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).

TransCon IL-2 ß/γ: a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2 variant with improved pharmacokinetics and potent activation of cytotoxic immune cells for the treatment of cancer.

BACKGROUND: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe toxicities including cytokine storm and vascular leak syndrome (VLS). IL-2 promotes antitumor function of IL-2Rß/γ+ natural killer (NK) cells and CD8+, CD4+ and gamma delta (γδ) T cells. However, IL-2 also potently activates immunosuppressive IL-2Rα+ regulatory T cells (Tregs) and IL-2Rα+ eosinophils and endothelial cells, which may promote VLS. Aldesleukin is rapidly cleared requiring frequent dosing, resulting in high Cmax likely potentiating toxicity. Thus, IL-2 cancer immunotherapy has two critical drawbacks: potent activation of undesired IL-2Rα+ cells and suboptimal pharmacokinetics with high Cmax and short half-life. METHODS: TransCon IL-2 ß/γ was designed to optimally address these drawbacks. To abolish IL-2Rα binding yet retain strong IL-2Rß/γ activity, IL-2 ß/γ was created by permanently attaching a small methoxy polyethylene glycol (mPEG) moiety in the IL-2Rα binding site. To improve pharmacokinetics, IL-2 ß/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon (transient conjugation) linker creating a prodrug, TransCon IL-2 ß/γ, with sustained release of IL-2 ß/γ. IL-2 ß/γ was characterized in binding and primary cell assays while TransCon IL-2 ß/γ was studied in tumor-bearing mice and cynomolgus monkeys. RESULTS: IL-2 ß/γ demonstrated selective and potent human IL-2Rß/γ binding and activation without IL-2Rα interactions. TransCon IL-2 ß/γ showed slow-release pharmacokinetics with a low Cmax and a long (>30 hours) effective half-life for IL-2 ß/γ in monkeys. In mouse tumor models, TransCon IL-2 ß/γ promoted CD8+ T cell and NK cell activation and antitumor activity. In monkeys, TransCon IL-2 ß/γ induced robust activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells, Tregs and eosinophils, with no evidence of cytokine storm or VLS. Similarly, IL-2 ß/γ enhanced proliferation and cytotoxicity of primary human CD8+ T cells, NK cells and γδ T cells. SUMMARY: TransCon IL-2 ß/γ is a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2. It has remarkable and durable pharmacodynamic effects in monkeys and potential for improved clinical efficacy and tolerability compared with aldesleukin. TransCon IL-2 ß/γ is currently being evaluated in a Phase 1/2 clinical trial (NCT05081609).

Phase 1 safety, tolerability, pharmacokinetics and pharmacodynamics results of a long-acting C-type natriuretic peptide prodrug, TransCon CNP.

AIM: TransCon CNP is a novel prodrug designed to provide sustained release of C-type natriuretic peptide (CNP) for once-weekly therapy, addressing the pathology leading to aberrant skeletal development in achondroplasia. This phase 1 trial was initiated to assess the safety, tolerability, pharmacodynamics (PD) and pharmacokinetics (PK) of TransCon CNP. METHODS: This randomized, placebo-controlled, single-ascending dose phase 1 trial was performed at two sites in Australia and enrolled 45 healthy adult males. Subjects received placebo or TransCon CNP (single-ascending dose cohorts [3, 10, 25, 75 or 150 µg CNP/kg]). The primary endpoint was frequency of adverse events and other safety outcomes. Other endpoints included PK and PD measured by cyclic guanosine-monophosphate (cGMP) and amino-terminal propeptide of CNP (NTproCNP). RESULTS: TransCon CNP provided continuous systemic exposure to CNP over at least 7 days post-dose. Plasma and urine levels of cGMP were significantly increased in subjects administered TransCon CNP at 75-150 µg CNP/kg, indicating target engagement of active CNP at the natriuretic peptide receptor-B (NPR-B) for at least 1 week post-dose. TransCon CNP was well-tolerated, with no serious treatment-emergent adverse events or discontinuations. Extensive cardiac safety assessments did not reveal any clinically relevant effects on electrocardiogram parameters, including heart rate, PR, QRS and QTcF intervals. CONCLUSIONS: Safety and PD data from this phase 1 trial support that TransCon CNP is well tolerated, with a PK profile compatible with a once-weekly dosing regimen. Further studies are ongoing to evaluate the potential of TransCon CNP to positively impact abnormal endochondral ossification in children with achondroplasia.

Weekly Lonapegsomatropin in Treatment-Naïve Children With Growth Hormone Deficiency: The Phase 3 heiGHt Trial.

CONTEXT: For children with growth hormone deficiency (GHD), treatment burden with daily somatropin injections [human growth hormone (hGH)] is high, which may lead to poor adherence and suboptimal overall treatment outcomes. Lonapegsomatropin (TransCon hGH) is an investigational long-acting, once-weekly prodrug for the treatment of GHD. OBJECTIVE: The objective of this study was to evaluate the efficacy and safety of once-weekly lonapegsomatropin vs daily somatropin. DESIGN: The heiGHt trial was a randomized, open-label, active-controlled, 52-week Phase 3 trial (NCT02781727). SETTING: This trial took place at 73 sites across 15 countries. PATIENTS: This trial enrolled and dosed 161 treatment-naïve, prepubertal patients with GHD. INTERVENTIONS: Patients were randomized 2:1 to receive lonapegsomatropin 0.24 mg hGH/kg/week or an equivalent weekly dose of somatropin delivered daily. MAIN OUTCOME MEASURE: The primary end point was annualized height velocity (AHV) at week 52. Secondary efficacy end points included change from baseline in height SD scores (SDS). RESULTS: Least squares (LS) mean (SE) AHV at 52 weeks was 11.2 (0.2) cm/year for lonapegsomatropin vs 10.3 (0.3) cm/year for daily somatropin (P = 0.009), with lonapegsomatropin demonstrating both noninferiority and superiority over daily somatropin. LS mean (SE) height SDS increased from baseline to week 52 by 1.10 (0.04) vs 0.96 (0.05) in the weekly lonapegsomatropin vs daily somatropin groups (P = 0.01). Bone age/chronological age ratio, adverse events, tolerability, and immunogenicity were similar between groups. CONCLUSIONS: The trial met its primary objective of noninferiority in AHV and further showed superiority of lonapegsomatropin compared to daily somatropin, with similar safety, in treatment-naïve children with GHD.

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.

The citrus-derived flavonoid naringenin exerts uterotrophic effects in female mice at human relevant doses.

Gavage administration of the citrus flavonoid naringenin, 3',4,5,7-tetrahydroxyflavanon for 4 consecutive days, to immature female mice (postnatal day 17-20) at 4 or 100 mg/kg b.wt. significantly increased uterine weights 3 and 4 times, respectively. Analysis of uterine oestrogen receptor alpha revealed that naringenin significantly increased the cytosolic concentration of oestrogen receptor alpha, whereas in nuclei the oestrogen receptor alpha concentration was significantly decreased as compared to the solvent control. This was in contrast to the positive control 17 beta-oestradiolacetate which acted as a true oestrogen by increasing the concentration of both total and nuclear oestrogen receptor alpha. Both naringenin and 17 beta-oestradiolacetate, however, significantly, induced nuclear oestrogen receptor alpha in the liver, suggesting a tissue specific effect of naringenin on oestrogen receptor alpha distribution. In order to investigate the tissue levels at which the uterotrophic effect was observed, the distribution of an oral dose of tritiated naringenin (4 mg/kg) was investigated in 3-week-old female mice. The radioactivity content (ng naringenin equivalents/g tissue) was found to be highest in the gastrointestinal-tract, followed by the kidneys and liver. Uterus and ovaries were also found to contain relatively high and approximately equal amounts of naringenin. The concentration of naringenin in uterus and ovaries was found to be ten times higher as compared to the mammary tissue. The urinary excretion of more than 25% of the administered dose, within 8 hr after dosing indicated that naringenin is absorbed extensively in mice. The plasma concentration of 0.5 microM found in the present study is similar to the peak plasma concentration of naringenin (0.6 microM) observed in man following ingestion of 400-760 ml of orange juice (Erlund et al. 2001). This could be taken to suggests that ingestion of orange juice and other citrus fruits and juices may give rise to sufficiently high tissue levels of naringenin in man to exert a biological effect.

Non-nutritive bioactive food constituents of plants: bioavailability of flavonoids.

Flavonoids are polyphenols widely distributed in the plant kingdom, and are present in fruits and vegetables regularly consumed by humans. In vitro metabolic studies of flavonoids in rat liver microsomes identified the 3',4'-dihydroxylated derivatives as the major metabolic endpoint. However, in vivo in rats almost none of this metabolite and only minor amounts of the 4'-monohydroxylated derivative was produced. Flavonoids with the 4'-monohydroxylated structure were generally not metabolised and were excreted unchanged in urine in higher amounts than other flavonoids investigated. It has for long been a controversy, whether flavonoids are absorbed as the intact glycoside or whether they have to be hydrolysed to the free aglycon prior to absorption. Recent data suggest that beta-glucosidases and maybe also lactase phlorizin hydrolase (LPH) in the small intestine are capable of hydrolysing flavonoid glucosides and these compounds are thus taken up as the free aglycon and not as the intact glycosides. LC-MS analyses of 12 dietary flavonoids in human urine showed that no flavonoid glycosides were excreted, and that the citrus flavanones and phloretin are excreted in higher amounts than the flavonols. Furthermore, total flavonoid excretion may be a useful biomarker for habitual fruit and vegetable consumption.