Hospital compounding to face shortage: A case study of the development of a lopinavir-ritonavir oral suspension during the first wave of SARS-COV-2 in France.

The potential usefulness of lopinavir-ritonavir on Covid 19 infection during the first wave of contamination in France had boosted Kaletra® syrup prescription to the point of causing its national shortage. In the intensive care units of Parisian hospitals in charge of patients with life-threatening viral contamination, caregivers had to resort to lopinavir-ritonavir-based tablets, crushing them and then dispersing the powder in milk to facilitate administration by nasogastric tube. The difficulties and poor control of this degraded mode, which does not always ensure control of the amount of the drug in the prepared dose and may induce insufficient antiviral exposure, led us to develop in a very short time, while ensuring quality control proportional to the risk, a liquid form as an alternative to Kaletra® oral solution shortage. For this purpose, we describe this compounding formulation and its preparation process, while justifying the quality control strategy adapted to the risk as well as its chemical and physical stability. Based on the chemical and physical studies, the preparation was showed to be stable during at least 2 months between +2°C and +8°C and 1 week at room temperature. This has resulted in the design of kits that include multi-dose packaging and a measuring device and contain the appropriate quantities of drugs to ensure at least one week's treatment for each patient, during which time the kit in use can be stored at room temperature. The intensive care team used this treatment under conditions that they considered well adapted until the imported specialty became available.

Progressive multifocal leukoencephalopathy despite immune recovery in a HIV/HCV co-infected patient.

In HIV patients, HCV co-infection has been associated with an increased risk of progressive multifocal leukoencephalopathy (PML). Furthermore, PML has also been described in patients with cirrhosis, whether related to HCV infection or not. We describe here the case of a HIV/HCV co-infected patient with cirrhosis who developed PML despite HIV suppression and CD4 cell count above 250/mm3 for 2 years. Immunological studies performed at onset of PML and before HCV therapy showed a decrease in naïve CD4 cells (CD45RA+CCR7+CD27+ CD4+ T cells - 23% cells, i.e. 75/mm3) and NK lymphopenia with abnormal and activated NK cells (CD3- CD16+ and/or CD56+) (5% lymphocytes, i.e. 58/mm3, CD69 91%, NKp30 26%). This impaired immunity, possibly related to HIV infection, or HCV infection or cirrhosis, or a combination thereof, could have led to the development of PML.

Extended stability of the rituximab biosimilar CT-P10 in its opened vials and after dilution and storage in polyolefin bags.

The stability of the rituximab biosimilar CT-P10, in 50mL vials at a concentration of 10mg/mL, and after dilution to final concentrations of 1 and 4mg/mL and storage in polyolefin bags at 4°C and 25°C was studied by several orthogonal and complementary methods. No significant change (as defined by a magnitude greater than the inter-batch variability) was observed, for each of the parameters characterizing physical and chemical stability studied, for the two concentrations and temperatures tested, or for any of the three batches tested. This implies that cold-chain rupture and exposure to room temperature up to 15 days both for vials and diluted bags have no deleterious consequence on the quality of the product. Moreover, this extended stability permits safe in-advance preparation, dose-banding or flat-dose, that to avoid unnecessary delays in the management of the patient, improvement of the pharmacy and nurse workload and money saving by avoiding non justified losses of this expensive drug.

One-month stability study of a biosimilar of infliximab (Remsima®) after dilution and storage at 4°C and 25°C.

There is currently only one monoclonal antibody for which there is a biosimilar: infliximab, which was released onto the French market in 2015. The SPC for the biosimilar (Remsima®) are superimposable on those of the original, including 24-hour stability at both 4 and 25°C. The aim of our study was to determine the stability of this biosimilar during one month at 4 and 25°C. Three different batches at two concentrations (0.7mg/mL or 1.6mg/mL) were used. Physicochemical stability was evaluated by the following methods: turbidity, UV spectrometry, DLS, ion chromatography (CEX), gel exclusion chromatography (SEC), and light microscopy. The analyses were performed in triplicate. All methods used have been demonstrated to be valid for measuring antibody stability. There were no signs of physicochemical instability after seven days (on D7) of storage at 4 or 25°C. From D15, we observed slight changes by ion (percentage distribution of the different isoforms) and gel exclusion chromatography (percentage distribution of different polymers, i.e. dimers, oligomers). However, the areas under the curves were unchanged, and the proportions of polymers remained lower than 0.5%. Tertiary structure analysis also showed a change from D15. All observed changes are consistent with progressive oligomerization by hydrophobic interactions. In conclusion, the reconstituted biosimilar is stable for seven days at 4 and 25°C. Gradual oligomerization is observed from D15 but appears to be less than 0.5%, suggesting instability, albeit very limited, in the longer term; the practical consequences of this remain to be evaluated.

Implementation of real-time identification analysis and quantification of chemotherapies preparations with a Multispec(®) analyser.

Post-production analytic control of chemotherapies preparations remains a challenge for hospital pharmacists. Indeed, to be feasible, this control needs to be reliable, fast and easy to implement and to use on real life. This is particularly true for teams not familiar with analytic methods. The Multispec(®) analyser has been specially manufactured for that purpose. After several years of daily use, we wanted to focus on its implementation, abilities and defects that should be corrected on the next analyser. Upon 24 months, 23,350 samples have been analysed. Four percent have been rejected on the first analysis, and finally only 0.37% with another sample after homogenization. Eighty-six preparations have been done another time for non-conformity purpose. Difficulties of implementation were in particular on anthracyclins, oxazophosphorins and monoclonal antibodies. However, compared to liquid chromatography for example, the ultraviolet and infrared combination allows a large number of drugs to be recognized and quantified fastly. As a conclusion this analyser is quite helpful and gives a serious alternative to post-production analytic control for chemotherapies preparations. Some points should however be improved, probably on the next analyser, for instance the sample volume necessary for analysis.

Physicochemical stability study of a new Trimix formulation for treatment of erectile dysfunction.

Currently, severe erectile dysfunction can be treated by intracavernous injections of solutions containing three active ingredients: prostaglandin E1 (PGE1), papaverine and urapidil. Very few data exist on this mixture where phentolamine has been replaced by Urapidil because Phentolamine is not used for this indication in France. The aim of our study was to assess the stability of this formulation and to extend its expiration to permit preparation of batches. Three batches of the preparation containing 15µg/mL PGE1, 15mg/mL of papaverine and 2mg/mL urapidil were made aseptically and then packed in polypropylene syringes stored at 4°C. The physico-chemical stability has been tested as follows: HPLC stability-indicating method, visual observation, measurement of pH and osmolarity. We found that the limiting factor was PGE1 and we exceeded the threshold of 10% loss after 55 days. Replacement of Urapidil by Phentolamine seems to have a slight detrimental effect on stability. Nevertheless, these results allow us to consider the advance preparation of this formulation and provide quality treatment to these patients by avoiding too frequent visits to the hospital.

Long-term stability of bevacizumab repackaged in 1mL polypropylene syringes for intravitreal administration.

INTRODUCTION: The anti-angiogenic monoclonal antibody, bevacizumab, is currently used by intravitreal administration as off-label drug to treat age-related macular degeneration or other ophthalmologic diseases. For this purpose, commercial bevacizumab is repackaged in 1mL polypropylene syringes under sterile conditions. However, no complete study on the stability of this hospital-based preparation is available. METHODS: Commercial bevacizumab (25mg/mL; Avastin(®)) was aseptically repackaged in 1mL polypropylene syringes, stored at 4°C, and analyzed within the preparation day (D0), after 30 days (D30) and 90 days (D90). Some syringes were kept for up to 8 months to observe possible instability. Several complementary and stability-indicating analytical methods were used to assess in details the primary, secondary and tertiary structure of the antibody during its conservation: ionic chromatography, size-exclusion chromatography, peptide mapping, 2nd derivative UV and IR spectroscopy, turbidimetry, diffraction laser spectroscopy, thermal denaturation curves, microscopic examination and image analysis. RESULTS: We clearly demonstrate that the commercial solution of bevacizumab can be safely repackaged in polypropylene syringes and stored up to 3 months at 4°C without alteration of its primary, secondary and tertiary structure. The only difference observed is the contamination of the syringe content by silicone oil microdroplets, which is quite immediate and does not change significantly during the storage in terms of number and size. CONCLUSION: Our results support the off-label use of repackaged bevacizumab by intravitreal administration, at least from a pharmaceutical point of view, with a validated stability of 3 months. This stability period is largely enough to practical situations and support current practices, such as in advance or batch preparations, which present major advantages in terms of GMP respect, workload optimization and financial savings.

HIV-specific T cell cytotoxicity mediated by RANTES via the chemokine receptor CCR3.

CC chemokines produced by CD8(+) T cells are known to act as HIV-suppressive factors. We studied the possible role of these chemokines in HIV-1-specific killing of target cells. We found that the activity of cytotoxic T lymphocytes (CTLs) in CTL lines or freshly isolated peripheral blood mononuclear cells from HIV-1-infected individuals is markedly enhanced by RANTES (regulated on activation, normal T cell expressed and secreted) and virtually abolished by an antibody neutralizing RANTES or the RANTES receptor antagonist RANTES(9-68). Lysis was mediated by CD8(+) major histocompatibility complex class I-restricted T cells and was obtained with target cells expressing epitopes of the HIV-1LAI proteins Gag, Pol, Env, and Nef. The cytolytic activity observed in the presence or absence of added RANTES could be abolished by pretreatment of the CTLs with pertussis toxin, indicating that the effect is mediated by a G protein-coupled receptor. The chemokines monocyte chemotactic protein (MCP)-3, MCP-4, and eotaxin acted like RANTES, whereas macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MCP-1, and stromal cell-derived factor 1 were inactive, suggesting a role for the eotaxin receptor, CCR3, and ruling out the involvement of CCR1, CCR2, CCR5, and CXCR4. CTL activity was abrogated by an antibody that blocks CCR3, further indicating that specific lysis is triggered via this chemokine receptor. These observations reveal a novel mechanism for the induction of HIV-1-specific cytotoxicity that depends on RANTES acting via CCR3.

[Reliable real-time analytical control of monoclonal antibodies chemotherapies preparations on Multispec automaton]. / Contrôle rapide et fiable des préparations de chimiothérapies à base d'anticorps monoclonaux à l'aide de l'automate Multispec.

The aim of our study was the implementation of a systematic control for preparations based upon monoclonal antibodies. Multispec automaton combines ultraviolet (UV) with infrared spectroscopy. Validation of the analytical method was carried out, in terms of qualitative results (with a correct recognition) and quantitative ones. Our first tests tend to show that Multispec automaton is not reliable enough to manage every antibody currently sold on the French market because of a weak correct recognition percentage (around 35 %). When taking into consideration only its own spectra, the improvement is low (50 % recognition). Only a new spectral library with restricted zones in the infrared domain and furthermore with another correlation calculation mode will be able to produce fast and reliable results. In our final library, spectral recognition is correct for 100 % of samples. Quantification achieved thanks to UV is correct in terms of exactness and precision, respectively 6.0 and 8.1 %. To date, we have settled this routine control for every monoclonal antibody available in our institution used in chemotherapies regimens (alemtuzumab, bevacizumab, cetuximab, gemtuzumab ozogamycin, panitumumab, rituximab and trastuzumab). The reliability of our antibody-specific library was demonstrated, likewise the original library for classical chemotherapies.

Stability of nivolumab in its original vials after opening and handing in normal saline bag for intravenous infusion.

OBJECTIVES: Nivolumab is an anti-programmed cell death-1 monoclonal antibody, approved for numerous indications in oncohaematological cancers. It is available as solution for infusion at 10 mg/ml. In accordance with the Summary of Product Characteristics (SmPCs), the product is stable for 24 h at 2-8 °C after dilution. However, to anticipate the needs and constraints related to the handling of the product, the aim was to obtain additional information that will contribute to the risk assessment in case of deviation. Potential changes in the stability of Opdivo® leftovers (10 mg/ml) and diluted nivolumab in normal saline solution (2 mg/ml) over a period exceeding 24 h, at different temperatures and after freezing/thawing cycles were studied. METHODS: Turbidimetry, Ultraviolet (UV)-spectroscopy, dynamic light scattering and chromatography were used to evaluate physicochemical stability. Potential pharmacological variations were monitored in vitro by a functional binding inhibition method. RESULTS: No change was detected after 1 month of storage at 2-8 °C neither after 7 days at 40 °C. Although slight changes were detected only after 3 months under 2-8 °C, major changes were found for the same period of time at 40 °C (variants in the distribution profile, slight increase in oligomers and fragments and UV spectral modifications). Physical instability was observed upon 3 freeze/thaw cycles, with the appearance of a new protein population associated with an increase in polydispersity index. CONCLUSION: In conclusion, our results provide additional rationale to the SmPCs, regarding the use of leftovers, reassignment of bags, pre-preparation or breaking the cold chain for Nivolumab.

What Is the most Important for Elite Control: Genetic Background of Patient, Genetic Background of Partner, both or neither? Description of Complete Natural History within a Couple of MSM.

BACKGROUND: We describe a homosexual man who strongly controlled HIV-1 for ten years despite lack of protective genetic background. METHODS: HIV-1 DNA was measured in blood and other tissues. Cell susceptibility was evaluated with various strains. HIV-1-specific (CD4 and CD8 activation markers and immune check points) and NK cells responses were assessed; KIRs haplotypes and HLA alleles were determined. FINDINGS: Two HIV-1 RNA copies/mL of plasma were detected in 2009, using an ultra-sensitive assay. HIV-DNA was detected at 1.1 and 2 copies/106 PBMCs in 2009 and 2015 respectively, at 1.2 copies/106 cells in rectal cells in 2011. WBs showed weak reactivity with antibodies to gp160, p55 and p25 from 2007 to 2014, remaining incomplete in 2017. CD4 T cells were susceptible to various strains including HIVKON, a primary isolate of his own CRF02_AG variant. CD8 T cells showed a strong poly-functional response against HIV-Gag, producing mainly IFN-γ; a robust capacity of antibody-dependant cell cytotoxicity (ADCC) was observed in NK cells. Case patient was group B KIR haplotype. Neutralizing antibodies were not detected. CD4 and CD8 blood T cells showed normal proportions without increased activation markers. Phylogenetic analyses identified the same CRF02_AG variant in his partner. The patient and his partner were heterozygous for the CCR5ΔD32 deletion and shared HLA-B*07, C*07 non-protective alleles. INTERPRETATION: This thorough description of the natural history of an individual controlling HIV-1 in various compartments for ten years despite lack of protective alleles, and of his partner, may have implications for strategies to cure HIV-1 infection.

Clinical impact of NK-cell reconstitution after reduced intensity conditioned unrelated cord blood transplantation in patients with acute myeloid leukemia: analysis of a prospective phase II multicenter trial on behalf of the Société Française de Greffe de Moelle Osseuse et Thérapie Cellulaire and Eurocord.

Unrelated cord blood transplantation (UCBT) after a reduced intensity conditioning regimen (RIC) has extended the use of UCB in elderly patients and those with co-morbidities without an HLA-identical donor, although post-transplant relapse remains a concern in high-risk acute myeloid leukemia (AML) patients. HLA incompatibilities between donor and recipient might enhance the alloreactivity of natural killer (NK) cells after allogeneic hematopoietic stem-cell transplantation (HSCT). We studied the reconstitution of NK cells and KIR-L mismatch in 54 patients who underwent a RIC-UCBT for AML in CR in a prospective phase II clinical trial. After RIC-UCBT, NK cells displayed phenotypic features of both activation and immaturity. Restoration of their polyfunctional capacities depended on the timing of their acquisition of phenotypic markers of maturity. The incidence of treatment-related mortality (TRM) was correlated with low CD16 expression (P=0.043) and high HLA-DR expression (P=0.0008), whereas overall survival was associated with increased frequency of NK-cell degranulation (P=0.001). These features reflect a general impairment of the NK licensing process in HLA-mismatched HSCT and may aid the development of future strategies for selecting optimal UCB units and enhancing immune recovery.

Autocrine interferon-beta synthesis for gene therapy of HIV infection: increased resistance to HIV-1 in lymphocytes from healthy and HIV-infected individuals.

OBJECTIVE: To explore the possibility of gene therapy of HIV infection based on the multiple antiretroviral activities of interferon (IFN)-beta. DESIGN: We introduced into HIV target cells an IFN-beta gene placed under an expression control ensuring a low and constitutive expression, sufficient to confer a permanent antiviral state without impeding normal cell function. METHODS: We transformed, with an efficacy ranging from 20-55%, peripheral blood lymphocytes (PBL) derived from healthy, seronegative donors, and from asymptomatic HIV-infected individuals by the HMB-KbHuIFN beta retroviral vector carrying the human IFN-beta coding sequence driven by a fragment of the murine H-2Kb gene promoter. RESULTS: The replication rate of the IFN-beta-expressing cells was no different from that of untransformed controls during the 21-day period of in vitro observation. When IFN-beta-transformed, purified CD4+ lymphocytes from healthy donors were HIV-1LAI-infected, virus replication was inhibited and most of the cells survived, in contrast to untransformed CD4+ cells which were all destroyed 12 days after infection. Protection of CD4+ cells from the same donors was also observed in suspensions of IFN-beta-transformed total PBL that were infected with HIV-1LAI. In IFN-beta-transformed PBL from four HIV-infected donors, endogenous HIV replication was decreased and 28-69% of the CD4+ cells survived at the end of the 21 days in culture. In the untransformed control PBL suspensions, all CD4+ cells were destroyed. In long-term experiments, HIV-infected, IFN-beta-transformed cell populations of the lymphocytic CEM and the promonocytic U937 line were kept in culture for 60 days, during which time they remained resistant to HIV infection. CONCLUSION: These results indicate that further exploration of autocrine IFN-beta production for somatic cell gene therapy of HIV infection is warranted.

Inhibition of human immunodeficiency virus transmission to CD4+ T cells after gene transfer of constitutively expressed interferon beta to dendritic cells.

CD34(+)-derived dendritic cells (DCs) can be infected by the T cell-tropic HIVLAI strain, but are poorly permissive for efficient virus production. However, HIVLAI-infected DCs are able to transmit a vigorous cytopathic infection to activated CD4(+) T cells. We show that DCs differentiated from CD34(+) cells can be efficiently transduced by a retroviral vector carrying the IFN-beta coding sequence. This results in resistance to infection by HIV as shown by a threefold reduction in the HIV DNA copy number per cell, and by inhibition of HIV transmission from DCs to CD4(+) T cells. Moreover, constitutive IFN-beta production by DCs increases the synthesis of IL-12 and IFN-gamma Th1-type cytokines and of the beta-chemokines MIP-1alpha, MIP-1beta, and RANTES. This indicates that IFN-beta transduction of DCs blocks HIV infection and viral transmission to CD4(+) T cells, and could favor cellular immune responses in HIV-infected patients.

Development of methods for somatic cell gene therapy directed against viral diseases, using retroviral vectors carrying the murine or human interferon-beta coding sequence: establishment of the antiviral state in human cells.

We are developing methods for somatic cell gene therapy directed against chronic and fatal virus infections, such as acquired immunodeficiency (AIDS), by transforming cells with a constitutively expressed interferon (IFN) coding sequence. Previous work from our laboratory has shown that stable antiviral expression (SAVE) can be obtained in murine BALB/c 3T3 cells and human U937 cells transformed with plasmids carrying either the murine or the human IFN-beta coding sequence placed under the expression control of a 0.6-kb Xho II-Nru I promoter region of the murine H-2Kb major histocompatibility complex (MHC) gene (Macé et al., 1991; Seif et al., 1991). In the present paper, we report the construction of murine (Mu) and human (Hu) IFN-beta-expressing retroviral vectors (pMPZen-MuIFN beta, pHMB-KbMuIFN beta) and the problems encountered. Because of the murine origin of commonly used packaging cells and the species specificity of IFN, it was evident that placing the murine IFN-beta sequence under constitutive expression control could result in the production of Mu IFN in the murine packaging system, and thereby lead to decreased vector production and also to enhanced resistance of target cells. Using a packaging cell line that releases a beta-galactosidase-expressing vector, we show that, as expected, Mu IFN-alpha/beta decreases vector production of murine packaging cells and also inhibits the transformation of target NIH-3T3 cells with this vector, but the presence of anti-Mu IFN antibodies rescues the viral titer of the packaging cells and restores the sensitivity of target cells to virus transformation. However, the same antibody treatment is unable to rescue the viral titer of psi-2 packaging cells producing autocrine Mu IFN-beta encoded by the pMPZen-MuIFN beta and pHMB-KbMuIFN beta vectors. Because of the species specificity of IFN, this problem is circumvented with the pMFG-HuIFN beta vector carrying the human IFN-beta sequence. In spite of the production of Hu IFN, murine psi-CRIP packaging cells are able to release retroviral vectors expressing Hu IFN-beta, and these amphotropic vectors can transform human MRC-5 cells and confer to these cells an enhanced resistance to vesicular stomatitis virus (VSV) infection.

Acquired constitutive expression of interferon beta after gene transduction enhances human immunodeficiency virus type 1-specific cytotoxic T lymphocyte activity by a RANTES-dependent mechanism.

CTL lines directed against HIV-1 antigens were generated from infected individuals and were transduced by the HMB-K(b)HuIFNbeta vector, resulting in low, constitutive expression of interferon beta (IFN-beta). The IFN-beta-transduced cells showed markedly increased HIV-1-specific, MHC class I-restricted CTL activity against HIV-1-LAI Gag, Pol, or Env antigens. This effect of IFN-beta was correlated with an overexpression of RANTES and completely abrogated by RANTES-blocking antibody. The present results provide the first evidence that IFN-beta transduction of CTL lines enhances HIV-specific cytotoxic activities through an upregulation of RANTES production. The efficient elimination of HIV-infected cells by IFN-beta-transduced CTL lines makes this gene therapy approach an attractive treatment for AIDS.

[Development of an anti-HIV gene therapy based on the antiviral properties of beta interferon]. / Développement d'une génothérapie anti-VIH basée sur les propriétés antivirales de l'interféron bêta.

The aim of our work is to explore the use of IFN-beta for gene therapy in the HIV-infection. Transduction of various HIV target cells with a retroviral vector that carries the Hu-IFN-beta coding sequence under constitutive low expression control, confers resistance to HIV without affecting cell replication or function. After transduction, lymphocytes from HIV-infected patients develop resistance to the endogenous virus, provided the cells are derived from individuals with a CD4 cell count higher than 200 per mm3.

Stable antiviral expression (SAVE) as an approach to somatic cell gene therapy directed against HIV infection.

We are developing methods for somatic cell gene therapy directed against infection with human immunodeficiency virus by enhancing the antiviral resistance of target cells through the constitutive production of interferon-beta. Cells that have been transformed by plasmids or retroviral vectors carrying the human interferon-beta gene placed under the expression control of a murine H2Kb promoter fragment become resistant to HIV infection. Part of this enhanced resistance is due to inhibition of virus entry into the transformed cells, a hitherto unreported mechanism of interferon action.

Analysis of CD16+CD56dim NK cells from CLL patients: evidence supporting a therapeutic strategy with optimized anti-CD20 monoclonal antibodies.

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FcγRIIIA) is well preserved in CD16(+)CD56(dim) cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FcγRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FcγR engagement in CLL patients.