Berenice Y. Reed

Treating a Patient with the Werner Syndrome and Osteoporosis Using Recombinant Human Insulin-like Growth Factor

The Werner syndrome is a rare autosomal recessive disorder that in many ways resembles premature aging [1, 2]. At an early age, patients commonly develop cataracts, atherosclerosis, malignancies, and osteoporosis [3]. Because of the frequency of osteoporosis, the Werner syndrome may provide an insight into the pathogenesis and treatment of age-related bone loss. We have previously described the histomorphometric and biochemical indices of bone in a 43-year-old woman with the Werner syndrome and severe osteoporosis [4]. This patient was shown to have a low circulating concentration of insulin-like growth factor 1 (IGF-1). Diminished IGF-1 synthesis may contribute to osteoblastic suppression and bone loss because IGF-1 stimulates bone formation by increasing osteoblastic activity [5, 6]. The impairment with age in the growth hormone-insulin-like growth factor axis and resulting decrease in circulating IGF-1 levels have been implicated in the age-related decrease in lean body mass and bone mass [7]. However, an exact causal relation between reduced serum IGF-1 levels and osteoporosis has not been established [8]. Because examination of bone biopsy specimens showed that our patient had reduced osteoblastic activity in addition to low serum IGF-1 levels, we felt she would be an ideal candidate in whom to assess both the safety of recombinant human IGF-1 (rhIGF-1) and its effect on bone metabolism. We report the results of 6 months of daily subcutaneous doses of rhIGF-1. Case Report Our patients clinical presentation has been previously described and shows the typical history and physical findings of patients with the Werner syndrome [4]. Although menopause occurred when the patient was 31 years old, she began receiving conjugated estrogens and progesterone a few months after her last menstrual period and continued to receive them up to and throughout the study. Initial evaluation at our institution excluded secondary causes of osteoporosis. Skeletal radiographs showed marked radiolucency and compression fractures of almost all thoracic and lumbar vertebrae. Bone mineral densities (measured by QDR-2000; Hologic, Waltham, Massachusetts) of the L2 to L4 vertebrae, femoral neck, and radial shaft were 2.38, 3.93, and 2.0 standard deviations lower than the means for age-matched normal women, respectively. A transcortical iliac crest bone biopsy was done after tetracycline labeling [9]. The histomorphometric data were consistent with a suppressed bone formation rate in light of the normal bone resorption rate [4]. Methods The Institutional Review Board of the University of Texas Southwestern Medical Center reviewed and approved the study protocol, and the patient gave informed consent for participation in the study. The patient underwent four study phases, each lasting 4 days in the inpatient setting of the general clinical research center. The control phase took place before rhIGF-1 treatment was initiated. The patient continued to receive routine medications, including calcium citrate (200 mg of elemental Ca twice a day) and estradiol transdermal system (0.05 mg twice a week). She was kept on a constant metabolic diet consisting of 800 mg of calcium, 800 mg of phosphorus, and 100 mEq of sodium. On days 1 to 3, urine was collected in three consecutive 24-hour pools for urinary calcium, phosphorus, sodium, hydroxyproline, creatinine, and pyridinoline cross-links. We obtained fasting venous blood samples on days 1 through 4 to measure routine chemistries and intact parathyroid hormone, osteocalcin, type I procollagen C-peptide, and IGF-1 levels [10]; on day 1, we obtained a blood sample to measure 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels [11]. We determined intestinal fractional calcium absorption using fecal recovery of orally administered Calcium-47 [12]. Because diurnal variations have been reported with serum procollagen levels [13], we obtained venous blood samples every 4 hours for 24 hours on day 2 to determine diurnal levels of serum type I procollagen C-peptide. We determined bone mineral densities of the L2 to L4 vertebrae and the femoral neck using quantitative digital radiography (Hologic QDR 2000) and the bone density of the distal one third of the radius using a Norland single-photon absorptiometer (Madison, Wisconsin). Standing anterior-posterior and lateral radiographs of the thoracic and lumbar spine were also done. After we completed the baseline evaluation (control phase), the patient was treated with rhIGF-1. (Genentech [South San Francisco, California] provided rhIGF-1 [IND#39397] free of charge but gave no other support.) The drug was administered subcutaneously starting at 30 g/kg of body weight per day. At this dose, serum IGF-1 levels increased from low to normal levels that were age- and sex-matched (Figure 1). At 6 weeks, the dose was increased at increments of 15 g/kg per day to 45 g/kg per day, at 8 weeks to 60 g/kg per day, and at 10 weeks to 75 g/kg per day. Although no side effects were found at the latter dose, the serum IGF-1 level was abnormally high at 1000 ng/mL; the dose was therefore reduced to 60 g/kg per day for weeks 19 to 26. Figure 1. Serum insulin-like growth factor type 1 (IGF-1) and IGF-1-binding protein-3 (IGF-1-BP-3) levels before and during treatment with recombinant human IGF-1. The patient was readmitted to the general clinical research center after 1 month, 3 months, and 6 months of treatment. Evaluations identical to those of the control phase were done. In addition to receiving inpatient evaluations, the patient was seen as an outpatient weekly during the first 6 weeks of therapy and every 2 weeks thereafter to monitor for side effects. Results Symptoms The patient tolerated the administration of rhIGF-1 with no identifiable side effects. Body weight remained stable during treatment with rhIGF-1. We found no evidence of increased intracranial pressure such as papilledema or hypoglycemic symptoms. She reported having less discomfort in her feet and an improved sense of well-being. Serum Insulin-like Growth Factor-1 and Insulin-like Growth Factor-Binding Protein-3 Concentration As described previously, a single daily subcutaneous dose of rhIGF-1 (30 g/kg per day) could increase circulating IGF-1 levels from low to normal (Figure 1). Doses greater than 30 g/kg per day resulted in proportionally higher levels of circulating IGF-1. Random measurements showed that the serum IGF-binding protein-3 level remained normal during therapy. Markers and Measures of Bone Metabolism Serum calcium, phosphorus, and alkaline phosphatase levels did not change from baseline (Table 1). Serum osteocalcin and type I procollagen C-peptide increased at 1 month and remained elevated throughout therapy. Diurnal changes in serum type I procollagen C-peptide showed that values at 3 and 6 months of treatment were greater than those at baseline (data not shown). The mean serum type I procollagen C-peptide concentrations over 24 hours while the patient received rhIGF-1 therapy were statistically significantly higher than the baseline values. Table 1. Serum, Urine, and Bone Mineral Density Values before and during Therapy with Recombinant Human Insulin-like Growth Factor 1* Twenty-four-hour urinary calcium, hydroxyproline, and pyridinoline levels were higher after treatment with rhIGF-1 than before treatment (Table 1). The urinary pyridinoline level was lower at 6 months than at 3 months but remained elevated over the baseline value during all treatment phases. Bone Density and Intestinal Calcium Absorption During 6 months of treatment, the bone mineral density of the L2 to L4 vertebrae increased 3% (Table 1). However, the bone mineral densities of the femoral neck and radial shaft did not change. No new spinal fractures were shown on radiographs done during the study. Intestinal calcium absorption was in the low normal range (normal, 40% to 60%) [14] at baseline and was the same after 6 months of rhIGF-1 treatment. Absorption was approximately 12% higher during the first and third months of treatment. No significant changes were noted in serum values for 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and parathyroid hormone levels. Other Tests Other biochemical measurements before and during therapy with rhIGF-1 are listed in Table 1. Fasting serum glucose, cholesterol, and triglyceride levels did not change. We found no disturbances in serum electrolytes, liver enzymes, or measures of renal function. Discussion The administration of rhIGF-1 increased several biochemical markers of bone turnover. Serum type 1 procollagen C-peptide and osteocalcin levels, both markers of bone formation, increased, although total serum alkaline phosphatase levels did not change. Urinary pyridinoline cross-links and hydroxyproline, both measures of bone resorption, increased with rhIGF-1 therapy compared with baseline values. Moreover, 24-hour urinary calcium levels increased in the absence of increased intestinal calcium absorption or serum 1,25-dihydroxyvitamin D, suggesting that bone was the source of increased urinary calcium. Two reports of 1 week of treatment with rhIGF-1 have described increased bone turnover in normal postmenopausal women and in a man with idiopathic osteoporosis [14, 15]. In our patient being treated with transdermal estrogen, exogenous IGF-1 overcame the antiresorptive action of estrogen. Despite the short duration of treatment, bone mineral density at the lumbar spine increased in excess of the coefficient of variation (2%) of the instrument used. Because bone mass measured at the femoral neck and radial shaft did not change compared with baseline values, the apparent gain in bone mass in the spine was probably not caused by bone redistribution but rather by increased bone formation in excess of resorption. A single daily dose of rhIGF-1 administered subcutaneously at a dosage of 30 to 75 g/kg per day was sufficient to increase the circulating IGF-1 level to normal and greater-th

Effects of high levels of fluoride on bone formation: An in vitro model system

In order to develop an in vitro model for the study of the effects of different agents on biomineralization, a three-dimensional cell culture system was investigated at different levels of fluoride. Rat fetal osteoblasts were seeded onto collagen discs and maintained in a culture medium for 40 days. Results showed that, at 40 days, the cultured matrices had a Ca:P ratio, mineral content and Fourier transform infrared (FTIR) spectrum that were close to those seen for normal rat bone. Viable cells, observed by light microscopy, were present in the matrix at 40 days. The formation of a mineralized matrix in this experimental set-up provided a model for exploring in vitro the effects of high levels of fluoride on bone. The fluoride content of the mineral formed in the cultures showed a dose-dependent increase in fluoride content with time. Also, an increase in the crystallinity of the apatite in the presence of fluoride, was observed by FTIR. The Ca:P ratio and percentage mineral by weight showed no apparent differences among the groups. The three-dimensional model used for this study has the potential to be a powerful tool in the study of time-dependent effects of drugs and other factors on osteoblast cell functions and subsequently on matrix mineralization.

Twenty-four hour growth hormone secretion in a patient with Werner's syndrome

OBJECTIVE To assess the 24-h endogenous secretory growth hormone (GH) profile and serum insulin-like growth factor-I (IGF-I) response to exogenous recombinant human growth hormone (rhGH) in a patient with Werners syndrome. DESIGN Blood sampling every 20 min for 24 h followed by three daily injections of growth hormone. SETTING General Clinical Research Center. PATIENTS Single patient with Werners syndrome. MEASUREMENTS Serum GH and IGF-I. RESULTS Growth hormone pulses were absent during the 24-h monitoring period. Likewise, integrated GH concentrations were very low at 0.25 mu min/mL, and no peaks occurred after sleep onset. Following single daily administration of rhGH, serum GH and IGF-I rose. CONCLUSIONS Our findings support previous but less extensive studies suggesting patients with Werners syndrome have reduced growth hormone levels. Preliminary investigations using rhGH in patients with Werners syndrome should be considered.

The Genetics of Stone Disease

The lifetime risk of stone formation is estimated at 5–10%; hence, stone disease represents one of the most frequent causes of hospitalization in the United States (1). Various intrinsic and extrinsic factors are associated with risk for stone formation. Among intrinsic factors are race, sex, and genetics (2,3). Over the past decade significant advances have occurred in our understanding of the underlying genetic lesions that are associated with many forms of stone disease. However, it is interesting to note that most advances in the identification of genetic defects have been made in the rarer forms of stone disease. Progress toward understanding the genetic contribution in the more common forms of calcium oxalate stone disease has been impeded by the fact that many forms of this disease are complex, associated with phenotypic variability, and further compounded by multifactorial inheritance. A recent review of the literature indicates that 27 individual chromosomal loci have been associated with various forms of urolithiasis (4). All modes of inheritance are represented among the various genetic stone diseases.

Comparison of Pediatric Stone-Formers with and without mutations in Soluable Adenylate Cyclase

Abstract Purpose Two-thirds of children with urolithiasis have hypercalciuria. Recently, a candidate gene for absorptive hypercalciuria (AH) was mapped to chromosome 1q23.3-24 in the adult population. In adults, the presence of up to 6 identified base substitutions in the soluble adenylate cyclase gene (sAC) was associated with a 2.2- to 3.5-fold increased risk for AH. We screened a pediatric population of stone formers for sAC sequence variations. Material and Methods Pediatric patients with stone disease were offered study participation if they met the following criteria: family history of stones; hypercalciuria (urine calcium/creatinine ratio > 0.21 mg/mg creatinine or 24 hour calcium excretion > 4 mg/kg/day); and/or calcium stones on stone analysis. Serum laboratories, spot and 24-hour urine, and bone mineral density (BMD) were collected. Patients were excluded for metabolic conditions predisposing to stone disease. Probands were genotyped for 6 previously identified base substitutions in the sAC gene. Comparisons were made between probands with and without sAC substitutions using Fishers exact test. Results Of 46 children recruited, sAC substitutions were identified in 19 (41%). Clinical parameters were similar in patients with and without substitutions: positive family history in 14/17 (82%) vs. 21/27 (78%); hypercalciuria in 13/19 (68%) vs. 22/27 (82%); and mean number of stone events 2.1 vs. 2.8. Overall, 5/16 (31%) had abnormal BMD (age-adjusted Z-scores Conclusions Of 46 pediatric stone formers, 31% had abnormal BMD scores, and 41% of probands had sAC gene base changes previously identified in adults to increase risk for stones and osteoporosis. This warrants further investigation for potential genetic screening and therapeutic interventions to prevent pediatric stone formation and long-term osteoporotic risk.