Sathy V. Balu-Iyer,1,2 Razvan D. Miclea,2 and Vivek S. Purohit3 1 University at Buffalo, State Un... more Sathy V. Balu-Iyer,1,2 Razvan D. Miclea,2 and Vivek S. Purohit3 1 University at Buffalo, State University of New York, Amherst, New York 2 Roswell Park Cancer Institute, Buffalo, New York 3 R&D, Eurand, Inc., Vandalia, Ohio ... Chapter Contents 1 Introduction 1.1 Recombinant ...
To determine the effect of dose, the anatomical site of injection, and the injection volume on su... more To determine the effect of dose, the anatomical site of injection, and the injection volume on subcutaneous absorption of rituximab in rats and to explore absorption mechanisms using pharmacokinetic modeling. Rituximab serum concentrations were measured following intravenous and subcutaneous administration at the back, abdomen, and foot of rats. Several pharmacokinetic models were developed that included linear and saturable absorption, and degradation and/or protective binding at the injection site. Rituximab exhibited linear kinetics following intravenous administration; however, bioavailability following subcutaneous injection was inversely related to the dose level. For the 1 mg/kg dose, bioavailability was approximately 70% at all tested injection sites, with faster absorption from the foot (T(max) = 12 h for foot vs. 4.6 days for back). Bioavailability for the 10 mg/kg dose was 44 and 31% for the abdomen and back sites and 18% for 40 mg/kg injected at the back. A pharmacokinetic model that included binding as part of the absorption mechanism successfully captured the nonlinearities in rituximab absorption. The anatomical site of subcutaneous injection influences the rate of absorption and bioavailability of rituximab in rats. Saturable binding may be a major determinant of the nonlinear absorptive transport of monoclonal antibodies.
Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy f... more 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, 1530% of patients develop inhibitory antibodies against administered rFVIII, which complicates the ...
The development of antidrug antibodies, also termed inhibitors, against administered factor VIII ... more The development of antidrug antibodies, also termed inhibitors, against administered factor VIII (FVIII) is one of the major complications in the clinical management of hemophilia A. Once formed, these inhibitory antibodies abrogate the activity of FVIII, resulting in loss of hemostatic efficacy and patients are subjected to increased risk of bleeding tendencies. Current treatment options after inhibitor development are expensive and ineffective in some cases. Therefore, treatment strategies that can prevent inhibitor formation is an effective approach in the management of hemophilia A.
A major complication of replacement therapy with Factor VIII (FVIII) for hemophilia A (HA) is the... more A major complication of replacement therapy with Factor VIII (FVIII) for hemophilia A (HA) is the development of unwanted immune responses. Previous studies showed that administration of FVIII in the presence of phosphatidyl serine (PS) reduced the development of anti-FVIII antibodies in HA mice. However, the impact of PS-mediated effects on immunological memory, such as generation of memory B-cells, is not clear. The effect of PS on memory B-cells was therefore investigated using adoptive transfer approach in FVIII(-/-) HA mice. Adoptive transfer of memory B-cells from a PS-FVIII-treated group to naïve mice followed by challenge of the recipient mice with FVIII showed a significantly reduced anti-FVIII antibody response in the recipient mice, compared with animals that received memory B-cells from free FVIII and FVIII-charge matched phosphatidyl glycerol (PG) group. The decrease in memory B-cell response is accompanied by an increase in FoxP3 expressing regulatory T-cells (Tregs). Flow cytometry studies showed that the generation of Tregs is higher in PS-treated animals as compared with FVIII and FVIII-PG treated animals. The PS-mediated hyporesponsiveness was found to be antigen-specific. The PS-FVIII immunization showed hyporesponsiveness toward FVIII rechallenge but not against ovalbumin (OVA) rechallenge, an unrelated antigen. This demonstrates that PS reduces immunologic memory of FVIII and induces antigen-specific peripheral tolerance in HA mice.
The role of aggregates in the immunogenicity of biologics is a major concern. A recent US FDA gui... more 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. Further...
The identification of immunosuppressive factors within human tumor microenvironments, and the abi... more The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients' anti-tumor 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 80nm. 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 immune suppressive vesicles in the microenvironments of ovarian tumors and our ability ...
Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progres... more Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to ...
Progress in Molecular Biology and Translational Science, 2008
ABSTRACT Proteins as therapeutic agents are highly specific and efficient; however, development o... more ABSTRACT Proteins as therapeutic agents are highly specific and efficient; however, development of therapeutic proteins as drugs presents difficulties. The difficulties are determined by the complexity of the protein structure, folding, and its sensitivity to the environmental conditions. Proteins are subjected to chemical and physical instability, which greatly affects their efficacy as therapeutic agents. Changes in protein conformation resulting in protein unfolding, aggregation, and denaturation can abolish the activity of the protein and additionally increase its immunogenicity and toxicity. Different factors during therapeutic protein development affect its stability and folding, for example, pH shift, temperature and pressure changes, presence of salts, metal ions and surfactants, pressure, shaking and shearing, absorption to surfaces, and protein concentration. Monitoring of stability and folding of therapeutic proteins and biosimilars by analytical techniques is extremely, important for their development and confirmation of their structure and function. The case study of recombinant factor VIII demonstrates how investigation of folding by a variety of biochemical and biophysical techniques led to the rational development of a more stable formulation of factor VIII.
Subcutaneous route of administration is highly desirable for protein therapeutics. It improves pa... more 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.
Immunogenicity against therapeutic proteins is a clinical problem in the successful treatment of ... more Immunogenicity against therapeutic proteins is a clinical problem in the successful treatment of many diseases and, as such, immunogenicity risk assessment in preclinical setting would be useful to improve safety and efficacy of protein-based therapeutics in the product development stages. Here, we attempted a mechanism-based in vitro study as screening tool to capture clinically observed antibody-based immune response against two representative therapeutic proteins: recombinant human Erythropoietin-alpha (rHuEPO)and recombinant Factor VIII (rFVIII). Flow-cytometry was used to determine the maturation level of dendritic cells (DCs), a primary initiator of T-cell responses. Studies to capture T-lymphocyte proliferation upon challenge with free rFVIII were performed and secretion of immunomodulatory cytokines was analyzed by ELISA assay. These in vitro techniques could be used as risk assessment tool to determine the immunogenic potential of formulations of recombinant proteins in preclinical setting.
The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailabi... more 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...
Sathy V. Balu-Iyer,1,2 Razvan D. Miclea,2 and Vivek S. Purohit3 1 University at Buffalo, State Un... more Sathy V. Balu-Iyer,1,2 Razvan D. Miclea,2 and Vivek S. Purohit3 1 University at Buffalo, State University of New York, Amherst, New York 2 Roswell Park Cancer Institute, Buffalo, New York 3 R&D, Eurand, Inc., Vandalia, Ohio ... Chapter Contents 1 Introduction 1.1 Recombinant ...
To determine the effect of dose, the anatomical site of injection, and the injection volume on su... more To determine the effect of dose, the anatomical site of injection, and the injection volume on subcutaneous absorption of rituximab in rats and to explore absorption mechanisms using pharmacokinetic modeling. Rituximab serum concentrations were measured following intravenous and subcutaneous administration at the back, abdomen, and foot of rats. Several pharmacokinetic models were developed that included linear and saturable absorption, and degradation and/or protective binding at the injection site. Rituximab exhibited linear kinetics following intravenous administration; however, bioavailability following subcutaneous injection was inversely related to the dose level. For the 1 mg/kg dose, bioavailability was approximately 70% at all tested injection sites, with faster absorption from the foot (T(max) = 12 h for foot vs. 4.6 days for back). Bioavailability for the 10 mg/kg dose was 44 and 31% for the abdomen and back sites and 18% for 40 mg/kg injected at the back. A pharmacokinetic model that included binding as part of the absorption mechanism successfully captured the nonlinearities in rituximab absorption. The anatomical site of subcutaneous injection influences the rate of absorption and bioavailability of rituximab in rats. Saturable binding may be a major determinant of the nonlinear absorptive transport of monoclonal antibodies.
Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy f... more 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, 1530% of patients develop inhibitory antibodies against administered rFVIII, which complicates the ...
The development of antidrug antibodies, also termed inhibitors, against administered factor VIII ... more The development of antidrug antibodies, also termed inhibitors, against administered factor VIII (FVIII) is one of the major complications in the clinical management of hemophilia A. Once formed, these inhibitory antibodies abrogate the activity of FVIII, resulting in loss of hemostatic efficacy and patients are subjected to increased risk of bleeding tendencies. Current treatment options after inhibitor development are expensive and ineffective in some cases. Therefore, treatment strategies that can prevent inhibitor formation is an effective approach in the management of hemophilia A.
A major complication of replacement therapy with Factor VIII (FVIII) for hemophilia A (HA) is the... more A major complication of replacement therapy with Factor VIII (FVIII) for hemophilia A (HA) is the development of unwanted immune responses. Previous studies showed that administration of FVIII in the presence of phosphatidyl serine (PS) reduced the development of anti-FVIII antibodies in HA mice. However, the impact of PS-mediated effects on immunological memory, such as generation of memory B-cells, is not clear. The effect of PS on memory B-cells was therefore investigated using adoptive transfer approach in FVIII(-/-) HA mice. Adoptive transfer of memory B-cells from a PS-FVIII-treated group to naïve mice followed by challenge of the recipient mice with FVIII showed a significantly reduced anti-FVIII antibody response in the recipient mice, compared with animals that received memory B-cells from free FVIII and FVIII-charge matched phosphatidyl glycerol (PG) group. The decrease in memory B-cell response is accompanied by an increase in FoxP3 expressing regulatory T-cells (Tregs). Flow cytometry studies showed that the generation of Tregs is higher in PS-treated animals as compared with FVIII and FVIII-PG treated animals. The PS-mediated hyporesponsiveness was found to be antigen-specific. The PS-FVIII immunization showed hyporesponsiveness toward FVIII rechallenge but not against ovalbumin (OVA) rechallenge, an unrelated antigen. This demonstrates that PS reduces immunologic memory of FVIII and induces antigen-specific peripheral tolerance in HA mice.
The role of aggregates in the immunogenicity of biologics is a major concern. A recent US FDA gui... more 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. Further...
The identification of immunosuppressive factors within human tumor microenvironments, and the abi... more The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients' anti-tumor 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 80nm. 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 immune suppressive vesicles in the microenvironments of ovarian tumors and our ability ...
Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progres... more Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to ...
Progress in Molecular Biology and Translational Science, 2008
ABSTRACT Proteins as therapeutic agents are highly specific and efficient; however, development o... more ABSTRACT Proteins as therapeutic agents are highly specific and efficient; however, development of therapeutic proteins as drugs presents difficulties. The difficulties are determined by the complexity of the protein structure, folding, and its sensitivity to the environmental conditions. Proteins are subjected to chemical and physical instability, which greatly affects their efficacy as therapeutic agents. Changes in protein conformation resulting in protein unfolding, aggregation, and denaturation can abolish the activity of the protein and additionally increase its immunogenicity and toxicity. Different factors during therapeutic protein development affect its stability and folding, for example, pH shift, temperature and pressure changes, presence of salts, metal ions and surfactants, pressure, shaking and shearing, absorption to surfaces, and protein concentration. Monitoring of stability and folding of therapeutic proteins and biosimilars by analytical techniques is extremely, important for their development and confirmation of their structure and function. The case study of recombinant factor VIII demonstrates how investigation of folding by a variety of biochemical and biophysical techniques led to the rational development of a more stable formulation of factor VIII.
Subcutaneous route of administration is highly desirable for protein therapeutics. It improves pa... more 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.
Immunogenicity against therapeutic proteins is a clinical problem in the successful treatment of ... more Immunogenicity against therapeutic proteins is a clinical problem in the successful treatment of many diseases and, as such, immunogenicity risk assessment in preclinical setting would be useful to improve safety and efficacy of protein-based therapeutics in the product development stages. Here, we attempted a mechanism-based in vitro study as screening tool to capture clinically observed antibody-based immune response against two representative therapeutic proteins: recombinant human Erythropoietin-alpha (rHuEPO)and recombinant Factor VIII (rFVIII). Flow-cytometry was used to determine the maturation level of dendritic cells (DCs), a primary initiator of T-cell responses. Studies to capture T-lymphocyte proliferation upon challenge with free rFVIII were performed and secretion of immunomodulatory cytokines was analyzed by ELISA assay. These in vitro techniques could be used as risk assessment tool to determine the immunogenic potential of formulations of recombinant proteins in preclinical setting.
The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailabi... more 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...
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