Detection of formaldehyde-induced crosslinking in soft elastic gelatin capsules using near-infrared spectrophotometry.

The purpose of this research was to monitor the migration of formaldehyde from a polyethylene glycol (PEG) fill into the gelatin shell of a soft elastic gelatin capsule (SEGC) using near-infrared (NIR) spectrophotometry. SEGCs were filled with five solutions of aqueous formaldehyde in PEG (0, 0.05, 0.10, 0.20, and 0.40 v/v%), stored at ambient conditions for 48 hr, emptied, and scanned in NIR spectrophotometer. Principal component regression (PCR) was employed to analyze the spectra of the empty capsules. Good correlation was established (r2 = 0.988) when actual concentrations of formaldehyde in the PEG fill of the capsules were regressed against the principal component (PC) values from NIR spectra of the emptied and washed capsules. The loadings of the first PC describe a baseline shift in the spectra that arises from a change in water concentration. Lower PC loadings reveal the presence of signals at 1780 and 2200 nm that are not due to water absorbance, confirming the hypothesis that chemical bonds are formed during the formaldehyde-induced crosslinking of the gelatin in SEGCs. Gelatin crosslinking, initiated by formaldehyde migration from the PEG fill into the shell of an SEGC, was detected by NIR spectrophotometry. When NIR was coupled to principal component analysis, a linear relationship was found between the NIR spectra of empty SEGCs and the amount of crosslinking induced by concentrations of formaldehyde in the original fill material.

Near-IR and IR imaging in lipid metabolism and obesity.

Approximately one-third of Americans are classified as obese. There has long been an interest in drug therapies for obesity. Interest in obesity research and in drug interventions in obesity has greatly increased since the discovery of a protein named leptin, one of apparently many competing biological signals in energy metabolism. The complexity of the obesity problem demands new non-invasive and non-destructive methods for monitoring lipid metabolism and energy expenditure to study the competing biological signals and their effects. A new computer algorithm for spectrometric imaging of living subjects is used to remove artifacts arising from subject motion from spectra and images. The algorithm is sufficiently simple to be implemented easily in hardware for real-time video processing. Because the algorithm can be applied to images, thermogenesis and lipid metabolism in interscapular adipose tissue can be observed directly in unrestrained and unanesthetized subjects using an InSb focal plane array video camera. The accuracy and precision of temperature and spectral measurements are established using laboratory references and prototype drugs in test subjects.

Relative bioavailability of itraconazole from an extemporaneously prepared suspension and from the marketed capsules.

The bioavailability of itraconazole from an extemporaneously prepared suspension was compared with its bioavailability from the commercially available capsules. Ten healthy volunteers were fed breakfast and were then randomly assigned to receive either 400 mg of itraconazole 40-mg/mL oral suspension or four 100-mg itraconazole capsules with 240 mL of water. They were not allowed to rest in a supine position for six hours, eat for four hours, or take any beverages for two hours post-dose. Blood samples were taken immediately after the subjects had eaten and at intervals up to 72 hours post-dose. Serum was separated and stored at -70 degrees C. Serum itraconazole and hydroxyitraconazole concentrations were measured by high-performance liquid chromatography. After 14 days, each subject was given the dosage form that he or she did not previously receive, and testing was repeated. Maximum concentration (Cmax) and time to reach maximum concentration (tmax) were determined, and the area under the serum concentration-versus-time curve from 0 to 72 hours (AUC0-72) was estimated. The suspension:capsule ratios of least-squares means for Cmax, tmax, and AUC0-72 for itraconazole were 0.15 (90% confidence interval [CI], 0.11-0.21), 0.95 (90% CI, 0.75-1.20), and 0.12 (9% CI, 0.06-0.23), respectively. The results for hydroxyitraconazole were similar: 0.19 (0.13-0.28), 0.95 (0.81-1.12), and 0.13 (0.07-0.23), respectively. The bioavailability of itraconazole from the extemporaneously prepared suspension is much lower than that from capsules.

Determination of extent of formaldehyde-induced crosslinking in hard gelatin capsules by near-infrared spectrophotometry.

PURPOSE: To predict the degree of crosslinking from formaldehyde-stressed hard gelatin capsules (HGCs) using near-infrared spectrophotometry (NIR). METHODS: HGCs were exposed to a 150 ppb atmosphere of formaldehyde for 2.25, 4.60, 9.42, 16.0 and 24.0 hours. The capsules were filled with fresh amoxicillin, placed in a 90 degrees conical reflector cone, and scanned in a NIR spectrophotometer. Principal component regression (PCR) was employed to analyze the spectra of the intact capsules. Dissolution profiles were then obtained for each experimental group. RESULTS: The dissolution of amoxicillin from the capsules at pH 1.2 was found to decrease with increasing time of exposure to the formaldehyde atmosphere. A set of principal components (PCs) was formed by a linear combination of the absorbance values at each wavelength scanned. A good correlation was established (r2 = 0.963) when PC values from the NIR spectra of the HGCs were regressed against percentage of amoxicillin dissolved at 45 minutes, at pH 1.2. Water content of the capsules was found to be the largest determinant in the variation between HGC spectra at each exposure time. CONCLUSIONS: NIR spectrophotometry, combined with PCR, was successful at not only predicting dissolution of HGCs exposed to formaldehyde, but also at determining which wavelengths contributed most to spectral variation of these stressed HGCs.

Bioequivalence of two orally administered nicardipine products.

The relative bioavailabilities of orally administered nicardipine (Zenith Laboratories) and nicardipine (Cardene Syntex Laboratories) were compared following a single 30 mg dose under fasted conditions using a two-way crossover study with 34 healthy adult male subjects. In a separate study the effect of food on the relative bioavailabilities of these products was assessed following an identical dose by comparing the Zenith product under fasted conditions, the Zenith product under fed conditions, and the Syntex product under fed conditions using a three-way crossover study with 17 healthy adult male subjects. In the fasted study, 90% confidence intervals surrounding ratios (Zenith/Syntex) of least-squares means derived from 1n-transformed data were 0.84-1.02 for AUCt, 0.85-1.04 for AUCinfinity, and 0.86-1.05 for Cmax, clearly demonstrating bioequivalence of the two products. In the food-effect study ratios of least-squares means (Zenith under fed conditions/Zenith under fasted conditions) were 0.62 for AUCt, 0.65 for AUCinfinity, and 0.40 for Cmax, with tmax delayed from 0.906 +/- 0.337 h (Zenith under fasted conditions) to 2.33 +/- 0.717 h (Zenith under fed conditions) and 2.84 +/- 0.834 h (Syntex under fed conditions). Findings indicate that the presence of food in the gastrointestinal tract reduces the bioavailability of orally administered nicardipine. However, ratios under fed conditions (Zenith/Syntex) were very close to unity for each metric, suggesting that the observed food effect is independent of the product formulation. Findings further suggested that food effects on conventional pharmacokinetic metrics might be attributed to alteration of extent, rather than rate, of gastrointestinal absorption. Finally, these results question the applicability of the peak plasma concentration (Cmax) as an index of absorption rate in nicardipine studies.

Bioequivalence of a highly variable drug: an experience with nadolol.

PURPOSE: To assess the bioequivalence of nadolol 40mg and 160mg tablets (Zenith-Goldline Pharmaceuticals) using Corgard 40mg and 160mg tablets (Bristol-Meyers Squibb) as reference products, to estimate the effect of food in the gastrointestinal tract on nadolol bioavailability, and to evaluate the effectiveness of standard pharmacokinetic metrics AUCt, AUC infinity, and Cmax in bioequivalence determinations. METHODS: Four bioequivalence studies were conducted as described in the FDA Guidance. Four additional studies of varying designs were conducted to establish bioequivalence of the 40mg tablet in terms of Cmax. RESULTS: Fasted and food-effect studies of the 160mg tablet clearly established bioequivalence and revealed an unexpected reduction in nadolol bioavailability from test and reference products in the presence of food. The food-effect study of the 40mg tablet (80mg dose) revealed a similar reduction in bioavailability from each product. Fasted studies of the 40mg tablet (80mg dose) established bioequivalence in terms of AUCt and AUC infinity. However, Cmax criteria proved extremely difficult to meet in the initial 40mg fasted study because of the large variability, leading to additional studies and ultimately requiring an unreasonable number of subjects. CONCLUSIONS: Final results clearly established bioequivalence of both strengths and characterized an unexpected food effect which did not appear to be formulation-related. However, the Cmax of nadolol is only slightly sensitive to absorption rate and the relatively large variability of Cmax reduces its effectiveness as a bioequivalence metric. Findings suggest that bioequivalence criteria for highly variable drugs should be reconsidered.

Investigation of passive venovenous shunts during the anhepatic period of canine hepatic transplantation.

The anhepatic period of canine orthotopic hepatic transplantation is usually accompanied by cardiovascular instability due to occlusion of both the portal and inferior systemic venous systems. The present study was undertaken in order to determine some of the hemodynamic and renal alterations that occur during a 2 hour anhepatic period in the dog and to investigate the use of a passive endoportal, endocaval venovenous shunt that places both the portal and infrahepatic vena caval limbs of the shunt directly into the divided ends of these vessels. Three groups of experimental animals were studied. One group had hepatectomy without a shunt and was compared with two other groups which had hepatectomy with some form of venovenous shunting. Systemic arterial blood pressure, portal and inferior vena caval pressure, urinary output, and renal histology were all better maintained when a shunt was used.

Cyclosporine disposition in the dog. Comparison of radioimmunoassay with high-performance liquid chromatographic assay and pharmacokinetics following intravenous administration.

Radioimmunoassay (RIA) and high performance liquid chromatography (HPLC) with ultraviolet absorbance detection have been compared as potential tools for cyclosporine pharmacokinetic studies in dogs. RIA clearly affords greater assay sensitivity, although crossreactivity with cyclosporine metabolites causes an over-estimation of parent drug concentrations with a subsequent reduction in the apparent values of clearance and volume of distribution. HPLC appears to be specific for parent cyclosporine. Thus, with the sacrifice of some sensitivity, HPLC-measured time-course data afford more reliable estimates of cyclosporine pharmacokinetic parameters. After the selection of a dosage regimen from preliminary studies, the pharmacokinetics of i.v.-administered cyclosporine were studied in six adult male mongrel dogs. Following administration of 20 mg/kg by constant-rate 30-min i.v. infusion the time courses of cyclosporine were studied in plasma and urine. Concentrations were measured by reversed-phase HPLC with ultraviolet absorbance detection. Data were fitted to triexponential equations using a digital computer with the CSTRIP and NONLIN programs, and pharmacokinetic parameters were calculated. Present findings suggest that cyclosporine is slowly yet extensively distributed into peripheral body regions that might serve as slowly releasing storage areas. Large volumes of distribution along with moderately slow clearances resulted in long half-lives for the disposition of cyclosporine. Less than 1% of the administered dose was recovered as parent cyclosporine in the urine, suggesting that renal clearance of cyclosporine was negligible. The potential relevance of present findings to cyclosporine therapy of transplant patients is discussed.

The plasma time course of orally-administered cyclosporine in the dog.

The plasma concentration time course of orally-administered cyclosporine has been studied in five adult male mongrel dogs. Cyclosporine concentrations were measured by high performance liquid chromatography with ultraviolet absorbance detection. Cyclosporine plasma concentrations were below assay sensitivity within 24 hours after administration. Peak plasma concentrations ranging from 411.8 to 1735.0 ng/ml were attained between post-administration times of 1 and 2.5 hours. Areas under plasma concentration time course curves (AUC = 4.63 +/- 5.60 micrograms/ml X hr, mean +/- SD) suggest that systemic availability is highly variable between animals. Present and previous findings discourage the oral route of cyclosporine administration for canine transplant research and suggest more extensive pharmacokinetic evaluation of cyclosporine following oral administration in humans.

Nifedipine: a potent inhibitor of contractions in the body of the human esophagus. Studies in healthy volunteers and patients with the nutcracker esophagus.

Nifedipine, a calcium channel blocker, inhibits lower esophageal sphincter pressure but has only minimal effect on esophageal contractions. We investigated the effects of nifedipine on esophageal contractions in 5 healthy volunteers and 10 patients with the nutcracker esophagus. Nifedipine (10, 20, 30 mg) or placebo was ingested as capsules in a double-blind design on 4 separate days. In volunteers, mean distal amplitude decreased 16.6%, 38.4%, and 49.0% as the nifedipine dose was increased. These changes were significantly (p less than 0.05) different from the placebo response and were sustained with higher doses. Patients with the nutcracker esophagus had a similar response, decreasing mean distal amplitude significantly (p less than 0.05) by 16.3%, 36.2%, and 54.2%. In both groups, nifedipine also had a significant (p less than 0.05) dose-dependent depressant effect on distal duration, although to a lesser degree than on amplitude. The percent decrease in distal amplitude showed good correlation (p less than 0.01) with plasma nifedipine concentrations at 60 min. These studies suggest nifedipine may be useful in the treatment of motility disorders of the esophageal body.

Effects of total body irradiation followed by bone marrow transplantation on the disposition kinetics of methotrexate in the rat.

The effects of total body irradiation followed by bone marrow transplantation on the disposition kinetics of intravenously-administered methotrexate have been studied in the Wistar-Furth rat. Eight test animals received total body irradiation (1000 rads) followed by intravenous administration of 3 X 10(8) bone marrow cells per kg body weight. Eight control animals were sham-irradiated and received an equal volume of blank suspension medium. One day after these treatments each rat received methotrexate (25 mg/kg) by rapid intravenous injection and serial blood samples were obtained over a 3 hour period. Serum methotrexate concentrations were measured by high performance liquid chromatography and pharmacokinetic parameters were calculated after NONLIN analysis of data. No significant differences were observed in total body clearances of test and control animals. As methotrexate in the rat is cleared predominantly by renal excretion of unchanged drug, these findings suggest that this process is not affected by radiation. Significantly larger volumes of distribution were observed in test animals. Increased extent of distribution in irradiated animals could be a result of a radiation-induced increase in membrane permeability and/or increased blood flow to irradiated areas. Future studies should assess the clinical significance of such findings.

Reversed-phase high performance liquid chromatographic determination of nifedipine in human plasma.

A reversed-phase high performance liquid chromatographic method is presented by which the calcium channel blocker, nifedipine (NFP), may be measured in human serum using 17-alpha-ethinylestradiol as an internal standard. A mobile phase of phosphate buffer (0.01 M, pH 6.1)/methanol/acetonitrile (20:35:45) passed through a muBondapak C-18 column at 1.0 ml/min produced symmetrical bands for nifedipine and internal standard. A rapid and simple chloroform extraction of NFP from 1 ml serum samples proved to be efficient and reproducible. Recovery over a concentration range of 5-100 ng/ml was 92.3 +/- 5.1% (mean +/- SD, n = 6). Ultraviolet absorbance detection at 235 nm was sensitive to serum NFP concentrations of 5 ng/ml. Between-day coefficients of variation at 100 and 5 ng/ml were 4.0 and 11.4%, respectively (n = 10). Serum concentration data from NFP-treated subjects are presented.

Effects of total body irradiation followed by bone marrow transplantation on the disposition kinetics of mitomycin-C in the rat.

The effects of total body irradiation followed by bone marrow transplantation on the disposition kinetics of intravenously-administered mitomycin-C have been studied in the Wistar-Furth rat. Five test animals received total body irradiation (1000 rads) followed by intravenous administration of 3 X 10(8) bone marrow cells per kg body weight. Five control animals were sham-irradiated and received an equal volume of blank suspension medium. One day after these treatments, each rat received mitomycin-C (10 mg/kg) by rapid intravenous injection and serial blood samples were obtained. Serum mitomycin-C concentrations were measured by high performance liquid chromatography and pharmcokinetic parameters were calculated after NONLIN analysis of data. Smaller total body clearances in test animals were probably due to radiation-induced inhibition of microsomal enzyme activity. Reduced volumes of distribution were observed in test animals although the reason for this is unclear. Future studies should assess the clinical significance of these results.

The stability and antitumor activity of recycled (intravesical) mitomycin C.

Although intravesical mitomycin C (MMC) is effective in the treatment of superficial bladder cancer, its expense is a major factor limiting its use. These authors have analyzed the antitumor activity and stability of MMC following 2-hour intravesical instillation in consideration of recycling the drug or using a smaller dose over a longer retention time. The first voided urine samples from 11 patients who received 40 mg MMC intravesically were measured for MMC content by high performance liquid chromatography (HPLC). An average of 50% of the parent drug was recovered. MMC from the urine samples inhibited the growth of a transplantable murine transitional cell carcinoma as effectively as stock drug. Moreover, MMC is relatively stable in human urine at body temperature. These findings suggest that recovery and reuse of the intravesically administered drug is possible and if sterility and appropriate concentrations can be established for the initial and subsequent doses, the drug may be able to be recycled.

Pharmacokinetics of mitomycin C in non-oat cell carcinoma of the lung.

The disposition kinetics of the cancer chemotherapeutic agent mitomycin C have been studied in six male patients receiving mitomycin C in combination with cisplatin and vinblastine for non-oat cell carcinoma of the lung. Following rapid IV administration of mitomycin C (10 mg/m2), serum concentration-time course data were biexponential, with biologic half-lives of 46.2 +/- 12.1 min (mean +/- SD). Pharmacokinetic analysis of data by the CSTRIP and NONLIN digital computer programs generated parameters which suggested extensive distribution (V area = 656.8 +/- 169.8 ml X kg-1, mean +/- SD) and, as reported for other alkylating agents, rapid elimination (total body clearance = 10.3 +/- 3.2 ml X kg-1 X min-1, mean +/- SD). Interpatient variations in pharmacokinetic parameters were relatively small, suggesting that close monitoring of mitomycin C therapy might be unnecessary in patients with normal renal and hepatic function.

Reversed-phase high-performance liquid chromatographic determination of mitomycin C in human serum.

A reversed-phase high-performance liquid chromatographic method is presented by which the cancer chemotherapeutic agent, mitomycin C, may be measured in human serum. A mobile phase of methanol:water (35:65) passed through a mu-Bondapak C-18 column at a rate of 1.0 ml/min produced a sharp, symmetrical band for mitomycin C. An improved serum extraction procedure, using a reversed-phase sample preparation cartridge, proved to be efficient and reproducible. Recovery over a concentration range of 10-100 ng/ml was 81.6% with a between-day coefficient of variation of 4.6% (n = 5). The within-day coefficient of variation at 50 ng/ml was 5.6% (n = 10). Ultraviolet detection at a wavelength of 365 nm was sensitive to serum concentrations of 10 ng/ml. Serum concentration-time course data from lung cancer patients receiving mitomycin C by rapid intravenous injection are presented.

Platinum kinetics in patients treated with cis-dichlorodiammine platinum (II).

The pharmacokinetics of platinum have been studied in six patients following treatment with cis-dichlorodiammine platinum (CDDP) for metastatic bladder cancer. Each patient received CDDP by 15 min intravenous infusion with total doses ranging from 90 to 120 mg (360 to 480 mg/h). Platinum disposition was biexponential with a relatively rapid distribution and slow elimination. Individual patient elimination half-lives varied from 16.1 to 53.3 h. Although normalized for body weight, individual distribution parameters, V1 and V area, varied from 0.17 to 0.70 L/kg and 0.67 to 1.47 L/kg, respectively. Total body clearance (TBC) also revealed considerable interpatient differences (7 to 40 ml/kg/h). Observed parameter variations could not be explained in terms of existing clinical data. These findings suggest that the pharmacokinetics of platinum in the individual patient might be difficult to predict, and thus serum platinum concentrations should be monitored during cis-dichlorodiammine platinum (II) therapy.

Reversed-phase high-pressure liquid chromatographic determination of serum methotrexate and 7-hydroxymethotrexate.

A reversed-phase high-pressure liquid chromatographic method for determining methotrexate and 7-hydroxymethotrexate in human serum is presented. A mobile phase of acetate buffer (0.2 M, pH 5.5 with 0.03 M ethylenediaminetetraacetate), methanol, and acetonitrile (85.3:8.4:6.3), passed through a mu Bondapak C18 column at 1.5 ml/min produced excellent resolution of sharp, symmetrical bands. An improved extraction process, using a sample preparation cartridge, resulted in analytical recoveries in excess of 90% for methotrexate and 70% for 7-hydroxymethotrexate, permitting the determination of serum concentrations of 2.20 and 2.13 x 10(-7) M, respectively, using only 200 microliter of serum. UV detection at 313 nm provided adequate sensitivity for each component. While the reproducibility for 7-hydroxymethotrexate was approximately equal to previous methods, that for methotrexate was greatly improved. Serum methotrexate data at selected time points following high dose methotrexate therapy are presented.