Biorelevant In Vitro Performance Testing of Orally Administered Dosage Forms—Workshop Report

Biorelevant in vitro performance testing of orally administered dosage forms has become an important tool for the assessment of drug product in vivo behavior. An in vitro performance test which mimics the intraluminal performance of an oral dosage form is termed biorelevant. Biorelevant tests have been utilized to decrease the number of in vivo studies required during the drug development process and to mitigate the risk related to in vivo bioequivalence studies. This report reviews the ability of current in vitro performance tests to predict in vivo performance and generate successful in vitro and in vivo correlations for oral dosage forms. It also summarizes efforts to improve the predictability of biorelevant tests. The report is based on the presentations at the 2013 workshop, Biorelevant In Vitro Performance Testing of Orally Administered Dosage Forms, in Washington, DC, sponsored by the FIP Dissolution/Drug Release Focus Group in partnership with the American Association of Pharmaceutical Scientists (AAPS) and a symposium at the AAPS 2012 Annual meeting on the same topic.     

Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), employs host-cell angiotensin-converting enzyme 2 (ACE2) for cell entry. Genetic analyses of ACE2 have identified several single-nucleotide polymorphisms (SNPs) specific to different human populations. Molecular dynamics simulations have indicated that several of these SNPs could affect interactions between SARS-CoV-2 and ACE2, thereby providing a partial explanation for the regional differences observed in SARS-CoV-2 infectivity and severity. However, the significance of population-specific ACE2 SNPs in SARS-CoV-2 infectivity is unknown, as no in vitro validation studies have been performed. Here, we analyzed the impact of eight SNPs found in specific populations on receptor binding and cell entry in vitro. Except for a SNP causing a nonsense mutation that reduced ACE2 expression, none of the selected SNPs markedly altered the interaction between ACE2 and the SARS-CoV-2 spike protein (SARS-2-S), which is responsible for receptor recognition and cell entry, or the efficiency of viral cell entry mediated by SARS-2-S. Our findings indicate that ACE2 polymorphisms have limited impact on the ACE2-dependent cell entry of SARS-CoV-2 and underscore the importance of future studies on the involvement of population-specific SNPs of other host genes in susceptibility toward SARS-CoV-2 infection.     

Adacolumn,an adsorptive carrier based granulocyte and monocyte apheresis device for the treatment of inflammatory and refractory diseases associated with leukocytes

Apheresis has been recognized both economically and therapeutically as a novel approach for the treatment of inflammatory diseases, and certain others, which respond poorly to drug therapy. This report is about Adacolumn, an adsorptive carrier based granulocyte and monocyte apheresis device with a volume of 335 mL, filled with about 220 g of cellulose acetate beads of 2 mm diameter as the column adsorptive carriers. Pre- and post-column leukocyte counts have shown that the carriers adsorb about 65% of granulocytes, 55% of monocytes and 2% of lymphocytes from the blood in the column. Additionally, after apheresis, there is a marked decrease in inflammatory cytokines (TNF-alpha, IL-1beta, IL-6 and IL-8) produced by blood leukocytes, together with down-modulation of L-selectin and the chemokine receptor CXCR3. Adacolumn has been used to treat patients with rheumatoid arthritis, ulcerative colitis and HIV infection. Typical apheresis sessions have been 4-10, at a frequency of one or two sessions per week. Treatment of patients with Adacolumn has been associated with very promising efficacy and safety data. Accordingly, in Japan, Adacolumn has been approved by the Ministry of Health for the treatment of ulcerative colitia. Furthermore, Adacolumn met the required quality and safety standards for medical devices and received an EC certification (CE-mark) from TUV in 1999. However, although Adacolumn carriers are very efficient in depleting excess and activated granulocytes and monocytes/macrophages, the clinical efficacy associated with Adacolumn apheresis cannot be fully explained on the basis of reducing granulocytes and monocytes per se. Hence, a long lasting effect on inflammatory cytokine generation, chemokine activities or immunomodulation is likely, but the precise mechanisms involved are not fully understood yet.     

Selective Thymic Localization of Murine Leukemia Virus-Related Antigens in C57BL/Ka Mice after Inoculation with Radiation Leukemia Virus

The tissue distribution and extent of virus-specific antigen expression were studied by immunofluorescence as a function of time and of lymphoma development in adult C57BL/Ka (Fv-1(b)) mice after intravenous injection of radiation leukemia virus, a B-tropic murine leukemia virus. Viral antigens were detected earlier in the thymus (1 week) than in the bone marrow, spleen, or lymph nodes (2-3 weeks). Despite an initial virus-induced thymic involution, the percentage of immunofluorescence-positive cells in the thymus rapidly increased thereafter to 65-80%, at which level it remained until 9 weeks, at which time increases in size and weight, histological changes, and an increased number of blastoid cells indicated the onset of lymphoma development in the thymus. In contrast, the percentage of immunofluorescence-positive cells in the bone marrow, spleen, and nodes remained low, and gradually decreased to zero within 8 weeks after thymectomy. The selective thymic localization of antigens induced by radiation leukemia virus in C57BL/Ka mice is in striking contrast to the previously reported ubiquitous tissue distribution of the Gross-AKR virus, an N-tropic virus, in its natural host, the Fv-1(n), AKR strain with a high incidence of leukemia.     

Successful Outcome of Mismatched Hematopoietic Stem Cell Transplantation from a Related Donor in an Infant with Acute Lymphoblastic Leukemia and 9;11 Translocation: Case Report and Review of the Literature

Although infants with acute lymphoblastic leukemia (ALL) and MLL gene rearrangements have a poor prognosis, those with acute myeloid leukemia (AML) have been shown to have a superior outcome with intensive chemotherapy alone despite the presence of MLL gene rearrangements. We report the case of an ALL infant with t(9;11), a common cytogenetic abnormality in infant AML, who after relapse underwent successful hematopoietic stem cell transplantation (HSCT) from her HLA 2-loci-mismatched mother. Analysis of the outcome among ALL infants with MLL gene rearrangements registered in the Japan Infant Leukemia Study between 1996 and 1999 showed the event-free survival of patients with t(9;11) was not different from that of those with other 11q23 translocations. Most of the patients with t(9;11) described in the reviewed literature also experienced either induction failure or early relapse after achievement of complete remission, but some of them were rescued with subsequent HSCT. These findings suggest that infant ALL with t(9;11) has features distinct from those of infant AML with the same karyotype and that the prognosis among these patients can be improved only with the combination of intensive chemotherapy and HSCT An appropriate strategy for the treatment of ALL infants with different 11q23 translocations must be clarified.     

Shuttling of initiating kinase between discrete aggregates of the high affinity receptor for IgE regulates the cellular response

Using defined oligomers of IgE, our group previously studied the quantitative relationship between the aggregation of the high affinity receptors for IgE (FcRI) and the earliest signals initiated by such aggregation: the phosphorylation of tyrosines on the receptor. Notably, at certain doses of the oligomers such phosphorylation reached a plateau level well before the aggregation of the receptors had reached a maximum. These findings and others led us to propose that aggregates of the receptor were competing for a limited amount of the critical kinase—thought to be Lyn in this system. This paper describes a test of this proposal. We incubated cells with two distinguishable IgEs and examined the effect of aggregating one or the other or both types on the phosphorylation. When receptors binding antigen-specific IgE were aggregated with polyvalent antigen, they became rapidly phosphorylated as expected. Remarkably, however, FcRI that had already been phosphorylated by the binding of dimers of IgE, became dephosphorylated simultaneously. Furthermore, when the antigen-driven aggregates were dissociated with hapten, the phosphorylation pattern reverted to that seen prior to the addition of antigen: as the antigen-driven aggregates became dephosphorylated, the receptors stably aggregated by the bound oligomers became rapidly rephosphorylated. Dephosphorylation of oligomer-driven aggregates was also partially reversed during the “spontaneous” dephosphorylation of the antigen-driven receptors seen at longer times after addition of antigen. Thus signal transduction in this system is in part regulated by the shuttling of limited amounts of the kinase that initiates the cascade of phosphorylations.     

Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome

Early-onset sarcoidosis (EOS) and inheritable Blau syndrome (BS) share characteristic clinical features of juvenile-onset systemic granulomatosis syndrome that mainly affects skin, joints, and eyes. However, no direct evidence has been shown for the possible common origin of these 2 diseases. Recent discovery of CARD15 mutations in BS families encouraged us to investigate similar CARD15 mutations in EOS patients. Among 10 EOS cases retrospectively collected in Japan, heterozygous missense mutations were found in 9 cases; 4 showed a 1000C>T (R334W in amino acid change) that has been reported in BS, 4 showed novel 1487A>T (H496L), 1538T>C (M513T), 1813A>C (T605P), and 2010C>A (N670K), and 1 case showed double 1146C>G (D382E)/1834G>A (A612T) mutations on different alleles. All 6 of these variants of CARD15 showed increased basal nuclear factor (NF)-kappaB activity. These findings indicate that the majority of EOS and BS cases share the common genetic etiology of CARD15 mutations that cause constitutive NF-kappaB activation.