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Objective: To describe the patterns of recurrence and the prognosis of patients with a recurrent TP53 mutated endometrial carcinoma treated initially by surgery., Methods: All patients with endometrial carcinoma, treated at hospital European Georges Pompidou between 2001 and 2021 were retrospectively included. Patients were separated into two groups: TP53-mutated and not TP53-mutated (POLE/ultramutated-like (POLEmut), dMMR (mismatch repair-deficient) and NSMP (No specific molecular profile)). We estimated survival using recurrence free survival, overall survival and overall survival from recurrence. The risk of recurrence according to TP53 status and the type of recurrence (locoregional recurrence, peritoneal recurrence, and metastasis) were also compared between the two groups., Results: Two hundred and ninety-one patients with endometrial carcinoma were included. Of these, 57 were TP53-mutated and 234 patients were not TP53-mutated. TP53 mutated patients had the worst recurrence free survival and overall survival (p < 0.001 for each). The hazard rate of recurrence was higher during the first three years for TP53 mutated endometrial carcinoma then tend to join the one of no TP53 mutated. There was a statistical difference between the two groups in terms of cumulative incidence of peritoneal recurrence (p = 0.002). There was, however, no statistical difference in overall survival from recurrence., Conclusions: TP53-mutated endometrial carcinoma were more likely to experience a recurrence during the first three years and most often peritoneal recurrence compared to not TP53-mutated. TP53 status in endometrial carcinoma could be useful to define follow-up. Further prospective studies are required to assess the predictive impact of TP53 mutation on chemotherapy benefit., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Phosphate buffer is predominantly used instead of the more physiological bicarbonate buffer, as the latter requires a technical solution of adequate gas mixing. Recent pioneering work on how bicarbonate buffer affected drug supersaturation revealed interesting effects that call for more mechanistic understanding. Therefore, this study used hydroxypropyl cellulose as a model precipitation inhibitor and real-time desupersaturation testing was conducted with the drugs bifonazole, ezetimibe, tolfenamic acid and triclabendazole. Specific buffer effects for the different compounds were noted and overall, statistical significance was found for the precipitation induction time (p = 0.0088). Interestingly, molecular dynamics simulation revealed a conformational effect of the polymer in the presence of the different buffer types. Subsequent molecular docking trials suggested a stronger interaction energy of drug and polymer in the presence of phosphate compared to bicarbonate buffer (p =0.0010). In conclusion, a better mechanistic understanding of how different buffers affect drug-polymer interactions regarding drug supersaturation was achieved. Further mechanisms may account for the overall buffer effects and additional research on drug supersaturation is certainly needed, but it can already be concluded that bicarbonate buffering should be used more often for in vitro testing in drug development., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Edmont Stoyanov is an employee of Nisso Chemical Europe GmbH., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lipid-based systems have many advantages in formulation of poorly water-soluble drugs but issues of a limited solvent capacity are often encountered in development. One of the possible solubilization approaches of especially basic drugs could be the addition of fatty acids to oils but currently, a systematic study is lacking. Therefore, the present work investigated apparently neutral and basic drugs in medium chain triglycerides (MCT) alone and with added either caproic acid (C6), caprylic acid (C8), capric acid (C10) or oleic acid (C18:1) at different levels (5 - 20%, w/w). A miniaturized solubility assay was used together with X-ray diffraction to analyze the residual solid and finally, solubility data were modeled using the conductor-like screening model for real solvents (COSMO-RS). Some drug bases had an MCT solubility of only a few mg/ml or less but addition of fatty acids provided in some formulations exceptional drug loading of up to about 20% (w/w). The solubility changes were in general more pronounced the shorter the chain length was and the longest oleic acid even displayed a negative effect in mixtures of celecoxib and fenofibrate. The COSMO-RS prediction accuracy was highly specific for the given compounds with root mean square errors (RMSE) ranging from an excellent 0.07 to a highest value of 1.12. The latter was obtained with the strongest model base pimozide for which a new solid form was found in some samples. In conclusion, targeting specific molecular interactions with the solute combined with mechanistic modeling provides new tools to advance lipid-based drug delivery., Competing Interests: Declaration of Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

The field of clear cell renal cell carcinoma (ccRCC) has undergone major changes in the last decade, both in terms of the understanding of the mechanisms of oncogenesis and the role of the tumor microenvironment in anti-tumor immunity, as well as in therapeutic developments. After the era of tyrosine kinase inhibitors (TKIs) targeting VEGFR and then the era of immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 pathway, we are now entering the era of combination therapy for first-line metastatic cancer (m-ccRCC), such as combinations including a TKI and a PD-1 inhibitor or combinations of PD-1 and CTLA-4 blockers. In this extremely dynamic environment, new molecules with various mechanisms of action will appear in the very near future: immune response modulators (other ICIs, pro-inflammatory cytokines, gut microbiota modulators), new anti-angiogenic agents (new TKIs, anti-HIF-1α antibodies), agents affecting cell metabolism (glutaminase inhibitors, tryptophan regulators or adenosine A2A receptor antagonists) or epigenetic regulators (HDAC inhibitors). In parallel, new strategies are being evaluated that could rapidly change the standards of management of advanced disease, including therapeutic intensification with triple combinations or, conversely, adaptive and/or alternative de-escalation regimens (SURF trial), and biomarker-driven treatments (BIONIKK trial). The main new molecules and strategies currently being evaluated are reviewed in this article., (Copyright © 2022 Elsevier Masson SAS. Tous droits réservés.)

Lipid based formulations (LBF) have shown to overcome food dependent bioavailability for some poorly water-soluble drugs. However, the utility of LBFs can be limited by low dose loading due to a low drug solubility in LBF vehicles. This study investigated the solubility and drug loading increases in LBFs using lipophilic counterions to form lipophilic salts of venetoclax. Venetoclax docusate was formed from venetoclax free base and verified by 1 H NMR. Formation of stable venetoclax-fatty acid associations with either oleic acid or decanoic acid were attempted, however, the molecular associations were less consistent based on 1 H NMR. Venetoclax docusate displayed a up to 6.2-fold higher solubility in self-emulsifying drug delivery systems (SEDDS) when compared to the venetoclax free base solubility resulting in a higher dose loading. A subsequent bioavailability study in landrace pigs demonstrated a 2.5-fold higher bioavailability for the lipophilic salt containing long chain SEDDS compared to the commercially available solid dispersion Venclyxto® in the fasted state. The bioavailability of all lipophilic salt SEDDS in the fasted state was similar to Venclyxto® in the fed state. This study confirmed that lipophilic drug salts increase the dose loading in LBFs and showed that lipophilic salt-SEDDS combinations may be able to overcome bioavailability limitations of drugs with low inherent dose loading in lipid vehicles. Furthermore, the present study demonstrated the utility of a LBF approach, in combination with lipophilic salts, to overcome food dependent variable oral bioavailability of drugs., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

New drug candidates often require bio-enabling formation technologies such as lipid-based formulations, solid dispersions, or nanosized drug formulations. Development of such more sophisticated delivery systems generally requires higher resource investment compared to a conventional oral dosage form, which might slow down clinical development. To achieve the biopharmaceutical objectives while enabling rapid cost effective development, it is imperative to identify a suitable formulation technique for a given drug candidate as early as possible. Hence many companies have developed internal decision trees based mostly on prior organizational experience, though they also contain some arbitrary elements. As part of the EU funded PEARRL project, a number of new decision trees are here proposed that reflect both the current scientific state of the art and a consensus among the industrial project partners. This commentary presents and discusses these, while also going beyond this classical expert approach with a pilot study using emerging machine learning, where the computer suggests formulation strategy based on the physicochemical and biopharmaceutical properties of a molecule. Current limitations are discussed and an outlook is provided for likely future developments in this emerging field of pharmaceutics., (Copyright © 2021 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Several approaches to predict and model drug solubility have been used in the drug discovery and development processes during the last decades. Each of these approaches have their own benefits and place, and are typically used as standalone approaches rather than in concert. The synergistic effects of these are often overlooked, partly due to the need of computational experts to perform the modeling and simulations as well as analyzing the data obtained. Here we provide our views on how these different approaches can be used to retrieve more information on drug solubility, ranging from multivariate data analysis over thermodynamic cycle modeling to molecular dynamics simulations. We are discussing aqueous solubility as well as solubility in more complex mixed solvents and media with colloidal structures present. We conclude that the field of computational pharmaceutics is in its early days but with a bright future ahead. However, education of computational formulators with broad knowledge of modeling and simulation approaches is imperative if computational pharmaceutics is to reach its full potential., (Copyright © 2020. Published by Elsevier Inc.)

Drug solubility screening in solvents and lipids is central for the development of lipid-based formulations (LBFs), and any guidance to reduce the experimental workload would be highly desirable. Solubility parameters are interesting as they can be predicted in silico for a drug but they are hardly predictable for complex lipids. This paper uses a new approach to convert an in silico drug solubility parameter to an estimated relative permittivity, ε r . Diverse solvents and lipid-based excipients were then experimentally tested for ε r and solubility using fenofibrate as model. The typical excipients and solvents used in LBFs showed an ε r range of about 2-24, and good solubility of fenofibrate was indeed evidenced in vicinity of its estimated relative permittivity 13.2 ± 2.7. Mixtures of promising excipients were studied subsequently, and the obtained ε r was predictable based on the known values of the individual components. The novel permittivity approach has demonstrated its usefulness, it has much potential in early development for ranking of suitable excipients, and it gives an initial orientation to design formulations. Future research may clarify further opportunities and limits of the novel approach for LBFs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

It is for the pharmaceutical sciences of vital importance to understand how drugs are solubilized in biorelevant media. However, the complexity of fasted state simulated intestinal fluid (FaSSIF) has so far hampered adequate solubility modeling. The present study focuses on apparently neutral compounds at physiological pH and a linear free energy relationship is introduced for biorelevant drug solubilization. Based on literature data of 40 compounds, the Abraham solvation descriptors were calculated from chemical structure to then predict the ratio of solubility enhancement log(SE) in FaSSIF compared to aqueous buffer solubility at pH 6.5. A suitable model was obtained with R 2 of 0.810 and notable were especially the positive effect of McGowan's characteristic volume and the negative effect of drug basicity. A negative influence on log(SE) was further evidenced for dipolarity/polarizability and for the excess molar refraction descriptor. A positive solubilization effect was obtained for drug acidity and hence the tendency for proton donation, which was likely due to the different proton-accepting moieties of taurocholic acid and lecithin that are both present in the mixed colloids of FaSSIF. Overall, an improved understanding was achieved regarding the molecular features that are driving drug solubilization in biorelevant media., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Nanosuspensions provide a drug delivery approach to cope with erratic absorption of poorly water-soluble compounds. Despite extensive research over the last years, there are still open pharmaceutical challenges so it is often unclear how quality attributes such as viscosity and physical stability are generated, which requires a more thorough study of the colloidal structures and interactions in nanosuspensions. In this study, diffusing wave spectroscopy and microfluidics-based rheology were used for the first time to assess pharmaceutical nanosuspensions that were obtained by wet milling. Further sample characterization following centrifugation was based on optical rotatory dispersion and conductivity experiments. Ketoconazole was selected as model drug in the presence of sodium dodecyl sulfate and hydroxypropyl cellulose as anionic and steric stabilizer, respectively. The results unexpectedly showed that the investigated nanosuspensions did not behave as Einstein-like suspensions because a viscosity decrease was evidenced for increased drug load. This effect was attributed to the polymer that formed a dominating network in the bulk solution from where adsorption occurred onto particle surfaces. This depletion of bulk polymer caused the observed rheological finding. Further colloidal research should be invested into different pharmaceutical nanosuspensions to gain a more complete structural understanding and to harness their full technological potential., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

This commentary reflects current developments in pediatric medicine. The underpinning legislation in both Europe and the United States has led to the initiation of an increased number of clinical trials in the pediatric population, but there are still a number of outstanding issues within this field. These include the differences in the physiology between adults and the very heterogeneous nature of pediatric patients. There is an ongoing scientific debate on the applicability of a Pediatric Biopharmaceutical Classification System to define when waivers for bioequivalence studies can be supported by in vitro dissolution. However, a challenge is that in vitro models should adequately mimic the physiology of different pediatric age-groups and dose definition is another critical aspect. There is a tendency for off-label use of established adult medicines, resulting in increased adverse events and decreased efficacy in the target population. Recent advances in physiologically based pharmacokinetic modelling may be used to provide valuable input into these discussions, but there are currently still many knowledge gaps. It is encouraging that there is a global recognition of these deficiencies and substantial funding in the field of basic research is being provided, for example, within Europe the Innovative Medicines Initiative consortium., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Despite advances in supportive therapy to prevent complications of sickle cell disease (SCD), access to care is not universal. Hematopoietic cell transplantation is, to date, the only curative therapy for SCD, but its application is limited by availability of a suitable HLA-matched donor and lack of awareness of the benefits of transplant. Included in this study are 1000 recipients of HLA-identical sibling transplants performed between 1986 and 2013 and reported to the European Society for Blood and Marrow Transplantation, Eurocord, and the Center for International Blood and Marrow Transplant Research. The primary endpoint was event-free survival, defined as being alive without graft failure; risk factors were studied using a Cox regression models. The median age at transplantation was 9 years, and the median follow-up was longer than 5 years. Most patients received a myeloablative conditioning regimen (n = 873; 87%); the remainder received reduced-intensity conditioning regimens (n = 125; 13%). Bone marrow was the predominant stem cell source (n = 839; 84%); peripheral blood and cord blood progenitors were used in 73 (7%) and 88 (9%) patients, respectively. The 5-year event-free survival and overall survival were 91.4% (95% confidence interval, 89.6%-93.3%) and 92.9% (95% confidence interval, 91.1%-94.6%), respectively. Event-free survival was lower with increasing age at transplantation (hazard ratio [HR], 1.09; P < .001) and higher for transplantations performed after 2006 (HR, 0.95; P = .013). Twenty-three patients experienced graft failure, and 70 patients (7%) died, with the most common cause of death being infection. The excellent outcome of a cohort transplanted over the course of 3 decades confirms the role of HLA-identical sibling transplantation for children and adults with SCD., (© 2017 by The American Society of Hematology.)

The generation of drug supersaturation close to the absorptive site is an important mechanism of how several formulation technologies enhance oral absorption and bioavailability. Lipid-based formulations belong to the supersaturating drug delivery systems although this is not the only mechanism of how drug absorption is promoted in vivo. Different methods to determine drug supersaturation and precipitation from lipid-based formulations are described in the literature. Experimental in vitro setups vary according to their complexity and proximity to the in vivo conditions and, therefore, some tests are used for early formulation screening, while others better qualify for a later stage of development. The present commentary discusses this rapidly evolving field of in vitro testing with a special focus on the advancements in analytical techniques and new approaches of mechanistic modeling. The importance of considering a drug absorption sink is particularly emphasized. This commentary should help formulators in the pharmaceutical industry as well as in academia to make informed decisions on how to conduct in vitro tests for lipid-based delivery systems and to decide on the implications of experimental results., (Copyright © 2016. Published by Elsevier Inc.)

The development of antibiotic resistance is a major problem for mankind and results in fatal consequences on a daily basis across the globe. There are a number of reasons for this situation including increasing globalization with worldwide travel, health tourism, over use and ineffective use (both in man and animals), and counterfeiting of the antimicrobial drug products we have available currently. Although there are huge economical, demographic, legal and logistic differences among the global communities, there are also differences regarding the best approach to dealing with antibiotic resistance. However, as resistant bacteria do not respect international borders, there is clearly a need for a global strategy to minimize the spread of antibiotic resistance, to optimize the use of antibiotics, and to facilitate the development of new and effective medications. This commentary provides an insight into the issues and some of the ongoing programs to ensure an effective treatment for the future., (Copyright © 2016. Published by Elsevier Inc.)

The development of novel systems with oral protein delivery as ultimate goal represents an important field of pharmaceutics. Prilling of protein-loaded polymeric solutions into lipid-based hardening baths could provide here an attractive formulating technology. As the obtained microgel dispersion can be directly capsule-filled, no drying step is required and thermal drug degradation is avoided. This study aims to find excipient combinations for the novel prilling process and investigate systematically diverse material and process factors. Bovine serum albumin and mono-N-carboxymethyl chitosan were selected as model protein and prilling polymer, respectively. The prilling suitability of 880 formulations was screened with 60 ternary phase diagrams comprising two co-solvents, 10 different glycerides, and three so-called complementary excipients. Preliminary capsule compatibility was tested for one month on 245 formulations in hard and soft capsules with different shell materials. Ternary phase diagrams' center points were used to evaluate morphology, encapsulation efficiency, and protein stability of the prilled microgels. As result, several formulations proved suitable for prilling and compatible for capsule filling. Statistical analysis using partial least square regression revealed significant factors regarding different quality attributes of microgel dispersions. Therefore, an improved understanding was obtained for this promising drug delivery approach., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Emulsions are widely used for pharmaceutical, food, and cosmetic applications. To guarantee that their critical quality attributes meet specifications, it is desirable to monitor the emulsion manufacturing process. However, finding of a suitable process analyzer has so far remained challenging. This article introduces diffusing wave spectroscopy (DWS) as an at-line technique to follow the manufacturing process of a model oil-in-water pharmaceutical emulsion containing xanthan gum. The DWS results were complemented with mechanical rheology, microscopy analysis, and stability tests. DWS is an advanced light scattering technique that assesses the microrheology and in general provides information on the dynamics and statics of dispersions. The obtained microrheology results showed good agreement with those obtained with bulk rheology. Although no notable changes in the rheological behavior of the model emulsions were observed during homogenization, the intensity correlation function provided qualitative information on the evolution of the emulsion dynamics. These data together with static measurements of the transport mean free path (l*) correlated very well with the changes in droplet size distribution occurring during the emulsion homogenization. This study shows that DWS is a promising process analytical technology tool for development and manufacturing of pharmaceutical emulsions., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Oral delivery of biologicals is a thriving field in pharmaceutics and the first challenge is to achieve a stable drug product. Interesting is prilling of a drug-containing polymeric solution as microgel into an aqueous hardening bath where crosslinking occurs. However, to deliver a final dosage form, for example, soft gelatin capsules, the aqueous hardening bath must be removed, thus leading to manufacturing processes that are potentially harmful for the active. The current work introduces a prilling method with a lipid-based hardening bath, which could theoretically be filled directly into capsules. Bovine serum albumin (BSA) and mono-N-carboxymethyl chitosan (MCC) were selected as model biological and encapsulating polymer, respectively. Several nonaqueous formulations of the receiving bath were investigated; calcium chloride was added to these formulations to allow the MCC gelling. The obtained microgels had average diameters of ∼300 μm and spherical to toroidal shapes, according to the hardening bath composition. Along with a high encapsulation efficiency (>85%), the microgels protected the BSA from any denaturing effect of the hardening bath. The release study showed a rather fast BSA release within the first 10 min from most microgels. This novel approach demonstrated technical viability for encapsulation of biologicals using lipid formulations regarding oral delivery., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The aim of the present study was to analyze the impact of lipid-based formulation (LBF) dispersion and digestion on the precipitation behavior of weakly basic drugs. Loratadine and carvedilol were formulated in a range of LBFs and drug solubilization was analyzed under simulated dispersive and digestive conditions (fasted state). The extent of supersaturation and drug precipitation as well as the solid-state properties and redissolution behavior of precipitated drugs were assessed. X-ray powder diffraction indicated that carvedilol precipitated in a crystalline form upon dispersion, but interestingly, this drug gave an amorphous precipitate during lipolysis. In contrast, loratadine precipitated as crystalline material during both formulation dispersion and digestion. No influence of the formulation composition on the type of precipitation was observed. These results suggested that in vitro conditions (dispersive versus digestive) largely influenced the solid-state properties of precipitating weak bases. Solid-state characterization of precipitated drugs under different experimental conditions should be routinely performed in formulation screening to better understand the biopharmaceutical behavior of LBFs. Hence, these findings are of high practical importance for the pharmaceutical development and in vitro assessment of LBFs using weakly basic drugs., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Quality by design is an important concept, but only limited research has been invested in concentrated pharmaceutical suspensions. A need exists for novel analytical tools to thoroughly characterize the drug as well as its aggregated particle structure in suspension. This work focuses on lipid-based pharmaceutical suspensions for filling of capsules. A rheological approach, namely the fractal concept of flocculation, is introduced to the pharmaceutical field. The model drug mebeverine hydrochloride was first physicochemically analyzed. A special aim was to study the surface energy profiles using inverse gas chromatography as a critical characteristic for the suspension's rheological behavior. Suspensions were manufactured in laboratory process equipment while applying different homogenization speeds. Flow curves of the final suspensions were measured using a cone-and-plate rheometer. As a result, surface energy profiles revealed differences from one mebeverine lot to another. Different homogenization intensities greatly affected the viscosity and the Mooney model was able to predict experimental values as a function of the drug volume fraction. The fractal concept of flocculation characterized mebeverine in suspension and a slight increase of fractal dimension was noted when homogenization speed was increased. It was concluded that the introduced concepts have large potential for designing quality into concentrated pharmaceutical suspensions., (Copyright © 2012 Wiley Periodicals, Inc.)

For decades, gelatin has been used in the rotary die process as a shell-forming material of soft capsules because of its unique physicochemical properties. However, with respect to the encapsulation of comparatively hydrophilic lipid-based formulations, gelatin has one considerable drawback: Immediately after production, the capsule shell contains a large amount of water (up to 35%). There is the potential for water to migrate from the capsule shell into the formulation, which will lead to a decrease in drug solubility and, in turn, the potential for drug crystallization. The present study introduces a novel capsule material that was obtained from extrusion. The starch-based polyvinyl alcohol thermoplastic capsules (S-PVA-C) mainly comprised a blend of starch and PVA. Gelatin and the novel material were used to encapsulate a hydrophilic lipid-based system of fenofibrate. Considerable water migration was observed from the soft gelatin shell to the hydrophilic formulation during drying and drug crystallization resulted in soft gelatin capsules. In contrast, S-PVA-C displayed no substantial water exchange or drug crystallization upon storage. The thermoplastic capsule material further exhibited more surface roughness and higher resistance to mechanical deformation compared with gelatin. In conclusion, S-PVA-C provided a robust drug product following encapsulation of a rather hydrophilic lipid-based formulation., (Copyright © 2012 Wiley Periodicals, Inc.)

There are currently no adequate process analyzers for nanoparticulate viscosity enhancers. This article aims to evaluate ultrasonic resonator technology as a monitoring tool for homogenization of nanoparticulate gels. Aqueous dispersions of colloidal microcrystalline cellulose (MCC) and a mixture of clay particles with xanthan gum were compared with colloidal silicon dioxide in oil. The processing was conducted using a laboratory-scale homogenizing vessel. The study investigated first the homogenization kinetics of the different systems to focus then on process factors in the case of colloidal MCC. Moreover, rheological properties were analyzed offline to assess the structure of the resulting gels. Results showed the suitability of ultrasound velocimetry to monitor the homogenization process. The obtained data were fitted using a novel heuristic model. It was possible to identify characteristic homogenization times for each formulation. The subsequent study of the process factors demonstrated that ultrasonic process analysis was equally sensitive as offline rheological measurements in detecting subtle manufacturing changes. It can be concluded that the ultrasonic method was able to successfully assess homogenization of nanoparticulate viscosity enhancers. This novel technique can become a vital tool for development and production of pharmaceutical suspensions in the future., (Copyright © 2011 Wiley-Liss, Inc.)

Transcranial Doppler (TCD) is used to detect children with sickle cell anemia (SCA) who are at risk for stroke, and transfusion programs significantly reduce stroke risk in patients with abnormal TCD. We describe the predictive factors and outcomes of cerebral vasculopathy in the Créteil newborn SCA cohort (n = 217 SS/Sβ(0)), who were early and yearly screened with TCD since 1992. Magnetic resonance imaging/magnetic resonance angiography was performed every 2 years after age 5 (or earlier in case of abnormal TCD). A transfusion program was recommended to patients with abnormal TCD and/or stenoses, hydroxyurea to symptomatic patients in absence of macrovasculopathy, and stem cell transplantation to those with human leukocyte antigen-genoidentical donor. Mean follow-up was 7.7 years (1609 patient-years). The cumulative risks by age 18 years were 1.9% (95% confidence interval [95% CI] 0.6%-5.9%) for overt stroke, 29.6% (95% CI 22.8%-38%) for abnormal TCD, which reached a plateau at age 9, whereas they were 22.6% (95% CI 15.0%-33.2%) for stenosis and 37.1% (95% CI 26.3%-50.7%) for silent stroke by age 14. Cumulating all events (stroke, abnormal TCD, stenoses, silent strokes), the cerebral risk by age 14 was 49.9% (95% CI 40.5%-59.3%); the independent predictive factors for cerebral risk were baseline reticulocytes count (hazard ratio 1.003/L × 10(9)/L increase, 95% CI 1.000-1.006; P = .04) and lactate dehydrogenase level (hazard ratio 2.78/1 IU/mL increase, 95% CI1.33-5.81; P = .007). Thus, early TCD screening and intensification therapy allowed the reduction of stroke-risk by age 18 from the previously reported 11% to 1.9%. In contrast, the 50% cumulative cerebral risk suggests the need for more preventive intervention.

The detection of circulating galactomannan (GM) in serum samples is an important step in the diagnosis of invasive aspergillosis (IA). The assay has been mainly explored in neutropenic patients, and is now used to monitor patients at high risk for IA. However, the performance of the assay varies greatly among studies. The objective of this study was to explore the impact of the neutrophil count on the GM serum index at the time of IA diagnosis. Ninety-nine episodes of proven or probable, microbiologically documented IA in 91 patients with haematological malignancies were studied retrospectively. Three groups were identified: groups 1-3, with <100 polymorphonuclear neutrophils (PMN)/mm(3) (n = 18), between 100 and 500 PMN/mm(3) (n = 21), or >500 PMN/mm(3) (n = 60), respectively. The mean GM index was significantly higher in group 1 than in the other groups (p <0.05). This finding did not change after stratifying the analysis with regard to the use of antibiotics likely to give false-positive GM results or with regard to treatment effective against fungi before the diagnosis of IA. This finding could be considered in the routine use of the GM antigenaemia test in non-neutropenic patients; a negative result or a low GM index should not eliminate the diagnosis of IA. This limitation calls for other microbiological tests, including analysis of bronchoalveolar lavage fluid, to establish a definitive diagnosis of IA.

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I-binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2-restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT1(37-45)-specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT1(37-45), have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

Allogeneic hematopoietic stem-cell transplantation (HSCT) is the only curative treatment for sickle cell disease (SCD); nevertheless, its use has been limited by the risk of transplantation-related mortality (TRM). Between November 1988 and December 2004, 87 consecutive patients with severe SCD ranging from 2 to 22 years of age received transplants in France. Cerebral vasculopathy was the principal indication for transplantation (55 patients). All the patients received grafts from a sibling donor after a myeloablative conditioning regimen (CR). The only change in the CR during the study period was the introduction of antithymocyte globulin (ATG) in March 1992. The rejection rate was 22.6% before the use of ATG but 3% thereafter. With a median follow-up of 6 years (range, 2.0 to 17.9 years), the overall and event-free survival (EFS) rates were 93.1% and 86.1%, respectively. Graft versus host disease (GVHD) was the main cause of TRM. Importantly, cord blood transplant recipients did not develop GVHD. No new ischemic lesions were detected after engraftment, and cerebral velocities were significantly reduced. The outcome improved significantly with time: the EFS rate among the 44 patients receiving transplants after January 2000 was 95.3%. These results indicate that HLA-identical sibling HSCT after myeloablative conditioning with ATG should be considered as a standard of care for SCD children who are at high risk for stroke.

Natural killer (NK) cell alloreactivity is reported to mediate strong GvL (graft versus leukemia) effect in patients after haploidentical stem-cell transplantation (SCT) for acute myeloid leukemia (AML). Because subsequent immune reconstitution remains a major concern, we studied NK-cell recovery in 10 patients with AML who received haplomismatched SC transplants, among whom no GvL effect was observed, despite the mismatched immunoglobulin-like receptor (KIR) ligand in the GvH direction for 8 of 10 patients. NK cells generated after SCT exhibited an immature phenotype: the cytotoxic CD3- CD56(dim) subset was small, expression of KIRs and NKp30 was reduced, while CD94/NKG2A expression was increased. This phenotype was associated to in vitro lower levels of cytotoxicity against a K562 cell line and against primary mismatched AML blasts than donor samples. This impaired lysis was correlated with CD94/NKG2A expression in NK cells. Blockading CD94/NKG2A restored lysis against the AML blasts, which all expressed HLA-E, the ligand for CD94/NKG2A. Our present study allows a better understanding of the NK-cell differentiation after SCT. These results revealed that the NK cells generated after haplomismatched SCT are blocked at an immature state characterized by specific phenotypic features and impaired functioning, having potential impact for immune responsiveness and transplantation outcome.

The use of peripheral blood stem cells (PBSCs) is rapidly growing in the allogeneic transplantation setting as an alternative to bone marrow (BM). We previously reported a higher incidence of chronic graft-versus-host disease (cGVHD) associated with allogeneic PBSC transplantation in a randomized trial. In this follow-up report, we analyzed the evolution of cGVHD in the patients (n = 101) enrolled on this study. At a median follow-up of 45 months (range, 31-57 months), we found that the 3-year cumulative incidence of cGVHD was 65% (95% confidence interval [CI] 51%-78%) in the PBSC group and 36% (95% CI 23%-49%) in the BM group (P =.004). We also found that extensive cGVHD was more frequent in the PBSC group (44% [95% CI 30%-58%] vs 17% [95% CI 7%-27%]; P =.004). The prevalence of cGVHD was always higher in the PBSC arm. Ocular involvement was more frequent in PBSC recipients (P =.02). Cutaneous and liver involvement was similar among BM and PBSC recipients. Chronic GVHD required multiple courses of immunosuppressive therapy in addition to cyclosporine and corticosteroids during longer periods (P =.03). Altogether, this translated into longer periods of hospitalization after transplantation in the PBSC group (P =.04). Finally, we also confirm that cGVHD after PBSC transplantation is associated with an antileukemic effect that is at least as potent as after BM. However, to date, this has not translated into a survival difference, possibly due to the early-stage leukemic status of these patients or to the relatively small size of the study population.

We have recently shown that the use of allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood hematopoietic stem cell transplantation (PBHSCT), as compared with bone marrow transplantation (BMT), is associated with increased titers of antibodies (Abs) directed against red blood cell ABO antigens. To further evaluate the influence of a G-CSF-mobilized PBHSCT graft on alloimmune Ab responses, we examined the frequency of anti-HLA Abs after transplantation in the setting of the same randomized study, comparing PBHSCT with BMT in adults. Anti-HLA Ab presence was determined by complement-dependent cytotoxicity assay (CDC) and flow cytometry in the recipient before and 30 days after transplantation as well as in the donor before graft donation. The use of PBHSCT was significantly associated with increased detection of anti-HLA immunoglobulin G (IgG) Abs early after transplantation as evidenced by flow cytometry (11 of 24 versus 4 of 27 transplant recipients, P =.03) and, less so, by CDC (5 of 24 versus 1 of 27 transplant recipients, P =.09). The difference between PBHSCT and BMT was further heightened when analysis was restricted to anti-HLA IgG Ab-negative donor/recipient pairs. In such a setting, early anti-HLA Ab was never detected after BMT but was repeatedly detected after PBHSCT (flow cytometry, 6 of 18 versus 0 of 17 transplant recipients, P =.02; CDC, 4 of 23 versus 0 of 26 transplant recipients, P =.04). Importantly, the PBHSCT-associated increase in anti-HLA Ab detection was observed despite a reduction in the median number of platelet-transfusion episodes per patient in PBHSC transplant versus BM transplant recipients (3 platelet-transfusion episodes [range, 1-21] in PBHSCT group vs 6 platelet-transfusion episodes [range, 3-33] in the BMT group; P =.02). In conclusion, this study strongly suggests that G-CSF-mobilized PBHSCT results in an increased incidence of circulating anti-HLA Abs and further confirms that the use of such a graft alters alloimmune Ab responses.

Several acute hemolysis episodes, sometimes lethal, have been recently described after transplantation of allogeneic peripheral blood hematopoietic stem cells (PBHSCs). Hemolysis resulted from the production of donor-derived antibodies (Abs) directed at ABO antigens (Ags) present on recipient red blood cells (RBCs). A multicenter randomized phase III clinical study comparing allogeneic PBHSC transplantation (PBHSCT) versus bone marrow hematopoietic stem cell transplantation (BMHSCT) has been conducted in France. In the course of this study, serum anti-A and/or anti-B Ab titers were compared before the conditioning regimen and on day +30 after transplantation in 49 consecutive evaluable PBHSCT (n = 21) or BMHSCT (n = 28) recipients. PBHSCT resulted in a higher frequency of increased anti-A and/or anti-B Ab titers 30 days after transplantation as compared to BMHSCT: 8 (38%) of 21 versus 3 (11%) of 28 (P =.04). In PBHSCT recipients, increased titers were observed mostly after receiving a minor ABO mismatch transplant: 5 of 7 versus 3 of 14 in the absence of any minor ABO mismatch (P =.05), whereas this was not the case after BMHSCT: 1 of 8 versus 2 of 20. Anti-A and/or anti-B serum Abs detectable at day +30 after PBHSCT were always directed against A and/or B Ags absent both on donor and recipient RBCs. Finally, 3 of 21 PBHSCT versus 0 of 28 BMHSCT recipients developed anti-allogeneic RBC Abs other than ABO (P =.07). Overall, the data strongly suggest that immunohematologic reconstitution differs significantly after granulocyte colony-stimulating factor-mobilized PBHSCT when compared to BMHSCT. Such a difference could contribute to the acute hemolysis described after PBHSCT as well as to distinct alloreactivity after PBHSCT.

The pressure susceptibility (chip), which is defined as the decrease of porosity (epsilon) under pressure was investigated. Of special interest are compacts obtained at very low pressures, because of the transition between the state of a powder and the state of a tablet. This range was found to be critical in respect to a diverging pressure susceptibility. Above a critical porosity (epsilonc) or below the corresponding relative density (rhoc), no pressure susceptibility can be defined, because of no rigid structure exists. To take this into account, a simple function was proposed for the pressure susceptibility: chip approximately 1/(epsilonc - epsilon). This proposal leads to a new porosity vs pressure relationship. The new model was compared to the Heckel equation that involves a constant pressure susceptibility. Various polymers were tested from "out of die" measurements, and the new relationship was found superior to the Heckel equation. As a conclusion, the pressure susceptibility exhibits a curvature that can be called critical at low relative densities. Consequently, a better understanding evolves as to why the Heckel equation is not valid at low pressures. The new model has proven to be adequate for polymer tablets but, so far it is not clear whether other substances exhibit the same performance. Especially tableting materials exhibiting brittle fracture will be of interest considering their importance in compaction technology.

Autologous stem-cell transplantation has become a widely used therapy in Hodgkin's disease (HD). To appreciate the early and late risks associated with this procedure, its lethal toxicity and effects on the incidence of secondary cancers were studied. Data related to 467 French patients grafted from 1982 to 1995 for primary sensitive disease (PSD, 22%), primary refractory disease (PRD, 18%), first relapse (R1, 45%), or subsequent relapses (R2, 15%) were analyzed. Grafted patients (PSD, PRD, and R1; n = 393) were matched (3 controls for 1 case) on age, gender, clinical stage, B symptoms, and time at risk with 1179 conventionally treated patients issued from international databases. The proportional hazards (Cox) model was used to assess relative risks (RR). Among grafted patients, 8% died of toxicity related to the procedure, and 18 secondary cancers occurred leading to a 5-year cumulative incidence rate of 8.9%. In this series, risk factors for second cancer were age >/=40 years (RR = 3.73, P = .007) and the use of peripheral blood stem cells as source of graft (RR = 3.10, P = .03). Among grafted and matched ungrafted patients, risk factors for the development of secondary cancer were age >/=40 years (RR = 2.90, P < .001), relapse versus no relapse (RR = 5.22, P = .006), PRD versus other patients (RR = 3.86, P = .033), and grafted versus ungrafted patients (RR = 2.04, P = . 024). Solid tumors were more frequent in grafted than in ungrafted patients (RR = 5.19, P = .001) although the incidence of myelodysplasia and acute myeloid leukemia was similar in the two groups. We conclude that high-dose chemotherapy administered as first-line treatment or after relapse is associated with an acceptable toxic death rate. The risk of secondary myelodysplasia or acute myeloid leukemia is not significantly increased after autologous stem-cell transplantation for HD, whereas an increased risk of solid tumors exists. The peripheral blood stem-cell-associated risk of secondary cancer among grafted patients needs further investigations., (Copyright 1998 by The American Society of Hematology.)

Early recommendations on prophylactic transfusion of thrombocytopenic patients involved a standard platelet dose of about 0.5 x 10(11)/10 kg body weight. Given the lack of data supporting this dose, we prospectively studied the dose response to platelet transfusions in adults and children with hematologic malignancies. Each patient received, in similar clinical conditions, a medium, high, and very high dose of fresh (< 24 hours old) ABO-compatible platelets, in the form of apheresis platelet concentrates (APC). For the adults, the medium dose was defined as APC containing between 4 and 6 x 10(11) platelets, the high dose between 6 and 8 x 10(11), and the very high dose > 8 x 10(11); for the children, the three doses corresponded to 2 to 4, 4 to 6, and > 6 x 10(11) platelets. The end points were the platelet increment, platelet recovery, and the transfusion interval, and the results were compared with a paired t-test. Sixty-nine adults and 13 children could be assessed. Recoveries in the adults were similar with the three doses (from 28% to 30%), but the high and very high doses led to a significantly better platelet increment (52 and 61 x 10(9)/L, respectively) than the medium dose (33 x 10(9)/L, P < .01). The main difference was in the transfusion interval, which increased with the dose of platelets transfused, from 2.6 days with the medium dose to 3.3 and 4.1 days with the high and very high doses, respectively (P < .01). The positive effect of the high dose was observed regardless of pretransfusional clinical status, but was more marked in patients with no clinical factors known to impair platelet recovery. In these patients, a platelet dose of 0.07 x 10(11) per kg of body weight led to a transfusion interval of more than 2 days in 95% of cases. In patients with clinical factors favoring platelet consumption, the proportion of transfusions yielding an optimal platelet increment and transfusion interval increased with the dose of platelets. The platelet dose-effect was also significant in the children, in whom the high and very high doses led to 1.5-fold to twofold higher posttransfusion platelet counts and transfusion intervals. We conclude that transfusion of high platelet doses can reduce the number of platelet concentrates required by thrombocytopenic patients and significantly reduce donor exposure., (Copyright 1998 by The American Society of Hematology.)

We report on 71 consecutive patients with de novo myelodysplastic syndromes referred to physicians belonging to the Société française de greffe de moelle from 1982 through 1991 and transplanted with marrow from HLA-identical siblings. There were 16 cases of refractory anemia, 27 of refractory anemia with excess of blast cells, and 28 of refractory anemia with excess of blast cells in transformation. Seventeen patients had received cytoreductive chemotherapy before the graft. The disease progressed in 17 patients between diagnosis and grafting. Twenty-three patients are alive with a median follow-up of 6 years, whereas 24 died from relapse and 24 from transplant-related complications. Kaplan-Meier estimates of event-free survival, relapse and transplant-related mortality at 7 years were 32%, 48%, and 39%, respectively. The log-rank test and Cox's model revealed better outcome among young patients, patients in an early stage of the French-American-British (FAB) classification or with a low percentage of marrow blasts before transplantation, patients who did not undergo cytoreductive chemotherapy before transplantation, and patients conditioned with total body irradiation and cyclophosphamide. The high rate of relapse in advanced FAB stages has led us to graft patients earlier in the course of the disease, and we are currently conducting a multicenter, randomized study to determine the value of intensive chemotherapy before grafting in patients with an excess of marrow blasts.

We report a case of mild, clinically asymptomatic, immune thrombocytopenia after allogenic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML) caused by the presence of a recipient-origin Br(a) antibody that recognized the donor platelets. Although the antibody titer decreased, it remained detectable more than 3 years after BMT. Chimerism studies were performed combining cytogenetics, blood cell phenotype studies, and genomic amplification of hypervariable sequences. Cytogenetic studies and molecular analysis of peripheral blood cells, purified B- and T-lymphocyte subpopulations, and bone marrow colonies showed the hematopoiesis to be of donor origin, but absorption-elution experiments with peripheral RBCs showed a small amount of recipient RBCs. The CML chimeric transcript was also detected by means of polymerase chain reaction on samples collected until day +867 post-BMT. This case shows that recipient-origin platelet alloantibodies can cause thrombocytopenia after BMT and that the persistence of small numbers of recipient cells (even leukemic) is not necessarily associated with hematologic relapse.

The toxicity of autologous bone marrow transplantation (ABMT) is correlated to neutropenia. Although recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) seems to hold promise in accelerating neutrophil recovery, few analyses from randomized studies are presently available. Ninety-one patients with non-Hodgkin's lymphoma receiving high-dose ablative chemotherapy followed by ABMT with unpurged or purged marrow were included in a randomized, double-blind, placebo-controlled trial. Forty-four patients received 250 micrograms rhu GM-CSF (Escherichia coli)/m2 and 47 patients received placebo. Treatment was administered daily as continuous infusion from day of ABMT until the absolute neutrophil count (ANC) reached 0.5 x 10(9)/L for 7 days or until day 30, whichever was first. With rhu GM-CSF, 50% of the patients reached an ANC count greater than 0.5 x 10(9)/L at day 14 as opposed to day 21 with placebo (P less than .0001). Patients transplanted with marrow purged by mafosfamide also recovered earlier when treated with rhu GM-CSF (16 v 20.5 days, P = .013). The hospitalization duration was shorter in the rhu GM-CSF group (median, 23 v 28 days, P less than .05). No difference was observed in fever, number of infections, and antibiotic administration between the two groups. The major adverse event ascribed to rhu GM-CSF was a capillary leak syndrome in three patients graded as severe in two patients, moderate in one, and reversible in all three patients. In addition, one patient in the rhu GM-CSF group died suddenly with no explanation. In long term follow-up, the relapse rate was identical in both groups and there was no significant difference in the number of deaths at 1 year (12 with rhu GM-CSF v 9 with placebo), although deaths seemed to occur slightly earlier in the rhu GM-CSF group. We conclude that after ABMT with purged or unpurged marrow, rhu GM-CSF (E coli) significantly reduces neutropenia duration and hospitalization stay. A positive causative relation between the study drug and/or its mode of application with an increased toxicity as compared with GM-CSF from other sources and/or other modes of application cannot be deduced from the experiences in this study. Additional randomized trials would be necessary for an appropriate answer.

We have used DNA sequence polymorphism analysis to document engraftment after T cell-depleted bone marrow transplantation (BMT), with a selected panel of four DNA probes. In contrast to nondepleted BMT recipients, the patients who received T cell-depleted marrow exhibited a mixed blood chimerism. This mosaicism was observed before graft failure or relapse in six patients. However, in five other patients, this mixed chimerism was not followed by these complications with a follow-up of 9 to 31 months after transplantation. Our results support the hypothesis that transplanted bone marrow T cells may help to maintain engraftment by eliminating host cells that can cause graft failure.