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Current management guidelines for urea cycle disorders (UCDs) offer clear strategies, incorporating both authorized and non-authorized medicinal products (including intravenous formulations and products regulated as food). These varying product categories are subject to specific accessibility challenges related to availability, reimbursement, and pricing. The aim of this study is to identify potential obstacles to optimal UCD treatment implementation in European clinical practice. A survey aimed at metabolic healthcare professionals (HCPs) managing patients with UCDs in Europe was disseminated through the European Reference Network for Hereditary Metabolic Disorders and the European registry and network for intoxication type metabolic diseases. Forty-eight survey responses were collected from 21 European countries. In 16 of these countries, at least one metabolic HCP reported challenges in accessing UCD products. Reimbursement issues were reported for most products (8/10), including both authorized and non-authorized products. Availability-related challenges were also reported for 8/10 products, although unavailability was limited to non-authorized products. Prices impacted accessibility for all authorized products (3/3) and one non-authorized IV product. The accessibility of UCD treatment products varied across Europe, although no clear regional variations could be discerned. Survey data revealed that metabolic HCPs experience challenges in accessing both authorized and non-authorized products for UCD management in the majority of European countries. This indicates that registering unauthorized products may not resolve all issues. Improved reimbursement policies and fair pricing models, as well as (adjusted) authorization procedures may help address these concerns, thereby optimizing treatment access for UCD patients., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

1. We investigated why angiotensin (Ang) I and II induce vasoconstriction with similar potencies, although Ang I-II conversion is limited. 2. Construction of concentration-response curves to Ang I and II in porcine femoral arteries, in the absence or presence of the AT(1) or AT(2) receptor antagonists irbesartan and PD123319, revealed that the approximately 2 fold difference in potency between Ang I and II was not due to stimulation of different AT receptor populations by exogenous and locally generated Ang II. 3. Measurement of Ang I and II and their metabolites at the time of vasoconstriction confirmed that, at equimolar application of Ang I and II, bath fluid Ang II during Ang I was approximately 18 times lower than during Ang II and that Ang II was by far the most important metabolite of Ang I. Tissue Ang II was 2.9+/-1.5% and 12.2+/-2.4% of the corresponding Ang I and II bath fluid levels, and was not affected by irbesartan or PD123319, suggesting that it was located extracellularly. 4. Since approximately 15% of tissue weight consists of interstitial fluid, it can be calculated that interstitial Ang II levels during Ang II resemble bath fluid Ang II levels, whereas during Ang I they are 8.8 - 27 fold higher. Consequently at equimolar application of Ang I and II, the interstitial Ang II levels differ only 2 - 4 fold. 5. Interstitial, rather than circulating Ang II determines vasoconstriction. Arterial Ang I, resulting in high interstitial Ang II levels via its local conversion by ACE, may be of greater physiological importance than arterial Ang II.

Using a combination of RT - PCR and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-HT(1B) receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an open reading frame of 390 amino acids showing a 95% similarity with the human 5-HT(1B) receptor. The recombinant porcine 5-HT(1B) cDNA was expressed in monkey Cos-7 cells and its pharmacological profile was determined by radioligand binding assay using [(3)H]-GR125743. The affinities of several agonists (L694247>ergotamine > or =5-carboxamidotryptamine=dihydroergotamine=5-HT>CP122638=zolmitriptan>sumatriptan) and putative antagonists (GR127935>methiothepin>SB224289>>ritanserin>ketanserin > or =BRL15572) correlated highly with those described for the recombinant human 5-HT(1B) receptor. In membranes obtained from cells co-expressing the porcine 5-HT(1B) receptor and a mutant G(alphao)Cys(351)Ile protein, 5-HT and zolmitriptan increased, while the 5-HT(1B) receptor antagonist SB224289 decreased basal [(35)S]-GTPgammaS binding, thus showing inverse agonism. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 7.64+/-0.04) agreed with its affinity in displacing the antagonist [(3)H]-GR125743 (pK(i): 7.36+/-0.07). The 5-HT(1B) receptor mRNA was observed by RT-PCR in several blood vessels, cerebral cortex, cerebellum and trigeminal ganglion. In situ hybridization performed in frontal cerebral cortex sections revealed the expression of 5-HT(1B) receptor mRNA in pyramidal cells. In conclusion, we have cloned and established the amino acid sequence, ligand binding profile and location of the porcine 5-HT(1B) receptor. This information may be useful in exploring the role of 5-HT(1B) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

A cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine 5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT(1D) receptor. Nonetheless, a selective antagonist at the human 5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine 5-HT(1D) receptor and rat G(alphail)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [(35)S]-GTPgammaS binding. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT(1D) receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor-ligand interaction as well as the role of 5-HT(1D) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.