HEALTH TECHNOLOGY ASSESSMENT IN EVALUATION OF PHARMACEUTICALS IN THE CZECH REPUBLIC.

OBJECTIVES: In the Czech Republic, the health technology assessment (HTA) approaches have been implemented in evaluation of medicinal products since 2008. The aim of this study was to provide an overview of the implementation of HTA and different levels thereof in the evaluation process conducted by the State Institute for Drug Control (SUKL) and to describe the impact of HTA on the entrance of new medicinal entities into out-patient healthcare system including highly innovative and orphan drugs. METHODS: Materials supporting this overview were collected using the records in the database of administrative proceedings of SUKL, in-house standard operating procedures, and the legislation in force. Based on these sources as well as the hands-on knowledge of the current practice, a brief description of the general rules of administrative proceedings involving HTA of varying complexity was elaborated. Characteristic features of the individual types of proceedings, basic differences in the complexity of HTA employed, and its most important challenges were summarized. RESULTS: In Czech Republic, HTA in the formal administrative proceedings ensures a transparent process of introduction of new medicinal products into clinical practice and leaves space for restriction of reimbursement conditions to minimize budget impact. CONCLUSIONS: As a robust as well as pragmatic HTA methodology has been implemented by SUKL, relevant stakeholders (marketing authorization holders, Health Care Funds, clinical expert groups) are now able to influence reimbursement of new technologies.

The Arf GAP ASAP1 provides a platform to regulate Arf4- and Rab11-Rab8-mediated ciliary receptor targeting.

Dysfunctional trafficking to primary cilia is a frequent cause of human diseases known as ciliopathies, yet molecular mechanisms for specific targeting of sensory receptors to cilia are largely unknown. Here, we show that the targeting of ciliary cargo, represented by rhodopsin, is mediated by a specialized system, the principal component of which is the Arf GAP ASAP1. Ablation of ASAP1 abolishes ciliary targeting and causes formation of actin-rich periciliary membrane projections that accumulate mislocalized rhodopsin. We find that ASAP1 serves as a scaffold that brings together the proteins necessary for transport to the cilia including the GTP-binding protein Arf4 and the two G proteins of the Rab family--Rab11 and Rab8--linked by the Rab8 guanine nucleotide exchange factor Rabin8. ASAP1 recognizes the FR ciliary targeting signal of rhodopsin. Rhodopsin FR-AA mutant, defective in ASAP1 binding, fails to interact with Rab8 and translocate across the periciliary diffusion barrier. Our study implies that other rhodopsin-like sensory receptors may interact with this conserved system and reach the cilia using the same platform.

Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments.

The biogenesis of cilia-derived sensory organelles, the photoreceptor rod outer segments (ROS), is mediated by rhodopsin transport carriers (RTCs). The small GTPase Rab8 regulates ciliary targeting of RTCs, but their specific fusion sites have not been characterized. Here, we report that the Sec6/8 complex, or exocyst, is a candidate effector for Rab8. We also show that the Qa-SNARE syntaxin 3 is present in the rod inner segment (RIS) plasma membrane at the base of the cilium and displays a microtubule-dependent concentration gradient, whereas the Qbc-SNARE SNAP-25 is uniformly distributed in the RIS plasma membrane and the synapse. Treatment with omega-3 docosahexaenoic acid [DHA, 22:6(n-3)] causes increased co-immunoprecipitation and colocalization of SNAP-25 and syntaxin 3 at the base of the cilium, which results in the increased delivery of membrane to the ROS. This is particularly evident in propranolol-treated retinas, in which the DHA-mediated increase in SNARE pairing overcomes the tethering block, including dissociation of Sec8 into the cytosol. Together, our data indicate that the Sec6/8 complex, syntaxin 3 and SNAP-25 regulate rhodopsin delivery, probably by mediating docking and fusion of RTCs. We show further that DHA, an essential polyunsaturated fatty acid of the ROS, increases pairing of syntaxin 3 and SNAP-25 to regulate expansion of the ciliary membrane and ROS biogenesis.

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4.

Dysfunctions of primary cilia and cilia-derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans-Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase-activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post-TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1-mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4-based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

Assay for in vitro budding of ciliary-targeted rhodopsin transport carriers.

Primary cilia and cilia-derived sensory organelles are cell's antennas that contain sensory receptors and signal transduction modules. Defects in the expression and targeting of ciliary proteins to this specialized cellular compartment lead to human disorders collectively known as ciliopathies. To examine the molecular basis for the ciliary targeting of the light receptor rhodopsin, we have developed a cell-free assay that reconstitutes its packaging into the specific post-Golgi rhodopsin transport carriers (RTCs). This assay accurately reproduces the in vivo process of carrier budding, while allowing examination of individual components of the macromolecular complexes, thus providing insight into a more general mechanism for the regulation of ciliary membrane targeting. Examples are shown for the use of this assay in rhodopsin trafficking. The cell-free assay is applicable to other ciliary-targeted sensory molecules.

Cochlear function in young and adult Fischer 344 rats.

Fischer 344 (F344) rats are often used as an animal model for investigation of the mechanisms underlying age-related hearing loss. The aim of this study was to assess cochlear function in young (1-month-old) and adult (6-month-old) F344 rats using recording of otoacoustic emissions and auditory brainstem responses (ABRs). The results were compared with control groups of Long Evans (LE) rats of the same ages. The results demonstrate a significant increase in the hearing threshold in F344 rats in comparison with LE rats, expressed mainly at low frequencies (1-2 kHz). In F344 rats, transient evoked otoacoustic emissions were not measurable and distortion product otoacoustic emissions could be detected within a frequency range of 2.4-6.3 kHz. Tympanometric measurements did not reveal any differences in middle ear parameters between F344 and LE rats. The amplitudes of click-evoked ABRs were significantly lower in 6-month-old F344 rats than in LE rats, but other parameters of the ABRs were almost identical in both rat strains. The results demonstrate a significant deficit in low-frequency hearing and altered otoacoustic emissions in both young and adult F344 rats, suggesting a defect of the inner ear sensory epithelium at the apical part of the cochlea.

Auditory function in presbycusis: peripheral vs. central changes.

The hearing abilities of a group of 30 elderly (67-93 yr of age) subjects were compared with those of a group of 30 young (19-27 yr of age) normal hearing volunteers with the aim of characterizing the changes in the peripheral and central parts of the auditory system. In elderly subjects the pure-tone thresholds were typically represented by a gradually sloping curve with a significantly greater decline in men than in women at frequencies of 3 and 4 kHz. In spite of pure tone threshold elevation in the elderly, the difference limen for intensity at 1 and 3 kHz were not significantly smaller than in the young subjects. The incidence and levels of spontaneous, transient and distortion product otoacoustic emissions were low, which would suggest the involvement of outer hair cell pathology. Also, contralateral suppression was less marked in elderly than in young subjects. Speech audiometry in the elderly revealed serious difficulties in understanding speech. Deteriorated temporal resolution, as demonstrated by increased gap detection thresholds, correlated significantly with increased speech recognition thresholds. The results support the view that presbycusis represents a combination of deteriorated function of the auditory periphery with deteriorated function of the central auditory system.