Novel bone-targeted parathyroid hormone-related peptide antagonists inhibit breast cancer bone metastases.

Patients with advanced breast cancer often develop bone metastases. Treatment is limited to palliative care. Parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) antagonists for bone metastases failed clinically due to short half-life and inadequate concentration in bone. We synthesized two novel PTHrP antagonists fused to an inert bacterial collagen binding domain (CBD) that directs drugs to bone. PTH(7-33)-CBD is an N-terminal truncated PTHrP antagonist. [W2]PTH(1-33)-CBD is an PTHrP inverse-agonist. The aim of this study was to assess PTH(7-33)-CBD to reduce breast cancer bone metastases and prevent osteolytic destruction in mice and to assess both drugs for apoptosis of breast cancer cells in vitro and inhibition of PTH receptor (PTHR1). PTH(7-33)-CBD (1000 µg/kg, subcutaneous) or vehicle was administered 24 h prior to MDA-MB-231 breast cancer cell inoculation into the tibia marrow. Weekly tumor burden and bone density were measured. Pharmacokinetic analysis of PTH(7-33)-CBD in rat serum was evaluated. Drug effect on cAMP accumulation in SaOS-2 osteosarcoma cells and apoptosis of MDA-MB-231 cells was assessed. PTH(7-33)-CBD reduced MDA-MB-231 tumor burden and osteolytic destruction in mice 4-5 weeks post-treatment. PTH(7-33)-CBD (1000 µg/kg i.v. and subcutaneous) in rats was rapidly absorbed with peak concentration 5-min and terminal half-life 3-h. Bioavailability by the subcutaneous route was 43% relative to the i.v. route. PTH(7-33)-CBD was detected only on rat periosteal bone surfaces that stained positive for collagen-1. PTH(7-33)-CBD and [W2]PTH(1-33)-CBD (10-8M) blocked basal and PTH agonist-induced cAMP accumulation in SaOS-2 osteosarcoma cells. Both drugs induced PTHR1-dependent apoptosis of MDA-MB-231 cells in vitro. Novel bone-targeted PTHrP antagonists represent a new paradigm for treatment of breast cancer bone metastases.

Central Precocious Puberty as a Complication of Therapy with Adrenocorticotropin (ACTH) and an Aromatase Inhibitor for Refractory Nephrotic Syndrome.

Glucocorticoids are typically prescribed for the treatment of idiopathic nephrotic syndrome of childhood. In selected patients with refractory focal segmental glomuerulosclerosis (FSGS), adrenocorticotropin (ACTH) can be used to induce remission and decrease the progression of the disease. We report a 6 8/12-year-old girl with recurrent proteinuria, resistant to standard immunotherapy. She underwent related renal transplant but again developed proteinuria and was started on ACTH. She subsequently developed peripheral precocious puberty (PPP), presumably from peripheral aromatization of adrenal androgens. She was started on an aromatase inhibitor, and her ACTH dose was slowly decreased. She then developed central precocious puberty (CPP). We hypothesize that treatment of her peripheral precocious puberty with an aromatase inhibitor may have triggered central precocious puberty.

Incidence and Outcomes of Pituitary Microadenomas in Children with Short Stature/Growth Hormone Deficiency.

BACKGROUND/AIMS: Patients with short stature (SS)/growth hormone deficiency (GHD) and precocious puberty (PP) undergo brain MRI to evaluate for structural brain abnormalities or pituitary lesions, and pituitary microadenomas are a common finding. Theoretically, a mass effect from these lesions could cause GHD and growth hormone treatment could cause them to enlarge, but they should not cause PP, at least in females. METHODS: We investigated if pituitary microadenomas cause GHD by comparing their incidence in patients with SS/GHD to that in females with PP. We performed a retrospective chart review of patients with these disorders who had a brain MRI between 2000 and 2013. RESULTS: The incidence of microadenoma was high in both groups, 18.5% for SS (n = 346) and 21.1% for PP females (n = 194), but did not differ between groups (p = 0.46). In patients with microadenomas, repeat imaging showed resolution in 58% (SS, n = 33) and 67% (PP females, n = 21). Importantly, none of the lesions grew, even in patients treated with growth hormone. CONCLUSIONS: Pituitary microadenomas are common in children with GHD/SS and PP, but it does not appear that they are a cause of GHD. They appear to be of limited clinical significance and should not be considered a contraindication to growth hormone therapy.

Commentary on the Impact of Obesity on PediatricDiabetes.

While conventionally most children diagnosed with diabetes are thought to have type 1 diabetes mellitus (T1DM), with the increased prevalence of obesity, more are being affected by type 2 (T2) DM. Obesity leads to increased insulin resistance, which over time can lead to progressive ß-cell failure and ultimately T2DM. However, patients developing T1DM may also be obese, making both the proper classification and management of diabetes in children more challenging. In this commentary, the authors discuss the impact ofobesity on the presentation of pediatric diabetes, how to differentiate between T1DM and T2DM, and the proper management of both diseases.

Treatment of Vitamin D Deficiency in Predominantly Hispanic and Black Adolescents: A Randomized Clinical Trial.

OBJECTIVES: To compare 3 different treatment regimens for vitamin D deficiency in minority adolescents and to explore factors that impact treatment efficacy. STUDY DESIGN: We conducted an 8-week, prospective, open-label, randomized clinical trial in an urban, academic, children's hospital. A total of 183 vitamin D-deficient adolescents, mean 25-hydroxyvitamin D or 25(OH)D 13.7 ± 3.9 ng/mL; mean age 16.6 ± 2.2 years, were randomized into 3 vitamin D3 (cholecalciferol) treatment arms: 50,000 IU/wk; 5000 IU/d; and 1000 IU/d. Serum 25(OH)D and vitamin D binding protein (VDBP) levels were measured pre-and posttreatment; 122 (67%) participants completed posttreatment measures. Complete-case and multiple-imputation, intention-to-treat analyses were performed. RESULTS: Mean change in 25(OH)D level posttreatment was significantly different among the 3 arms, 24.9 ± 15.1 vs 21.0 ± 15.2 vs 6.2 ± 6.5 ng/mL, for 50,000 IU, 5000 IU, and 1000 IU doses, respectively, P < .001. Both high-dose treatments were effective in increasing the 25(OH)D level out of deficiency range (≥ 20 ng/mL) in more than 80% of participants, and 60% remained deficient after low-dose treatment. Only 72%, 56%, and 2% achieved vitamin D sufficiency (>30 ng/mL) with 50,000 IU, 5000 IU, and 1000 IU doses, respectively, P < .001. Obese participants had substantially less mean change in 25(OH)D level after treatment than normal-weight participants, 13.7 ± 10.7 vs 21.9 ± 16.9 ng/mL, P < .001. Mean baseline VDBP level was almost twice as high in Hispanic compared with black participants (P < .001) and did not alter treatment response or change with treatment. CONCLUSIONS: Adult-sized adolescents require 8 weeks of high-dose cholecalciferol, at least 5000 IU/d, to correct deficiency. Obese adolescents have poorer response to treatment and may need higher doses than nonobese youth. Hispanic and black adolescents have different VDBP levels but similar treatment responses. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01784029.

Therapy for Alopecia Areata in Mice by Stimulating the Hair Cycle with Parathyroid Hormone Agonists Linked to a Collagen-Binding Domain.

UNLABELLED: Alopecia areata is a common disorder in which autoimmune destruction of hair follicles results in patchy hair loss. Currently there is no adequate therapy, although immune modulator therapies are currently in development. Parathyroid hormone (PTH) is a hair cycle stimulator which shows promise in treating various forms of alopecia, although its short half-life limits its clinical use. PTH-CBD is a PTH analog which binds collagen, prolonging retention in skin. We tested effects of PTH-CBD in C3H/HeJ-engrafted mice, the animal model for alopecia areata, on hair growth and found that a significant proportion of animals had reduced hair loss (PTH-CBD: 13/21, 62% vs. CONTROL: 3/10, 30%; P<0.01). Histological analysis showed no change in immune response, but there was increased number of anagen hair follicles and increased production of beta-catenin, a factor which initiates the anagen phase of the hair cycle. PTH-CBD thus shows promise as a therapy for alopecia areata, either alone or in conjunction with immune modulation therapy.

A new technique for quantitative analysis of hair loss in mice using grayscale analysis.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Parathyroid hormone-related peptide and the hair cycle - is it the agonists or the antagonists that cause hair growth?

While the effects of PTHrP have been studied for almost 20 years, most of these studies have focused on effects on the termination of the anagen phase, giving an incomplete picture of the overall effect of PTHrP on the hair cycle. PTHrP was determined in several experimental models to promote transition of hair follicles from anagen to catagen phase, which by itself would suggest that PTHrP blockade might prolong the anagen phase and promote hair growth. However, clinical trials with topically applied PTHrP antagonists have been disappointing, leading to a reconsideration of this model. Additional studies performed in mouse models where hair follicles are damaged (alopecia areata, chemotherapy-induced alopecia) suggest that PTHrP has effects early in the hair cycle as well, promoting hair follicles' entry into anagen phase and initiates the hair cycle. While the mechanism of this has yet to be elucidated, it may involve activation of the Wnt pathway. Thus, the overall effect of PTHrP is to stimulate and accelerate the hair cycle, and in the more clinically relevant models of hair loss where hair follicles have been damaged or become quiescent, it is the agonists, not the antagonists, which would be expected to promote hair growth.

Parathyroid hormone linked to a collagen binding domain promotes hair growth in a mouse model of chemotherapy-induced alopecia in a dose-dependent manner.

Chemotherapy-induced alopecia is a major source of psychological stress in patients undergoing cancer chemotherapy, and it can influence treatment decisions. Although there is currently no therapy for alopecia, a fusion protein of parathyroid hormone and collagen binding domain (PTH-CBD) has shown promise in animal models. The aim of this study was to determine whether there are dose-dependent effects of PTH-CBD on chemotherapy-induced alopecia in a mouse model. C57BL/6J mice were waxed to synchronize hair follicles; treated on day 7 with vehicle or PTH-CBD (100, 320, and 1000 mcg/kg subcutaneous injection); and treated on day 9 with vehicle or cyclophosphamide (150 mg/kg intraperitoneally). Mice were photographed every 3-4 days and killed on day 63 for histological analysis. Photographs were quantified by gray scale analysis to assess hair content. Mice not receiving chemotherapy showed regrowth of hair 2 weeks after waxing and normal histology after 2 months. Mice receiving chemotherapy alone showed marked hair loss after chemotherapy, which was sustained for 10 days and was followed by rapid regrowth of a normal coat. Histological analysis revealed rapid cycling dystrophic anagen/catagen follicles. Animals receiving chemotherapy and PTH-CBD showed decreased hair loss and more rapid regrowth of hair than that seen with chemotherapy alone (increased hair growth by gray scale analysis, P<0.05), and the effects were dose dependent. Histologically, hair follicles in animals receiving the highest dose of PTH-CBD were in a quiescent phase, similar to that in mice that did not receive chemotherapy. Single-dose subcutaneous administration of PTH-CBD showed dose-dependent effects in minimizing hair loss and speeding up recovery from chemotherapy-induced alopecia.

Pharmacokinetics in rats of a long-acting human parathyroid hormone-collagen binding domain peptide construct.

The pharmacokinetics of a hybrid peptide consisting of the N-terminal biologically active region of human parathyroid hormone (PTH) linked to a collagen-binding domain (CBD) were evaluated in female Sprague-Dawley rats. The peptide, PTH-CBD, consists of the first 33 amino acids of PTH linked as an extension of the amino acid chain to the CBD peptide derived from ColH collagenase of Clostridium histolyticum. Serum concentrations arising from single dose administration by the subcutaneous and intravenous routes were compared with those measured following route-specific mole equivalent doses of PTH(1-34). Population-based modeling demonstrated similar systemic absorption kinetics and bioavailability for both peptides. Exposure to PTH-CBD was sixfold higher because of a systemic clearance of approximately 20% relative to PTH(1-34); however, these kinetics were consistent with more than 95% of a dose being eliminated from serum within 24 h. Results obtained support continued investigation of PTH-CBD as a bone-targeted anabolic agent for the treatment of postmenopausal osteoporosis.

Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model.

Alopecia is a psychologically devastating complication of chemotherapy for which there is currently no effective therapy. PTH-CBD is a collagen-targeted parathyroid hormone analog that has shown promise as a therapy for alopecia disorders. This study compared the efficacy of prophylactic versus therapeutic administration of PTH-CBD in chemotherapy-induced alopecia using a mouse model that mimics the cyclic chemotherapy dosing used clinically. C57BL/6J mice were treated with a single subcutaneous injection of PTH-CBD (320 mcg/kg) or vehicle control before or after hair loss developing from three courses of cyclophosphamide chemotherapy (50-150 mg/kg/week). Mice receiving chemotherapy alone developed hair loss and depigmentation over 6-12 months. Mice pretreated with PTH-CBD did not develop these changes and maintained a normal-appearing coat. Mice treated with PTH-CBD after development of hair loss showed a partial recovery. Observations of hair loss were confirmed quantitatively by gray scale analysis. Histological examination showed that in mice receiving chemotherapy alone, there were small, dystrophic hair follicles mostly in the catagen phase. Mice receiving PTH-CBD before chemotherapy showed a mix of normal-appearing telogen and anagen hair follicles with no evidence of dystrophy. Mice receiving PTH-CBD therapy after chemotherapy showed intermediate histological features. PTH-CBD was effective in both the prevention and the treatment of chemotherapy-induced alopecia in mice, but pretreatment appears to result in a better cosmetic outcome. PTH-CBD shows promise as an agent in the prevention of this complication of chemotherapy and improving the quality of life for cancer patients.

Clinical response to combined therapy of cyclosporine and prednisone.

Reported is a patient with severe alopecia areata, multiple autoimmune diseases (chronic lymphocytic thyroidis, primary ovarian failure), and Down syndrome. She had a poor response to topical treatment with glucocorticoids and minoxidil, but showed some improvement with glucocorticoid injections. At the time of evaluation, she had hair loss on 85-90% of her scalp. She was treated initially with oral prednisone 50 mg per day for 2 weeks, followed by a 3-month course of prednisone 10 mg per day and cyclosporine 125 mg (4 mg kg(-1)) two times per day. She responded well with excellent regrowth of hair on the scalp, and prednisone was tapered and ultimately discontinued. Importantly, her parents noted marked improvement in sense of well-being. Several months after discontinuing treatment, she developed hyperpigmentation on the trunk consistent with confluent and reticulated papillomatosis; she has several known risk factors for this disorder, but it is not clear if this is related to her previous treatment.

Therapy for alopecia areata in mice using parathyroid hormone agonists and antagonists, linked to a collagen-binding domain.

Alopecia areata is a common form of hair loss in which autoimmune-mediated destruction of hair follicles causes patchy hair loss, for which there is no adequate therapy. Parathyroid hormone (PTH) induces the hair cycle and promotes hair growth. PTH-CBD is a fusion protein of PTH and a bacterial collagen-binding domain (CBD), leading to targeted delivery to and retention in the skin collagen. We tested the effects of a single dose of PTH-CBD (low or high dose) on an animal model for alopecia areata, the C3H/HeJ engrafted mouse. In all the treated animals, there was a rapid (1-4 days) increase in hair growth, with sustained effects observed over a 2-month period (7/10 total treated mice<40% hair loss based on gray scale analysis, vs. 2/5 in vehicle control animals). Histological examination revealed massive stimulation of anagen VI hair follicles in treated animals despite an ongoing immune response. PTH-CBD thus shows promise as a therapy for alopecia areata, likely in conjunction with a mild immune suppressant, such as hydrocortisone cream.

A single injection of the anabolic bone agent, parathyroid hormone-collagen binding domain (PTH-CBD), results in sustained increases in bone mineral density for up to 12 months in normal female mice.

Parathyroid hormone (PTH) is the most effective osteoporosis treatment, but it is only effective if administered by daily injections. We fused PTH(1-33) to a collagen binding domain (PTH-CBD) to extend its activity, and have shown an anabolic bone effect with monthly dosing. We tested the duration of action of this compound with different routes of administration. Normal young C57BL/6J mice received a single intraperitoneal injection of PTH-CBD (320 µg/kg). PTH-CBD treated mice showed a 22.2 % increase in bone mineral density (BMD) at 6 months and 12.8 % increase at 12 months. When administered by subcutaneous injection, PTH-CBD again caused increases in BMD, 15.2 % at 6 months and 14.3 % at 12 months. Radiolabeled PTH-CBD was concentrated in bone and skin after either route of administration. We further investigated skin effects of PTH-CBD, and histological analysis revealed an apparent increase in anagen VI hair follicles. A single dose of PTH-CBD caused sustained increases in BMD by >10 % for 1 year in normal mice, regardless of the route of administration, thus showing promise as a potential osteoporosis therapy.

Reversed seasonal variation in maternal vitamin D levels in southern Louisiana.

Vitamin D is critical in bone and mineral homeostasis, particularly in the prevention of rickets in children. Levels of vitamin D in cord blood were measured in a population from New Orleans as an index of maternal vitamin D status at the time of delivery. Cord blood samples from infants born during the summer and winter showed lower 25-hydroxyvitamin D levels compared with those from infants born during fall and spring, indicating an unusual pattern of seasonality where vitamin D levels were among the lowest in the season with the greatest sunlight. It is important to establish screening and supplementation guidelines based on observed regional trends and risk factors, in addition to considerations based on global recommendations.

Treatment for chemotherapy-induced alopecia in mice using parathyroid hormone agonists and antagonists linked to a collagen binding domain.

Parathyroid hormone (PTH) agonists and antagonists have been shown to improve hair growth after chemotherapy; however, rapid clearance and systemic side-effects complicate their usage. To facilitate delivery and retention to skin, we fused PTH agonists and antagonists to the collagen binding domain (CBD) of Clostridium histolyticum collagenase. in-vitro studies showed that the agonist fusion protein, PTH-CBD, bound collagen and activated the PTH/parathyroid hormone-related peptide receptor in SaOS-2 cells. The antagonist fusion proteins, PTH(7-33)-CBD and PTH([-1]-33)-CBD, also bound collagen and antagonized PTH(1-34) effect in SaOS-2 cells; however, PTH(7-33)-CBD had lower intrinsic activity. Distribution studies confirmed uptake of PTH-CBD to the skin at 1 and 12 hr after subcutaneous injection. We assessed in vivo efficacy of PTH-CBD and PTH(7-33)-CBD in C57BL/6J mice. Animals were depilated to synchronize the hair follicles; treated on Day 7 with agonist, antagonist, or vehicle; treated on Day 9 with cyclophosphamide (150 mg/kg i.p.) or vehicle; and sacrificed on Day 39. Normal mice (no chemo and no treatment) showed rapid regrowth of hair and normal histology. Chemo+Vehicle mice showed reduced hair regrowth and decreased pigmentation; histology revealed reduced number and dystrophic anagen/catagen follicles. Chemo+Antagonist mice were grossly and histologically indistinguishable from Chemo+Vehicle mice. Chemo+Agonist mice showed more rapid regrowth and repigmentation of hair; histologically, there was a normal number of hair follicles, most of which were in the anagen phase. Overall, the agonist PTH-CBD had prominent effects in reducing chemotherapy-induced damage of hair follicles and may show promise as a therapy for chemotherapy-induced alopecia.

A treatment trial of vitamin D supplementation in breast-fed infants: universal supplementation is not necessary for rickets prevention in Southern Louisiana.

This study was conducted to determine if vitamin D supplementation is required to prevent rickets in breast-fed infants. Breast-feeding rates are increasing, and there are concerns about whether the vitamin D content of breast milk is sufficient. There are a few treatment trials of vitamin D supplementation in breast-fed infants; these were conducted in northern climates. The authors therefore performed a prospective clinical trial comparing vitamin D supplementation with placebo as control in southern Louisiana. Blood samples and questionnaires were collected at birth, 2, 4, and 6 months of age. There were no cases of rickets observed, and no differences in alkaline phosphatase levels between groups. Thus, there was no evidence that vitamin D supplementation reduced rickets risk in the authors' study population. This suggests that the current recommendations for universal vitamin D supplementation of breast-fed infants throughout the United States may need to be revised.

A new technique for precisely and accurately measuring lumbar spine bone mineral density in mice using clinical dual energy X-ray absorptiometry (DXA).

Dual Energy X-ray Absorptiometry (DXA) is effective in measuring bone mineral density (BMD) in mice for early detection of osteoporosis. However, scanners designed for use with small animals (i.e. PIXImus) are very expensive. Used human DXA machines are cheaper to obtain, but analysis of scans from these instruments is operator-dependent. Obtaining reliable data depends on having a single operator analyze the scans in a blinded fashion. Scan quality is improved by excising the bone prior to scanning, which does not allow serial measurements. This study describes a novel method of analyzing lumbar spine BMD in mice using whole body DXA. This non-invasive technique has a high degree of precision and reproducibility, with good correlation between multiple observers. Inter-observer variability (0.063 +/- 0.00317 g/cm(2) [mean +/- SD], 5.05 [% coefficient of variation (CV)], repeat scan variability (0.063 +/- 0.00364 g/cm(2) [mean +/- SD], 5.94 [%CV]) were very low compared to variability between different animals (0.063 +/- 0.00588 g/cm(2) [mean +/- SD], 9.64 [%CV]) and variability seen in same animal over time (0.011 +/- 0.00885 g/cm(2) [mean +/- SD], 80.68 [%CV]). The measurement error is thus smaller than the biological variation. Accuracy was determined by comparing average peak BMD from two scans per mouse in-vivo (0.066 g/cm(2)) versus excised spine (0.065 g/cm(2)). Furthermore, correlation between bone ash weights and whole body lumbar spine BMD measurements (p < 0.0001) was highly significant. This technique thus shows a high degree of precision and accuracy, even with multiple observers, for measuring BMD in mice using a DXA machine designed for clinical use.