Cost minimization analysis of brolucizumab in the treatment of neovascular age-related macular degeneration.

INTRODUCTION: Brolucizumab is a novel anti-vascular endothelial growth factor (anti-VEGF) drug administered in a fixed regimen of 8 or 12 weeks which, in the HAWK and HARRIER studies, was shown not to be inferior to aflibercept with respect to the best corrected visual acuity, with a less burdensome treatment regimen. The aim of the analysis was to compare the direct healthcare costs of both anti-VEGF as a treatment in patients with neovascular age-related macular degeneration. MATERIAL AND METHODS: A cost minimization analysis was performed under a 25-year time horizon and considering the drug costs, administration, follow-up tests, and management of adverse events. Resource use was obtained from the related literature and validated by clinical experts. Various scenario analysis were carried out to check the robustness of the results. RESULTS: Brolucizumab resulted in a lower cost per patient compared with aflibercept, considering the number of injections derived from the HAWK and HARRIER studies. This result was maintained in the different scenarios analysed, except for the number of injections of the flexible aflibercept regimen of the ARIES study, since the lower discontinuation of treatment with brolucizumab implies maintaining the treatment of more patients. Considering the same discontinuation, brolucizumab maintained the results observed in the base case of the analysis. CONCLUSIONS: This study shows how the fixed administration regimen of brolucizumab can help reduce both healthcare and patients' burden.

A checklist for critical appraisal of indirect comparisons.

The aim of this study was to develop a user-friendly checklist for critical appraisal of indirect comparisons of drugs, considering clinical, methodological/statistical and quality aspects, mainly to be applied in drug evaluation in the decision-making context. After conducting a review of the literature, we used group consensus to establish the key points of the checklist, focusing mainly on indirect comparisons, but including topics related to network meta-analysis or multiple treatment comparisons. The coordinating group elaborated the first draft, which was reviewed by external experts, re-evaluated by the coordinating group and finally assessed by 23 drug evaluation experts trained in indirect comparisons, who applied the checklist to one study. The Kappa index of agreement was calculated and the final checklist was developed by group consensus including the external experts. The checklist has two parts. The first consists of three eliminatory key questions while the second includes 17 items: 5 regarding quality, 5 regarding clinical issues and 7 dealing with methodology/statistics. The median kappa values of the 23 evaluations were 0.83 (range 0.67-0.93), 0.61 (0.54-0.91) and 0.36 (0.22-1) with regard to quality, clinical aspects and methodology/statistics, respectively. A structured checklist was developed to facilitate critical appraisal of key issues in indirect comparisons, including comments for assessing the consequences of its application to drug evaluation in the decision-making context. Agreement between reviewers in clinical and quality items was good, but weaker in methodology/statistics ones.

Acute and chronic effects of morphine and naloxone on the phosphorylation of neurofilament-H proteins in the rat brain.

Increased amounts of phosphorylated neurofilaments (pNF-H and pNF-M) are found in postmortem brains of opioid addicts. Because of the potential relevance of aberrant pNF in opioid addiction (alterations of neuronal cytoskeleton and associated functions), the effects of opiate drugs on pNF-H were investigated in rat brain. Acute morphine (30 mg/kg, 2 h) induced a marked increase in the immunodensity of pNF-H in the cerebral cortex (93%). Chronic morphine (10-100 mg/kg for 5 days) followed by opiate withdrawal resulted in a time-dependent decline in pNF-H (induction of tolerance). Thus, 2 h after the last dose of morphine, the abundance of pNF-H was still increased (27%), which was followed (6-24 h) by down-regulation of pNF-H (5% increase at 6 h; 5% decrease at 12 h, and 29% decrease at 24 h). The acute (10 mg/kg for 2 h) and chronic (2 x 10 mg/kg for 14 days) treatments with naloxone, an opioid receptor antagonist, did not alter pNF-H in the cerebral cortex, suggesting that the opioid receptors (probably the mu-type) modulating the phosphorylation state of NF-H are not tonically activated by endogenous opioids. The results indicate that morphine addiction is associated with an aberrant hyperphophorylation of NF-H in the rat brain.

Regulation of G protein-coupled receptor kinase 2 in brains of opiate-treated rats and human opiate addicts.

The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8-22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18-25%). These results suggest that GRK2 is involved in the short-term regulation of mu-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.

Marked decrease of immunolabelled 68 kDa neurofilament (NF-L) proteins in brains of opiate addicts.

NEUROFILAMENT (NF) proteins, the major components of the neuronal cytoskeleton, have been shown to represent previously unknown targets for the chronic effects of morphine in rats. This study was designed to evaluate the abundance of immunoreactive NF-L (68 kDa) proteins in post-mortem brains of chronic opiate addicts who had died of a heroin or methadone overdose. Levels of NF-L proteins were assessed by immunoblotting techniques. Levels of immunoreactive NF-L proteins were markedly decreased (47%, n = 17) in the frontal cortex. The reduced abundance of brain NF-L proteins was not related to the post-mortem delay or to the plasma concentrations of opiates, suggesting that the observed changes represent a specific long-term effect of opiate drugs. Because of the functions associated with NF proteins (e.g. axonal transport), this finding suggests that opiate drugs may induce neuronal damage after chronic abuse in humans.

Modulation of immunoreactive protein kinase C-alpha and beta isoforms and G proteins by acute and chronic treatments with morphine and other opiate drugs in rat brain.

The abundance of protein kinase C-alpha and beta isoforms (PKC-alphabeta), PKC-alpha messenger (m) RNA and guanine nucleotide-binding G protein subunits (G alpha(i1/2), G alpha(o), and G beta) were quantitated in the rat cerebral cortex after acute and chronic treatments with various opiate drugs. Acute (100 mg/kg for 2 h) and chronic (10 to 100 mg/kg for 5 days) treatment with morphine decreased similarly the immunoreactivity of PKC-alphabeta (28% and 32%, respectively). Acute (2 h) and chronic treatment (5 days) with other mu-agonists heroin (30 mg/kg and 10 to 30 mg/kg) and methadone (30 mg/kg and 5 to 30 mg/kg) also induced similar decreases of PKC-alphabeta (acute: 25% and 23%; chronic: 28% and 18%). After the chronic treatments, spontaneous (48 h) or naloxone (2 mg/kg)-precipitated opiate withdrawal (2 h) resulted in up-regulation of PKC-alphabeta above control levels (30-38%), and in the case of morphine withdrawal in a concomitant marked increase in the expression of PKC-alpha mRNA levels (2.3-fold). Acute (2 h) treatments with pentazocine (80 mg/kg, mixed kappa/delta-agonist and mu-antagonist), spiradoline (30 mg/kg, selective kappa-agonist) and [D-Pen2, D-Pen5] enkephalin (14 nmol i.c.v., selective delta-agonist) induced significant decreases of PKC-alphabeta (19-33%). Chronic (5 days) treatment with pentazocine (10 to 80 mg/kg), but not spiradoline (2 to 30 mg/kg), also induced a similar decrease of PKC-alphabeta (35%). In pentazocine- or spiradoline-dependent rats, naloxone (2 mg/kg) did not induce up-regulation of brain PKC-alphabeta. Acute (10 mg/kg for 2 h) and chronic (2x10 mg/kg for 5 and 14 days) treatment with naloxone did not alter PKC-alphabeta immunoreactivity. Chronic, but not acute, treatment with mu-agonists (morphine, heroin and methadone) increased the immunoreactivities of G alpha(i1/2) (33-37%), G alpha(o), (25-41%) and G beta (10-33%) protein subunits. In heroin- and methadone-dependent rats naloxone (2 mg/kg)-precipitated withdrawal (2 h) did not modify the up-regulation of these G proteins induced by chronic mu-opiate treatment. In marked contrast to mu-agonists, chronic treatment with high doses of pentazocine and spiradoline or acute treatment with [D-Pen2, D-Pen5] enkephalin did not result in up-regulation of these G protein subunits. After chronic treatment with mu-agonists, significant negative correlations were found when the percentage changes in immunoreactivity of PKC-alphabeta were related to the percentage changes in immunoreactivity of G alpha(i1/2), (r = -0.53, n = 29) and G beta (r = -0.41, n = 24) in the same brains. PKC-alphabeta abundance did not correlate significantly with the density of G alpha(o) (r = -0.21, n = 28). Together the results indicate that the brain PKC-alphabeta system may play a major regulatory role in opiate tolerance and dependence. Moreover, the possible in vivo cross-communication between this regulatory enzyme and specific inhibitory G proteins may also be of relevance in the cellular and molecular processes of opiate addiction.

Naloxone-precipitated withdrawal in morphine-dependent rats increases the expression of alpha 2a-adrenoceptor mRNA in brain.

Opiate withdrawal has been associated with up-regulation of alpha 2-adrenoceptors (mainly the alpha 2A-subtype) in brain. The modulation of these inhibitory receptors regulating norepinephrine release appears to be a relevant mechanism by which the opiate abstinence syndrome might be counteracted. The aim of this study was to investigate possible changes in alpha 2a-adrenoceptor gene expression as the molecular mechanism underlying the opiate withdrawal-induced up-regulation of alpha 2A-adrenoceptors. In morphine-dependent rats (10-100 mg/kg for 5 days), naloxone (2 mg/kg)-precipitated withdrawal induced a rapid (2 h) and marked up-regulation (111%, P < 0.001) in the expression of alpha 2a-adrenoceptor mRNA (Northern and dot-blot analyses) in the cerebral cortex. Acute and chronic morphine treatments did not alter significantly the expression of cortical alpha 2a-adrenoceptor mRNA. The results indicate that the opiate abstinence syndrome is associated with a transcriptional activation of the alpha 2a-adrenoceptor mRNA which can explain the up-regulation of brain alpha 2A-adrenoceptors during opiate withdrawal.

Regulation of immunolabelled mu-opioid receptors and protein kinase C-alpha and zeta isoforms in the frontal cortex of human opiate addicts.

To assess the status of opioid receptors in the human brain during the process of opiate addiction, the abundance of immunoreactive mu-opioid receptors was quantitated in postmortem brains of chronic opiate addicts who had died of a heroin or methadone overdose. The immunoreactive levels of the associated enzyme protein kinase C (PKC-alpha and zeta isoforms) and G proteins (G alpha(i1/2) subunits) were also assessed in the same brains. In the frontal cortex of opiate addicts, the abundance of mu-opioid receptors was not different from that obtained in matched controls. The level of Ca2+-dependent PKC-alpha was decreased (25%), whereas that of the atypical PKC-zeta remained unchanged. The density of G alpha(i1/2) proteins also was found to be increased (40%). The results indicate that opiate addiction in humans does not appear to be associated with a reduced density of brain mu-opioid receptors. The sustained down-regulation of PKC-alpha in the brain of opiate addicts would allow the up-regulation of G alpha(i1/2) proteins aimed at compensating the postulated desensitization of the mu-opioid receptor system.

Age-dependent increases in protein kinase C-alpha beta immunoreactivity and activity in the human brain: possible in vivo modulatory effects on guanine nucleotide regulatory G(i) proteins.

In the postmortem human brain (20 specimens of frontal cortex, Brodmann area 9) the abundance of immunoreactive protein kinase C (PKC-alpha beta) and the activity of PKC (calcium, phosphatidylserine, and phorbol ester-dependent enzymes) were determined to study the effect of aging (range 1 month to 89 years) on this regulatory enzyme. Also, the abundance of immunoreactive G protein subunits (G alpha i1/2, G alpha i3, G alpha o, G alpha s and G beta) were assessed in parallel to investigate possible relationships with PKC-alpha beta. The abundance of PKC-alpha beta was positively correlated with aging (r = 0.62, n = 20, P < 0.005). Moreover, PKC activity also showed a significant positive correlation with the age of the subject at death (r = 0.55, n = 14, P < 0.05). Because of the known in vitro modulatory role of PKC-alpha beta on G(i) proteins, the existence of an in vivo effect of brain PKC-alpha beta on various G proteins was assessed through correlation analyses. In the brain of the same subjects, there were significant negative correlations between the immunoreactivity of PKC-alpha beta and the immunoreactivities of G alpha i1/2 (r = -0.78, n = 13, P < 0.005) and G alpha i3 (r = -0.58, n = 15, P < 0.005). In the same brains, similar negative, although non-significant, correlations were found between the levels of PKC-alpha beta and those of G alpha o, G alpha s and G beta. The results demonstrate an up-regulation of brain PKC-alpha beta with aging and suggest the existence of a relevant in vivo modulatory role of this regulatory enzyme on inhibitory Gi proteins in the human brain during the process of aging.

Decreased density of I2-imidazoline receptors in the postmortem brain of heroin addicts.

The aim of this study was to quantitate imidazoline receptors in postmortem brains of heroin addicts who died of an opiate overdose. The density of I2-imidazoline receptors ([3H]idazoxan binding in the presence of adrenaline) and the immunoreactivity of the related 29/30 kDa imidazoline receptor protein were decreased (39% and 28%, respectively) in the frontal cortex. The density of brain I2-imidazoline receptors was also decreased in heroin-dependent rats (27%). This novel finding indicates that opiate addiction induces down-regulation of I2-imidazoline receptors in astrocytes, and presumably down-regulation of the functions associated with these receptors.