Early Results of Extreme Hypofractionation Using Stereotactic Body Radiation Therapy for High-risk, Very High-risk and Node-positive Prostate Cancer.

AIMS: Stereotactic body radiotherapy (SBRT) in low- and intermediate-risk prostate cancer has shown encouraging results. However, its use in high-risk patients is limited due to lack of data regarding adequate radiotherapy dose, need for pelvic nodal treatment and androgen deprivation therapy. Herein we report our experience of SBRT in this subgroup. MATERIALS AND METHODS: Analysis of a prospectively maintained database of 68 consecutive patients of the National Comprehensive Cancer Network (NCCN) high-risk, very high-risk and node-positive adenocarcinoma prostate treated with SBRT was undertaken. All patients were treated with rotational intensity-modulated radiotherapy with daily image guidance. The dose delivered to the prostate and gross node was 35-37.5 Gy in 5 alternate day fractions. Node-positive patients received 25 Gy to pelvic nodal regions until the common iliac nodes. Treatment was delivered in 7-10 days. All patients received long-term androgen deprivation therapy (79% medical and 21% surgical). RESULTS: Most patients (65%) had a Gleason score ≥ 8. The median prostate-specific antigen was 42. Twenty patients were high risk (30%), 11 (16%) very high risk and 37 (54%) node positive. No acute Radiation Therapy Oncology Group grade ≥ 3 genitourinary or gastrointestinal toxicity was noted. Acute grade 2 genitourinary and gastrointestinal toxicity were 12% and 3%, respectively. Late grade 3 genitourinary and gastrointestinal toxicity was 3% and 0%, respectively. There was no increase in acute or late gastrointestinal toxicity with prophylactic pelvic nodal radiotherapy. Prior transurethral resection of prostate (n = 11) did not increase toxicity. At a median follow-up of 18 months, 97% patients were alive and 94% were biochemically controlled. CONCLUSION: SBRT is safe in the treatment of high-risk, very high-risk and node-positive prostate cancer, even with prophylactic pelvic radiotherapy or prior transurethral resection of prostate. Longer follow-up is required to determine efficacy.

A randomized, crossover, assessor-blind study of the bioequivalence of a single oral dose of 200 mg of four formulations of phenytoin sodium in healthy, normal Indian volunteers.

The objective of the study was to compare the bioavailability of a single oral 200-mg dose of four brands of phenytoin sodium available in the Indian market. Dilantin, Epsolin, and M-toin were compared with Eptoin, which was taken as the reference standard. A randomized, assessor-blind, four-way crossover study was done in 12 healthy Indian volunteers. The study was conducted at a clinical pharmacology ward at King Edward VII Memorial Hospital, a tertiary referral center in Mumbai (Bombay). All 12 subjects received a single oral 200-mg dose of all the formulations with a 2-week washout period between the formulations. Blood samples for plasma phenytoin levels were collected at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 48, and 72 hours. Safety was measured by pretreatment and posttreatment biochemical investigations, physical examination, and ECG. The pharmacokinetics of the four brands of phenytoin were calculated by maximum plasma concentration (C(max)), time to reach C(max) (t(max)), area under the concentration versus time curve for time 0 to 72 hours (AUC(0-72)), and from time 0 to infinity (AUC(0- infinity)). For all brands, 90% CI of all untransformed and log transformed pharmacokinetic parameters failed to remain within prescribed limits of 80% to 120% for untransformed data and 80% to 125% for log transformed data. Since phenytoin obeys Micheles Mentens kinetics, the AUC methodology used for comparison would give only an approximate indication of relative bioavailability. M-toin was shown to be bioinequivalent to Eptoin. The other comparisons indicate but do not prove bioinequivalence of the other brands. The results of the study show that in India switching phenytoin brands could have significant implications and is not advisable once a patient is carefully titrated on one formulation.

Variability in the bioavailability of phenytoin capsules in males and females.

PURPOSE: To determine inter-lot and intra-subject variability in the bioavailability of the 100 mg extended phenytoin sodium capsules. In addition, to determine the effect of gender and menstrual cycle on phenytoin bioavailability. METHODS: Three different lots of extended phenytoin sodium capsules were given to 12 healthy male and 12 healthy female subjects in a crossover fashion. One of the lots was also given a second time to each subject. Plasma phenytoin was determined, using an HPLC assay, in samples collected over a 73-hr period after each dose. RESULTS: The mean Cmax for the four administrations ranged from 1.71-1.79 microg/ml and mean AUC(0-infinity) values from ranged 53.0-54.1 microg*hr/ml. The elimination half-life was 3 hr shorter, and the AUC(0-infinity) adjusted for the mg/kg dose was 30% lower for females. Average bioequivalence was demonstrated between the three lots for both Cmax and AUC(0-infinity) based on the BE limit of 80-125%. Further, all confidence intervals of AUC(0-infinity) fell within the limit of 90-111%. There were no differences in the confidence limits for Cmax and AUC(0-infinity) determined separately for males and females. Also, there was no difference in the mean Cmax or AUC(0-infinity) for females when analyzed as a function of the week of their menstrual cycle. Individual bioequivalence was demonstrated between three lots of phenytoin using the constant-scaled method, but not the reference-scaled method. CONCLUSIONS: There was very little difference in the bioavailability of the three lots of phenytoin. Females exhibited a lower AUC(0-infinity) than males after adjustment of dose for body weight, but their inclusion in the study did not affect the assessment of bioequivalence. When dose was not adjusted for body weight, no difference in AUC(0-infinity) was seen between males and females.

The effect of gelatin cross-linking on the bioequivalence of hard and soft gelatin acetaminophen capsules.

PURPOSE: To determine if changes in the in vitro dissolution of hard and soft gelatin acetaminophen capsules, which result from gelatin crosslinking, are predictive of changes in the bioavailability of the capsules in humans. METHODS: Both hard and soft gelatin capsules were "stressed" by a controlled exposure to formaldehyde, resulting in unstressed, moderately stressed and highly stressed capsules. In vitro dissolution studies were conducted using water or SGF with and without pepsin as the media. Separate 24-subject, 3-way crossover human bioequivalence studies were performed on the unstressed and stressed acetaminophen capsules. Plasma acetaminophen was determined by high performance liquid chromatography (HPLC) for 12 hr after each dose. RESULTS: The in vitro rate of dissolution of hard and soft gelatin capsules was decreased by crosslinking. The bioequivalence studies showed that both hard and soft gelatin capsules, which failed to meet the USP dissolution specification in water, but complied when tested in SGF containing pepsin, were bioequivalent to the unstressed control capsules. The capsules that were cross-linked to the greatest extent were not bioequivalent to the unstressed control capsules, based on Cmax. A trend toward an increase in Cmax with increased level of cross-linking was observed, but this was only significant for the severely stressed capsules. CONCLUSIONS: On the basis of this study a two-tier in vitro dissolution test was developed using enzymes to distinguish between bioequivalent and bioinequivalent gelatin capsules.

Strategies for locating disulfide bonds in a monoclonal antibody via mass spectrometry.

The location of the disulfide bonds in a recombinant monoclonal antibody was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). A non-reduced Endoproteinase Lys-C (Endo Lys-C) digest of the antibody was analyzed directly by MALDI-TOFMS. The sample was then reduced on-plate by depositing dithiothreitol (DTT) on the sample spot and re-analyzed by MALDI-TOFMS. The disulfide bonds were assigned based on the disappearance of certain mass ions in the non-reduced digest and the appearance of product ions in the reduced digest. A rapid LC/ESI-MS protocol was also developed to determine the location of the disulfide bonds. The peptides generated from the Endo Lys-C digest of the antibody were partially separated on a high performance liquid chromatography (HPLC) column by utilizing a steep gradient and analyzed by ESI-MS. The masses of the partially resolved peptides were determined by deconvoluting the mass spectra.

The relative bioavailability and in vivo-in vitro correlations for four marketed carbamazepine tablets.

PURPOSE: To determine if three marketed generic carbamazepine tablets were bioequivalent to the innovator formulation, as well as to each other. In addition, to examine in vivo-in vitro relationships among the four formulations. METHODS: Each formulation was given as a single dose to 18 healthy male and female subjects using a crossover design. Blood samples were collected for 169 hr. Carbamazepine was assayed by HPLC with UV detection. RESULTS: In vivo fraction absorbed plots indicated that the three generic formulations were absorbed more rapidly than the innovator product, and the mean time of maximum plasma concentration was 6-7 hr sooner for the generic formulations. The mean maximum plasma concentration ranged from 17-19 percent higher for the generic products compared to the innovator, and the 90% confidence limits for Cmax data ranged from 11 1% to 126%. The mean AUC(0-infinity) for the generic products ranged from 101-104% compared to the innovator, and the confidence limits for AUC ranged from 97-108%. CONCLUSIONS: The generic products were all more rapidly absorbed than the innovator, but simulations of steady-state concentrations indicated that it would be unlikely that these differences would have any significant clinical effect. An excellent association was seen between the Cmax and the percent of drug dissolved in vitro. The correlation was used to accurately predict the Cmax of four other 200 mg tablets evaluated in an earlier study.

Lack of in vivo/in vitro correlations for 50 mg and 250 mg primidone tablets.

PURPOSE: To determine if large differences in the in vitro dissolution profiles for primidone tablets would result in significant bioavailability differences. METHODS: Two separate bioavailability studies were conducted. The first study used 18 healthy subjects and compared the bioavailability of an old 50 mg tablet formulation, a new 50 mg tablet formulation, and a suspension containing 50 mg/ml of primidone. The second study enrolled 24 subjects who were to receive a new 250 mg tablet formulation, two lots of an old 250 mg tablet formulation and a 250 mg tablet from a second manufacturer. In vitro dissolution was conducted over 90 minutes, using USP 23 Apparatus 2 at 50 rpm, with 900 ml of water. RESULTS: Dissolution at 90 minutes for the old and new 50 mg tablets was approximately 20% and 100%, respectively. The dissolution of the four 250 mg tablets ranged from approximately 30% to 100%. The 50 mg tablet that dissolved slower had a longer Tmax and a 14% lower Cmax than the more rapidly dissolving tablet, but the AUC(0-infinity) values differed by only 3%. Only nine subjects completed the 250 mg study because of side effects. The differences in Cmax and AUC(0-infinity) among the four 250 mg tablets were less than 7%. CONCLUSIONS: Even though there were large differences in the in vitro dissolution of the 50 mg and the 250 mg primidone tablets, the two 50 mg tablets were shown to be bioequivalent, as were the four 250 mg tablets.

Characterization of antigen-antibody complexes by size-exclusion chromatography coupled with low-angle light-scattering photometry and viscometry.

In this paper, the molecular masses (M(r)s) of the complexes of monoclonal anti-BSA (antibody to bovine serum albumin) (clone: 33) and monomer BSA were determined on-line by using size-exclusion chromatography (SEC) coupled with a low-angle laser light-scattering (LALLS) detector and two concentration detectors, ultraviolet (UV) and refractive index (RI) (SEC-LALLS/UV/RI system). Also, the size and M(r)s of the complexes were evaluated by the SEC-LALLS/UV/viscometer (VISC) system. This study demonstrated that, for small size macromolecules, the combination of light scattering and viscosity detection was a suitable choice for determining their M(r)s and sizes.

Rapid purification and monitoring of immunoglobulin M from ascites by perfusion ion-exchange chromatography.

A purification and on-line monitoring procedure for IgM was developed. Perfusion ion-exchange chromatography was used for rapid purification of IgM from ascites fluid and hybridoma supernatant. Crude ascites was directly loaded onto an ion exchanger. Due to the complexity of IgM, a two-step ion-exchange procedure had to be developed. This procedure involved a rapid cation-exchange chromatography capture step followed by further purification using anion-exchange chromatography. High linear velocities, in excess of 3500 cm/h, enabled separations to be performed under 5 min. Purity of the final product by SDS-PAGE was shown to be greater than 95%. Furthermore, the antibodies retained biological activity as measured by indirect immunofluorescence (IIF) and ELISA. The IgM peak was also monitored on-line using a novel peak tracking approach. This involved placing an antibody column (specific to the IgM) prior to the ion-exchange column and operating the ion-exchange column with and without the antibody column in-line. The missing peak that is identified by comparing the two chromatograms indicates where the IgM elutes.

Capillary electrophoresis-based immunoassay for cortisol in serum.

A competitive immunoassay for cortisol based on capillary electrophoresis (CE) and laser-induced fluorescence is described. The work involved the production of assay reagents and the development of separation conditions allowing for routine analysis of serum samples. Fluorescein-labeled cortisol was synthesized and purified. Fab fragments were produced from mouse monoclonal anticortisol antibody and purified using a POROS cation exchange chromatography column. After incubation of these reagents with serum, free and bound labeled antigen were separated by CE with high reproducibility. No prior sample cleanup of the serum samples was necessary. Serum calibration curves were established and used for the quantitation of cortisol in serum. The results demonstrate feasibility for a cortisol assay based on CE operating directly on serum samples.

Determination of on-line differential refractive index and molecular weight via gradient HPLC interfaced with low-angle laser light scattering, ultraviolet, and refractive index detection.

Gradient ion-exchange chromatography (IEC) was interfaced with a low-angle laser light scattering photometer (LALLS) and ultraviolet (UV) and refractive index (RI) detectors connected in series for on-line determination of the differential refractive index (dn/dc) of proteins and eventually their molecular weights (Mw's). Interfacing of gradient HPLC with a RI detector was made possible by using two isorefractive buffers, which helped generate stable baselines for the LALLS and RI detectors. An optically modified, laser based RI detector was used for determination of dn/dc. On-line determinations of dn/dc required smaller amounts of sample compared to off-line determinations (3-4 mg vs 200 mg). Also, dn/dc could be measured by making one repetitive injection of the sample, if the molar absorptivity of the sample was known. dn/dc values determined on-line were in close agreement with those determined off-line. Additionally, percent recovery of the sample was calculated on-line, and this provided true concentration detected at the UV detector, which was then used for determination of Mw.

Interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry for analysis of proteins.

Molecular weights (MWs) of different proteins were determined by interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry (IEC-LALLS). A high-performance strong cation-exchange column was used to elute proteins using fast (5 min) and conventional (15-30 min) gradients. The eluted proteins were characterized on-line by determining their MWs using LALLS. The specific refractive index (RI) increment (dn/dc) and the RI of the solvent used over the gradient range were determined off-line and used to calculate the absolute weight-average MWs. Four proteins, ribonuclease A, alpha-chymotrypsinogen A, trypsinogen and beta-lactoglobulin A (beta-LACT) were studied. Accurate MWs were obtained for all the proteins using fast and conventional gradients, except for beta-LACT, which aggregated as a function of the gradient employed. The degree of aggregation of beta-LACT increased as the rapidity of the gradient was increased over a fixed gradient range. This study indicated that it is possible to separate and characterize proteins rapidly using IEC-LALLS.

Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection.

The determination of molecular weights for certain proteins has been performed. This has involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-micron, non-porous, Monosphere RP-C8 column has been used in order to perform fast and conventional RP-HPLC gradients (5-45 min). Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for ribonuclease A, lysozyme, and bovine serum albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5-10 min of fast gradient elution, each protein appears to exhibit unusual Mw values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.

Pharmacologic disposition of chlorozotocin in mice.

Cholorozotocin is a water-soluble chloroethylnitrosourea with the cytotoxic group attached to the C2 position of glucose. The distribution of the alkylating and carbamoylating moieties of the chlorozotocin molecule was determined in mice following the ip administration of an LD10 dose: 20 mg/kg. The half-life (T 0.5) for the plasma disappearance of intact drug was 5 minutes. The plasma disappearance of the ethyl-14C group was biphasic up to 120 minutes after administration; the T 0.5 of the initial phase was 17.5 minutes and the T 0.5 of the prolonged second phase was107 minutes. The disappearance of glucose-14C chlorozotocin followed kinetics similar to the chloroethyl-labeled compound. Fifteen minutes after administration, ethyl-14C drug concentrated maximally in the liver (194 nmols/g of tissue) and the kidney (131 nmols/g of tissue). Uptake into the bone marrow at 60 minutes after ip administration of the ethyl-labeled drug was 6.6 pmols of the ethyl-14C group covalently bound to proteins and nucleic acids per 10(7) nucleated cells. The concentration of ethyl-14C drug in the brain remained at 4 mnols/g of tissue up to 2 hours after administration, reflecting the water-soluble property of this new nitrosourea.