Cabazitaxel for Hormone-Relapsed Metastatic Prostate Cancer Previously Treated With a Docetaxel-Containing Regimen: An Evidence Review Group Perspective of a NICE Single Technology Appraisal.

As part of its single technology appraisal (STA) process, the National Institute for Health and Care Excellence (NICE) invited the company that manufactures cabazitaxel (Jevtana®, Sanofi, UK) to submit evidence for the clinical and cost effectiveness of cabazitaxel for treatment of patients with metastatic hormone-relapsed prostate cancer (mHRPC) previously treated with a docetaxel-containing regimen. The School of Health and Related Research Technology Appraisal Group at the University of Sheffield was commissioned to act as the independent Evidence Review Group (ERG). The ERG produced a critical review of the evidence for the clinical and cost effectiveness of the technology based upon the company's submission to NICE. Clinical evidence for cabazitaxel was derived from a multinational randomised open-label phase III trial (TROPIC) of cabazitaxel plus prednisone or prednisolone compared with mitoxantrone plus prednisone or prednisolone, which was assumed to represent best supportive care. The NICE final scope identified a further three comparators: abiraterone in combination with prednisone or prednisolone; enzalutamide; and radium-223 dichloride for the subgroup of people with bone metastasis only (no visceral metastasis). The company did not consider radium-223 dichloride to be a relevant comparator. Neither abiraterone nor enzalutamide has been directly compared in a trial with cabazitaxel. Instead, clinical evidence was synthesised within a network meta-analysis (NMA). Results from TROPIC showed that cabazitaxel was associated with a statistically significant improvement in both overall survival and progression-free survival compared with mitoxantrone. Results from a random-effects NMA, as conducted by the company and updated by the ERG, indicated that there was no statistically significant difference between the three active treatments for both overall survival and progression-free survival. Utility data were not collected as part of the TROPIC trial, and were instead taken from the company's UK early access programme. Evidence on resource use came from the TROPIC trial, supplemented by both expert clinical opinion and a UK clinical audit. List prices were used for mitoxantrone, abiraterone and enzalutamide as directed by NICE, although commercial in-confidence patient-access schemes (PASs) are in place for abiraterone and enzalutamide. The confidential PAS was used for cabazitaxel. Sequential use of the advanced hormonal therapies (abiraterone and enzalutamide) does not usually occur in clinical practice in the UK. Hence, cabazitaxel could be used within two pathways of care: either when an advanced hormonal therapy was used pre-docetaxel, or when one was used post-docetaxel. The company believed that the former pathway was more likely to represent standard National Health Service (NHS) practice, and so their main comparison was between cabazitaxel and mitoxantrone, with effectiveness data from the TROPIC trial. Results of the company's updated cost-effectiveness analysis estimated a probabilistic incremental cost-effectiveness ratio (ICER) of £45,982 per quality-adjusted life-year (QALY) gained, which the committee considered to be the most plausible value for this comparison. Cabazitaxel was estimated to be both cheaper and more effective than abiraterone. Cabazitaxel was estimated to be cheaper but less effective than enzalutamide, resulting in an ICER of £212,038 per QALY gained for enzalutamide compared with cabazitaxel. The ERG noted that radium-223 is a valid comparator (for the indicated sub-group), and that it may be used in either of the two care pathways. Hence, its exclusion leads to uncertainty in the cost-effectiveness results. In addition, the company assumed that there would be no drug wastage when cabazitaxel was used, with cost-effectiveness results being sensitive to this assumption: modelling drug wastage increased the ICER comparing cabazitaxel with mitoxantrone to over £55,000 per QALY gained. The ERG updated the company's NMA and used a random effects model to perform a fully incremental analysis between cabazitaxel, abiraterone, enzalutamide and best supportive care using PASs for abiraterone and enzalutamide. Results showed that both cabazitaxel and abiraterone were extendedly dominated by the combination of best supportive care and enzalutamide. Preliminary guidance from the committee, which included wastage of cabazitaxel, did not recommend its use. In response, the company provided both a further discount to the confidential PAS for cabazitaxel and confirmation from NHS England that it is appropriate to supply and purchase cabazitaxel in pre-prepared intravenous-infusion bags, which would remove the cost of drug wastage. As a result, the committee recommended use of cabazitaxel as a treatment option in people with an Eastern Cooperative Oncology Group performance status of 0 or 1 whose disease had progressed during or after treatment with at least 225 mg/m2 of docetaxel, as long as it was provided at the discount agreed in the PAS and purchased in either pre-prepared intravenous-infusion bags or in vials at a reduced price to reflect the average per-patient drug wastage.

Disulfide cross-links in the interaction of a cataract-linked alphaA-crystallin mutant with betaB1-crystallin.

A number of alphaA-crystallin mutants are associated with hereditary cataract including cysteine substitution at arginine 49. We report the formation of affinity-driven disulfide bonds in the interaction of alphaA-R49C with betaB1-crystallin. To mimic cysteine thiolation in the lens, betaB1-crystallin was modified by a bimane probe through a disulfide linkage. Our data suggest a mechanism whereby a transient disulfide bond occurs between alphaA- and betaB1-crystallin followed by a disulfide exchange with cysteine 49 of a neighboring alphaA-crystallin subunit. This is the first investigation of disulfide bonds in the confine of the chaperone/substrate complex where reaction rates are favored by orders of magnitude. Covalent protein cross-links are a hallmark of age-related cataract and may be a factor in its inherited form.

Effect of glycation on alpha-crystallin structure and chaperone-like function.

The chaperone-like activity of alpha-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of alpha-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of alpha-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of alpha-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of alpha-crystallin. Modification of alpha-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [N(epsilon)-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of alpha-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of alpha-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, alpha-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of alpha-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describe the effects of glycation on the structure and chaperone-like activity of alpha-crystallin.

Analysis of betaB1-crystallin unfolding equilibrium by spin and fluorescence labeling: evidence of a dimeric intermediate.

A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in betaB1-crystallin targeting the domain interface within the fold of a subunit. Global unfolding was monitored by tryptophan fluorescence while concomitant structural changes at the dimer interface were monitored by fluorescence and spin labels. Both spectral probes report explicit evidence of multi-state unfolding equilibrium. The biphasic nature of the unfolding curves was more pronounced at higher protein concentration. Distinct shifts in the midpoint of the second transition reflect the population of a dimeric intermediate. This intermediate may be a critical determinant for the life-long stability of the beta-crystallins and has important consequences on interactions with alpha-crystallin.

Specificity of alphaA-crystallin binding to destabilized mutants of betaB1-crystallin.

To elucidate the structural and energetic basis of attractive protein interactions in the aging lens, we investigated the binding of destabilized mutants of betaB1-crystallin to the lens chaperones, alpha-crystallins. We show that the mutations enhance the binding affinity to alphaA- but not alphaB-crystallin at physiological temperatures. Complex formation disrupts the dimer interface of betaB1-crystallin consistent with the binding of a monomer. Binding isotherms obtained at increasing concentrations of betaB1-crystallin deviate from a classic binding equilibrium and display cooperative-like behavior. In the context of betaB1-crystallin unfolding equilibrium, these characteristics are reflective of the concentration-dependent change in the population of a dimeric intermediate that has low affinity to alphaA-crystallin. In the lens, where alpha-crystallin binding sites are not regenerated, this may represent an added mechanism to maintain lens transparency.

Chaperone-like activity and hydrophobicity of alpha-crystallin.

alpha-Crystallin, a prominent member of small heat shock protein (sHsp) family and a major structural protein of the eye lens is a large polydisperse oligomer of two isoforms, alphaA- and alphaB-crystallins. Numerous studies have demonstrated that alpha-crystallin functions like a molecular chaperone in preventing the aggregation of various proteins under a wide range of stress conditions. The molecular chaperone function of alpha-crystallin is thus considered to be vital in the maintenance of lens transparency and in cataract prevention. alpha-Crystallin selectively interacts with non-native proteins thereby preventing them from aggregation and helps maintain them in a folding competent state. It has been proposed and generally accepted that alpha-crystallin suppresses the aggregation of other proteins through the interaction between hydrophobic patches on its surface and exposed hydrophobic sites of partially unfolded substrate protein. However, a quantifiable relationship between hydrophobicity and chaperone-like activity remains a matter to be concerned about. On an attentive review of studies on alpha-crystallin chaperone-like activity, particularly the studies that have direct or indirect implications to hydrophobicity and chaperone-like activity, we found several instances wherein the correlation between hydrophobicity and its chaperone-like activity is paradoxical. We thus attempted to provide an overview on the role of hydrophobicity in chaperone-like activity of alpha-crystallin, the kind of evaluation done for the first time.

Alphab-crystallin-assisted reactivation of glucose-6-phosphate dehydrogenase upon refolding.

Alphab-crystallin, a small heat-shock protein has been shown to prevent the aggregation of other proteins under various stress conditions. We have investigated the role of alphaB-crystallin in the reactivation of denaturant [GdmCl (guanidinium chloride)]-inactivated G6PD (glucose-6-phosphate dehydrogenase). Studies indicate that unfolding and inactivation of G6PD by GdmCl proceeds via formation of a molten globule-like state at low concentrations of GdmCl, which was characterized by having maximum surface hydrophobicity and no catalytic activity. At high concentrations of GdmCl, G6PD was completely unfolded, which upon dilution-induced refolding yielding 35% of original activity. In contrast, no activity was recovered when G6PD was refolded from a molten globule-like state. Interestingly, refolding of completely unfolded G6PD in the presence of alphaB-crystallin resulted in 70% gain of the original activity, indicating that alphaB-crystallin assisted in enhanced refolding of G6PD. Intriguingly, alphaB-crystallin was unable to reactivate G6PD from a molten globule-like state. Size-exclusion chromatography data indicate that alphaB-crystallin-assisted reactivation of completely unfolded G6PD is concomitant with the restoration of the native structure of G6PD. Nonetheless, alphaB-crystallin failed to reactivate G6PD from preformed aggregates. Moreover, methylglyoxal-modified alpha-crystallin, which occurs in aged and diabetic cataract lenses, was less efficient in the reactivation of denaturant inactivated G6PD. Diminished chaperone-like activity of alpha-crystallin due to post-translational modifications may thus result in the accumulation of aggregated/inactivated proteins.

Insights into hydrophobicity and the chaperone-like function of alphaA- and alphaB-crystallins: an isothermal titration calorimetric study.

Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.

alpha-Lipoic acid ameliorates altered colonic contractility and intestinal transit in STZ-diabetic rats.

alpha-Lipoic acid treatment (100 mg/kg/day for 2 weeks after 6 weeks of untreated diabetes) of streptozotocin diabetic rats partially but significantly reversed both reduced contractile response of distal colon to acetylcholine and delayed transit of charcoal meal in small intestine compared to diabetic control. These effects of alpha-Lipoic acid were associated with complete reversal of diabetes induced increased plasma lipid peroxidation level. alpha-Lipoic acid had no effect on any of the parameters measured in non-diabetic rats. These findings demonstrate contribution of oxidative stress in the development of physiological changes of gut in diabetes.

Nanosuspensions as the most promising approach in nanoparticulate drug delivery systems.

Over the years, controlled drug delivery as well as site-specific delivery have made considerable advances. One area that contributed significantly to this progress is the rapidly developing field of colloidal drug delivery systems. Nanoparticles, one of the colloidal drug delivery systems, may enable new possibilities for therapy that presently have not been investigated. Recent advances in nanoparticle research are discussed here. The present review highlights new and upcoming developments such as nanosuspensions and solid lipid nanoparticles.

Effect of dicarbonyl-induced browning on alpha-crystallin chaperone-like activity: physiological significance and caveats of in vitro aggregation assays.

Alpha-crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of alpha-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine alpha-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified alpha-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified alpha-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified alpha-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of alpha-crystallin in high proportions in the insoluble fraction. Furthermore, soluble alpha-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of alpha-crystallin.

Effect of long-term dietary manipulation on the aggregation of rat lens crystallins: role of alpha-crystallin chaperone function.

PURPOSE: To investigate the effect of food, protein, and vitamin restriction on the susceptibility of lens crystallins to aggregation and chaperone activity of alpha-crystallin. METHODS: Thirty day old Wistar/NIN rats were maintained on regular rodent diet (C), 50% food restriction (FR), 75% protein restriction (PR), and 50% vitamin restriction (VR) diet for 20 weeks. At the end, alpha-, beta-, and gamma-crystallins were isolated from the lenses of these animals and subjected to in vitro aggregation induced by oxidation, UV irradiation and heat. Aggregation and chaperone activity was assessed by light scattering methods. RESULTS: Dietary restriction has been shown to extend the mean and maximum life span and retard age-related diseases, including cataract. In this study, we demonstrate that while beta- and gamma-crystallins isolated from FR and PR groups were less susceptible to in vitro induced aggregation, beta- and gamma-crystallins from the VR group were more susceptible, compared to controls. Alpha-crystallin from any of the groups did not shown a considerable amount of aggregation. On the other hand, the chaperone activity of alpha-crystallin from FR and PR groups was not significantly different from controls. However, alpha-crystallin from the VR group demonstrated substantially higher chaperone activity than controls. CONCLUSIONS: These results indicate that while food and protein restriction appear to lower the susceptibility of beta- and gamma-crystallins towards aggregation, vitamin restriction tends to increase the aggregation. Chaperone activity of alpha-crystallin is affected (improved) by only vitamin restriction.