In vivo models and decision trees for formulation development in early drug development: A review of current practices and recommendations for biopharmaceutical development.

The ability to predict new chemical entity performance using in vivo animal models has been under investigation for more than two decades. Pharmaceutical companies use their own strategies to make decisions on the most appropriate formulation starting early in development. In this paper the biopharmaceutical decision trees available in four EFPIA partners (Bayer, Boehringer Ingelheim, Bristol Meyers Squibb and Janssen) were discussed by 7 companies of which 4 had no decision tree currently defined. The strengths, weaknesses and opportunities for improvement are discussed for each decision tree. Both pharmacokineticists and preformulation scientists at the drug discovery & development interface responsible for lead optimization and candidate selection contributed to an overall picture of how formulation decisions are progressed. A small data set containing compound information from the database designed for the IMI funded OrBiTo project is examined for interrelationships between measured physicochemical, dissolution and relative bioavailability parameters. In vivo behavior of the drug substance and its formulation in First in human (FIH) studies cannot always be well predicted from in vitro and/or in silico tools alone at the time of selection of a new chemical entity (NCE). Early identification of the risks, challenges and strategies to prepare for formulations that provide sufficient preclinical exposure in animal toxicology studies and in FIH clinical trials is needed and represents an essential part of the IMI funded OrBiTo project. This article offers a perspective on the use of in vivo models and biopharmaceutical decision trees in the development of new oral drug products.

Long-term results of intraoperative electron beam radiotherapy for primary and recurrent retroperitoneal soft tissue sarcoma.

PURPOSE: This study assesses the long-term outcome of patients with retroperitoneal sarcoma treated by preoperative external beam radiotherapy, resection, and intraoperative electron beam radiation (IOERT). METHODS AND MATERIALS: From 1980 to 1996, 37 patients were treated with curative intent for primary or recurrent retroperitoneal soft tissue sarcoma. All patients underwent external beam radiotherapy with a median dose of 45 Gy. This was followed by laparotomy, resection, and IOERT, if feasible. Twenty patients received 10-20 Gy of IOERT with 9-15 MeV electrons. These patients were compared to a group of 17 patients receiving preoperative irradiation without IOERT. RESULTS: The 5-yr actuarial overall survival (OS), disease-free survival, local control (LC), and freedom from distant disease of all 37 patients was 50%, 38%, 59%, and 54%, respectively. After preoperative irradiation, 29 patients (78%) underwent gross total resection. For 16 patients undergoing gross total resection and IOERT, OS and LC were 74% and 83%, respectively. In contrast, these results were less satisfactory for 13 patients undergoing gross total resection without IOERT. For these patients, OS and LC were 30% and 61%, respectively. Four patients experienced treatment-related morbidity. CONCLUSIONS: In selected patients, IOERT results in excellent local control and disease-free survival with acceptable morbidity.

The bioavailability of oral fludarabine phosphate is unaffected by food.

INTRODUCTION: A prospective, open and randomized, two-way crossover study was conducted to evaluate the pharmacokinetics and bioavailability of oral fludarabine phosphate when taken on a full versus an empty stomach. The effectiveness of therapy was also assessed after two cycles of treatment, four weeks apart MATERIALS AND METHODS: Patients with chronic lymphocytic leukemia or low-grade non-Hodgkin's lymphoma were randomly assigned to two groups, both of which received two cycles of treatment with 90 mg of oral fludarabine phosphate administered when either fed or fasted. Patients in Group 1 (n = 8) received oral treatment on a full stomach for the first cycle then on a fasted stomach for the second, while those in Group 2 (n = 10) received their treatment in the reverse sequence. Oral fludarabine phosphate was administered on the first day of the two study cycles and intravenous fludarabine phosphate was administered on days 3-6. RESULTS AND CONCLUSION: Of 22 patients recruited, 18 (CLL n = 10; NHL n = 8) were eligible for efficacy and safety evaluation, and 16 for bioavailability and pharmacokinetic analyses. The response to oral 2-F-ara-AMP was rapid: by two treatment cycles, 12 out of 18 patients (66.7%) had achieved partial response. Of the six patients who did not respond, five patients (27.7%) had stable disease. There was no notable difference in the rate of response between patients with B-CLL and lg-NHL. There was a marginal increase in total systemic availability of fludarabine phosphate when administered orally on a fed stomach (2-F-ara-A AUC((0-24 h)) = 3.28 +/- 1.48 microg.h/ml) compared to a fasted stomach (2-F-ara-A AUC((0-24 h)) = 3.05 +/- 1.56 microg.h/ml). Time to peak plasma concentration was slightly extended by the presence of food (2.2 +/- 1.0 versus 1.3 +/- 0.74 h) but the terminal half-life was unaffected. The minor differences in the pharmacokinetics of oral fludarabine phosphate when taken after food were not statistically significantly different and seem unlikely to be clinically relevant. The efficacy and safety data closely paralleled previous experience with the intravenous formulation.

Predicting the oral bioavailability of 19-nortestosterone progestins in vivo from their metabolic stability in human liver microsomal preparations in vitro.

It was the aim of this study to investigate whether assessment of the metabolic stability of selected progestins of the 19-nortestosterone type in human microsomal liver preparations was a suitable approach to predict the oral bioavailability of these drugs in humans. The Michaelis-Menten parameters Vmax and KM,app for norethisterone, levonorgestrel, gestodene, desogestrel, 3-keto-desogestrel, norgestimate, and dienogest were determined in in vitro incubations with human liver microsomes. Using these data, both the in vitro intrinsic clearance (CLint) and, after application of a suitable scaling factor, the scaled in vivo CLint were calculated. For progestins for which human in vivo data were available, the in vitro results were correlated with in vivo CLint values and oral bioavailability. A comparison of the scaled in vivo CLint values with the corresponding in vivo CLint values showed a reasonable correlation, although the latter values were generally approximately 2-fold higher than the former. Excluding desogestrel, which is subject to substantial intestinal metabolism in vivo, there was a linear relationship (r = -0.986) between increasing in vitro CLint values for the progestins and decreasing bioavailability in vivo. Other methods of assessing the metabolic stability of the progestins in vitro, such as evaluation of metabolic half-lives at single initial concentrations, showed either no correlation or a less satisfactory correlation with bioavailability data.

A pilot study of concomitant boost accelerated superfractionated radiotherapy for stage III cancer of the uterine cervix.

PURPOSE: Retrospective studies suggest that prolonged treatment time adversely affects control rates of squamous carcinomas managed by radiotherapy. From 1989 to 1994 a prospective clinical trial was conducted to assess the feasibility and efficacy of concomitant boost accelerated superfractionated (CBASF) radiotherapy for advanced uterine cervical carcinoma. METHODS AND MATERIALS: Twenty newly diagnosed patients with FIGO stage III squamous cell carcinoma of the cervix were irradiated using a CBASF regimen. Patients received 45 Gy administered to the whole pelvis in 25 fractions in 5 weeks. On Monday, Wednesday, and Friday of the last 3 weeks, an additional 1.6 Gy boost was given 6 hours after the whole pelvis treatment. The 9 boost treatments, totaling 14.4 Gy, were given via lateral fields encompassing the parametria and primary tumor for a cumulative tumor dose of 59.4 Gy. A single low-dose rate brachytherapy procedure was performed within 1 week after the external beam radiotherapy to raise the point A dose to 85-90 Gy in 42 days. Primary endpoints of analysis were local control, complications, and patterns of failure. Results are compared with the outcomes of 21 patients treated with conventionally fractionated (CF) radiotherapy during the same years. RESULTS: Median total treatment time was 46 days in the CBASF group (range 37-62). Median follow-up interval among surviving CBASF patients is 3.8 years. The four-year actuarial local control rates are 78% and 70% in the CBASF and CF groups, respectively (p = ns). Only 2 CBASF patients required a treatment break because of acute toxicity, but severe late complications occurred in 8/20 CBASF patients for a crude rate of 40%. Distant failure was more common than local failure in the CBASF group, and para-aortic node failure occurred in six of the eight CBASF patients with distant failure. CONCLUSIONS: In the management of stage III cervix cancer, the CBASF regimen produced a trend toward improved local control when compared with the CF regimen, shifting the patterns of failure toward a higher rate of isolated distant failures. The high frequency of para-aortic node failure warrants consideration of elective treatment to this region in stage III patients treated with curative intent. Although the high local control rate of the CBASF regimen supports further investigation of accelerated treatment regimens for locally advanced cervix cancer, the unacceptable risk of late complications necessitates refinement in technique and scheduling to improve the therapeutic ratio.

Development of a V79 cell line expressing human cytochrome P450 2D6 and its application as a metabolic screening tool.

Expression of human cytochrome P450 (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP2D6-VAL(374) was inserted into an expression vector containing the strong mycloproliferative sarcoma virus promotor in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP2D6 cDNA was confirmed by polymerase chain reaction analysis. The protein expression was shown by Western blotting. Functional expression could be demonstrated by O-demethylation of dextromethorphan to dextrorphan in live cells. The enzymatic activity of 154 ± 16 pmol min(-1) mg(-1) protein was comparable with dextromethorphan-O-demethylation activities of human liver. The metabolism of two dopaminergic ergoline derivatives was investigated in whole recombinant V19 cells. Both lisuride and terguride were monodeethylated; in case of lisuride a correlation to the in vivo situation was demonstrated comparing poor and extensive metabolizers.

Clinical use of a digital simulator for rapid setup verification in high dose rate brachytherapy.

PURPOSE: Fractionated high dose rate (HDR) brachytherapy provides a number of technical advantages over conventional implant therapy in that (a) it can be carried out on an outpatient basis, (b) personnel exposure is reduced to insignificant levels, and (c) patient motion during irradiation is minimized, resulting in a more accurate delivery of the planned radiation dose distribution to the target and critical structures. The patient discomfort associated with the repeated applicator insertions and/or treatment setups can be alleviated to the extent that the setup time is held to a minimum. This work describes the use of a prototype digital simulator to obtain fast, high-quality digital images for rapid setup verification. METHODS AND MATERIALS: The digital imaging system of the prototype simulator consists of a charge-coupled device (CCD) camera, which views the x-ray image optically transmitted from a conventional phosphor screen. Treatment is carried out with a remote afterloading HDR unit immediately after setup verification with the patient on the simulator stretcher. The high-resolution digital images are processed and displayed in about 5 s, as opposed to a minimum of approximately 2 min for film. RESULTS: The imaging system has been evaluated for a variety of implant types, both intracavitary and interstitial. The digital radiographs provided permanent high-resolution images as required in most cases for precise applicator positioning. The gray scale manipulation capabilities were found to be useful for imaging in regions of different density, such as lung and soft tissue, in the same radiograph. The advantages of short image acquisition and display times were observed in all cases, but were most evident in the intraluminal procedures, which sometimes involved several pretreatment applicator adjustments at a time of considerable patient discomfort. CONCLUSION: Pretreatment imaging is necessary to fully exploit the technical advantages of HDR brachytherapy. High-quality digital radiography offers unique advantages in HDR setup and verification by providing fast high-resolution, undistorted images with software manipulation capabilities and permanent storage of images.

Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives.

Expression of human cytochrome (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP3A4 (PCN1) was inserted into an expression vector containing the strong myeloproliferative sarcoma virus promoter in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP3A4 cDNA cell clones was confirmed by polymerase chain reaction analysis. Transcription was detected by reverse transcribed polymerase chain reaction analysis. Functional expression could be demonstrated by conversion of testosterone to the specific 6beta-hydroxylated product. In recombinant V79 cells expressing CYP3A4 about 6% of the substrate was converted to 6beta-hydroxytestosterone. The metabolism of two dopaminergic ergot derivatives was investigated in live recombinant V79 cells. Both lisuride and terguride were monodeethylated.

Studies on the biotransformation of lonazolac, bromerguride, lisuride and terguride in laboratory animals and their hepatocytes.

1. Metabolic patterns and the extents of metabolism of four drugs, namely [14C]lonazolac (LON), [14C]bromerguride)BRO), [14C]lisuride (LIS) and [3H]terguride (TER) have been studied in three experimental models, namely hepatocyte suspensions of rat, guinea pig, beagle dog and cynomolgus monkey, isolated perfused liver of rat and guinea pig and intact animals (rat, guinea pig, dog and monkey). 2. Selection of compounds was based on differences in phase I metabolic pathways. LON is exclusively hydroxylated in the N-substituting aromatic ring, BRO is mainly N-deethylated in the urea moiety, and LIS and TER are both degraded into numerous metabolites. 3. The decrease in unchanged drug levels in hepatocyte suspensions was characterized by half-lives, with LON as the most stable and LIS as the least stable compound. Marked interspecies differences were found. De-ethylation and aromatic hydroxylation were much slower in rat hepatocytes than in the liver cells of other species; BRO was slowly biodegraded in dog hepatocytes while LIS was broken down extremely quickly. 4. Liver perfusion experiments and studies in vivo were evaluated for the extents of metabolism of each drug. 5. Metabolism studies in hepatocytes did not show any quantitative correlation to those of metabolism in vivo. The suitability of evaluating parameters for in vitro studies is discussed.