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This study was undertaken to analyze the hematotoxicity of paclitaxel (Taxol) and to test whether transduction of repopulating hematopoietic cells with a retroviral vector (SF1m) expressing the human multidrug resistance 1 gene (MDR1) would permit dose intensification following bone marrow transplantation (BMT). While the regimen chosen (8 x 20 mg/kg i.p. within 12 days) produced a non-lethal, reversible hematotoxicity in mice with steady-state hematopoiesis, only 35.3% (6/17) of control mice survived when treated starting 14 days post BMT. In contrast, 83.3% (15/18) of mice transplanted with SF1m-transduced cells survived, owing to a significant protection against severe acute myelotoxicity (as determined by neutrophil counts, white and red blood cell counts and values for hemoglobin and hematocrit). After recovery from chemotherapy, an increase of myeloid cells that were resistant to colchicine and effluxed the fluorochrome Rhodamine 123 was observed in SF1m-mice, but not in controls. These results reveal that the lethal, dose-limiting hematotoxicity of an intensified post-transplantation chemotherapy with paclitaxel can be prevented by retroviral transfer of the MDR1 gene to a minor proportion of repopulating cells. Our mouse model, mimicking clinically achievable gene transfer rates, thus suggests that bone marrow chemoprotection may widen the therapeutic window and permit an earlier onset of post-transplantation chemotherapy., (Copyright 2002 Wiley-Liss, Inc.)

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).

Objective: Hematopoietic progenitor cells are a promising source for generation of genetically modified dendritic cells. A prerequisite for using these cells in therapeutic approaches is stable vector-mediated transgene expression during and after cell maturation. We investigated the expression of enhanced green fluorescence protein (EGFP) mediated by retroviral vectors in dendritic cells and other hematopoietic cells differentiated in vitro., Material and Methods: CD34(+) cells were efficiently transduced with retroviral vector constructs known to mediate different expression levels due to distinct cis-acting elements. EGFP(+) cells were purified by cell sorting and differentiated to monocytes, granulocytes, dendritic cells, and erythrocytes. Coexpression of EGFP and cell type-specific markers was analyzed by flow cytometry., Results: Transgene expression from various retroviral vectors was silenced exclusively in dendritic cells, but not in other mature myeloid cells. Loss of EGFP was most pronounced in cells initially displaying low expression levels. This was confirmed by using a retroviral vector coding for a variant of EGFP with significantly reduced half-life. In contrast, a majority of dendritic cells showed stable expression when a self-inactivating retroviral construct using an internal cytomegalovirus promotor was used., Conclusions: We suggest that expression from the retroviral long terminal repeat is silenced during dendritic cell differentiation in vitro. High levels of stable transgene product in progenitor cells may mask a loss of expression. An improvement of retroviral vectors mediating stable transgenic expression is necessary for therapeutic approaches using gene-modified dendritic cells.

Myelosuppression is the main side effect of cancer chemotherapy. An improved rate of retroviral vector-mediated gene transfer to hematopoietic stem cells, shown in more recent clinical trials, has created the basis to test the concept of myeloprotective gene therapy. We transplanted clinical-scale human peripheral blood progenitor cell grafts (n = 2) transduced with retroviral vector SF91m3, which contains the human multidrug resistance 1 gene (MDR1), into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Engrafted mice of one cohort were protected from paclitaxel toxicity (p < 0.05) and we noted a similar trend in the second cohort. In paclitaxel-treated mice that had received gene-transduced cells we found a significant increase in gene marking (p < 0.05 - p < 0.01) or P-glycoprotein expression (p < 0.01) compared with their chemotherapy-naive counterparts. This is the first report showing that cytostatic drug resistance gene therapy can mediate chemoprotection of human clinically relevant stem cell populations with marrow engraftment potential.

Background: For many inherited and acquired diseases of the blood system, gene transfer into hematopoietic cells is a promising strategy to alleviate disease-related symptoms or even correct genetic alterations. In clinical gene therapy applications, low transduction efficiencies have been a major limitation mainly because of insufficient effective titers of the retroviral supernatants used. Thus, optimization of clinical-grade vector production under current 'Good Manufacturing Practice' (GMP) conditions is a prerequisite for successful gene therapy trials., Methods: We established stable retroviral producer clones with single integrations of a retroviral vector encoding for the multidrug-resistance gene 1 (MDR1). Optimization of vector production in multi-tray cell factories (MTCFs) was studied with particular regard to harvest medium, cell density and harvest time point., Results: We demonstrated that high-titer vector stocks could be produced in serum-free medium. By reducing the volume of harvest medium, titers could be increased up to four-fold. Plating optimal cell densities of 1 x 10(4) cells/cm2, repetitive harvests of vector supernatant were feasible over four consecutive days. Combining the most advantageous culture and harvest parameters tested, we were able to produce large quantities of serum-free vector supernatant in 40-tray MTCFs. Highly efficient gene transfer into primary human CD34+ progenitor cells demonstrated the quality of these vector stocks., Conclusion: The large-scale vector-production protocol in MTCFs described here is easy to handle, is applicable to a wide range of adherent producer cell lines and, most importantly, complies with current GMP guidelines.

Using retroviral vectors encoding enhanced green fluorescent protein (EGFP), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (MOI) and on the half-life of the encoded protein. We show that an elevation of the MOI not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of C57BL/6J mice analyzed 13 weeks after transplantation). This suggests that the MOI has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. The impact of protein stability is demonstrated by comparing vectors expressing EGFP or a destabilized variant with a C-terminal PEST-sequence, d2EGFP. The loss of expression with d2EGFP was more pronounced in terminally differentiated cells of the peripheral blood (>30 fold) than in progenitor cells (five- to 10-fold), indicating a stronger transcription of the retroviral promoter in progenitor cells and a predominant role of protein inheritance over de novo synthesis of transgenic protein in mature blood cells. This analysis reveals an important and differentiation-dependent contribution of protein half-life to the expression of retroviral vectors in hematopoietic cells, establishes d2EGFP as a more accurate reporter for determination of vector transcription, and also suggests that preclinical data obtained under conditions of high transduction rates or with vectors expressing stable reporter proteins require careful interpretation.

We describe the functional analysis of a novel retroviral vector, SF91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (MDR1), in hematopoietic cells. SF91m3 combines several promising features. The vector backbone lacks viral coding sequences and AUG-start codons 5' of the MDR1 cDNA. A point mutation of a cryptic splice acceptor of the MDR1 cDNA increases the probability of transferring an intact provirus. The titer of a PG13 packaging cell clone containing a single proviral integration is high (>2 x 10(6) particles/ml from frozen stocks of serum-free vector harvests). Human hematopoietic cells transduced with SF91m3 reliably express MDR1 before and after passage through NOD/SCID mice, as shown by quantitative PCR and efflux assays with rhodamine 123 or Hoechst 33342. Finally, SF91m3 mediates resistance to escalated doses of cytotoxic agents, as shown by survival and differentiation of transduced colony-forming cells in the presence of colchicine at 48 ng/ml (>10 x IC(50)). Thus, SF91m3 may represent an interesting candidate for future trials addressing the safety and utility of MDR1 gene transfer; moreover, this study demonstrates that sequence alterations improving post-transcriptional processing of retroviral vectors have a substantial impact for gene expression in hematopoietic cells.

We have adapted a recently published protocol for retroviral gene transfer into hematopoietic cells [A. J. Schilz et al. (1998) Blood 92: 3163-3171] with respect to clinical requirements such as large-volume vector stock generation, adequate cell source, high cell numbers, and serum-free conditions. We present data on transduction efficacy and expression of the multidrug resistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral blood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector. Using a 1-day cytokine-mediated prestimulation, consisting of human interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO), followed by a 3-day transduction procedure, we were able to detect up to 51% CD34(+) cells expressing MDR1. Xenotransplantation of transduced cells into NOD/LtSz-scid/scid (NOD/SCID) mice resulted in a mean engraftment level of 23% (0.1 to 87%). As shown by quantitative PCR analysis, a mean of 12.7% (range 0.3 to 55%) of the engrafted human cells in the bone marrow of chimeric mice contained the MDR1 cDNA. Furthermore, enhanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population. Our data suggest that NOD/SCID repopulating cells derived from mobilized PB can be transduced efficiently with existing retroviral vector systems under clinically applicable conditions.

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.

Genetic transfer and expression of drug-resistance functions into haematopoietic stem and progenitor cells is a promising means to overcome both the acute and longterm side-effects of cytotoxic drugs in bone marrow. Here, we describe a functional analysis of a retroviral vector that co-expresses human cDNAs for multidrug resistance 1/P-glycoprotein (MDR1) and a double mutant of O(6)-alkylguanine-alkyltransferase (hATPA/GA) to high levels. The hATPA/GA protein contains two amino acid substitutions that render it resistant to compounds such as O(6)-benzylguanine that inhibit the wild-type protein which is often overexpressed in resistant tumour cells. Evidence for simultaneous drug resistance of genetically modified primary murine progenitor cells to colchicine or the podophyllotoxin etoposide, both covered by MDR1-mediated efflux activity, and the nitrosourea BCNU, which is counteracted by hATPA/GA, is presented using in vitro colony assays.

Transplantation of suicide gene modified allogeneic T lymphocytes is an approach to prevent T cell mediated GVHD while preserving the 'graft-versus-leukemia' (GVL) effect of an allograft. A prerequisite for such a therapy is the efficient transduction of T cells with suitable vectors. Since existing techniques allow only insufficient transduction of T cells, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 10(6) cells/ml) by retroviral infection. The presented protocol allowed us to obtain transduction rates of more than 70% of CD3+ cells after two cycles of infection. It is based on the use of FBS-free media for both the production of retrovirus-containing supernatant, as well as the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large scale conditions, it may easily be adapted to clinical needs and 'good manufacturing practice' (GMP) standards.

Retroviral vectors are currently the most important and best characterized tools for ex vivo genetic modification of hematopoietic progenitor/stem cells. As a prerequisite for clinical applications, large volumes of high-titer vector supernatants have to be generated in compliance with 'GMP' guidelines. This goal can be reached using a carefully selected producer cell clone and a conventional large-scale cell culture system. The retroviral vector SF1m provides efficient expression of the human multidrug resistance 1 (MDR1) gene in hematopoietic progenitor/stem cells in vitro and in NOD/SCID mouse repopulating human cells in vivo. Currently, a clinical phase I/II study is in preparation to test whether intensified consolidation chemotherapy is enabled by autologous transplantation of peripheral blood progenitor/stem cells that have been genetically modified with SF1m. Using multi-tray cell factories >19 l of serum-free vector containing supernatant were generated from cells of a previously established SF1m-producer clone, based on the PG13 packaging cell line. Testing of the final samples revealed sufficient quality (>1.5 x 10(6) infectious particles/ml) for clinical scale transduction of CD34+ cells. Results from the production runs and the applied biosafety concept are described.

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)

Ventricular septal defect following cardiac trauma: percutaneous closure with the Amplatzer septal occluder. HISTORY AND CLINICAL FINDINGS: A 36-year old roofer fell 8 m and suffered a severe polytrauma. A complicating pericardial tamponade was relieved as an emergency and myocardial fissure of the left ventricle about 1 cm in length sutured. 2 weeks later, a severe mitral insufficiency due to rupture of the papillary muscle occurred, that was cared by the implantation of a bioprosthesis. 6 weeks later dyspnoea and restricted physical capability were clinically impressive. INVESTIGATIONS AND DIAGNOSIS: Echocardiography demonstrated a posttraumatic muscular ventricular septal defect. Doppler echocardiography and heart catheterization showed a ventricular septal defect still restrictive with a left-to-right shunt (pulmonary-to-systemic flow ratio Qp/Qs 1.8:1). Under exercise, there was a significant increase in mean pulmonary arterial pressure from 27 to 60 mmHg. TREATMENT AND COURSE: The patient who had already been operated twice before was treated by the percutaneous occlusion of the ventricular septal defect from arterio- to venofemoral, a guide catheter was inserted transseptally into the left ventricle. An Amplatzer Septal Occluder, a self-expandable, self-centering wire-mesh double disc with a connecting central stent part, was loaded and then implanted in the ventricular septal defect. The intervention was controlled by fluoroscopy and echocardiography. Post intervention, only a trivial residual shunt was seen. The pumping efficacy of the left ventricle increased, in particular of the septal and apical segments. Clinically, the patient was markedly more load-bearing, the exercise-induced dyspnoea reduced. CONCLUSIONS: Following a cardiac trauma, various complications may occur that can manifest themselves clinically at two or more times. A posttraumatic ventricular septal defect of a patient already operated was successfully occluded by an Amplatzer Septal Occluder. Alongside established surgical methods, the non-operative implantation of new occlusion systems could mean an effective treatment option for muscular ventricular septal defects.

Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (DeltaLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and 'good manufacturing practice' (GMP) standards.

We have developed a retroviral vector coexpressing the multidrug-resistance 1 (MDR1) cDNA for inducing cancer drug resistance and the truncated version of the low-affinity nerve growth factor receptor (DeltaLNGFR) for cell-surface marking of transduced cells. The vector is based on the FMEV backbone which mediates high levels of gene expression in hematopoietic cells. To achieve optimal expression levels of both cDNAs, untranslated regions from MDR1 and DeltaLNGFR were removed and three different connections were tested: retroviral splice signals, an internal ribosomal entry site (IRES) from encephalomyocarditis virus, and an internal promoter from the chicken beta-actin gene. As determined by two-color flow cytometry, the best correlation of the expression of both cDNAs was obtained using the vector SF1mSdelta which utilized retroviral splice signals for co-expression. Simultaneous expression of both cDNAs at the single cell level was also shown by confocal laser microscopy. Lymphoid and hematopoietic progenitor cells, including primary human CD34+ cells, transduced with SF1mSdelta acquired dominant multidrug resistance. Transduced primary CD34+ cells could be enriched in vitro based on expression of DeltaLNGFR, avoiding exposure to cytostatic agents. Thus, monitoring the selection of chemotherapy-resistant cells and analyzing their biological properties may be alleviated, both in vitro and in vivo.

FMEV retroviral vectors combine the long terminal repeat of Friend mink cell focus-forming viruses with the 5' untranslated leader region of the murine embryonic stem cells virus. These modules were connected to achieve high transgene expression in hematopoietic progenitor and stem cells. Here, we report the cloning of safety-improved and versatile FMEV vectors allowing module-wise exchange of crucial elements for comparative studies. By transfer and expression of four different marker genes (neomycin phosphotransferase, lacZ, enhanced green fluorescent protein and truncated low affinity nerve growth factor receptor), we formally demonstrate that both the long terminal repeat and the leader contribute to the high expression of FMEV in transduced hematopoietic cells. Most prominent are the data recorded in the absence of selection in myelo-erythroid progenitor cells. Here, FMEV vectors mediate up to two orders of magnitude increased transgene expression levels when compared with vectors based on the Moloney murine leukemia virus.

The polycythemic strain of the Friend spleen focus-forming virus (SFFVp) is a replication-defective, acutely transforming retrovirus inducing a bistage erythroleukemia in susceptible mice. The first stage of the disease is an acute polyclonal erythroblastosis induced by the proliferation-promoting effect of gp55. gp55 is expressed from a spliced subgenomic message of SFFVp and stimulates the cellular receptor for erythropoietin. Using a selectable SFFVp that otherwise mimics the specificity of the disease, we demonstrate that the kinetics of the polyclonal expansion depends on the transcriptional strength of the retroviral cis-active elements. By exchanging gp55 for apathogenic genes, we show that SFFVp enhancer and splice signals can be successfully utilized for the development of retroviral vectors mediating very efficient transgene expression in hematopoietic cells. Apathogenic selectable SFFVp-based vectors carrying distinct enhancer alterations are a valuable tool to analyze transcriptional control of leukemia viruses in the absence of oncogenic proteins. Moreover they might have therapeutic potential.

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.

Therapeutic gene transfer into hematopoietic cells is critically dependent on the evolution of methods that allow ex vivo expansion, high-frequency transduction, and selection of gene-modified long-term repopulating cells. Progress in this area needs elaboration of defined culture and transduction conditions for long-term repopulating cells and improvement of gene transfer systems. We have optimized retroviral vector constructions based on murine leukemia viruses (MuLV) to overcome the transcriptional repression encountered with the use of conventional Moloney MuLV (MoMuLV) vectors in early hematopoietic progenitor cells (HPC). Novel retroviral vectors, termed FMEV (for Friend-MCF/MESV hybrid vectors), were cloned that mediate greatly improved gene expression in the myeloerythroid compartment. Transfer of the selectable marker multidrug resistance 1 (mdr1), FMEV, in contrast to conventional MoMuLV-related vectors currently in use for clinical protocols, mediated background-free selectability of transduced human HPC in the presence of myeloablative doses of the cytostatic agent paclitaxel in vitro. Furthermore, FMEV also greatly improved chemo-protection of hematopoietic progenitor cells in a murine model system in vivo. Finally, when a second gene was transferred along with mdr1 in an FMEV-backbone, close to 100% coexpression was observed in multidrug-resistant colonies. These observations have significant consequences for a number of ongoing and planned gene therapy trials, for example, stem cell protection to reduce the myelotoxic side effects of anticancer chemotherapy, correction of inherited disorders involving hematopoietic cells, and antagonism of HIV infection.

Friend mink cell focus-forming (FMCF) viruses are recombinants between the Friend murine leukemia virus (F-MuLV) and endogenous polytropic retroviruses involved in a number of retrovirus-induced malignancies of the myelo-erythroid compartment. To analyze the contribution of the viral cis regulatory elements to the host range determinants within the hematopoietic system, we performed a series of marker gene experiments using both transient transfection and retroviral-mediated stable transduction of indicator cell lines representing distinct developmental stages. According to our data, the U3 region in the long terminal repeat (LTR) of FMCF viruses possesses an enhancer assembly that allows efficient transcription in both early and late myelo-erythroid stem and progenitor cells. Retroviral gene expression, however, is subjected to stage-dependent transcriptional controls during blood cell maturation. We obtained evidence that a repressor element overlapping with the primer binding site in the viral leader region compromises U3-mediated gene expression in a stage-dependent manner, with the strongest restriction observed in the most primitive cells analyzed, FDCP-mix. In addition, our data indicate a second hurdle for retroviral gene expression in early hematopoietic cells that is independent of the primer binding site and most likely related to inefficient utilization of U3-located enhancers. These data shed light on the mechanisms of host range restriction within the hematopoietic system and define a basis for the design of retroviral vectors aimed to overcome transcriptional inefficiency in early hematopoietic cells. Thus, we developed novel retroviral vectors combining FMCF-type U3 regions with a permissive leader from the murine embryonic stem cell virus. These vectors are highly efficient for gene transfer and expression in both early and late myelo-erythroid cells, indicating that they will be of great use for a variety of experimental and therapeutic applications.

We present data that retroviral gene expression in early hematopoietic cells is subjected to transcriptional controls similar to those previously described for embryonic stem cells. Transient transfection experiments revealed that both the viral enhancer region in the U3 region of the long terminal repeat as well as a repressor element coincident with the primer binding site of Moloney leukemia viruses are limiting for expression in hematopoietic cells in a differentiation-dependent manner. Within the group of Moloney leukemia virus-related viruses, only the myeloproliferative sarcoma virus showed high enhancer activity in myeloid (including erythroid) cells. In contrast, enhancer regions related to the Friend mink cell focus-forming viruses mediate much higher gene expression levels in both multipotent and lineage-committed myeloid cells. In addition, transcriptional repression related to sequences in the primer binding site of Moloney leukemia virus-derived vectors is also found in early hematopoietic cells and can be overcome by using the corresponding sequences of the murine embryonic stem cell virus. On the basis of these results, two types of novel retroviral hybrid vectors were developed; they combine the U3 regions of either the Friend mink cell focus-forming virus family or the myeloproliferative sarcoma virus with the primer binding site of the murine embryonic stem cell virus. When used to express the human multiple drug resistance gene, these vectors substantially improve protection to cytostatic drugs in transduced hematopoietic cell lines FDC-Pmix, TF-1, and K-562 in comparison with Moloney leukemia virus-derived vectors presently used for the stem cell protection approach in somatic gene therapy.

Twenty two healthy males participated in a randomised, placebo-controlled, double blind, cross-over study to investigate the influence of simvastatin on the pharmacokinetics of ramipril and its active metabolite (ramiprilat), and on the ACE-inhibiting effect of ramiprilat. During two study periods, each of 7 days, subjects received daily either simvastatin 20 mg at 19.00 h or placebo; ramipril (5 mg) was given on Day 5 of each of the periods. Plasma concentrations of ramipril and ramiprilat and ACE-activity were measured in sequential blood specimens, and ramipril and ramiprilat concentrations were measured in urine. Blood and urine collections for pharmacokinetic and pharmacodynamic assessment were made up to 72 h after the dose of ramipril. The mean AUC of ramipril for ramipril+placebo (R+P) and ramipril+simvastatin (R+S) was 22.2 and 21.3 ng.h.ml-1, respectively; for ramiprilat the corresponding figures were 61.3 and 57.6 ng.h.ml-1. The urinary excretion of ramipril+metabolites for (R+P) and (R+S) was 25.2 and 24.1% of dose. The maximum percentage inhibition of ACE-activity for (R+P) was 94.6%, and for (R+S) it was 94.1%. It is concluded that concomitant administration of simvastatin and ramipril has no clinically relevant effect on the pharmacokinetics or ACE-inhibition of the latter drug and its metabolites.

The pharmacokinetics of cefpirome were studied in healthy male subjects following single (0.5, 1.0 and 2.0 g) and multiple (1.0 g every 12 h for 3.5 days) intramuscular injections. High pressure liquid chromatography was used to determine cefpirome concentrations in plasma and urine. Cefpirome was absorbed rapidly, mean peak times were 1.6-2.3 h. Pharmacokinetics were linear over the 0.5 to 2.0 g range with mean total body clearance ranging from 148 to 154 mL/min. The peak plasma concentration and area under the curve increased in a dose proportional manner. The terminal half-life (2 h) was not influenced by dose or duration of dosing. There was no drug accumulation after multiple in administrations. About 70-80% of an administered dose was excreted in the urine as unchanged cefpirome. Cefpirome was well tolerated, slight to moderate pain being reported in less than 30% of the injections.

The studies were performed in healthy male rats and in one male dog after oral and intravenous administration of [2-carbamoyl-14C]HOE 077. HOE 077 wa rapidly and completely absorbed after oral administration. In blood, tmax was at about 0.5 h and cmax 2.63 +/- 0.92 micrograms equivalents/g in rats (5 mg/kg), and 18.1 micrograms equivalents/g in the dog (15 mg/kg). The predominant half-lives for total radioactivity were in the range of 1 h in the rats and 2 h in the dog, independent of the route of administration. The radioactivity was distributed throughout the body. The highest concentrations were detected in kidneys and liver. Urine (75% of dose) was the main route of excretion after oral and intravenous administration. The radioactivity was almost entirely eliminated 2 days after administration. The compound was intensively metabolized by rat and dog. While the parent compound was the major component in plasma at early time after dosage, more than ten metabolites, accompanied by only small amounts of original substance, were detected in the urine of the first study day. In faeces only metabolites were found. The known metabolites are assumed to be formed by oxidative degradation of the alkylic side chains of the molecule, preferably that in the 2-position of the pyridine ring. The main metabolite in the dog urine was a 2-hydroxyethyl derivative (M4), and in the rat a hippuric acid analogue of HOE 077 (M6). The pyridine carboxylic acid in 2-position of the side chain of HOE 077 (M2) was the predominant metabolite in faeces of rat and dog. In both animal species, more than 80% of the administered radioactivity had been identified. In rats with liver damage caused by treatment with CCl4 the amount of parent compound increased and the rates of formation of metabolites were lower than in normal rats. This can be interpreted as a consequence of the diminished number of hepatocytes able to metabolize HOE 077.