Bridging the Gap With Clinical Pharmacology in Innovative Rare Disease Treatment Modalities: Targeting DNA to RNA to Protein.

Rare diseases are frequently caused by inherited 'monogenic' defects. Treatment interventions that target a specific genetic location or that replaces a specific protein provide rational therapeutic approaches. The current review discusses innovative targeted therapies that act or modulate at the level of DNA, RNA, or protein. They include DNA gene editing, small interference RNA (siRNA), antisense oligonucleotide (ASO), small molecule RNA splicing modifier, and bispecific antibody. With limited numbers of patients, testing multiple dose levels and regimens prior to making an informed dose decision remains one of the major challenges in rare disease drug development. Clinical pharmacology strategically bridges the gap to support drug development and regulatory approvals. Pharmacokinetic drug exposures are driven by absorption, distribution, metabolism, elimination, and in some cases immunogenicity. Drug responses are measured by pharmacodynamic biomarkers that are linked to either short- or long-term clinical outcomes. Understanding the drug exposure-response relationship lies at the heart of bridging the gap that enables a dose decision by balancing effectiveness and safety. Furthermore, and importantly, understanding the influence of intrinsic and extrinsic factors on drug pharmacokinetics enables dose adjustment decisions based on drug exposures. Case examples include the identification of doses and regimens without a formal dose-finding study, the support of new doses and regimens without conducting additional studies, and the extrapolation of adult drug-drug interaction (DDI) studies to pediatrics without performing a pediatric DDI study. With increasing discoveries of innovative treatment modalities, the responsibility of clinical pharmacologists is expected to grow and enhance the development of novel treatments for rare diseases.

Immunogenicity Considerations for Therapeutic Modalities Used in Rare Diseases.

New therapeutic modalities carry with them great promise for the treatment of rare diseases. They also present unique development challenges including immunogenicity, which can impact the safety and efficacy of those new modalities. In this review, an overview of the basic function of the immune system and its possible interaction with new therapeutic modalities is presented. A juxtaposition of immunogenicity in the rare disease space versus traditional clinical programs is hereby being proposed. A clinical pharmacology viewpoint of immunogenicity, proposed approaches to account for immunogenicity in clinical data, bioanalytical considerations, and effects of route of administration and production changes on immunogenicity are discussed.

Mechanistic Modeling of the Effect of Recombinant Human Hyaluronidase (rHuPH20) on Subcutaneous Delivery of Cetuximab in Rats.

PURPOSE: To evaluate the duration of effect of rHuPH20 on SC absorption of cetuximab and to develop a mechanistic pharmacokinetic model linking the kinetics of rHuPH20 action with hyaluronan (HA) homeostasis and absorption of cetuximab from the SC space. METHODS: Serum pharmacokinetics of cetuximab was evaluated after IV and SC dosing at 0.4 and 10 mg/kg (control groups). In test groups, SC cetuximab was administered simultaneously with rHuPH20 (Co-Injection) or 12 h after injection of rHuPH20 (Pre-Injection). Mechanistic pharmacokinetic model was developed to simultaneously capture cetuximab kinetics in all groups. RESULTS: Administration of rHuPH20 resulted in a faster absorption of cetuximab; the difference between co-injection and pre-injection groups appeared to be dependent on the dose level. The model combined three major components: kinetics of rHuPH20 at SC site; HA homeostasis and its disruption by rHuPH20; and cetuximab systemic disposition and the effect of HA disruption on cetuximab SC absorption. The model provided good description of experimental data obtained in this study and collected previously. CONCLUSIONS: Proposed model can serve as a potential translational framework for capturing the effect of rHuPH20 across multiple preclinical species and in human studies and can be used for optimization of SC delivery of biotherapeutics.

Safety and pharmacokinetics of docetaxel in combination with pegvorhyaluronidase alfa in patients with non-small cell lung cancer.

This open-label, phase Ib study (NCT02346370) assessed the effect of pegvorhyaluronidase alfa (PVHA; PEGPH20) on the plasma pharmacokinetics (PKs) and safety of docetaxel in 15 patients with stage IIIB/IV non-small cell lung cancer (NSCLC). The docetaxel PK profile from this study was consistent with simulations from a published docetaxel population PK model, and did not demonstrate an effect of PVHA on docetaxel PK. A maximum a posteriori Bayesian fit of the literature PK model to the docetaxel PK appeared unbiased. Adverse events (AEs) were generally consistent with previous reports for docetaxel monotherapy in NSCLC, except for higher incidence of musculoskeletal events, including myalgias, with PVHA plus docetaxel. The most common AEs were fatigue (87%), muscle spasms (60%), and myalgia (53%). Four patients experienced thromboembolic events (27%), three leading to treatment discontinuation. PVHA appeared to demonstrate an acceptable safety profile when given with docetaxel without significantly changing the plasma PK of docetaxel in patients with stage IIIB/IV NSCLC.

The Effect of Small Oligomeric Protein Aggregates on the Immunogenicity of Intravenous and Subcutaneous Administered Antibodies.

The role of aggregates in the immunogenicity of biologics is a major concern. A recent US FDA guidance on the issue suggests that a gap in knowledge exists regarding the type and size of aggregates involved in the immunogenicity of biologics. Furthermore, the guidance suggests that current techniques cannot capture the crucial stages of protein aggregation. Using a protein unfolding model developed earlier, we generated and classified aggregates of two therapeutic antibodies based on size and conformation. The immunogenic potential of these aggregates were then tested in a murine model. Our findings show that small native-like oligomeric aggregates (<100 nm) are more immunogenic toward the native protein than monomer and large non-native aggregates in the micron-size range, irrespective of route of administration [intravenous (i.v.) vs. subcutaneous (s.c.)]. Those smaller oligomeric aggregates represented 5%-20% of the total protein concentration in the test formulations. Furthermore, in vitro data suggest that TNF-α production by bone marrow-derived dendritic cells could serve as a predictive marker for increased immunogenic risk of aggregates after s.c. administration. The use of orthogonal techniques such as fluorescence anisotropy and quasielastic light scattering may be useful to detect these oligomeric aggregates.

Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine-Dependent Arrest in the T-cell Signaling Cascade.

The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients' antitumor immune responses. We previously established that an unidentified factor, or factors, present in ovarian tumor ascites fluids reversibly inhibited the activation of T cells by arresting the T-cell signaling cascade. Ultracentrifugation of the tumor ascites fluid has now revealed a pellet that contains small extracellular vesicles (EV) with an average diameter of 80 nm. The T-cell arrest was determined to be causally linked to phosphatidylserine (PS) that is present on the outer leaflet of the vesicle bilayer, as a depletion of PS-expressing EV or a blockade of PS with anti-PS antibody significantly inhibits the vesicle-induced signaling arrest. The inhibitory EV were also isolated from solid tumor tissues. The presence of immunosuppressive vesicles in the microenvironments of ovarian tumors and our ability to block their inhibition of T-cell function represent a potential therapeutic target for patients with ovarian cancer.

Anatomical, physiological, and experimental factors affecting the bioavailability of sc-administered large biotherapeutics.

Subcutaneous route of administration is highly desirable for protein therapeutics. It improves patient compliance and quality of life (McDonald TA, Zepeda ML, Tomlinson MJ, Bee WH, Ivens IA. 2010. Curr Opin Mol Ther 12(4):461-470; Dychter SS, Gold DA, Haller MF. 2012. J Infus Nurs 35(3):154-160), while reducing healthcare cost (Dychter SS, Gold DA, Haller MF. 2012. J Infus Nurs 35(3):154-160). Recent evidence also suggests that sc administration of protein therapeutics can increase tolerability to some treatments such as intravenous immunoglobulin therapy by administering it subcutaneously (subcutaneous immunoglobulin therapy SCIG), which will reduce fluctuation in plasma drug concentration (Kobrynski L. 2012. Biologics 6:277-287). Furthermore, sc administration may reduce the risk of systemic infections associated with i.v. infusion (McDonald TA, Zepeda ML, Tomlinson MJ, Bee WH, Ivens IA. 2010. Curr Opin Mol Ther 12(4):461-470; Dychter SS, Gold DA, Haller MF. 2012. J Infus Nurs 35(3):154-160). This route, however, has its challenges, especially for large multidomain proteins. Poor bioavailability and poor scalability from preclinical models are often cited. This commentary will discuss barriers to sc absorption as well as physiological and experimental factors that could affect pharmacokinetics of subcutaneously administered large protein therapeutics in preclinical models. A mechanistic pharmacokinetic model is proposed as a potential tool to address the issue of scalability of sc pharmacokinetic from preclinical models to humans.

Effects of hypertonic buffer composition on lymph node uptake and bioavailability of rituximab, after subcutaneous administration.

The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailability after s.c. administration remains a major challenge. In this work we investigated the effects of excipient dependent hyperosmolarity on lymphatic uptake and plasma exposure of rituximab as a model protein. Using Swiss Webster (SW) mice as the animal model, we compared the effects of NaCl, mannitol and O-phospho-L-serine (OPLS) on the plasma concentration of rituximab over 5 days after s.c. administration. An increase was observed in plasma concentrations in animals administered rituximab in hypertonic buffer solutions, compared with isotonic buffer. Bioavailability, as estimated by our pharmacokinetic model, increased from 29% in isotonic buffer to 54% in hypertonic buffer containing NaCl, to almost complete bioavailability in hypertonic buffers containing high dose OPLS or mannitol. This improvement in plasma exposure is due to the improved lymphatic trafficking as evident from the increase in the fraction of dose trafficked through the lymph nodes in the presence of hypertonic buffers. The fraction of the dose trafficked through the lymphatics, as estimated by the model, increased from 0.05% in isotonic buffer to 13% in hypertonic buffer containing NaCl to about 30% for hypertonic buffers containing high dose OPLS and mannitol. The data suggest that hypertonic solutions may be a viable option for improving s.c. bioavailability.

O-phospho-l-serine mediates hyporesponsiveness toward FVIII in hemophilia A-murine model by inducing tolerogenic properties in dendritic cells.

The clinical use of therapeutic proteins can be complicated by the development of anti-product antibodies. We have previously observed that O-phospho-l-serine (OPLS) reduced antibody response to FVIII in Hemophilia-A (HA) mice. However, the mechanism underlying this observation is not clear. We hypothesize that OPLS reduces immunogenicity by inducing tolerogenic properties in dendritic cells (DCs). We tested this hypothesis using in vivo, in vitro, and ex vivo methods. Naive HA mice that were pre-exposed to FVIII in the presence of OPLS showed substantially lower antibody response following rechallenge with OPLS free FVIII as compared with dexamethasone-pretreated mice. Exposure of OPLS to bone-marrow-derived dendritic cells (BMDCs) in culturing conditions resulted in an increase in the regulatory cytokine TGF-ß and a decrease in proinflammatory cytokines TNF-α and IL12p70. This was accompanied by a significant reduction in upregulation of costimulatory marker CD40, as measured by flow cytometry. Furthermore, ex vivo matured BMDCs in the presence of FVIII and OPLS failed to elicit a robust immune response in HA mice compared with FVIII-treated BMDCs. Our data suggest that OPLS modulates the immune response by altering the function and maturation of DCs, resulting in the induction of tolerogenic properties. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3457-3463, 2014.

Immunogenicity of subcutaneously administered therapeutic proteins--a mechanistic perspective.

The administration of therapeutic proteins via the subcutaneous route (sc) is desired for compliance and convenience, but could be challenging due to perceived immunogenic potential or unwanted immune responses. There are clinical and preclinical data supporting as well as refuting the generalized notion that sc is more immunogenic. We provide a mechanistic perspective of immunogenicity of therapeutic proteins administered via the sc route and discuss strategies and opportunities for novel therapeutic approaches to mitigate immunogenicity.

Immunogenicity and pharmacokinetic studies of recombinant factor VIII containing lipid cochleates.

Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15-30% of patients develop inhibitory antibodies against administered rFVIII, which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previous studies developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. It was hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. This study investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as a delayed release delivery vehicle with an input peak of cochleates showed limited RES uptake and associated rFVIII displayed a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.