Ranjitha Katikaneni

Treating osteoporosis by targeting parathyroid hormone to bone

Osteoporosis is a major public health problem despite widespread use of bisphosphonate therapy. PTH(1-34) is a more effective treatment; but its use has been limited by side effects (hypercalcemia, tumor risk) and inconvenient dosing (daily injection). Long-acting forms of PTH are also effective but cause severe hypercalcemia, presumably from effects in kidney. We hypothesized that targeted delivery of PTH to bone using a collagen binding domain (PTH-CBD) could reduce hypercalcemia. PTH-CBD is cleared from serum within 12hours after subcutaneous administration. In ovariectomized rats, monthly administration of PTH-CBD increased spinal BMD by 14.2% with no associated hypercalcemia. Such bone-targeted anabolic agents may ultimately allow the superior efficacy of anabolic therapy to be obtained with the dosing convenience of bisphosphonates.

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.

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.

Breastfeeding Does Not Protect Against Urinary Tract Infection in the First 3 Months of Life, but Vitamin D Supplementation Increases the Risk by 76%:

Our goal was to determine if breastfeeding provides any protection against urinary tract infection (UTI) and if vitamin D supplementation imposes any additional risks for UTI in infants < 3 months of age. In this study, 40% of the children who had urine cultures were breastfed, and 18.7% of the children were exclusively breastfed. Twenty percent of all of the urine cultures tested positive, and this number was greater in females (22.5%) than in males (18.1%, P < .05). There was no significant difference between the rates of positive urine cultures in exclusively breastfed (22% vs 21%, nonsignificant [NS]) formula-fed infants. The relative risk of UTI with breastfeeding versus formula feeding was 1.03 (0.58-1.82), and any breastfeeding versus no breastfeeding was 0.92 (0.58-1.45). Vitamin D supplementation increased the UTI risk, with a relative risk of 1.76 (1.07-2.91, P < .05). However, only formula-fed infants showed an increased risk of UTI after vitamin D supplementation.

Prevention of chemotherapy-induced osteoporosis by cyclophosphamide with a long-acting form of parathyroid hormone

Background: Most chemotherapeutics reduce bone mineral density (BMD) and increase risk for fractures by causing gonadal suppression, which in turn increases bone removal. Cyclophosphamide (CYP) also has a direct effect of inhibiting bone formation and removal, making the resulting bone loss particularly difficult to treat with antiresorptive therapy. Aim: We tested whether a single dose of the anabolic agent PTH linked to a collagen binding domain (PTH-CBD) could prevent the effects of CYP-induced bone loss. Methods: Mice received either buffer alone, CYP, or CYP+ PTH-CBD. BMD and alkaline phosphatase were measured every 2 weeks for a total of 8 weeks. Results: After 6 weeks, mice treated with CYP showed expected reductions in BMD (increase from baseline: 7.4±6.9 vs 24.35±4.86% in mice without chemotherapy, p<0.05) and decrease in alkaline phosphatase levels (42.78±6.06 vs 60.62±6.23 IU/l in mice without chemotherapy, p<0.05), consistent with osteoporosis from impaired bone formation. Administration of a single dose of PTH-CBD (320 µg/kg ip) prior to CYP treatment improved BMD (change from baseline: 23.4±5.4 vs 7.4±6.9%, CYP treatment alone, p<0.05) and increased alkaline phosphatase levels (50.14±4.86 vs 42.78±6.06 IU/l in CYP treatment alone, p<0.05). BMD values and alkaline phosphatase levels were restored to those seen in mice not receiving chemotherapy. Conclusions: A single dose of PTH-CBD prior to chemotherapy reversed CYP-induced suppression of bone formation and prevented CYP-induced bone loss in mice.

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/cm2 [mean ± SD], 5.05 [% coefficient of variation (CV)], repeat scan variability (0.063 ± 0.00364 g/cm2 [mean ± SD], 5.94 [%CV]) were very low compared to variability between different animals (0.063 ± 0.00588 g/cm2 [mean ± SD], 9.64 [%CV]) and variability seen in same animal over time (0.011 ± 0.00885 g/cm2 [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/cm2) versus excised spine (0.065 g/cm2). 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.

Adjuvant liraglutide and insulin versus insulin monotherapy in the closed-loop system in type 1 diabetes: A randomized open-labeled crossover design trial

Background: The closed-loop (CL) system delivers insulin in a glucose-responsive manner and optimal postprandial glycemic control is difficult to achieve with the algorithm and insulin available. We hypothesized that adjunctive therapy with liraglutide, a once-daily glucagon-like peptide-1 agonist, would be more effective in normalizing postprandial hyperglycemia versus insulin monotherapy in the CL system, in patients with type 1 diabetes. Methods: This was a randomized, controlled, open-label, crossover design trial comparing insulin monotherapy versus adjuvant subcutaneous liraglutide 1.2 mg and insulin, using the CL system in 15 patients. Blood glucose (BG), insulin, and glucagon concentrations were analyzed. Results: The liraglutide arm was associated with overall decreased mean BG levels (P = .0002). The average BG levels from 8:00 pm (day 1) to 9:00 pm (day 2) were lower in the liraglutide arm (144.6 ± 36.31 vs 159.7 ± 50.88 mg/dl respectively; P = .0002). Two-hour postbreakfast and lunch BG profiles were better in the liraglutide arm (P < .05) and the insulin and glucagon assay values were lower (P < .0001). Postprandially, the area under the curve (AUC) for 2-hour postbreakfast and lunch BG levels were significant (P = .01, P = .03) and the AUC for glucagon, postbreakfast (P < .0001) and lunch (P < .05), was also significant. The incidence of hypoglycemia did not differ between arms (P = .83, Fisher’s exact test). Overall, adjunct liraglutide therapy plus CL was well tolerated even with expected side effects. Conclusion: This is a proof-of-concept study showing liraglutide can be a potential adjunctive therapy in addition to CL with insulin to reduce postprandial hyperglycemia in type 1 diabetes.

Use of Sitagliptin With Closed-Loop Technology to Decrease Postprandial Blood Glucose in Type 1 Diabetes:

Background: Postprandial hyperglycemia poses a challenge to closed-loop systems. Dipeptidyl peptidase-4 (DPP-4) inhibitors, like sitagliptin, reduce postprandial glucose concentrations in patients with type 2 diabetes. The objective of this study was to assess sitagliptin’s role in type 1 diabetes (T1DM) as an adjunct therapy in reducing postprandial blood glucose with an insulin-only closed-loop system. Methods: This was a randomized, double-blinded, placebo controlled, crossover design trial. The participants were18-35 years old, had T1DM, and an HbA1c of ≤ 8.5%. A dose determination study included eight subjects with T1DM. There were three study visits. Four hours after receiving study drug (placebo, sitagliptin 50 mg, sitagliptin 100 mg), subjects underwent a mixed meal tolerance test with assessment of hormone concentrations. In a second study, 15 subjects underwent two visits receiving either placebo or 100 mg of sitagliptin plus an insulin only closed-loop system for 25 hours with timed meals. Blood glucose and other hormone concentrations were analyzed using repeated measures ANOVA. Results: For the dose determination study, sitagliptin 100 mg resulted in reduced postprandial blood glucose (P = .006). For the closed-loop study, glucose concentrations were lower in the treatment group, most prominently during the first two study meals (P = .03). There was no difference in glucagon concentrations, but insulin concentrations and insulin delivery were lower in the treatment group. Conclusions: Sitagliptin may be considered as an adjunct therapy in a closed-loop setting. Larger studies are needed to determine the role of oral agents like sitagliptin to lower postprandial hyperglycemia with closed loop.

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.

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.