The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Ritlecitinib, a Janus Kinase 3 and Tyrosine-Protein Kinase Family Inhibitor, in Humans.

Ritlecitinib is an oral once-daily irreversible inhibitor of Janus kinase 3 and tyrosine-protein kinase family being developed for the treatment of moderate-to-severe alopecia areata. This study examined the disposition of ritlecitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite profiles. The results indicated ritlecitinib had a systemic clearance of 43.7 L/h, a steady state volume of distribution of 73.8 L, extent of absorption of 89%, time to maximum plasma concentration of ~0.5 hour, and absolute oral bioavailability of 64%. An observed long terminal half-life of total radioactivity was primarily attributed to ritlecitinib binding to plasma albumin. Ritlecitinib was the main circulating drug species in plasma (~30%) with one major pharmacologically inactive cysteine conjugated metabolite (M2) at >10%. Oxidative metabolism (fractional clearance 0.47) and glutathione related conjugation (fractional clearance 0.24) were the primary routes of elimination for ritlecitinib with the greatest disposition of radioactivity shown in the urine (~71%). In vitro phenotyping indicated ritlecitinib cytochrome P450 fraction of metabolism assignments of 0.29 for CYP3A, 0.09 for CYP2C8, 0.07 for CYP1A2, and 0.02 for CYP2C9. In vitro phenotyping in recombinant human glutathione S-transferases indicated ritlecitinib was turned over by a number of cytosolic and microsomal enzyme isoforms. Significance Statement This study provides a detailed understanding of the disposition and metabolism of ritlecitinib, a JAK3 and TEC family kinase inhibitor for alopecia areata, in humans, as well as characterization of clearance pathways and PK of ritlecitinib and its metabolites. As an AMS-based ADME study design, we have expanded on reporting the standard ADME endpoints, providing key pharmacokinetic parameters like clearance, volume of distribution and bioavailability allowing for a more comprehensive understanding of drug disposition.

Pharmacokinetic Profile of Brepocitinib with Topical Administration in Atopic Dermatitis and Psoriasis Populations: Strategy to Inform Clinical Trial Design in Adult and Pediatric Populations.

INTRODUCTION: Topical brepocitinib, a tyrosine kinase (TYK)2/Janus kinase (JAK)1 inhibitor, is in development for psoriasis (PsO) and atopic dermatitis (AD). Quantitative analyses of prior clinical trial data were used to inform future clinical trial designs. METHODS: Two phase 2b studies in patients with AD and PsO were used to characterize the amount of topical brepocitinib and the resultant systemic trough concentration (CTrough) using a linear mixed-effects regression (LMER). This model was used to predict brepocitinib systemic CTrough for higher treated body surface areas (BSAs) in adults and children. Information from non-clinical and clinical trials with oral brepocitinib was leveraged to set safety thresholds. This combined approach was used to inform future dose-strength selection and treated BSA limits. RESULTS: Data from 256 patients were analyzed. Patient type, dose strength, and frequency had significant impacts on the dose-exposure relationship. Systemic concentration in patients with PsO was predicted to be 45% lower than in patients with AD from the same dose. When topically applied to the same percentage BSA, brepocitinib systemic exposures are expected to be comparable between adults and children. The systemic steady-state exposure after 3% once daily and twice daily (2 mg/cm2) cream applied to less than 50% BSA in patients with AD and PsO, respectively, maintains at least a threefold margin to non-clinical safety findings and clinical hematologic markers. CONCLUSION: The relationship between the amount of active drug applied and brepocitinib systemic CTrough, described by LMER, may inform the development strategy for dose optimization in the brepocitinib topical program.

Moving Beyond Boundaries: Utilization of Longitudinal Exposure-Response Model for Bounded Outcome Score to Inform Decision Making in the Accelerated Drug Development Paradigm.

BACKGROUND AND OBJECTIVES: As drug development scientists strive to accelerate availability of therapies for patients, model-informed drug development (MIDD) plays an important role in contextualizing existing information and facilitating decision making. This paper describes an example of MIDD, where modeling and simulation informed decision making in the circumstance of a combined phase 2b and single pivotal study for ritlecitinib (JAK3/TEC family kinases inhibitor). METHODS: Longitudinal exposure-response (ER) modeling was conducted to describe ritlecitinib efficacy in alopecia areata patients. The Severity of Alopecia Tool (SALT) score (a continuous bounded outcome [CBO] score [0-100]) was used as the efficacy response. The average concentration during the time interval between two adjacent SALT scores was used as the exposure metric driving efficacy. RESULTS: The developed model well described the longitudinal SALT profile of ritlecitinib as well as the frequency of boundary data. The CBO model indicated tested doses in the phase 2b/3 clinical trial are in the ascending region of ER and contextualized a loading dose effect that impacted onset of efficacy without long-term benefit. It also identified disease severity as the only covariate impacting efficacy. The model-based simulation further informed impact of treatment interruption on the loss of efficacy in the absence of a dedicated treatment withdrawal study. Results indicated temporary treatment interruption ≤ 6 weeks is not expected to result in significant loss of efficacy. CONCLUSION: The CBO modeling approach and simulation supported the single pivotal trial strategy and guided dose selection in the accelerated drug development program of ritlecitinib, which can be applied to many indications where efficacy is measured on a bounded scale.

Utilization of Rosuvastatin and Endogenous Biomarkers in Evaluating the Impact of Ritlecitinib on BCRP, OATP1B1, and OAT3 Transporter Activity.

PURPOSE: Ritlecitinib, an inhibitor of Janus kinase 3 and tyrosine kinase expressed in hepatocellular carcinoma family kinases, is in development for inflammatory diseases. This study assessed the impact of ritlecitinib on drug transporters using a probe drug and endogenous biomarkers. METHODS: In vitro transporter-mediated substrate uptake and inhibition by ritlecitinib and its major metabolite were evaluated. Subsequently, a clinical drug interaction study was conducted in 12 healthy adult participants to assess the effect of ritlecitinib on pharmacokinetics of rosuvastatin, a substrate of breast cancer resistance protein (BCRP), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporter 3 (OAT3). Plasma concentrations of coproporphyrin I (CP-I) and pyridoxic acid (PDA) were assessed as endogenous biomarkers for OATP1B1 and OAT1/3 function, respectively. RESULTS: In vitro studies suggested that ritlecitinib can potentially inhibit BCRP, OATP1B1 and OAT1/3 based on regulatory cutoffs. In the subsequent clinical study, coadministration of ritlecitinib decreased rosuvastatin plasma exposure area under the curve from time 0 to infinity (AUCinf) by ~ 13% and maximum concentration (Cmax) by ~ 27% relative to rosuvastatin administered alone. Renal clearance was comparable in the absence and presence of ritlecitinib coadministration. PK parameters of AUCinf and Cmax for CP-I and PDA were also similar regardless of ritlecitinib coadministration. CONCLUSION: Ritlecitinib does not inhibit BCRP, OATP1B1, and OAT3 and is unlikely to cause a clinically relevant interaction through these transporters. Furthermore, our findings add to the body of evidence supporting the utility of CP-I and PDA as endogenous biomarkers for assessment of OATP1B1 and OAT1/3 transporter activity.

Evaluation of the effect of ritlecitinib on the pharmacokinetics of caffeine in healthy participants.

AIMS: This clinical study was conducted to evaluate the impact of ritlecitinib on the pharmacokinetics of caffeine, a cytochrome P450 1A2 (CYP1A2) substrate. METHODS: In this single-centre, single-arm, open-label, fixed-sequence study, healthy participants received a single 100-mg dose of caffeine on 2 separate occasions: on Day 1 of Period 1 as monotherapy and on Day 8 of Period 2 after oral administration of ritlecitinib 200 mg once daily for 8 days. Serial blood samples were collected and analysed using a validated liquid chromatography-mass spectrometry assay. Pharmacokinetic parameters were estimated by using a noncompartmental method. Safety was monitored by physical examination, vital signs, electrocardiograms and laboratory assessments. RESULTS: Twelve participants were enrolled and completed the study. Coadministration of caffeine 100 mg in the presence of steady-state levels of ritlecitinib (200 mg once daily) increased caffeine exposure compared with caffeine given alone. Area under the curve to infinity and maximum concentration of caffeine increased by approximately 165 and 10%, respectively, when coadministered with ritlecitinib. The ratios of the adjusted geometric means (90% confidence interval) for caffeine area under the curve to infinity and maximum concentration were 265.14% (234.12-300.26%) and 109.74% (103.90-15.91%), respectively, when caffeine was coadministered with steady-state ritlecitinib (test) compared with its administration alone (reference). Multiple doses of ritlecitinib when coadministered with a single dose of caffeine were generally safe and well tolerated in healthy participants. CONCLUSION: Ritlecitinib is a moderate inhibitor of CYP1A2 and can increase systemic exposures of CYP1A2 substrates.

Population PK and PD Analysis of Domagrozumab in Pediatric Patients with Duchenne Muscular Dystrophy.

Myostatin, a negative regulator of skeletal muscle growth, is a therapeutic target in muscle-wasting diseases. Domagrozumab, a humanized recombinant monoclonal antibody, binds myostatin and inhibits activity. Domagrozumab was investigated in a phase II trial (NCT02310763) as a potential treatment for boys with Duchenne muscular dystrophy (DMD). Pharmacokinetic/pharmacodynamic (PK/PD) modeling is vital in clinical trial design, particularly for determining dosing regimens in pediatric populations. This analysis sought to establish the PK/PD relationship between free domagrozumab and total myostatin concentrations in pediatric patients with DMD using a prior semimechanistic model developed from a phase I study in healthy adult volunteers (NCT01616277) and following inclusion of phase II data. The refined model was developed using a multiple-step approach comprising structural, random effects, and covariate model development; assessment of model adequacy (goodness-of-fit); and predictive performance. Differences in PKs/PDs between healthy adult volunteers and pediatric patients with DMD were quantitatively accounted for and evaluated by predicting myostatin coverage (the percentage of myostatin bound by domagrozumab). The final model parameter estimates and semimechanistic target-mediated drug disposition structure sufficiently described both domagrozumab and myostatin concentrations in pediatric patients with DMD, and most population parameters were comparable with the prior model (in healthy adult volunteers). Predicted myostatin coverage for phase II patients with DMD was consistently > 90%. Baseline serum myostatin was ~ 65% lower than in healthy adult volunteers. This study provides insights into the regulation of myostatin in healthy adults and pediatric patients with DMD. Clinicaltrials.gov identifiers: NCT01616277 and NCT02310763.

Randomized phase 2 trial and open-label extension of domagrozumab in Duchenne muscular dystrophy.

We report results from a phase 2, randomized, double-blind, 2-period trial (48 weeks each) of domagrozumab and its open-label extension in patients with Duchenne muscular dystrophy (DMD). Of 120 ambulatory boys (aged 6 to <16 years) with DMD, 80 were treated with multiple ascending doses (5, 20, and 40 mg/kg) of domagrozumab and 40 treated with placebo. The primary endpoints were safety and mean change in 4-stair climb (4SC) time at week 49. Secondary endpoints included other functional tests, pharmacokinetics, and pharmacodynamics. Mean (SD) age was 8.4 (1.7) and 9.3 (2.3) years in domagrozumab- and placebo-treated patients, respectively. Difference in mean (95% CI) change from baseline in 4SC at week 49 for domagrozumab vs placebo was 0.27 (-7.4 to 7.9) seconds (p = 0.94). There were no significant between-group differences in any secondary clinical endpoints. Most patients had ≥1 adverse event in the first 48 weeks; most were mild and not treatment-related. Median serum concentrations of domagrozumab increased with administered dose within each dose level. Non-significant increases in muscle volume were observed in domagrozumab- vs placebo-treated patients. Domagrozumab was generally safe and well tolerated in patients with DMD. Efficacy measures did not support a significant treatment effect. Clinicaltrials.gov identifiers: NCT02310763 and NCT02907619.

Phase 1 study of crisaborole in Japanese healthy volunteers and patients with atopic dermatitis.

Crisaborole ointment, 2%, is a non-steroidal phosphodiesterase 4 inhibitor for the treatment of mild to moderate atopic dermatitis (AD). This parallel-cohort, phase 1 study was conducted to investigate skin irritation potential and safety of crisaborole in healthy Japanese adults (cohort 1) and the safety and pharmacokinetic profile of crisaborole and metabolites AN7602 and AN8323 in Japanese adults with mild to moderate AD (cohort 2). In cohort 1, 20 healthy volunteers received single applications of crisaborole and vehicle simultaneously on separate locations under 48-h occlusion. In cohort 2, 12 patients with mild to moderate AD received crisaborole (n = 10) or vehicle (n = 2) twice daily for 8 days. Skin irritation and safety were assessed in cohort 1. Pharmacokinetics and safety were assessed in cohort 2. Skin irritation index (scale 0-400) was 40.0 for crisaborole and 5.0 for vehicle. No treatment-emergent adverse events (TEAE) were reported in cohort 1. The most common TEAE in the crisaborole group in cohort 2 were application site irritation (n = 7) and application site pain (n = 4). Crisaborole was rapidly absorbed, with limited systemic exposure between days 1 and 8 that was comparable with that seen in US-based participants in previous trials. Crisaborole had higher skin irritation than vehicle under occlusion in healthy Japanese adults and had an acceptable safety profile in Japanese adults with mild to moderate AD.

Crisaborole and atopic dermatitis skin biomarkers: An intrapatient randomized trial.

BACKGROUND: Crisaborole ointment 2% is a nonsteroidal phosphodiesterase 4 inhibitor for the treatment of mild-to-moderate atopic dermatitis (AD). The mechanism of action of crisaborole and its effects on lesional measures of disease severity are not yet well defined. OBJECTIVE: This phase 2a, single-center, vehicle-controlled, intrapatient study was designed to further characterize the mechanism of action of crisaborole through evaluation of clinical efficacy and changes in skin biomarkers in adults (n = 40) with mild-to-moderate AD. METHODS: Two target lesions were randomized in an intrapatient (1:1) manner to double-blind crisaborole/vehicle applied twice daily for 14 days. Patients then applied crisaborole (open-label) to all affected areas for 28 days. Punch biopsy specimens were collected for biomarker analysis at baseline, day 8 (optional), and day 15. RESULTS: Crisaborole treatment resulted in early improvement in lesional signs/symptoms versus vehicle, with improvement in pruritus (pruritus numeric rating scale) observed as early as 24 hours after the first application. Crisaborole-treated lesions showed significant percentage improvement from baseline in lesional transcriptomic profile compared with vehicle at day 8 (91.15% vs 36.02%, P < 10-15) that was sustained until day 15 (92.90% vs 49.59%, P < 10-15). Crisaborole significantly modulated key AD biomarkers versus vehicle, including TH2 and TH17/TH22 pathways and epidermal hyperplasia/proliferation. Molecular profiles and epidermal pathology normalized toward nonlesional skin and correlated with clinical changes in lesion severity and barrier function. CONCLUSION: Crisaborole reversed biomarker profiles of skin inflammation and barrier function, with associated improvements in clinical efficacy measures, highlighting the therapeutic utility of targeting phosphodiesterase 4 in patients with AD.

Systemic Tofacitinib Concentrations in Adult Patients With Atopic Dermatitis Treated With 2% Tofacitinib Ointment and Application to Pediatric Study Planning.

Atopic dermatitis is a chronic eczematous, pruritic, inflammatory skin condition affecting children and adults. Tofacitinib is a Janus kinase inhibitor. The efficacy, safety, and pharmacokinetics of 2% tofacitinib ointment twice daily have been evaluated in a 4-week phase 2a multisite randomized, double-blind, vehicle-controlled, parallel-group study (NCT02001181) in adult patients with mild to moderate atopic dermatitis and 2% to 20% body surface area (BSA) involvement. Tofacitinib ointment demonstrated significantly greater efficacy versus vehicle for all efficacy end points and had an acceptable safety profile. Predose and postdose pharmacokinetic samples were collected in week 2 and week 4. The objective of this analysis was to assess if predicted mean tofacitinib concentrations with topical application at higher treated BSA across age groups would exceed relevant concentration thresholds based on oral doses of tofacitinib. In this analysis, the pharmacokinetic concentrations were characterized using a linear mixed-effects model. The model was used to predict concentrations for adults with higher (>20%) treatable BSA. Adult concentrations were used to extrapolate concentrations to a pediatric population (2 to 17 years) using allometric principles. The predicted systemic concentrations for 2% tofacitinib ointment in both adult and pediatric populations at treated BSA ≤50% for a mild to moderate atopic dermatitis population did not exceed those reported for the 10th percentile of observed oral tofacitinib 5-mg twice-daily doses in patients with moderate to severe plaque psoriasis. The methodology described will enable analysis and prediction of systemic concentrations for topical agents.

FDA Public Meeting Report on "Drug Interactions With Hormonal Contraceptives: Public Health and Drug Development Implications".

Potential drug interactions with hormonal contraceptives are an important public health concern. A public meeting on "Drug Interactions With Hormonal Contraceptives: Public Health and Drug Development Implication" was hosted by the United States Food and Drug Administration (FDA). The meeting endeavored to provide an opportunity for the FDA to seek input from experts on the public health concerns associated with the use of hormonal contraceptives and interacting drugs that might affect efficacy and safety, including pharmacokinetic/pharmacodynamic considerations, in the design of drug interaction studies of hormonal contraceptives for drug development and approaches to translating the results of drug interaction information into informative labeling and communication. The input received could be used to refine FDA's thinking on hormonal contraceptives drug interaction study design and interpretation and labeling communication of drug interaction risk. This meeting benefited from strong and diverse participation from the Center for Drug Evaluation and Research at the FDA, Centers for Disease Control and Prevention, National Institutes of Health, Swedish Medical Products Agency, pharmaceutical industry, and representatives of academia. This report provides a summary of the key discussion based on the presentations and panel discussion.

Intravenous administration of Factor VIII-O-Phospho-L-Serine (OPLS) complex reduces immunogenicity and preserves pharmacokinetics of the therapeutic protein.

Hemophilia A is a bleeding disorder caused by the deficiency of an important coagulation factor; Factor VIII (FVIII). Replacement therapy using exogenously administered recombinant FVIII is the most commonly used method of treatment. However, approximately 30% of Hemophilia A patients develop neutralizing antibodies (Nabs) against the recombinant protein. Nabs abolish FVIII activity and drastically influence efficacy of the protein. The immunogenic epitopes of FVIII reside predominantly in the C2 domain of FVIII. However, the C2 domain also contains a lipid binding region. O-Phospho-L-Serine (OPLS) which is the head-group moiety of phosphatidylserine, interacts with the lipid binding region of FVIII. Previous studies have shown that FVIII complexed with OPLS lowered Nab development against FVIII following subcutaneous administration. In dendritic cell-T-cell co-culture studies, OPLS treatment increased the secretion of immunosuppressive cytokines (Transforming Growth Factor-ß and Interleukin-10), and simultaneously decreased pro-inflammatory IL-17 cytokine. Here, we investigated FVIII immune response and pharmacokinetics upon intravenous administration of FVIII-OPLS complex. We studied the effect of FVIII-OPLS complex on the interaction between a professional antigen presenting cell; dendritic cell and T-cell, and T-cell clonal expansion. Pharmacokinetics parameters were estimated following intravenous administration of FVIII and FVIII-OPLS. The results suggest that OPLS lowers FVIII immune response following intravenous administration. OPLS also hinders FVIII-specific T-cell clonal proliferation and preserves FVIII PK profile. Thus, the ease of protein-lipid complexation, preservation of FVIII activity and in vivo behavior, and improved in vitro FVIII stability, makes OPLS an attractive excipient in the preparation of next generation or biosimilar FVIII products with improved safety profile.

A phase 2 study of filibuvir in combination with pegylated IFN alfa and ribavirin for chronic HCV.

OBJECTIVES: Filibuvir is a non-nucleoside inhibitor of hepatitis C virus (HCV) polymerase. This study evaluated the safety and efficacy of filibuvir plus pegylated interferon alfa-2a (pegIFN)/ribavirin. MATERIAL AND METHODS: Treatment-naïve, HCV genotype-1 patients were randomized to receive filibuvir 300 or 600 mg twice daily (BID) or placebo plus pegIFN (180 µg/wk) and ribavirin (1,000/1,200 mg BID) for 24 weeks. Filibuvir patients who achieved defined response through week 24 discontinued therapy at week 24. All other patients continued on open-label pegIFN/ribavirin through week 48. The primary endpoint was the proportion of patients who achieved sustained virologic response (SVR) defined as HCV RNA < 15 IU/mL at end of treatment (weeks 24 or 48) and week 72. RESULTS: Overall, 288 patients were randomized and treated. SVR was achieved by 41.7, 39.6, and 45.8% of patients in the filibuvir 300 mg, 600 mg, and placebo arms, respectively. While the addition of filibuvir to pegIFN/ribavirin improved on-treatment virologic response parameters, this did not translate into improved SVR rates due to a high rate of virologic relapse following completion of therapy (300 mg: 35.9%; 600 mg: 42.9%; placebo: 25.4%). The most commonly reported adverse events were nausea, fatigue, headache, and insomnia, and were reported at similar rates across arms. CONCLUSIONS: Filibuvir plus pegIFN/ribavirin did not improve the percentage of patients achieving SVR compared with administration of pegIFN/ribavirin alone. However, the agent was well tolerated and was associated with higher on-treatment virologic response parameters. Further evaluation of filibuvir in combination with other direct-acting antiviral agents may be considered.

Biopharmaceutic planning in pediatric drug development.

Pediatric drug development is a required consideration for all drug development programs. Age-appropriate formulations such as suspensions, chewable tablets, oral disintegrating tablets, etc., are typically developed and used in the pediatric clinical studies. However, it is not uncommon to use enabling formulations in the pivotal pediatric clinical study followed by bridging bioavailability and/or bioequivalence studies. Development of age-appropriate formulations is an essential part of pediatric drug development and adds additional biopharmaceutical considerations to an already complex problem. Careful planning of biopharmaceutic data collection during the adult and pediatric development program can contribute significantly to the biopharmaceutic risk assessment and planning of appropriate clinical studies leading to successful development of pediatric formulations.

Antiviral activity of the hepatitis C virus polymerase inhibitor filibuvir in genotype 1-infected patients.

UNLABELLED: More effective and better-tolerated therapies are needed for chronic hepatitis C virus (HCV) infection. Among the direct-acting anti-HCV agents in development is the nonstructural 5B protein (NS5B polymerase) non-nucleoside inhibitor filibuvir. We investigated the antiviral activity, pharmacokinetics, safety, and tolerability of multiple doses of filibuvir in treatment-naive and treatment-experienced patients who were chronically infected with HCV genotype 1 in two phase 1b clinical studies (study 1 was a randomized, placebo-controlled dose escalation study and study 2 was a nonrandomized, open-label study). The filibuvir doses evaluated ranged from 200-1400 mg daily, and the duration of dosing ranged from 3-10 days. Genotypic changes in the NS5B nucleotide sequence following short-term filibuvir therapy were also assessed. Filibuvir potently inhibited viral replication in a dose-dependent manner. Mean maximum HCV RNA change from baseline ranged from -0.97 log(10) IU/mL with filibuvir given at 100 mg twice daily to -2.30 log(10) IU/mL with filibuvir given at 700 mg twice daily in treatment-naive patients. In treatment-experienced patients, an HCV RNA reduction of 2.20 log(10) IU/mL was achieved with filibuvir given at 450 mg twice daily. Filibuvir was well tolerated in both studies. Adverse events were mild or moderate in severity. No discontinuations, serious adverse events, or deaths were reported. NS5B sequencing identified residue 423 as the predominant site of mutation after filibuvir dosing. CONCLUSION: Filibuvir administration resulted in significant reductions in HCV RNA concentrations at doses that were well tolerated in patients infected with HCV genotype 1. Filibuvir is currently being evaluated in combination with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients.

IL-12 delivered intratumorally by multilamellar liposomes reactivates memory T cells in human tumor microenvironments.

Using a novel loading technique, IL-12 is reported here to be efficiently encapsulated within large multilamellar liposomes. The preclinical efficacy of the cytokine loaded liposomes to deliver IL-12 into human tumors and to reactive tumor-associated T cells in situ is tested using a human tumor xenograft model. IL-12 is released in vivo from these liposomes in a biologically active form when injected into tumor xenografts that are established by the subcutaneous implantation of non-disrupted pieces of human lung, breast or ovarian tumors into immunodeficient mice. The histological architecture of the original tumor tissue, including tumor-associated leukocytes, tumor cells and stromal cells is preserved anatomically and the cells remain functionally responsive to cytokines in these xenografts. The local and sustained release of IL-12 into the tumor microenvironment reactivates tumor-associated quiescent effector memory T cells to proliferate, produce and release IFN-gamma resulting in the killing of tumor cells in situ. Very little IL-12 is detected in the serum of mice for up to 5 days after an intratumoral injection of the IL-12 liposomes. We conclude that IL-12 loaded large multilamellar liposomes provide a safe method for the local and sustained delivery of IL-12 to tumors and a therapeutically effective way of reactivating existing tumor-associated T cells in human solid tumor microenvironments. The potential of this local in situ T cell re-stimulation to induce a systemic anti-tumor immunity is discussed.

Phosphatidylserine containing liposomes reduce immunogenicity of recombinant human factor VIII (rFVIII) in a murine model of hemophilia A.

Factor VIII (FVIII) is a multidomain protein that is deficient in hemophilia A, a clinically important bleeding disorder. Replacement therapy using recombinant human FVIII (rFVIII) is the main therapy. However, approximately 15-30% of patients develop inhibitory antibodies that neutralize rFVIII activity. Antibodies to epitopes in C2 domain, which is involved in FVIII binding to phospholipids, are highly prevalent. Here, we investigated the effect of phosphatidylserine (PS)-containing liposomes, which bind to C2 domain with high affinity and specificity, upon the immunogenicity of rFVIII. Circular dichroism studies showed that PS-containing liposomes interfered with aggregation of rFVIII. Immunogenicity of free- versus liposomal-rFVIII was evaluated in a murine model of hemophilia A. Animals treated with s.c. injections of liposomal-rFVIII had lower total- and inhibitory titers, compared to animals treated with rFVIII alone. Antigen processing by proteolytic enzymes was reduced in the presence of liposomes. Animals treated with s.c. injections of liposomal-rFVIII showed a significant increase in rFVIII plasma concentration compared to animals that received rFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between PS-containing bilayers and rFVIII may provide a basis for designing lipidic complexes that improve the stability, reduce the immunogenicity of rFVIII formulations, and permit administration by s.c. route.

O-phospho-L-serine, multi-functional excipient for B domain deleted recombinant factor VIII.

Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain, A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, approximately 15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients' plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid binding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free- vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDrFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.