Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction.

BACKGROUND: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events. METHODS: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice. RESULTS: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions. CONCLUSIONS: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

Development, implementation and evaluation of an optimized cross-sectoral rehabilitation program after acute hip fracture surgery: A mixed-methods study.

Fall-related hip fracture (HF) is a frequent trauma in Scandinavia with a yearly incidence of 8,000 among ≥65-year-old citizens in Denmark. The rising incidence and global predictions are alarming since a HF is a major, and potentially fatal, trauma to the citizen, requiring acute surgery, a multimodal approach and post-operative crosssectoral rehabilitation. However, continuity of the rehabilitation program is frequently interrupted in the transition between sectors, compromising optimal recovery of frail citizens. Thus, there is a need to develop and implement optimized cross-sectoral rehabilitation after HF. The purpose of this explorative study was to develop, implement and evaluate an optimized cross-sectoral rehabilitation program (OCRP) after HF surgery using validated theoretical frameworks. OCRP was developed, implemented and evaluated in one municipality using a pragmatic user-centered approach, quantitative and qualitative data collection and theoretical frameworks including the Behavior Change Wheel (BCW) and RE-AIM. Results of OCRP showed optimized rehabilitation based on motivated health professionals, high patient satisfaction and tendencies of improved levels of physical function. No re-referrals to rehabilitation were reported after OCRP. The BCW, RE-AIM and user-centered approach to program development, implementation and evaluation are useful to apply in program development and evaluation processes across sectors, professions, and medical specialties.

Gamete preservation: knowledge, concerns and intentions of Israeli and Danish students regarding egg and sperm freezing.

RESEARCH QUESTION: What do Danish and Israeli students of both sexes know about age in relation to fertility and gamete preservation, and what are their concerns and intentions for the future in this regard? DESIGN: A cross-sectional comparative study of male and female Danish and Israeli students was conducted between November 2018 and April 2019. A total of 1010 students, 508 from Denmark and 502 from Israel, completed questionnaires assessing knowledge, perceptions and intentions regarding gamete preservation. RESULTS: More than 70% of both genders in Israel thought that women start experiencing fertility decline at age 35 and up. A total of 60% of Danish women and 51% of Danish men chose 29-34 as the time where fertility decline starts. Some 95% of Danish students chose 20-29 as the best age for egg freezing, while the corresponding number in Israel was 85%, regardless of gender. In total, 51% of Israeli women said they are extremely or very worried about future infertility, compared with 31% of Danish women, 26% of Israeli men and 12% of Danish men. Regarding preservation intentions, no gender differences were found. Some 3% of Israeli students said they would consider gamete preservation, as compared with 14% of Danish students. CONCLUSIONS: These findings suggest a widespread worry among Danish and Israeli women about their future fertility. Danish students report more awareness of age-related fertility decline. Unique to this study is the inclusion of male students. The preliminary findings reveal that men are less worried about their reproductive future.

Design and Preclinical Development of TransCon PTH, an Investigational Sustained-Release PTH Replacement Therapy for Hypoparathyroidism.

Hypoparathyroidism (HP) is a condition of parathyroid hormone (PTH) deficiency leading to abnormal calcium and phosphate metabolism. The mainstay of therapy consists of vitamin D and calcium supplements, as well as adjunct Natpara (PTH(1-84)). However, neither therapy optimally controls urinary calcium (uCa) or significantly reduces the incidence of hypercalcemia and hypocalcemia. TransCon PTH, a sustained-release prodrug of PTH(1-34) in development for the treatment of HP, was designed to overcome these limitations. To determine the pharmacokinetics and pharmacodynamics of TransCon PTH, single and repeat s.c. dose studies were performed in rats and monkeys. TransCon PTH demonstrated a half-life of 28 and 34 hours in rats and monkeys, respectively. After repeated dosing, an infusion-like profile of the released PTH, characterized by low peak-to-trough levels, was obtained in both species. In intact rats and monkeys, daily subcutaneous administration of TransCon PTH was associated with increases in serum calcium (sCa) levels and decreases in serum phosphate levels (sP). In monkeys, at a single dose of TransCon PTH that increased sCa levels within the normal range, a concurrent decrease in uCa excretion was observed. In 4-week repeat-dose studies in intact rats and monkeys, uCa excretion was comparable to controls across all dose levels despite increases in sCa levels. Further, in a rat model of HP, TransCon PTH normalized sCa and sP levels 24 hours per day. This was in contrast to only transient trends toward normalization of sCa and sP levels with an up to 6-fold higher molar dose of PTH(1-84). After repeated dosing to HP rats, uCa excretion transiently increased, corresponding to increases in sCa above normal range, but at the end of the treatment period, uCa excretion was generally comparable to sham controls. TransCon PTH was well tolerated and the observed pharmacokinetics and pharmacodynamics were in line with the expected action of physiological replacement of PTH. © 2019 American Society for Bone and Mineral Research.

TransCon CNP, a Sustained-Release C-Type Natriuretic Peptide Prodrug, a Potentially Safe and Efficacious New Therapeutic Modality for the Treatment of Comorbidities Associated with Fibroblast Growth Factor Receptor 3-Related Skeletal Dysplasias.

TransCon CNP is a C-type natriuretic peptide (CNP-38) conjugated via a cleavable linker to a polyethylene glycol carrier molecule, designed to provide sustained systemic CNP levels upon weekly subcutaneous administration. TransCon CNP is in clinical development for the treatment of comorbidities associated with achondroplasia. In both mice and cynomolgus monkeys, sustained exposure to CNP via TransCon CNP was more efficacious in stimulating bone growth than intermittent CNP exposure. TransCon CNP was well tolerated with no adverse cardiovascular effects observed at exposure levels exceeding the expected clinical therapeutic exposure. At equivalent dose levels, reductions in blood pressure and/or an increase in heart rate were seen following single subcutaneous injections of the unconjugated CNP-38 molecule or a daily CNP-39 molecule (same amino acid sequence as Vosoritide, USAN:INN). The half-life of the daily CNP-39 molecule in cynomolgus monkey was estimated to be 20 minutes, compared with 90 hours for CNP-38, released from TransCon CNP. C max for the CNP-39 molecule (20 µg/kg) was approximately 100-fold higher, compared with the peak CNP level associated with administration of 100 µg/kg CNP as TransCon CNP. Furthermore, CNP exposure for the daily CNP-39 molecule was only evident for up to 2 hours postdose (lower limit of quantification 37 pmol/l), whereas TransCon CNP gave rise to systemic exposure to CNP-38 for at least 7 days postdose. The prolonged CNP exposure and associated hemodynamically safe peak serum concentrations associated with TransCon CNP administration are suggested to improve efficacy, compared with short-lived CNP molecules, due to better therapeutic drug coverage and decreased risk of hypotension. SIGNIFICANCE STATEMENT: The hormone C-type natriuretic peptide (CNP) is in clinical development for the treatment of comorbidities associated with achondroplasia, the most common form of human dwarfism. The TransCon Technology was used to design TransCon CNP, a prodrug that slowly releases active CNP in the body over several days. Preclinical data show great promise for TransCon CNP to be an effective and well-tolerated drug that provides sustained levels of CNP in a convenient once-weekly dose, while avoiding high systemic CNP bolus concentrations that can induce cardiovascular side effects.

Long-Term Safety of PEGylated Coagulation Factor VIII in the Immune-Deficient Rowett Nude Rat.

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26- and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ~75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immune-deficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals.

Evaluation of Nonacog Beta Pegol Long-term Safety in the Immune-deficient Rowett Nude Rat (Crl:NIH-Foxn1rnu).

Nonacog beta pegol is a 40-kDa polyethylene glycosylated (PEGylated) human recombinant coagulation factor IX, intended for the treatment of hemophilia B. Human coagulation factors are immunogenic in animals; therefore, to evaluate the long-term toxicity of nonacog beta pegol, an immune-deficient, athymic rat (Rowett nude; Crl:NIH-Foxn1(rnu)) was used. Rats (n = 216) were given intravenous nonacog beta pegol 0, 40, 150, 600, or 1,200 IU/kg every 5th day for 26 weeks. To avoid infections, the animals were housed in a full-barrier environment with sterilized food and bedding. Standard toxicity end points were unaffected by treatment. All treated animals were exposed to nonacog beta pegol throughout the study, and no animals developed antidrug antibodies. Immunohistochemical staining revealed PEG in choroid plexus epithelial cells in a dose-dependent manner. Transmission electron microscopy showed that PEG was distributed in cytoplasmic vesicles of these cells, with no apparent effect on cellular organelle structures. Fourteen (6.5%) animals were euthanized or died prematurely due to nontreatment-related infections in the urogenital system and skin. In conclusion, the athymic rat is a suitable model for testing chronic toxicity of human proteins that are immunogenic in animals. Nonacog beta pegol was generally well tolerated, with no adverse effect of PEG on choroid plexus epithelial cells.

Long-term trends of nitrogen and phosphorus mass balances on New York State dairy farms.

The whole-farm nutrient mass balance (NMB) is an adaptive management tool that can be used to identify areas for improvement in nutrient management and to monitor progress over time. The objectives of this study were to (1) evaluate the trends of nitrogen and phosphorus mass balances of 27 New York State dairy farms over 6 to 10 yr, (2) identify specific management changes made by 4 case study farms that improved NMB over time by shifting NMB up or down depending on the initial NMB, and (3) evaluate the potential of key indicators to identify opportunities for improvement in NMB. During the study period, milk price fluctuated whereas costs associated with feed and fertilizer increased substantially. Of the 27 farms, 67 to 74% (depending on the nutrient) decreased NMB per hectare over time, whereas 63 to 67% decreased NMB per megagram of milk over time. In general, changes in NMB were directionally correct, with 43 to 56% of farms operating in the optimum operational zone (with both NMB per hectare and per megagram of milk below the feasible levels suggested for New York) toward the end of the study versus 22 to 26% in the first 2 yr of the assessments. The 4 case study farms improved their NMB, whole-farm nutrient use efficiencies, and feed nutrient use efficiencies while maintaining or increasing milk production per cow. The case study farmers made the largest changes in precision feed management, reducing protein and P in purchased feed by replacing concentrates with blends with lower nutrient concentrations. Total nutrient imports, feed imports, the percentage of homegrown feed and nutrients, the concentration of nutrients in the purchased feed, fertilizer imports, and overall crop yields were useful in identifying potential areas for improvement in NMB.

Changes in nutrient mass balances over time and related drivers for 54 New York State dairy farms.

Whole-farm nutrient mass balances (NMB) can assist producers in evaluation and monitoring the nutrient status of dairy farms over time. Most of the previous studies that report NMB for dairy farms were conducted over 1 to 3 yr. In this study, annual N, P, and K mass balances were assessed on 54 dairy farms in New York State for 4 to 6 yr between 2005 and 2010 with the objectives to (1) document changes in NMB over time and drivers for change, and (2) identify nutrient use efficiency parameters that predicted the potential for improvement in NMB. The study farms varied in size (42 small, 12 medium and large) and management practices. Phosphorus, K, and 2 N balances (N1 without N2 fixation, and N2 including N2 fixation) were calculated. In general, farms with high initial NMB levels reduced them over time whereas farms with negative NMB tended to increase their NMB, demonstrating a tendency across all farms to move toward more optimal NMB levels over time. Sixty-three to 76% of farms (depending on the nutrient) reduced their NMB per hectare over the 4 to 6 yr, and 55 to 61% of these farms were able to do so while increasing milk production per cow. Across all farms, the overall reduction in NMB per hectare averaged -22kg of N/ha for N1 (29% reduction), -16kg of N/ha for N2 (15% reduction), -4kg of P/ha (36% reduction), and -10kg of K/ha (29% reduction). Change in feed imports was the most important driver for change in N and P balances across farms, whereas adjustments in both feed and fertilizer imports affected the K balances. Key predictors of potential areas for improvement in NMB over time include total nutrient imports, feed imports, animal density, percentage of farm-produced feed and nutrients, and feed nutrient use efficiency. Overall, this study highlights the opportunities of an adaptive management approach that includes NMB assessments to evaluate and monitor changes in nutrient use efficiency and cost-efficiency over time.

Characterization of nitrogen, phosphorus, and potassium mass balances of dairy farms in New York State.

A whole-farm nutrient mass balance (NMB) is a useful measure of the nutrient status of a dairy farm. Research is needed to define and determine a feasible NMB range for dairy farm systems in New York State (NY). The objectives of this study were to (1) document the distribution of N, P, and K mass balances of 102 NY dairy farms (including 75 small, 15 medium, and 12 large farms); (2) establish initial NMB benchmarks based on what 75% of the farms achieved; (3) determine the maximum animal density that allows an example NY dairy farm to balance cow P excretions and crop P removal without exporting crops or manure; and (4) identify opportunities to improve NMB over time. Nutrient mass balances of the 102 farms ranged from -39 to 237 kg of N/ha for N without including N2 fixation (N1), from -14 to 259 kg of N/ha when N2 fixation was included (N2), from -7 to 51 kg of P/ha, and from -46 to 148 kg of K/ha. Seventy-five percent of the farms were operating at NMB less than 118 kg of N/ha for N1, 146 kg of N/ha for N2, 13 kg of P/ha, and 41 kg of K/ha (75% benchmarks). Farms with the highest nutrient use efficiencies (lowest NMB per unit of milk produced) operated with less than 8.8 kg of N/Mg of milk for N1, 11.8 kg of N/Mg of milk for N2, 1.1 kg of P/Mg of milk, and 3.0 kg of K/Mg of milk. The biggest contributor to the NMB was the amount of imported nutrients, primarily feed purchases. The example farm assessment (assuming no export of crops or manure) suggested that, when 70% of the feed is produced on the farm and P in feed rations does not exceed 4 g of P/kg of DM, cow P excretion and crop P removal were balanced at a maximum animal density of 2.4 animal units (AU)/ha (~0.97 AU/acre). Dairy farms operating with animal densities <2.4 AU/ha typically had NMB below the 75% benchmark, whereas most dairies with more than 2.4 AU/ha needed to export manure or crops to meet the 75% benchmark. Opportunities to reduce NMB on many farms, independent of size and without changes in animal density, are possible by more tightly managing fertilizer and feed imports, increasing the percentage of farm-produced nutrients, implementing precision feeding, and exporting crops or manure.

Circulating cytokine concentrations in dogs with different degrees of myxomatous mitral valve disease.

Cytokines have been associated with the progression of congestive heart failure (CHF) in humans and may be implicated in the pathophysiology of myxomatous mitral valve disease (MMVD) in dogs. The aim of this study was to determine the serum concentrations of cytokines in dogs with MMVD. The study included 16 Cairn terriers with no or minimal mitral regurgitation (MR), 41 Cavalier King Charles Spaniels (CKCS) with different degrees of MR and 11 dogs of different breeds with CHF due to MMVD. Granulocyte-macrophage colony-stimulating factor, interferon-γ, interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, keratinocyte-derived chemokine, interferon-γ-induced protein and monocyte chemoattractant protein-1 (MCP-1) were measured using a canine-specific multiplex immunoassay. CHF dogs had significantly higher MCP-1 concentrations than dogs with no or minimal MR. Among the CKCS, IL-2 and IL-7 decreased with increasing left atrial size and IL-7 also decreased with increasing MR. IL-8 decreased with increasing left ventricular end-systolic internal dimensions. MCP-1 was increased in CHF dogs compared to healthy control dogs and IL-2, IL-7 and IL-8 decreased with increasing indices of disease severity. The results suggest a role for these cytokines in canine MMVD and CHF.

Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs.

Elevations in the plasma concentrations of natriuretic peptides correlate with increased severity of myxomatous mitral valve disease (MMVD) in dogs. This study correlates the severity of MMVD with the plasma concentrations of the biomarkers N-terminal fragment of the pro-brain-natriuretic peptide (NT-proBNP) and its second messenger, cyclic guanosine monophosphate (cGMP). Furthermore, the L-arginine:asymmetric dimethylarginine (ADMA) ratio was measured as an index of nitric oxide availability. The study included 75 dogs sub-divided into five groups based on severity of MMVD as assessed by clinical examination and echocardiography. Plasma NT-proBNP and cGMP concentrations increased with increasing valve dysfunction and were significantly elevated in dogs with heart failure. The cGMP:NT-proBNP ratio decreased significantly in dogs with heart failure, suggesting the development of natriuretic peptide resistance. Although the l-arginine:ADMA ratio decreased with increasingly severe MMVD, this was largely due to the older age of the dogs with heart failure.

Evaluation of plasma and urinary levels of 6-keto-prostaglandin F1alpha as a marker for asymptomatic myxomatous mitral valve disease in dogs.

Endothelial dysfunction might be involved in the pathogenesis of myxomatous mitral valve disease (MMVD). The aims of this study were (1) to validate an enzyme immunoassay (EIA) for canine 6-keto-prostaglandin (PG)F(1alpha) (prostacyclin metabolite and marker for endothelial function) and (2) to compare plasma and urinary 6-keto-PGF(1alpha) in dogs with asymptomatic MMVD. The study included two breeds predisposed to MMVD and two control groups (Cairn terriers and dogs of different breeds). Echocardiography was used to estimate the severity of MMVD. The intra- and inter-assay coefficients of variation were between 3.1% and 24.5% in the assay range. No echocardiographic parameter was correlated with plasma or urinary 6-keto-PGF(1alpha) (P>0.05), but all control dogs had lower urinary 6-keto-PGF(1alpha) (P<0.02) and the Cairn terriers had higher plasma 6-keto-PGF(1alpha) (P<0.02). The EIA appeared valid for measuring canine 6-keto-PGF(1alpha) in plasma and urine. It is suggested that 6-keto-PGF(1alpha) levels are related to breed and not MMVD in asymptomatic stages.