Evolving regulatory perspectives on digital health technologies for medicinal product development.

Digital health technology tools (DHTTs) present real opportunities for accelerating innovation, improving patient care, reducing clinical trial duration and minimising risk in medicines development. This review is comprised of four case studies of DHTTs used throughout the lifecycle of medicinal products, starting from their development. These cases illustrate how the regulatory requirements of DHTTs used in medicines development are based on two European regulatory frameworks (medical device and the medicinal product regulations) and highlight the need for increased collaboration between various stakeholders, including regulators (medicines regulators and device bodies), pharmaceutical sponsors, manufacturers of devices and software, and academia. As illustrated in the examples, the complexity of the interactions is further increased by unique challenges related to DHTTs. These case studies are the main examples of DHTTs with a regulatory assessment thus far, providing an insight into the applicable current regulatory approach; they were selected by a group of authors, including regulatory specialists from pharmaceutical sponsors, technology experts, academic researchers and employees of the European Medicines Agency. For each case study, the challenges faced by sponsors and proposed potential solutions are discussed, and the benefit of a structured interaction among the different stakeholders is also highlighted.

Biomarker and Companion Diagnostics-A Review of Medicinal Products Approved by the European Medicines Agency.

Background: An increasing number of medicines authorised in Europe recommend or require biomarker-based patient selection. For some of these the use of a companion diagnostic (CDx), a subset of in vitro diagnostics (IVDs), to identify patient populations eligible for a specific medicinal product may be required. The information and recommendations of use of a medicinal product for which a CDx is required is particularly important to healthcare professionals for correct patient identification. Methods: We reviewed the existing information in SmPCs and European Public Assessment Reports (EPARs) of EU medicinal products approved via the centralised procedure at EMA where reference was made to biomarker testing, including by CDx, for patient selection. Results: The results show that varying levels of detail are provided for the biomarker and the diagnostic test, including variability in where the information was presented. The overall results demonstrate transparent but sometimes heterogeneous reporting of CDx in the SmPC and EPAR. Conclusions: With the introduction of the new Regulation (EU) 2017/746 on in vitro diagnostic medical devices, medicines regulatory authorities' will be required to be consulted during the review of CDx conformity assessment and so, there is opportunity for more consistent and transparent information on CDx to be provided in the SmPC and EPAR.

Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus.

Real time PCR technology was applied to the development of assays for detection and quantitation of porcine endogenous retrovirus (PERV) RNA and DNA sequences in tissues and cells of human or animal origin. A plasmid construct encoding the PERV-pol gene or the in vitro transcribed RNA derived from the plasmid (cRNA) serves as a standard template for amplification of a 178 bp fragment. This study showed that the detection of this target sequence was linear over a range from 20 copies to 2 million copies of the plasmid and from 100 copies to 1 million copies of the cRNA. In addition, amplification of the target sequence was not inhibited by the presence of exogenous genomic DNA. These results demonstrate that a real time (TaqMan-based) PCR or RT-PCR assay can provide a sensitive, reproducible, and robust method for detecting and quantifying PERV DNA or RNA sequences in samples of human or guinea pig origin.

Effects of boosted tipranavir and lopinavir on body composition, insulin sensitivity and adipocytokines in antiretroviral-naive adults.

OBJECTIVES: Thymidine-based nucleoside analogue reverse transcriptase inhibitors and some protease inhibitors of HIV are associated with lipoatrophy, relative central fat accumulation and insulin resistance. The latter associations have not been well evaluated prospectively in adults commencing antiretroviral therapy. We studied the effects of protease inhibitor-based antiretroviral regimens on body composition, insulin sensitivity and adipocytokine levels. DESIGN: 48-week substudy of a randomized, open-label, three-arm trial. SETTING: Hospital and community HIV clinics. PARTICIPANTS: 140 HIV-infected adults naive to antiretroviral therapy. INTERVENTION: Tipranavir/ritonavir [500/200 mg twice a day (TPV/r200)] or [500/100 mg twice a day (TPV/r100)] or lopinavir/ritonavir [400/100 mg twice a day (LPV/r)], each with tenofovir + lamivudine. MAIN OUTCOME MEASURES: Body composition [dual-energy x-ray absorptiometry for limb fat; L4, abdominal computed tomography for visceral adipose tissue (VAT)]; and fasting metabolic parameters. The primary analysis was change in limb fat mass in each TPV/r group vs. LPV/r. RESULTS: Limb fat increased in all three groups: LPV/r (1.17 kg) versus TPV/r200 (0.83 kg; P = 0.16) and TPV/r100 (0.41 kg; P = 0.07). VAT decreased in all groups: LPV/r (-3 cm) vs. TPV/r200 (-9 cm; P = 0.04) and TPV/r100 (-6 cm; P = 0.40). No significant change in insulin sensitivity was observed, including by oral glucose tolerance testing. The increase in leptin levels was significantly correlated with the increase in limb fat mass (r = 0.67; P < 0.0001). Despite increased limb fat, adiponectin levels increased, but significantly more with TPV/r200 (+6010 ng/ml; P < 0.0001) or TPV/r100 (+4497 ng/ml; P = 0.002) when compared with LPV/r (+1360 ng/ml). CONCLUSION: Unlike many other antiretroviral regimens, TPV/r or LPV/r with tenofovir-lamivudine increased subcutaneous fat without evidence for increasing visceral fat or insulin resistance over 48 weeks.

Porcine endogenous retrovirus infects but does not replicate in nonhuman primate primary cells and cell lines.

Porcine endogenous retroviruses (PERV) can infect human cell lines in vitro; hence, there is a presumed risk of viral exposure to a recipient when pig cells are transplanted into humans (xenotransplantation). Nonhuman primates (NHP) are considered a potential permissive animal model to study the risk of in vivo infection of PERV after xenotransplantation. We set out to determine whether PERV can infect and replicate in NHP primary cells or established cell lines from African green monkey, rhesus macaque, and baboon. We confirm that the NHP cell lines under investigation were infected with PERV as measured by detection of viral DNA and RNA by PCR and reverse transcription (RT)-PCR, respectively, indicating that a functional receptor must be present on the cell surface. However, the load of detectable viral DNA in infected NHP cells declined over time, and the cells never had detectable reverse transcriptase activity. Utilizing quantitative real-time TaqMan PCR we found detectable levels of unintegrated DNA intermediates, but the levels were approximately 100-fold lower compared to HEK 293 cells infected with PERV. Virions released from infected NHP cells could productively infect naïve human cell lines, HEK 293 and HeLa, as shown by RT-PCR and RT assay. However, naïve NHP cells remained negative in RT-PCR and RT assay after exposure to virions from infected NHP cells. Together our data demonstrate that NHP cells are not permissive to productive replication by PERV, presumably due to inefficient cell entry and replication. In light of these observations, the appropriateness of NHP as suitable animal models to study PERV infection in vivo needs to be reevaluated.

Sequence analysis of porcine endogenous retrovirus long terminal repeats and identification of transcriptional regulatory regions.

Porcine cells express endogenous retroviruses, some of which are infectious for human cells. To better understand the replication of these porcine endogenous retroviruses (PERVs) in cells of different types and animal species, we have performed studies of the long terminal repeat (LTR) region of known gammaretroviral isolates of PERV. Nucleotide sequence determination of the LTRs of PERV-NIH, PERV-C, PERV-A, and PERV-B revealed that the PERV-A and PERV-B LTRs are identical, whereas the PERV-NIH and PERV-C LTRs have significant sequence differences in the U3 region between each other and with the LTRs of PERV-A and PERV-B. Sequence analysis revealed a similar organization of basal promoter elements compared with other gammaretroviruses, including the presence of enhancer-like repeat elements. The sequences of the PERV-NIH and PERV-C repeat element are similar to that of the PERV-A and PERV-B element with some differences in the organization of these repeats. The sequence of the PERV enhancer-like repeat elements differs significantly from those of other known gammaretroviral enhancers. The transcriptional activities of the PERV-A, PERV-B, and PERV-C LTRs relative to each other were similar in different cell types of different animal species as determined by transient expression assays. On the other hand, the PERV-NIH LTR was considerably weaker in these cell types. The transcriptional activity of all PERV LTRs was considerably lower in porcine ST-IOWA cells than in cell lines from other species. Deletion mutant analysis of the LTR of a PERV-NIH isolate identified regions that transactivate or repress transcription depending on the cell type.