Detection and consequences of recombinant protein isoforms: implications for biological potency.

Various types of structural variants have been observed in recombinant DNA - derived products. These isoforms include variations in post translational carbohydrate modifications where variations in site occupancy or unoccupied sites may occur. In addition, varying degrees of C-terminal processing and N-terminal substitutions have been observed. Isoforms may also be generated during processing and can include aggregated and/or chemically modified forms of the protein. Sophisticated analytical techniques exist for the identification and characterization of these structural variants. Several strategies have been used to isolate or enrich the isoform before molecular characterization. However, the effect these structural variations have on the biological activity of the product is less well understood. This may, in part, be due to the specificity and variability of the bioassay employed. This presentation describes the isolation and characterization of specific molecular isoforms for a monoclonal antibody product as well as an assessment of effects on biological activity.

Correlation between histologic staging, hepatitis C virus genotypes and clinical features in HCV chronic hepatitis: evidence of a new pattern.

Genome heterogeneity may be related to the wide variability of clinical and pathological features in hepatitis C virus (HCV)-related chronic liver disease. This paper addresses the possible association between HCV subtypes and clinical and histological features of chronically infected patients. Sixty-eight consecutive liver biopsies of chronic hepatitis constituted the basis of the study. HCV genotyping was performed on frozen tissue. Grading of necroinflammatory activity and staging of fibrosis were histologically assessed. Serologic HCV-RNA and liver function were assessed at the same time. All information was compared with clinical data including age, sex, HCV serology, and probable data and route of infection. Two cases were excluded as inadequate tissue was available. Five cases were negative to HCV-RNA in both serum and tissue. In 61 cases HCV RNA was present at the same time in serum and liver tissue. Forty-four patients were men (72%) and 17 (28%) were women. Two peaks of age were observed: 1 in the 4th decade of life, the 2nd in the 7th. The 2 groups had different HCV genotypes. Patients with genotypes 1b (mean age 50.7 years), 2c (mean age 61.3 years), and a subgroup of coinfections (mean age 60 years) were older than patients with genotypes 1a (mean age 35.5 years), 3 (mean age 36 years), and a subgroup of coinfections (mean age 33 years). Patients with genotypes 1b, 2, or 2c and a subgroup of coinfections more frequently had a history of blood transfusion and or surgical intervention dating up to 49 years previously. Patients with HCV 1a, 3, and a subgroup of coinfections frequently admitted a period of intravenous drug abuse. Patients with advanced liver disease, i.e., severe fibrosis and cirrhosis, showed the same 2 peaks of incidence: in the 4th and 7th decades of life, the first group mainly comprising patients with HCV types 1a and 3, the second, patients with HCV types 1b and 2c. Both these groups shared a clinical history of a long-standing infection. Two profiles of patients emerged. The largest group was composed of elderly patients, infected by HCV genotypes 1b or 2c, with a history of blood transfusion and/or surgery, presenting an advanced stage of liver disease (namely, severe fibrosis or cirrhosis). The second group was composed of younger patients, mainly in the 4th decade of life, infected by HCV types 3 or 1a, often presenting with chronic hepatitis in the stage of severe fibrosis or cirrhosis. The latter could be the profile of HCV infection in the near future.

Fibrinogen storage disease without hypofibrinogenaemia associated with acute infection.

AIMS: The presence of ground glass hepatocytes in a liver biopsy may be related to different conditions, including fibrinogen storage disease. Three types of fibrinogen storage disease have been described, namely types I, II and III. Type I is an hereditary hypofibrinogenaemia genetically characterized by a mutant variant of the fibrinogen molecule designated as fibrinogen Brescia, consistent with a gamma284 Gly-->Arg mutation. Only rare cases of types II and III fibrinogen storage disease have been described. The purpose of the present paper is to describe two cases of fibrinogen storage disease without associated hypofibrinogenaemia, which appeared during acute infectious diseases. METHODS AND RESULTS: Both patients were female, aged 77 and 73 years, who developed high transaminases during an infectious disease. In each case blood coagulation tests were within the normal range, and despite clinical and laboratory investigations no possible cause for liver disease could be found. Liver biopsies were performed; in both cases weakly eosinophilic cytoplasmic inclusions were observed. Using immunohistochemistry the inclusions were found to be due to fibrinogen accumulation. At ultrastructural level features corresponding to type II inclusions were observed. Molecular studies, performed in case 2, excluded the mutation typical of type I fibrinogen storage disease. Both patients also presented features of chronic hepatitis. In case 1, giant cell granulomas were additionally present. No close relatives of the patients presented any clinical or laboratory features of liver disease. In both patients altered liver function test values gradually, spontaneously, returned to within normal ranges after infectious disease was resolved. CONCLUSIONS: These cases suggest that, on rare occasions, hepatocytes may accumulate fibrinogen during an infectious disease.

Detection and consequences of recombinant protein isoforms: implications for biological potency.

Protein isoforms commonly occur in nature and in products produced by recombinant DNA technologies. Some isoforms may be generated as a consequence of the manufacturing process. Bioassay methods may not be sufficiently sensitive enough or specific enough to determine the biological effect of having a new or increased level of a particular isoform. Sophisticated analytical techniques exist for the structural characterization of protein isoforms. The biological implications of these isoforms are not easy to determine. Additional "Biological Characterization" work may need to be done to evaluate the biological consequences of isoforms. An understanding of the product's mechanism of action and the disease mechanism of action is essential for a thorough evaluation.

[Demonstration of TT virus in liver tissue fixed in formalin and embedded in paraffin]. / Dimostrazione del virus TT in tessuto epatico fissato in formalina ed incluso in paraffina.

INTRODUCTION: TT virus has been recently isolated in Japan in patients with acute and chronic non-A/non-G hepatitis. Its possible etiopathogenetic role in causing hepatitis has been initially taken in consideration. On the contrary, more recent studies deny the importance of TT virus in causing liver damage. Most of the studies are based on serological data or on viral detection from frozen liver tissue. AIM OF THE STUDY: In the present paper we describe a method to detect viral genome from formalin-fixed and paraffin-embedded liver tissue. MATERIALS AND METHODS: Twelve needle biopsies from liver were studied. Six cases were selected on the basis of serological negativity for HBV and HCV markers. Five cases of HCV-related chronic hepatitis and one HCV- and HIV-positive intravenous drug abuser were also included. All patients underwent liver biopsy, performed with a 14-G needle. Liver specimens were formalin-fixed and paraffin-embedded as routine. From each block, sections were cut and stained for histopathologic examination. Additional 5 microns sections were employed to extract DNA for nested PCR. RESULTS: In 2 of 12 cases studied, TT virus genome was found. In both cases the presence of viral DNA was confirmed by sequencing. Both patients were male. The first patient was a 39-year-old HIV- and HCV-positive intravenous drug abuser. The second patient was a 60-year-old heavy alcohol drinker. In both cases the presence of TT virus apparently did not affect the histological picture. CONCLUSION: It is possible to detect TT virus genome from formalin-fixed and paraffin-embedded tissue. This method offers the possibility to perform retrospective studies.

Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4.

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.

[Albumin mRNA and pCEA in the histopathologic diagnosis of hepatocellular carcinoma]. / mRNA dell'albumina e pCEA nella diagnosi istopatologica del carcinoma epatocellulare.

Extrahepatic neoplasms metastatic to the liver histologically are often indistinguishable from hepatocellular carcinoma (HCC). The differential diagnosis between HCC and metastatic liver tumours can be even more difficult in ultrasound guided fine-needle biopsies. Purpose of the present study was to investigate the utility of immunohistochemical staining with polyclonal anticarcinoembryonic antigen (pCEA) antibody and of in situ hybridization (ISH) revealing human albumin mRNA, with emphasis on tissues obtained via fine-needle procedure. Cases consisted of 52 primary HCC; 2 HCC metastatic to vertebral bones; 18 tumours metastatic to the liver; 24 non-hepatocellular tumours metastatic to the skin, lymph nodes and brain; 2 immature teratomas with areas of hepatoid differentiation. Forty-seven HCC (90%) and 7 liver metastases (38%) were obtained by ultrasound guided fine-needle biopsies (21 g needle was used). All the remaining cases were surgical specimens. All the cases were studied with immunohistochemistry for pCEA and ISH using a cRNA probe for human albumin mRNA. The immunohistochemical staining using pCEA showed a canalicular type of positivity in 37 cases of HCC (71%), in one HCC metastatic to vertebral bone and in the hepatoid areas of one immature teratoma. No canalicular type of positivity was obtained in non-hepatocellular neoplasms metastatic to the skin, brain, lymph-nodes and liver. Albumin mRNA was detected in 51 (98%) primary HCC, in both HCC bone metastases, and in the hepatoid areas of both immature teratomas. No positivity was obtained in non-hepatocellular tumours. The data here obtained indicate that immunostaining with pCEA and ISH revealing human albumin mRNA are markers of hepatocellular differentiation and confirm their diagnostic utility. Detection of albumin mRNA showed a higher sensitivity. In addition the cRNA probe here used seems more sensitive that the oligonucleotide probes employed in previous studies.

Albumin gene expression in adenocarcinomas with hepatoid differentiation.

Two cases of hepatoid adenocarcinomas were studied with an in situ hybridization technique (ISH) using a RNA probe for human albumin mRNA. In case 1 the urinary bladder of a 67-year-old woman was affected; in case 2 the tumour was located in the gastric antrum of an 80-year-old woman. In neither case had alpha fetoprotein (AFP) been determined preoperatively. Histologically these cases showed adenocarcinomatous features intermingled with hepatoid areas. These latter areas were characterized by cords of polygonal cells, each with an oval nucleus and prominent nucleoli, separated by a fine network of sinusoids. In the hepatoid areas the immunohistochemical profile was similar to that observed in hepatocellular carcinomas, in that the tumour cells were positive with AFP, alpha-1-antitrypsin (A1AAT) and albumin antisera and there was a canalicular type of reactivity with polyclonal anti-CEA (pCEA) antibody. ISH revealed albumin mRNA in virtually all hepatoid cells in case 1, and in about 50% of those in case 2. In addition, in case 2 occasional cells in the adenocarcinomatous areas showed albumin transcripts of ISH. Our findings confirm that ISH for albumin mRNA probe is a valuable method of establishing hepatocellular differentiation, and that hepatoid adenocarcinomas are tumours with true extrahepatic hepatocellular differentiation.

Identification of mitochondria in liver biopsies. A study by immunohistochemistry, immunogold and Western blot analysis.

Hepatocytes are rich in mitochondria, which play an important role in hepatic metabolism. In certain pathologic conditions (most often alcoholic liver disease) mitochondria became enlarged; nevertheless, even in these conditions they are hardly detectable on light microscopy. Recently an antimitochondrial antibody (mAM), which recognizes a 60-kDa protein, has been characterized. The purpose of the present study was to study immunoreactivity of this antibody in a series of liver biopsies. We studied 146 liver biopsies using an mAM. In 8 cases an ultrastructural study was also done, and in 2 cases Western blot analysis was performed. Cases were divided as follows: alcoholic liver disease (ALD, 31); steatosis (8); nonalcoholic steatohepatitis (NASH, 1); hepatitis C virus (HCV)-related hepatitis (83); hepatitis B virus (HBV)-related hepatitis (6); primary biliary cirrhosis (1); sclerosing cholangitis (1); haemosiderosis (1); sarcoidosis (1); alpha-1-antitrypsin deficiency (1); nonspecific findings (12). All the patients were investigated for alcohol or drug abuse, pharmacological treatment, hyperlipidaemia, hypercholesterolaemia and diabetes. Immunoreactivity was diffuse in cases of ALD, NASH and steatosis, and in patients with drug abuse. Electron microscopic immunogold and Western blot analysis confirmed that in the conditions examined the protein recognized by the mAM showed greater expression. Immunohistochemical staining was helpful in demonstrating a toxic or a metabolic insult even in cases in which the histological picture was blurred by viral infection.

Lamivudine treatment for acute hepatitis B after liver transplantation.

BACKGROUND/AIMS: Acute hepatitis caused by recurrent or de novo hepatitis B virus (HBV) infection after liver transplantation frequently induces aggressive disease leading to liver failure. The aim of this study was to determine the efficacy and safety of lamivudine treatment in post-transplant acute hepatitis B. METHOD: Twelve patients with acute hepatitis B were started on lamivudine 100 mg p.o. daily within 8 weeks of the appearance of HBsAg. One patient was excluded after 1 month because of hepatocellular carcinoma recurrence. Patients were followed for an average of 68.6 weeks (range 32-108), and were clinically and biochemically evaluated on a monthly basis. They had a histological assessment at baseline, after at least 6 months, and whenever clinically indicated. RESULTS: Basal HBV-DNA ranged between 13 and 1288 pg/ml and serum alanine aminotransferase between 97 and 1036 U/l. HBV-DNA became undetectable within 8 weeks and transaminases normalized within 24 weeks in all cases. At the last visit, eight patients (73%) remained HBV-DNA negative by liquid hybridization and had normal or close to normal alanine aminotransferase. Five patients (45%) were also HBsAg negative and HBV-DNA negative by polymerase chain reaction. HBV-DNA and transaminase breakthrough occurred in three patients (27%). Histology after 6-9 months showed chronic hepatitis in seven patients. Lamivudine was well tolerated without serious adverse reactions. CONCLUSIONS: These results indicate that lamivudine treatment induces sustained inhibition of viral replication and normalization of transaminases in the majority of post-transplant patients with acute hepatitis B. HBsAg loss may be achieved in a considerable number of cases. Although viral resistance is relatively frequent, early initiation of lamivudine appears to be effective and safe.

Effect of glass transition temperature on the stability of lyophilized formulations containing a chimeric therapeutic monoclonal antibody.

PURPOSE: The purpose of this study is to highlight the importance of knowing the glass transition temperature, Tg, of a lyophilized amorphous solid composed primarily of a sugar and a protein in the interpretation of accelerated stability data. METHODS: Glass transition temperatures were measured using DSC and dielectric relaxation spectroscopy. Aggregation of protein in the solid state was monitored using size-exclusion chromatography. RESULTS: Sucrose formulation (Tg approximately 59 degrees C) when stored at 60 degrees C was found to undergo significant aggregation, while the trehalose formulation (Tg approximately 80 degrees C) was stable at 60 degrees C. The instability observed with sucrose formulation at 60 degrees C can be attributed to its Tg (approximately 59 degrees C) being close to the testing temperature. Increase in the protein/sugar ratio was found to increase the Tgs of the formulations containing sucrose or trehalose, but to different degrees. CONCLUSIONS: Since the formulations exist in glassy state during their shelf-life, accelerated stability data generated in the glassy state (40 degrees C) is perhaps a better predictor of the relative stability of formulations than the data generated at a higher temperature (60 degrees C) where one formulation is in the glassy state while the other is near or above its Tg.

The relationship between protein aggregation and molecular mobility below the glass transition temperature of lyophilized formulations containing a monoclonal antibody.

PURPOSE: To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. METHODS: The temperature dependence of relaxation time constant, tau, was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, beta, in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. RESULTS: For both sucrose and trehalose formulations, the variation in tau with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited < 2% aggregation at t/tau values < 10, where t is the time of storage. CONCLUSIONS: Since the relaxation times for sucrose and trehalose formulations at 5 degrees C are on the order of 10(8) and 10(6) hrs, it is likely that both formulations would undergo very little (< 2%) aggregation in a practical time scale under refrigerated conditions.

[Oral pefloxacin in the treatment of acute gastroenteritis]. / La pefloxacina per os nel trattamento delle gastroenteriti acute.

UNLABELLED: In the case of travellers' diarrhoea, in order to reduce to a minimum the period of sickness and temporary invalidity, suitable therapy needs to be undertaken, involving the reintegration of fluids and electrolytes, as well as antibiotic treatment whose spectrum of action covers the most commonly isolated microorganisms. The aim of the study was to verify the rapidity of the antibacterial action of pefloxacin in the treatment of this type of gastrointestinal infection. MATERIALS AND METHODS: The patients enrolled were treated for 5 days with pefloxacin in tablets containing 400 mg of the active agent, given orally. Treatment involved a first dose of 800 mg (2 tablets in a single administration), followed by 400 mg twice a day (1 tablet every 12 hours). Administration was carried out prevalently at mealtimes. At the end of treatment a two-week follow-up period was scheduled. At the beginning of the study, and after 5 days' treatment, a coproculture was carried out, with identification of the germ responsible for the infection. The study was carried out in accordance with the principles of the Helsinki declaration and its revisions, in particular each patient gave his/her informed consent to participate in the trial. RESULTS: Thirty patients enrolled in the study (16 males, 14 females), of mean age 48.2 years (18-82 min-max), mean weight of 66.3 kg (46-88 min-max), suffering from acute gastroenteritis. The coproculture carried out at the baseline for all patients led to the isolation in 10 patients, of 10 pefloxacin-sensitive GRAM-negative bacterial strains: Salmonella spp. in 6 cases, Shigella spp. in 2, Escherichia coli in 1 and Yersinia enterocolitica in 1. At the end of treatment and follow-up the coproculture indicated that all the germs isolated initially had been eradicated and no cases of superinfection or reinfection occurred. The number of daily evacuations gradually decreased from a mean of 6.8 at the baseline to 1.1 on the fifth day (p<0.0001). The consistency of faeces had increased in a significant fashion by the third day and at the end of treatment 93.3% of patients had well-formed faeces. In the course of treatment abdominal pain, nausea and vomiting decreased progressively and body temperature returned within normal limits by the third day. Safety was excellent in 93.3% of patients, with one case of nausea and one of erythema, which resolved spontaneously in the course of treatment. At the end of treatment with pefloxacin, the physician expressed his final judgment of efficacy, which was excellent in 86.7% of cases (26/30) and good in 13.3% (4/30), in accordance with the clinical evaluation of recovery in 93.3% of cases (28/30) and of improvement in 6.7% (2/30). CONCLUSIONS: In forms of acute gastroenteritis in which coproculture shows the presence of germs sensitive to pefloxacin, the use of this quinolone guarantees a high percentage of success with a short course of treatment.

[Duodenal somatostatinoma associated with von Recklinghausen's neurofibromatosis]. / Somatostatinoma duodenale associato a neurofibromatosi di von Recklinghausen.

This case report describes a 27-year-old man with von Recklinghausen's neurofibromatosis, manifested as cutaneous cafè au lait spots and neurofibromas, associated with duodenal somatostatinoma. The patient presented with ultrasonographic evidence of dilatation of the biliary and pancreatic ducts, in absence of clinical symptoms. The reason for the performance of ultrasonography was to identify the cause of an increase of hepatic enzymes during the last two years. Diagnostic ERCP showed an ulcerated tumor in the papillary region and pathological findings were compatible with somatostatinoma. Endoscopic sphincterotomy with placement of endoprostheses was successful in achieving biliary and pancreatic drainage. Subsequently a curative resection of the tumor was performed by the Whipple procedure and provocative tests demonstrated normal plasma somatostatin concentrations.

Tight binding affinities determined from thermodynamic linkage to protons by titration calorimetry.

A general titration calorimetry method is described that can be used to determine the affinity of tight binding interactions with proteins. The method is based on the thermodynamic linkage between ligand binding and coupled protonation reactions. The protons linked to a given ligand-binding reaction are measured by titration calorimetry, and integration of the resulting data set yields the pH dependence of the binding affinity based on thermodynamic relationships developed elsewhere. When the pH dependence of the binding affinity is combined with the absolute affinity determined independently at a pH at which the affinity can be conveniently measured, the absolute binding affinity over the entire pH range is determined. The method is well suited for determining high-affinity binding interactions of protein antigens with antibodies, but is applicable to any macromolecular ligand-binding reaction that is coupled to protonation.

Standardized reporting of histological diagnoses for non-neoplastic liver conditions in needle biopsies.

The importance of standardizing surgical pathology reports is emerging from the literature. The use of checklists has recently been proposed for diagnosing the major tumour types, but no attention has been given to non-neoplastic conditions. In this paper a checklist for standard reports of liver needle biopsies for non-neoplastic conditions is presented.

[Transcatheter arterial chemoembolization technique in cirrhotic patients with hepatocarcinoma. Considerations on the procedure and evaluation of survival]. / Chemioembolizzazione arteriosa trans-catetere dell'epatocarcinoma su cirrosi con tecnica globale. Considerazioni sul procedimento e valutazione della sopravvivenza.

Two hundred and eight cirrhotic patients with HCC underwent TACE with a standardized technique. Kaplan-Meier survival rates and 12, 24 at 36 months were 62%, 44% and 25%, respectively. Compared with 407 untreated patients, our series had a longer life expectancy, i.e., from 15 months after treatment on. Life experience was statistically different with the L-R test between the groups selected by Child-Pugh cirrhosis staging (p = 0.00000); all 8 Child-Pugh C patients died within 7 months; a high statistical difference was found between Child-Pugh A and B groups (p = 0.00012). Life experience was statistically different with the L-R test between the four groups selected by tumor size and spread (p = 0.012); statistical significance was not reached between contiguous groups in group vs. group comparisons. The patients with monofocal tumors, regardless of size, survive longer than those with multifocal and infiltrative (p = 0.0010) and those with multifocal (p = 0.0029) lesions. Hazard analysis, according to the stratified Cox model, proved tumor-size and Child-Pugh staging to be prognostic factors (p = 0.00027; p = 0.00000) which exhibit a highly significant correlation with each other (p = 0.00000). With the proportional hazard Cox model, tumor characteristics and Child-Pugh stage resulted to be highly significant independent prognostic factors (p = 0.013 and p = 0.000, respectively). Patient survival rates were graphically plotted against literature rates in 407 untreated patients classified by tumor size and by the Child-Pugh method: the two-year survival rates were higher in the subgroups of patients submitted to TACE. To conclude, TACE is an effective treatment not only for multifocal HCCs, but also for large monofocal and infiltrative HCCs. In contrast, TACE is quite useless in the patients with Child-Pugh C cirrhosis.

Combined ranitidine and pirenzepine in the treatment of duodenal ulcer: a multicentre double-blind study using endoscopy.

The efficacy of a combination of ranitidine and pirenzepine in the short-term treatment of duodenal ulcer was evaluated in a double-blind trial. In a multicentre study, 352 patients with active duodenal ulcers were randomly allocated to be treated with 300 mg/day ranitidine plus placebo (group I), 300 mg/day ranitidine plus 50 mg/day pirenzepine (group II), or 300 mg/day ranitidine plus 100 mg/day pirenzepine (group III) for 4 weeks. The respective healing rates assessed using endoscopic examination after 2 and 4 weeks' treatment were 40% and 70% in group 1, 44% and 82% in group II, and 37% and 77% in group III. The differences between the treatment groups were not significant, although 300 mg/day ranitidine plus 50 mg/day pirenzepine tended to be superior to the other treatments. Analgesic activity was the same in the three groups with 33%, 34% and 33% reductions, respectively, in the numbers of patients experiencing pain after 2 weeks. Side-effects (mainly dry mouth and blurred vision) were significantly more frequent in group III patients.