Inhalation of [123I]alpha1-protease inhibitor: toward a new therapeutic concept of alpha1-protease inhibitor deficiency?

UNLABELLED: The alpha1-protease inhibitor (alpha1-Pi) is separated from human serum and is therefore extremely expensive. Because only 2%-3% concentrates in the lung after intravenous administration, inhalational therapy for alpha1-Pi deficiency would seem likely to be better. The aims of this study were therefore to determine the pattern of deposition of inhaled alpha1-Pi labeled with 123I and measure the amount deposited in the lungs. METHODS: Eighteen patients with congenital severe alpha1-Pi deficiency were enrolled in the study. The low-specific-activity 123I-labeled alpha1-Pi aerosol (median particle size +/- SD, 3.9 +/- 2.5 microm) was generated by an air pressure-driven nebulizer. The patients inhaled for an average of 23.6 +/- 8.9 min. Static scintigrams in two projections were acquired immediately after (T1) and 1 (T2), 4 (T3), and 24 h (T4) after inhalation. The patients were divided into the following three groups according to their forced expiratory volume in 1 s (FEV1): group I, < or =40% of predicted normal (n = 8); group II, 40% < FEV1 < or = 60% of predicted normal (n = 4); group III, >60% of predicted normal (n = 6). RESULTS: The absolute percentage uptake values of alpha1-Pi in group I were 12.4 for T1, 7.3 for T2, 4.6 for T3, and 1.2 for T4; in group II the values were 13.0, 9.6, 6.2, and 2.0, respectively; and in group III, 14.6, 11.4, 6.5, and 3.6, respectively. Differences between the groups were generally statistically significant. Between T1 and T2, the probability value was <0.05 for group I versus group II, <0.006 for group I versus group III, and <0.39 for group II versus group III. Between T1 and T3, the probability value was <0.29 for group I versus group II, <0.22 for group I versus group III, and <0.94 for group II versus group III. Retention (between T1 and T4) was also dependent on the grade of the disease: P < 0.2 for group I versus group II, P < 0.001 for group I versus group III, and P < 0.02 for group II versus group III. Grading of the uptake pattern by three independent experienced investigators (87% agreement) revealed a peripheral deposition that was group dependent. We found that greater peripheral deposition corresponded with lower lung functional impairment: P < 0.5 for group I versus group II, P < 0.01 for group I versus group III, and P < 0.08 for group II versus group III. Degradation also corresponded with functional impairment: P < 0.05 for group I versus group II, P < 0.006 for group I versus group III, and P < 0.3 for group II versus group III. CONCLUSION: The results of this study show that sufficient amounts of alpha1-Pi can be deposited in the periphery of the lung by inhalation at least in patients with low-grade disease. Inhalation of alpha1-Pi may thus represent a new and more convenient route of drug administration.

On the value of large multicenter drug intervention trials.

Many large multicenter clinical drug trials are outcome trials with several thousand patients, therefore also called megatrials, numerous of which encompassing the cardiovascular field. Usually designed to test and compare the efficacy of a specific therapy with the major aim being improved outcome at hard end-points, specifically morbidity and mortality, they are representative of typical intervention trials. Considering also the risk of failure, examples of large multicenter therapy trials are examined as to their value to the patients, the society, the health care providers, and--finally--also the manufacturers of the respective drugs. The 'Syst-Eur Trial' (Systolic Hypertension in Europe) representing a very recent intervention trial to treat isolated high systolic blood pressure in elderly patients with treatment based on a calcium channel blocker, nitrendipine (CAS 39562-70-4, Bayotensin, Baypress), serves to indicate that besides the major end-point results, here total stroke rate, a number of important additional efficacy and safety results may be obtained with large trials, such as in this case favourable data on total mortality, myocardial infarctions, cancer, bleeding.

Examination of the biological safety of a drug derived from mammalian organs.

In order to assess the safety of a biological drug, a variety of factors have to be examined and then brought into an overall context considering the specific aspects of each individual product. Quoting Trasylol, the aprotinin (CAS 9087-70-1) drug extracted from bovine lungs as an example for such an approach, the complete procedure is discussed. The rationale of a safety concept, its implementation including safety related validation studies, and the combinatorial evaluation of results from these validations with underlying specificities for manufacture allow for an overall safety assessment. Validation of the removal/inactivation capacity of the manufacturing process for bovine spongiform encephalopathy (BSE) and various viruses showed high reduction potentials. These results constitute the cornerstone for the conclusion that Trasylol is safe with regard to BSE and viruses.

Reduction of the infectivity of scrapie agent as a model for BSE in the manufacturing process of Trasylol.

The Trasylol manufacturing process was investigated with respect to its capacity for the inactivation/removal of infectivity causing bovine spongiform encephalopathy (BSE). Four process steps were selected for this investigation and scaled down to laboratory scale. Authentic samples of bovine lungs used in the Trasylol manufacturing plant were taken and spiked in laboratory scale experiments with high infectious titres of the rodent adapted scrapie strain ME 7 which served as model for BSE. After performing the respective process steps the output samples collected were tested in C57BL mice carrying the Sinc gene. An overall reduction of the infectious agent in the order of 18 log10 was observed, indicating a very high capacity of the Trasylol process for the inactivation/removal of the BSE/scrapie agent. The discussed safety strategy for the product leads to the conclusion that Trasylol is BSE safe.

Hemopoietic regeneration in murine spleen following transfusion of normal and irradiated marrow: different response of granulocyte/macrophage and erythroid precursors.

To investigate cell proliferation in regenerating spleen, bone marrow of normal and gamma-irradiated donor mice (3 weeks after 5 Gy) was transfused into lethally irradiated recipients. In the donors and in the recipient spleens numbers of CFU-S and progenitor cells were determined. In the irradiated donors the progenitors were at control level after 3 weeks of recovery although CFU-S were still at 50% of control. Recipients of the irradiated marrow received therefore an increased proportion of progenitors. CFU-C appeared to be self-renewing and/or increased in number due to enhanced CFU-S differentiation, but not the erythroid progenitors. CFU-S self-renewal was reduced after 5 Gy. The data suggest that cell differentiation and maturation proceed during early splenic regeneration. The quantity of CFU-C does not necessarily mirror the situation in the stem cell compartment.

Production of human monoclonal antibodies from immobilized cells in the Opticell culture system.

An EBV-transformed human B cell line, which produces monoclonal IgM antibodies, was cultured in an immobilized state on a ceramic matrix in the Opticell system. Cell growth, metabolic activity and product formation of these cells were optimized in a single semi-continuous culture of 10 inductions for 29 d. All in all, 19.5 g of IgM were harvested in 193 l of culture supernatant. The average production of IgM in the Opticell unit of 20 l was 0.7 g/d which was obtained even in serum-reduced medium (1%). Under optimal conditions IgM yields were enhanced to 1.5-2.0 g/d. These results indicate that the Opticell is a suitable system for the large scale production of monoclonal antibodies, particularly because the capacity for aeration and pumping of one Opticell unit is sufficient to control 3 growth chambers running in parallel.

Reculturing of cells from primary CFU-C colonies.

This study was aimed at investigating whether cells of CFU-C derived colonies could form secondary colonies. Bone marrow cultures of volumes of agar medium between 25 microliter and 75 microliter contained in glass capillaries were stimulated with mouse lung-conditioned medium (MLCM) containing granulocyte/macrophage colony-stimulating factor (GM-CSF). Agar gels with colonies of up to greater than or equal to 20 were blown out into identical culture medium, completely dispersed on a whirl-mix to single cell suspensions, and used for establishing secondary agar cultures. In these secondary cultures considerable numbers of secondary granulocytic, mixed granulocytic/macrophage and macrophage colonies as well as numerous clusters arose. In contrast, when single colonies were recultured, only few secondary cell aggregates were formed. When primary cultures containing up to greater than or equal to 20 cell aggregates were used for serial reculture at intermittent intervals of 3 and 4 days, a 2-7-fold increase of colony-forming cells was found in tertiary cultures as was monitored by 7 day colony counts. And by use of different kinds of CSF-containing media, an over 4-fold increase of secondary over primary colonies was obtained with bovine lung-conditioned medium (BLCM) in primary and L-cell-conditioned medium (LCCM) in secondary 7 day cultures. Primary capillary cultures were found to be devoid of CFU-S. Also, setting up bone marrow cultures in petri dishes and stimulating with MLCM, growth of primary as well as secondary colonies was obtained. The results indicate some self-renewal potential of CFU-C in vitro.

Long-term residual radiation effect in the murine hematopoietic stem cell compartment.

For the measurement of long-term residual radiation effect in the murine hematopoietic system a test system was developed that quantifies the proliferation ability of progeny of spleen repopulating cells by the proliferation factor (PF). The PF expresses the ratios of 125IUdR incorporation in the recipient spleens at days 3 and 5 following cell transfusion, thus measuring the relative increase in number of proliferating cells. Following 500 rad whole-body gamma irradiation, PF recovered up to 6 months and remained thereafter, on the average, at 80% of control. Recovery of the number of 7-day CFU-S was similar to recovery of PF. Various studies were aimed at elucidating the reasons for reduction in PF. Loss of incorporated 125IUdR activity from spleens between days 3 and 5 after cell transfusion indicates loss of mature labeled cells. When the doubling time of proliferating cells of CFU-S progeny (td) is corrected for cell loss, td for control bone marrow approaches mitotic cycle time in normal bone marrow as was found elsewhere. Following 500 rad, both cell loss and td were initially increased and recovered in parallel with PF and number of CFU-S. Reduction of PF could be brought about by radiation-induced increase in transient CFU-S with the consequence of increased loss of mature cells between days 3 and 5. This possibility was excluded by the observation that 1 year after 500 rad the number of colonies per spleen did not decrease from day 7 to day 12 after cell transfusion, as was expected from a higher proportion of transient CFU-S, but increased more than in the controls. Measurement of these 12-day colonies showed a significantly reduced size. Average progeny from irradiated CFU-S, apparently, grow more slowly. It is concluded that sublethal injury resides in stem cells, increases mitotic cycle time, and causes precocious loss of cells from spleens probably by enhanced differentiation and maturation due to interference with endocellular control of cell proliferation and differentiation. Probably the observed recovery proceeds via replacement of injured stem cells by less injured or normal stem cells.

Expression of assayable residual stem cell damage in erythroid differentiation.

In rodents, residual damage is inducible in hematopoietic stem cells by exposure to ionizing radiation or alkylating agents. This damage can be assayed in mice by transferring bone marrow into lethally irradiated syngeneic recipients and subsequently measuring the incremental increase of 5-(125I)iodo-2'-deoxyuridine incorporation in spleens. In this study, bone marrow from mice treated 3 weeks previously with Methylnitrosourea (50 mg/kg) or 450 rad was injected into recipients in order to determine possible residual effects of treatment on erythroid cell differentiation following stem cell seeding. Such effects were detected by a reduced amount of 59Fe incorporation into spleens, thus indicating transfer of residual stem cell damage to differentiating cells.

Influence of valtrate/isovaltrate on the hematopoiesis and metabolic liver activity in mice in vivo.

Recently, cytotoxic effects of valepotriates with an epoxide moiety have been described on mouse bone marrow early progenitor cells IN VITRO. Consequently, the possible IN VIVO toxicity of valtrate on hematopoietic precursor cells was investigated. Mice were treated i.p. with 45 and 65 mg/kg or p.o. with 45 and 1350 mg/kg of the drug. Three days after treatment, colony formation of progenitor cells (CFC-S, GM-CFC, E-CFC) was not significantly different for control and experimental groups. Furthermore, the effect of valtrate on the ability of the liver to metabolize [ (14)C]methacetin was investigated by measuring the (14)CO (2) exhalation (breath test). There was a distinct reduction of the initial exhalation of (14)CO (2) following i.p. injection of 50 mg/kg of valtrate, but no effect was found after 50 or 1500 mg/kg of p.o. These results suggest that toxicity of valtrate in vivo is restricted because the distribution of the drug via circulation is obviously small.

Proliferative compensation of residual radiation damage in the compartment of hematopoietic early progenitor cells of the mouse.

The rate of cell entry from the compartment of hematopoietic early progenitor cells into differentiation was determined in sublethally irradiated mice. By use of the criterion of repopulating ability, transplantation of 5-(125I) iodo-2'-deoxyuridine labeled bone marrow cells into fatally irradiated syngeneic recipients allows to measure the relative number of early progenitor cells lodging in the spleen and the turnover of these cells in the donors. Following 450 rad the relative number of transplantable early progenitor cells in S-phase recovers to normal within 2 weeks and stabilizes after 5 weeks. At this time, the labeled progenitors turn over with a half-time of 1.4-2.2 days; the respective times for unirradiated mice are 1.5-1.8 days. Thus, quantitative and qualitative residual radiation damage that is known to exist in the compartment of CFU-S, is disguised within 2-5 weeks after irradiation by proliferative compensation in the entirety of early hemopoietic precursor cells which are here defined by their capacity of self renewal and delivery of differentiated cells and of seeding to spleens of lethally irradiated recipients.

Demand-control of proliferative activity in the hemopoietic system of lethally irradiated mice during transfusion-induced regeneration.

The extent of cell proliferation in the hemopoietic system after bone marrow transfusion of fatally irradiated mice depends on the regeneration of proliferative capacity. This may be modified by the demand for differentiated cells in the peripheral blood. This demand was suppressed by induction of transfusion plethora prior to 800 rad whole body irradiation and bone marrow transfusion. Controls were non-plethoric recipients. For 6 days the following parameters were measured: hemopoietic proliferation by the 125-iodo-deoxyuridine (125-IUdR) incorporation technique, CFU-S content and spleen colony histology. There are three general observations from spleen and marrow with respect to 125-IUdR uptake in plethoric mice: (1) initial higher 125-IUdR uptake, (2) reduced rate of increase of 125-IUdR incorporation, (3) this rate of increasing 125-IUdR uptake in spleen was more depressed than in marrow. On day 6 cellularity and CFU-S in spleen was below, and in marrow above that of the control. These data suggest that initially after fatal irradiation of control mice differentiation of transfused CFU-S predominates over proliferation. Later as the mice become anemic and erythropoietin is produced the stimulation to proliferate is greater in the control than in the plethoric mice in which erythrocytic proliferation is suppressed. These data suggest that there are multiple feedback loops that regulate regeneration in the spleen and the bone marrow. These differences may be connected with the microenvironment that preferentially initiates erythropoiesis in the spleen before the marrow and granulopoiesis in the marrow before the spleen.

Relationship between number of spleen colonies and 125IdUrd incorporation into spleen and femur.

Graded numbers of bone marrow (BM) cells were injected into fatally irradiated mice. Eight days later the mice were given 3.0 microCi (1 Ci = 3.7 X 10(10) Bq) of 125IdUrd to label proliferating cells in the spleen and BM. On day 9 the mice were killed and the spleens and femurs were removed for splenic colony assay and measurement of radioactivity in the spleen and femurs. The number of splenic colonies shows a linear relationship with dose of marrow cells injected from 10(4) to 10(5) cells. The slope of the curve of spleen colonies versus number of cells injected is less than 1, implying that the fraction seeded in spleen decreases with number of cells injected. Above 10(5) and below 10(4) there is a striking departure from the simple linearity. Below 2 X 10(3) cells injected, the logarithm of the observed colony yield is linear with logarithm of the number of cells injected. Poisson calculation of the average number of pluripotent stem cells that should be present with numbers of marrow cells injected below 2 X 10(3) followed closely the actual observations. The data show that there is no detectible proliferation in the BM until the dose of marrow cells exceeds 3.5 X 10(4) cells. Induction of cells into cycle increases the seeding into the BM, and thymidine cytocide drastically reduces seeding in the BM, leading us to conclude that the BM is repopulated almost exclusively by stem cells in DNA synthesis.

Drug-induced residual damage of murine hematopoietic stem cells measured by a new assay.

A recently developed assay for the measurement of residual stem cell damage after gamma-irradiation was tested to see if it could detect residual drug-induced stem cell damage. In this assay, the proliferative ability of transfused donor bone marrow is determined in the spleens of lethally irradiated recipients by the incremental increase of 125iodo-deoxyuridine (125IUdR) incorporation from day 3 to day 5. Three drugs were used to treat the donor: methylnitrosourea (MNU) 50 mg/kg; busulfan (BU) 20 mg/kg; cyclophosphamide (CP) 200 mg/kg. Residual damage of stem cells was detected 3 weeks after treatment in all mice. The measurable damage induced by MNU disappeared within 9 weeks, while damage caused by BU and CP was still present after 9 and 15 weeks. It is concluded that the new assay is suitable for the detection of proliferative defects in the stem cells induced by at least three alkylating agents.

Residual radiation effect in the murine spleen. In situ measurement by 125-iodo-deoxyuridine retention.

To investigate whether residual radiation damage in hematopoietic tissue is measurable in situ by a change in cell turnover, the retention of the thymidine analogue 5-(125-I)iodo-2'-deoxyuridine (125-IUdR) following incorporation into DNA of cells in bone marrow and spleen of mice was measured 35 days after 0-500 rad whole body gamma irradiation. In the bone marrow a rapid and a slow turnover component of 125-IUdR retention were found. Both components were almost identical for unirradiated and irradiated mice. In the spleen the 125-IUdR retention curves exhibited three components with increasingly prolonged half-times. In the second component the half-time was longer in irradiated than in unirradiated mice. This was dose-dependent. The increased half-time of 125-IUdR retention in irradiated spleens may be caused by direct cellular damage of long-lived cells (lymphocytes, early hematopoietic progenitor cells) or/and by diminished stimulation of proliferation by microenvironmental or long-range factors.