Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs.

The optimization of a silica-based trypsin bioreactor and its use in the quality control of biotechnological drugs like peptides and proteins is described. Five bioreactors based on monolithic material have been prepared, with different amount of bound trypsin. The performances of these bioreactors were compared to the proteolytic activity of a bioreactor based on silica material. The trypsin-based chromatographic columns were coupled on-line with an LC/ESI/MS/MS system for digestion and identification of proteins. First, human serum albumin has been used as test protein to compare the ability of the bioreactors to hydrolyse high-molecular-weight proteins. The best chromatographic material (epoxy monolithic silica) and the optimum amount of enzyme bound (7.13 mg) have been identified to obtain the highest protein recovery and an analytical reproducibility of the whole digestion, separation and identification process. The optimized enzyme-reactor has been used for the on-line digestion of some biotechnological drugs such as somatotropin. Somatotropin for parentheral use has been analyzed, without sample pre-treatment, with both an on-line procedure and the traditional off-line procedure described in the European Pharmacopoeia. It was found that the cleavage efficiency (aminoacidic recovery, %AA) achieved within minutes by the developed protocol is at least comparable or even better than the conventional 4h consuming method.

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins.

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.

Bactericidal activity of a single-dose combination of ofloxacin plus minocycline, with or without rifampin, against Mycobacterium leprae in mice and in lepromatous patients.

To develop a fully supervisable, monthly administered regimen for treatment of leprosy, the bactericidal effect of a single-dose combination of ofloxacin (OFLO) and minocycline (MINO), with or without rifampin (RMP), against Mycobacterium leprae was studied in the mouse footpad system and in previously untreated lepromatous leprosy patients. Bactericidal activity was measured by the proportional bactericidal method. In mouse experiments, the activity of a single dose of the combination OFLO-MINO was dosage related; the higher dosage of the combination displayed bactericidal activity which was significantly inferior to that of a single dose of RMP, whereas the lower dosage did not exhibit a bactericidal effect. In the clinical trial, 20 patients with previously untreated lepromatous leprosy were treated with a single dose consisting of either 600 mg of RMP plus 400 mg of OFLO and 100 mg of MINO or 400 mg of OFLO plus 100 mg of MINO. The OFLO-MINO combination exhibited definite bactericidal activity in 7 of 10 patients but was less bactericidal than the RMP-OFLO-MINO combination. Both combinations were well tolerated. Because of these promising results, a test of the efficacy of multiple doses of ROM in a larger clinical trial appears justified.

High relapse rate among lepromatous leprosy patients treated with rifampin plus ofloxacin daily for 4 weeks.

Fifty-one lepromatous leprosy patients, all of whom had relapsed after previous dapsone (DDS) monotherapy, were treated between 1990 and 1991 with 600 mg of rifampin (RMP) plus 400 mg of ofloxacin (OFLO) daily for 4 weeks, and the great majority of the patients were followed up at least once a year after completion of the treatment. After only 173 patient-years of follow-up, 5 relapses had been detected; the overall relapse rate was 10.0% (confidence limits, 1.7 and 18.3%), or 2.9 relapses (confidence limits, 0.4 and 5.4) per 100 patient-years. The unacceptably high relapse rate indicated that 4 weeks of treatment with daily RMP-OFLO was unable to reduce the number of viable Mycobacterium leprae organisms to a negligible level. In addition, the M. leprae from one of the relapses were proved to have multiple resistance to DDS, RMP, and OFLO. To avoid further relapses, the follow-up was terminated and the great majority of the patients were retreated with the standard 2-year multidrug therapy from 1994. No further relapse has been diagnosed since the beginning of retreatment.

Bactericidal activity of single dose of clarithromycin plus minocycline, with or without ofloxacin, against Mycobacterium leprae in patients.

Fifty patients with newly diagnosed lepromatous leprosy were allocated randomly to one of five groups and treated with either a month-long standard regimen of multidrug therapy (MDT) for multibacillary leprosy, a single dose of 600 mg of rifampin, a month-long regimen with the dapsone (DDS) and clofazimine (CLO) components of the standard MDT, or a single dose of 2,000 mg of clarithromycin (CLARI) plus 200 mg of minocycline (MINO), with or without the addition of 800 mg of ofloxacin (OFLO). At the end of 1 month, clinical improvement accompanied by significant decreases of morphological indexes in skin smears was observed in about half of the patients of each group. A significant bactericidal effect was demonstrated in the great majority of patients in all five groups by inoculating the footpads of mice with organisms recovered from biopsy samples obtained before and after treatment. Rifampin proved to be a bactericidal drug against Mycobacterium leprae more potent than any combination of the other drugs. A single dose of CLARI-MINO, with or without OFLO, displayed a degree of bactericidal activity similar to that of a regimen daily of doses of DDS-CLO for 1 month, suggesting that it may be possible to replace the DDS and CLO components of the MDT with a monthly dose of CLARI-MINO, with or without OFLO. However, gastrointestinal adverse events were quite frequent among patients treated with CLARI-MINO, with or without OFLO, and may be attributed to the higher dosage of CLARI or MINO or to the combination of CLARI-MINO plus OFLO. In future trials, therefore, we propose to reduce the dosages of the drugs to 1,000 mg of CLARI, 100 mg of MINO, and 400 mg of OFLO.

Bactericidal activities of combinations of new drugs against Mycobacterium leprae in nude mice.

The bactericidal activities of 12 regimens with various combinations of new drugs (clarithromycin [CLARI], minocycline [MINO], and ofloxacin [OFLO]) and the standard antileprosy drugs, especially rifampin (RMP), were compared in nude mice with established Mycobacterium leprae infection. The longest duration of treatment was 24 weeks for intermittent (once every 4 weeks) therapy and 8 weeks for daily therapy. Bactericidal effects were monitored by titrating the proportion of viable M. leprae isolates by subinoculating the organisms into the footpads of immunocompetent and nude mice. The results indicate that RMP was more bactericidal than any combination of the new drugs. A single dose of CLARI-MINO, with or without OFLO, displayed bactericidal activity as great as that of 4 weeks of daily treatment with dapsone (DDS) plus clofazimine (CLO); thus, intermittent CLARI-MINO, with or without OFLO, may replace DDS and CLO of the standard multidrug regimen, and these will become regimens that can be administered monthly and under full supervision. Additional evidence that this may be the case is provided by the finding that intermittent RMP-CLARI-MINO or RMP-CLARI-MINO-OFLO administered for 12 or 24 weeks was as active as the standard multidrug regimen. While the intermittent treatment always displayed significantly greater bactericidal activity than the same number of doses of daily treatment, daily treatment with CLARI-MINO and CLARI-MINO-OFLO were more active than the drugs given as intermittent treatment for the same duration; therefore, unless these combinations are to be administered together with intermittent RMP, they should be given daily, especially for the treatment of RMP-resistant cases of infection. Finally, 12 weeks of daily treatment with DDS-CLO was more bactericidal than had been expected, suggesting that it may not be necessary to administer the standard multidrug regimen for multibacillary leprosy for as long as 24 months in order to minimize the risk of developing RMP resistance.

Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem.

In Pseudomonas aeruginosa, resistance to imipenem is mainly related to a lack of protein OprD and resistance to fluoroquinolones is mainly related to alterations in DNA gyrase. However, strains cross resistant to fluoroquinolones and imipenem have been selected in vitro and in vivo with fluoroquinolones. We investigated the mechanisms of resistance to fluoroquinolones in 30 clinical strains of P. aeruginosa resistant to ciprofloxacin (mean MIC, >8 micrograms/ml), 20 of which were also resistant to imipenem (mean MIC, >16 micrograms/ml). By immunoblotting, OprD levels were markedly decreased in all of the imipenem-resistant strains. Plasmids carrying the wild-type gyrA gene (pPAW207) or gyrB gene (pPBW801) of Escherichia coli were introduced into each strain by transformation. MICs of imipenem did not change after transformation, whereas those of ciprofloxacin and sparfloxacin dramatically decreased (25- to 70-fold) for all of the strains. For 28 of them (8 susceptible and 20 resistant to imipenem), complementation was obtained with pPAW207 but not with pPBW801. After complementation, the geometric mean MICs of ciprofloxacin and sparfloxacin (MICs of 0.3 microgram/ml and 0.5 microgram/ml, respectively) were as low as those for wild-type strains. Complementation was obtained only with pPBW801 for one strain and with pPAW207 and pPBW801 for one strain highly resistant to fluoroquinolones. These results demonstrate that in clinical practice, gyrA mutations are the major mechanism of resistance to fluoroquinolones even in the strains of P. aeruginosa resistant to imipenem and lacking OprD, concomitant resistance to these drugs being the result of the addition of at least two independent mechanisms.

Clinical trial of ofloxacin alone and in combination with dapsone plus clofazimine for treatment of lepromatous leprosy.

Twenty-four patients with newly diagnosed lepromatous leprosy were allocated randomly to three groups and treated for 56 days with 400 mg of ofloxacin daily, 800 mg of ofloxacin daily, or 400 mg of ofloxacin, 100 mg of dapsone, and 50 mg of clofazimine daily plus 300 mg of clofazimine once every 28 days. The patients in all three groups demonstrated remarkable clinical improvements, accompanied by rapid decline of the morphological index in skin smears during treatment. More than 99, > 99.99, and > 99.99% of the viable Mycobacterium leprae organisms had been killed by 14, 28, and 56 days of treatment, respectively, as measured by inoculation into the footpads of immunocompetent and nude mice of organisms recovered from skin biopsy specimens obtained before and during treatment. Mild to moderate elevations of the serum glutamic pyruvic transaminase level were observed in four patients, all after 28 days of treatment, which returned to normal after the trial had been completed. Clinical improvement, bactericidal activity, and hepatotoxicity did not differ significantly among the three groups. Ofloxacin displayed powerful bactericidal activity against M. leprae in leprosy patients and may be an important component of new multidrug regimens for the treatment of leprosy. Its optimal dosage appears to be 400 mg daily, and combination with dapsone and clofazimine does not enhance its activity.

Further study of the effectiveness of single doses of clarithromycin and minocycline against Mycobacterium leprae in mice.

The anti-Mycobacterium leprae activities of single doses of rifampin (RMP), clarithromycin (CLARI), or minocycline (MINO) alone, and various combinations of CLARI + MINO were determined in immunocompetent mice by the kinetic method. A single dose of RMP 10 mg/kg, CLARI 100 mg/kg or 200 mg/kg, MINO 25 mg/kg or 50 mg/kg alone, or various combinations of CLARI & MINO were active. RMP was more active than the other treatments; the activity of CLARI 100 mg/kg was greater than that of 50 mg/kg, but did not differ significantly from that of 200 mg/kg; MINO 50 mg/kg was more active than 25 mg/kg; and none of the combinations of CLARI + MINO was more active than any of the stronger components administered alone. Therefore, both CLARI and MINO may be applied, either alone or in combination, as components of monthly administered, fully supervised, multidrug regimens for the treatment of multibacillary leprosy. Taking into account the effectiveness of the drugs and the comparative pharmacokinetic data, we propose that the optimal dosage in human trials is CLARI 1000 mg per month or MINO 200 mg per month.

Powerful bactericidal activities of clarithromycin and minocycline against Mycobacterium leprae in lepromatous leprosy.

Thirty-six patients with newly diagnosed lepromatous leprosy were allocated randomly to three groups and treated for 56 days with minocycline (100 mg daily), clarithromycin (500 mg daily), or clarithromycin (500 mg) plus minocycline (100 mg daily). All groups had rapid and remarkable clinical improvement and significant decline of the bacterial and morphologic indices in skin smears during treatment. More than 99% and > 99.9% of the viable Mycobacterium leprae had been killed by 28 and 56 days of treatment, respectively, as measured by inoculation of organisms recovered from skin samples, taken before and during treatment, into the footpads of immunocompetent and nude mice. Clinical improvement and bactericidal activity did not differ significantly among the three groups. Adverse reactions were rare and mild, and no laboratory abnormality was detected during the trial. Both clarithromycin and minocycline displayed powerful bactericidal activities against M. leprae in leprosy patients and may be considered important components of new multidrug regimens for the treatment of multibacillary leprosy.

Bactericidal activities of single or multiple doses of various combinations of new antileprosy drugs and/or rifampin against M. leprae in mice.

The bactericidal activities against Mycobacterium leprae of single or multiple doses of various combinations of new antileprosy drugs [minocycline (MINO), clarithromycin (CLARI), ofloxacin (OFLO), and sparfloxacin (SPFX)] and/or rifampin (RMP) were titrated in immunocompetent mice by the proportional bactericidal method. Drugs were administered by gavage at the following dosages (mg/kg) per dose: RMP 10, MINO 25, CLARI 100, OFLO 150, and SPFX 50. All 15 regimens exerted significant bactericidal activities, at least 96% of viables were killed. The activity of a single dose MINO + CLARI was only slightly inferior to that of RMP, and the activities of a single dose OFLO/SPFX + MINO + CLARI were similar to that of RMP. This suggests that either MINO + CLARI or OFLO/SPFX + MINO + CLARI may be administered once monthly together with RMP 600 mg for the treatment of multibacillary (MB) leprosy, and monthly administration of MINO + CLARI or OFLO/SPFX + MINO + CLARI may also be employed for the treatment of RMP-resistant MB leprosy. Because the killing effects of multiple doses of the combinations were so powerful, comparison of the bactericidal activities of these regimens was beyond the sensitivity of the immunocompetent mouse model, and are being tested in the nude mouse model. Although SPFX is more active against M. leprae than OFLO on a weight-to-weight basis, when both drugs were administered in mice at dosages equivalent to clinically tolerated dosages in humans, SPFX did not show more superiority than OFLO, and its real advantage over OFLO in the treatment of leprosy remains unclear.

Effectiveness of clarithromycin and minocycline alone and in combination against experimental Mycobacterium leprae infection in mice.

As determined by the proportional bactericide method, clarithromycin had strong bactericidal activity against Mycobacterium leprae. Clarithromycin was administered to mice by gavage as 20 daily doses at dosages of 12.5 to 50 mg/kg of body weight. At a dosage of 25 mg/kg, minocycline was more active than clarithromycin at a dosage of 50 mg/kg. Additive effects were displayed with the combination of clarithromycin (50 mg/kg) and minocycline (25 mg/kg), both of which were administered daily by gavage, and of clarithromycin and minocycline, both of which were administered daily by gavage at dosages of 25 mg/kg each, with rifampin at a single oral dose of 10 mg/kg.

Relationships between PGL-1 antigen in serum, tissue and viability of Mycobacterium leprae as determined by mouse footpad assay in multibacillary patients during short-term clinical trial.

In connection with a 56-day controlled clinical trial for comparing the therapeutic effects between pefloxacin and ofloxacin in 21 lepromatous patients, we have studied the relationships between PGL-1 antigen level in serum and in skin and serum PGL-1 antibody titre on the one hand, and the viability of Mycobacterium leprae, as measured by serial mouse footpad inoculations, and other bactericidal parameters on the other. Before and during treatment, significant correlation was found between serum PGL-1 level and the morphological index (MI), and with the number of viable organisms per mg skin tissue. However, neither serum PGL-1 antibody titre nor skin PGL-1 antigen level showed significant change during the 56-day trial. Because the reduction of serum PGL-1 level was well correlated but less pronounced as compared with the evolution of viable organisms during treatment, the serum PGL-1 antigen assay may be useful as an early indicator of response to chemotherapy in short-term clinical trial, but it is unlikely to replace mouse footpad inoculation for the evaluation of viability of M. leprae.

Clinical trial of pefloxacin and ofloxacin in the treatment of lepromatous leprosy.

Twenty-one previously untreated lepromatous patients were randomized into two groups and treated with either 800 mg pefloxacin (PEFLO) or 400 mg ofloxacin (OFLO) once daily. The trial consisted of two parts: monotherapy from day 0 to day 56; and combined with the World Health Organization multidrug therapy (WHO/MDT) regimen for multibacillary (MB) leprosy from day 57 to day 180. Four patients were removed from the trial because the organisms recovered from their pretreatment biopsies failed to infect mice. Among the remaining 17 cases, four (23.5%) had primary resistance to dapsone but all of them were susceptible to rifampin. The initial (day 0) proportion of viable organisms, as measured by mouse foot pad inoculation, varied tremendously from patient to patient despite randomization during admission. Definite clinical improvement was noticed in virtually all patients after 22 doses of treatment with either PEFLO or OFLO. A significant fall in the morphological index (MI) occurred as early as after 8 doses of PEFLO or after 22 doses of OFLO; the bacterial load also showed a moderate degree of reduction during the period of monotherapy. Although single-dose PEFLO or OFLO displayed only a modest degree of bactericidal effect against Mycobacterium leprae, about 99.9%, or 4 logs, of organisms viable on day 0 were killed by 22 doses of either PEFLO or OFLO. No significant difference in the therapeutic effect was detected between the two regimens. During PEFLO or OFLO monotherapy, except in one patient (case no. 10), the side effects were few and mild. Case no. 10 developed a psychic disorder after 27 days of PEFLO monotherapy, presumably due to the treatment with PEFLO. All of the patients tolerated the period of combined therapy extremely well, although some asymptomatic and transient laboratory abnormalities were observed. Because both PEFLO and OFLO displayed rapid bactericidal activities in human leprosy and were well tolerated by the patients, further clinical trials and field trials in evaluating the therapeutic effects of combined regimens containing both rifampin and PEFLO or OFLO are being organized. Since this is the first clinical trial in leprosy employing nude mice, in combination with normal mice, for monitoring the therapeutic effects of antimicrobials, the advantages, limitations and appropriate timing in using nude mice are discussed.

Effectiveness of pefloxacin in the treatment of lepromatous leprosy.

As a first clinical trial of a fluoroquinolone derivative in leprosy, ten previously untreated lepromatous leprosy patients, about two fifths of them with primary dapsone resistance but all susceptible to rifampin, were treated with pefloxacin 400 mg twice daily for 6 months. Definite clinical improvement was observed in all ten patients as early as 2 months after beginning treatment, and the morphological index was also drastically decreased to the baseline during the same period. The rapid bactericidal effects, as measured by serial mouse foot-pad inoculations, were demonstrated to the extent that about 99% of the bacilli were killed during the first 2 months of treatment. However, the bacterial load, in terms of the bacterial index and the number of acid-fast bacilli per mg of tissue, of the patients was only moderately reduced. The side effects were mild, and the patients tolerated the treatment well.

Activity of ofloxacin against Mycobacterium leprae in the mouse.

Mice inoculated with 4800 Mycobacterium leprae in the left hind foot pad were treated from day 62 to day 150 after infection with 50 mg or 150 mg of ofloxacin per kg body weight, 150 mg pefloxacin per kg, or 50 mg prothionamide per kg. These drugs were administered by esophageal cannula 5 days weekly with dapsone (0.01 g per 100 g diet). Multiplication of M. leprae in the treated and in untreated control mice was assessed by monthly harvests. The treatment of mice with the smaller dosage ofloxacin, with pefloxacin, prothionamide, or dapsone uniformly resulted in a delay of multiplication of 4 months, compared to the multiplication of M. leprae in the untreated controls. The delay of multiplication (4 months) being 1 month longer than the duration of drug administration (3 months), all of the treatments may be considered as bacteriopausal or moderately bactericidal. In contast with these results, treatment of mice with 150 mg ofloxacin per kg resulted in no growth of the organisms whatever as late as 18 months after inoculation, strongly suggesting that, in that dosage, ofloxacin had killed all of the M. leprae. Such a profound killing activity has been observed only with rifampin. Although the pharmacokinetic characteristics of ofloxacin are different in man from those in the mouse, the daily dosage of 150 mg ofloxacin per kg body weight in the mouse is equivalent to 400 mg per day in man which is the usual therapeutic dosage; thus, the results obtained in the mouse may be extrapolated to man. Therefore, ofloxacin appears a very promising drug for the chemotherapy of leprosy.

[Resistance of Mycobacterium leprae to dapsone and rifampicin: apropos of a survey carried out in the Cape Verde region (Senegal)]. / La résistance de Mycobacterium leprae à la dapsone et à la rifampicine: à propos d'une enquête effectuée dans la région du Cap-Vert (Sénégal).

Since 1983, primary resistance of M. leprae to DDS and rifampicin has been evaluated in new cases of lepromatous leprosy observed in the Cap-Verde region in Senegal. Out of the 13 strains isolated, 10 (77%) have been found resistant to DDS, 7 at low level, 2 at intermediate level, 1 at high level; all of them have been found sensible to rifampicin. Similar results have been obtained with 57 strains isolated from patients not yet treated coming from different geographical areas, seeing that 37, i.e. 65%, were resistant to DDS, 27 at low level, 5 at intermediate level and 5 at high level; all of them were sensible to rifampicin. Level of resistance to DDS is very different in case of acquired resistance. In 69 lepromatous patients treated for more than 5 years and showing a relapse, M. leprae was 62 times, i.e. 90%, resistant to DDS, 6 times at low level, 21 at intermediate level and 35 at high level; in addition, 13 times M. leprae was resistant to rifampicin. In order to avoid and to solve problems set by resistance of M. leprae to antibiotics, strict application of polychemotherapy of leprosy is compulsory.

Activities of pefloxacin and ciprofloxacin against Mycobacterium leprae in the mouse.

Because ciprofloxacin and pefloxacin are fluoroquinolones active against many mycobacterial species, both drugs were tested against Mycobacterium leprae in the mouse foot-pad system. Preliminary pharmacokinetic studies in the mouse showed that after a single oral dose of 150 mg/kg ciprofloxacin the peak serum concentration was 3.6 micrograms/ml, and after 50 mg/kg or 150 mg/kg pefloxacin peak serum concentrations were, respectively, 9.2 micrograms/ml and 16.9 micrograms/ml, the half-lives for serum elimination being about 2 hr for both drugs. The activity of daily 50 mg/kg and 150 mg/kg ciprofloxacin and pefloxacin against M. leprae was then tested in mice infected with 5 X 10(3) M. leprae. The growth of M. leprae was not prevented in mice treated continuously with either 50 mg/kg or 150 mg/kg ciprofloxacin, indicating that this drug had no or a limited bacteriostatic effect at the dosages used. In mice treated continuously with 50 mg/kg pefloxacin, growth of M. leprae was not prevented, but at monthly harvests the number of bacilli in the foot pads remained less than those of control mice (p less than 0.05). No growth of M. leprae occurred in mice treated continuously with 150 mg/kg pefloxacin. In mice treated for only 3 months with daily 150 mg/kg pefloxacin, the growth-delay that followed the stopping of the drug was 126 days, suggesting that approximately 99% of the M. leprae were killed. The pharmacokinetics of pefloxacin being more favorable in man than in the mouse, pefloxacin appears a possible drug for the chemotherapy of leprosy.