follow-up radiograph at 36 months. Incident clinical fractures, the secondary end point, included nonspine and clinical (symptomatic) vertebral fractures. All clinical fractures were confirmed with x-ray film reports or, in the case of clinical vertebral fractures, x-ray films. RESULTS: Overall, there was a 47% significant reduction in risk of new vertebral fractures in the alendronate group compared with the placebo group. The reduction in risk of new vertebral fracture was consistent across fracture risk categories including age (relative risk [RR], 0.49 in women < 75 years compared with 0.62 in those > or = 75 years), BMD (RR, 0.54 in women with a femoral neck BMD < 0.59 g/cm2 [median] compared with 0.53 in those with a BMD > or = 0.59 g/cm2), and number of preexisting vertebral fractures (RR, 0.58 in women with 1 vertebral fracture compared with 0.52 in those with > or = 2). The overall significant 28% reduction in risk of incident clinical fractures in the alendronate group compared with the placebo group was also observed within these subgroups. Compared with the number of lower-risk women, a similar or smaller number of high-risk women needed to be treated to prevent 1 fracture. For example, 8 women aged 75 years or older compared with 9 women younger than 75 years, or 4 women with 2 or more existing vertebral fractures compared with 16 women with 1 existing vertebral fracture, needed to be treated with alendronate for 5 years to prevent 1 new vertebral fracture. CONCLUSIONS: Alendronate effectively reduces fracture risk in postmenopausal women with vertebral fractures and low BMD, including those women at highest risk because of advanced age or severe osteoporosis. Since the risk reductions observed with alendronate treatment were consistent within fracture risk categories, more fractures were prevented by treating women at highest risk.

Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Fracture Intervention Trial Research Group.

BACKGROUND: Previous studies have shown that alendronate can increase bone mineral density (BMD) and prevent radiographically defined (morphometric) vertebral fractures. The Fracture Intervention Trial aimed to investigate the effect of alendronate on the risk of morphometric as well as clinically evident fractures in postmenopausal women with low bone mass. METHODS: Women aged 55-81 with low femoral-neck BMD were enrolled in two study groups based on presence or absence of an existing vertebral fracture. Results for women with at least one vertebral fracture at baseline are reported here. 2027 women were randomly assigned placebo (1005) or alendronate (1022) and followed up for 36 months. The dose of alendronate (initially 5 mg daily) was increased (to 10 mg daily) at 24 months, with maintenance of the double blind. Lateral spine radiography was done at baseline and at 24 and 36 months. New vertebral fractures, the primary endpoint, were defined by morphometry as a decrease of 20% (and at least 4 mm) in at least one vertebral height between the baseline and latest follow-up radiograph. Non-spine clinical fractures were confirmed by radiographic reports. New symptomatic vertebral fractures were based on self-report and confirmed by radiography. FINDINGS: Follow-up radiographs were obtained for 1946 women (98% of surviving participants). 78 (8.0%) of women in the alendronate group had one or more new morphometric vertebral fractures compared with 145 (15.0%) in the placebo group (relative risk 0.53 [95% Cl 0.41-0.68]). For clinically apparent vertebral fractures, the corresponding numbers were 23 (2.3%) alendronate and 50 (5.0%) placebo (relative hazard 0.45 [0.27-0.72]). The risk of any clinical fracture, the main secondary endpoint, was lower in the alendronate than in the placebo group (139 [13.6%] vs 183 [18.2%]; relative hazard 0.72 [0.58-0.90]). The relative hazards for hip fracture and wrist fracture for alendronate versus placebo were 0.49 (0.23-0.99) and 0.52 (0.31-0.87). There was no significant difference between the groups in numbers of adverse experiences, including upper-gastrointestinal disorders. INTERPRETATION: We conclude that among women with low bone mass and existing vertebral fractures, alendronate is well tolerated and substantially reduces the frequency of morphometric and clinical vertebral fractures, as well as other clinical fractures.

Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment.

To evaluate the clinical utility of recently developed biochemical markers of bone turnover to monitor the response of osteoporotic patients to antiresorptive therapy, we compared the results of three advanced assays for markers of bone resorption and four of bone formation to high pressure liquid chromatography (HPLC)-fluorometric assays for urinary pyridinoline and deoxypyridinoline. These assays were also used to resolve the uncertainties concerning the rate of bone turnover in late postmenopausal (late-PMP) osteoporotic women. The rate of bone turnover in 85 women (mean +/- SD age, 63 +/- 6 yr) with low bone mass and all more than 5 yr postmenopausal (mean +/- SD yr PMP, 16 +/- 7 yr) was compared to that in 46 premenopausal women (mean +/- SD age, 40 +/- 5 yr) randomly selected from a large cohort and all having a normal spine bone mineral density (BMD). The late-PMP osteoporotic patients were a subset of patients enrolled in a double blind, placebo-controlled, randomized study comparing the effects of several doses of oral alendronate, a potent and specific inhibitor of bone resorption. Periodically during the 2-yr study, the women's spinal BMD and the level of several markers of bone turnover were measured. Serum total and intact osteocalcin, bone-specific alkaline phosphatase, and carboxy-terminal propeptide of type I collagen measured by RIA were used to assess bone formation. To assess bone resorption, we measured the urinary excretion of total pyridinoline (HPLC Pyr) and deoxypyridinoline (HPLC D-Pyr) by HPLC, type I collagen cross-linked N-telopeptide and urinary free PYR (F-Pyr) by enzyme-linked immunosorbent assay, and the serum concentration of type I collagen cross-linked C-telopeptide (ICTP) by RIA. All bone formation markers, except carboxy-terminal propeptide of type I collagen, and all bone resorption markers, except ICTP, were significantly increased above normal (33-171%; P < 0.001) in late-PMP osteoporotic women. The long term within-patient variability assessed over a 15-month period in the placebo group was low and was somewhat lower for serum markers (12.5-17.4%) than for urinary markers (24-29%). Under treatment with alendronate, resorption markers decreased earlier than markers of bone formation, consistent with a direct action of the drug to inhibit osteoclastic bone resorption. With the exception of F-Pyr and ICTP, the levels of bone markers were reduced to the normal premenopausal range, and this steady state was maintained from 6-15 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Two homologs of the Drosophila polarity gene frizzled (fz) are widely expressed in mammalian tissues.

The frizzled (fz) locus of Drosophila encodes a protein (Fz) with a seven-transmembrane-domain profile characteristic of G-protein-coupled receptors. In Drosophila, genetic evidence suggests that Fz functions to transmit and transduce polarity signals in epidermal cells during hair and bristle development. We have isolated from a UMR 106 rat osteosarcoma cell library a cDNA (fz-1) encoding a predicted 641-residue protein (Fz-1) with 46% homology with Drosophila Fz. We also identified a second cDNA (fz-2) encoding a protein (Fz-2) of 570 amino acids that is 80% homologous with Fz-1, with divergence most evident in the extracellular domains. Southern blots of rat genomic DNA indicated that fz-1 and fz-2 represent distinct genes. Northern analysis revealed the presence of a single fz-1 mRNA (4.7 kilobases) and two fz-2 mRNAs (2.5 and 4.5 kilobases) in rat tissues. The fz-1 and fz-2 genes are widely expressed in rat tissues with the highest steady-state levels of mRNA in kidney, liver, heart, uterus, and ovary. fz-1 and -2 mRNA levels were greater in neonatal than in corresponding adult tissues. Treatment of UMR 106 cells with bone resorbing agents including parathyroid hormone, epidermal growth factor, and 1,25-dihydroxyvitamin D3 produced increases in fz-1 and -2 mRNA levels. We suggest that hormonal induction of Fz proteins in osteoblasts serves to promote intercellular signaling required for functional responses such as increased bone resorption. Fz-1 and Fz-2 may represent products of a gene family whose members serve as transducers or intercellular transmitters of signals required for normal morphogenesis and/or differentiated function in diverse tissues.

Features of the renal parathyroid hormone-parathyroid hormone-related protein receptor derived from structural studies of receptor fragments.

Our earlier results indicated that the binding moiety of the renal PTH receptor is an 85 kD protein that is susceptible to proteolytic cleavage to a 70 kD form that supports high-affinity binding and Gs coupling, and to a 50-55 kD form that contains the ligand binding domain but does not couple to Gs. In the present study we used [125I]hPTHrP-(1-34)amide and a chemical cross-linking technique to discern the structural features of the intact 85 kD PTH/PTHrP receptor that are retained in the proteolyzed forms to "structurally map" the receptor. The results of lectin chromatography and endoglycosidase treatment show that the partially proteolyzed receptor forms retain the complex, N-linked glycans present on the intact receptor. This conclusion is further supported by the finding that wheat germ agglutinin was equally effective at competitively inhibiting specific [125I]hPTHrP-(1-34)A binding to the 70 kD form and the intact 85 kD receptor. Specific binding of [125I]hPTHrP-(1-34)A to the intact 85 kD receptor or to the 70 kD form was completely abolished by treatment with disulfide reducing agents, and both partially proteolyzed receptor forms (70- and 50 kD) were shown to retain the small (less than or equal to 14 kD) labeled fragment that is released from the intact receptor by disulfide reduction. Lectin chromatography and endoglycosidase treatment revealed that the less than or equal to 14 kD receptor component is not glycosylated. The less than or equal to 14 kD fragment does not contain a transmembrane spanning region, as its release from the membrane can be affected without detergent solubilization. Identical partial proteolytic maps of the receptor were obtained whether the receptor was covalently labeled with [125I]hPTHrP-(1-34)amide or [125I]bPTH-(1-34). These results suggest a model of the renal PTH/PTHrP receptor binding moiety as a single-chain protein in which the sites of glycosylation, ligand binding, and the functionally critical disulfide bonds are in extracellular domains near one end of the protein and the sites of proteolysis reside near the other end of the protein. These studies also provide further confirmation that PTH and PTHrP bind to a structurally indistinguishable renal receptor and validate the use of PTHrP as a ligand for studies designed to characterize and purify the PTH receptor.

Structural properties of the renal parathyroid hormone receptor: hydrodynamic analysis and protease sensitivity.

The highly specific ligand [125I]bovine (b) PTH-(1-34) and a chemical cross-linking technique were used to explore structural features of the canine renal cortical PTH receptor. Membranes isolated under conditions designed to inhibit endogenous proteolysis displayed a major 85K labeled PTH receptor moiety on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linked receptors were solubilized with Lubrol-PX and partially purified by affinity chromatography on wheat germ agglutinin-agarose, and their hydrodynamic properties were assessed [Stokes radius = 7.3 +/- 0.1 nm; sedimentation coefficient = 6.4 +/- 0.2S; partial specific volume = 0.758 +/- 0.01 ml/g; frictional coefficient = 1.68 +/- 0.04; mol wt (Mr) = 216,000 +/- 14,000]. Corrections for detergent binding and for the presence of carbohydrate yielded an estimated Mr of 166,000 +/- 11,000 for the solubilized PTH receptor. Thus, the renal PTH receptor is oligomeric, with a Mr approximating that expected of a homodimer of 85K subunits. Peptide-mapping experiments revealed the presence within the 85K PTH receptor subunit of at least two major regions sensitive to proteolytic attack. Both elastase and an endogenous renal protease(s) cleaved the PTH receptor to a 70K form that is fully functional with respect to high affinity, guanyl nucleotide-sensitive PTH binding. Cleavage in a second domain by elastase, S. aureus V8 protease, or chymotrypsin generated a 50K labeled PTH receptor fragment. Cleavage at this second site was prevented by prior occupancy of the receptor with [125I]bPTH-(1-34), suggesting that this domain may be functionally important. Reduction of receptor disulfide bonds with dithiothreitol and beta-mercaptoethanol released a low Mr (less than or equal to 14K) labeled PTH receptor component, similar treatment of renal membranes abolished specific PTH binding, indicating that an intact disulfide bond(s) is essential for receptor function. These results provide new insights into the structural basis of PTH receptor function.

The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification.

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.

In vitro secretion of deoxycorticosterone by a benign Leydig cell tumor of the testis.

We evaluated the in vitro steroidogenic potential of a benign Leydig cell tumor of the testis. Tumor tissue was found to secrete deoxycorticosterone, progesterone, 17 alpha-hydroxyprogesterone, estradiol and testosterone into the medium. No corticosterone or aldosterone was detected. The ratio of progesterone to 17 alpha-hydroxyprogesterone was 4:1, consistent with a partial enzymatic block of 17 alpha-hydroxylase. The finding of deoxycorticosterone indicates the presence of 21-hydroxylase activity within the tumor. The high local levels of estrogen produced by Leydig cell tumors may induce this enzyme system and, together with the elevated concentrations of progesterone serving as substrate, may provide a favorable hormonal milieu for extra-adrenal deoxycorticosterone production by these neoplasms.

Current concepts in acromegaly: etiology, diagnosis, and treatment.

Acromegaly is an insidious disorder of somatic growth and metabolic derangements that results from the chronic hypersecretion of growth hormone (GH) in the adult. While its precise incidence in this country remains undocumented, a recent epidemiologic study of a stable population of 3.1 million in England suggested an annual incidence of approximately 0.3 cases per 100,000, with a prevalence of diagnosed cases approaching 4 per 100,000. In the majority of cases the diagnosis, once considered, is not difficult, although in recent years the spectrum of possible etiologies has been considerably broadened. As with most diseases, the problems of diagnosis arise with the more subtle or less classical examples of the syndrome. This review will briefly discuss the possible etiologies and clinical presentation of acromegaly, and then focus on the diagnosis and management of this potentially devastating disease.