From the bench to clinical practice: understanding the challenges and uncertainties in immunogenicity testing for biopharmaceuticals.

Unlike conventional chemical drugs where immunogenicity typically does not occur, the development of anti-drug antibodies following treatment with biologics has led to concerns about their impact on clinical safety and efficacy. Hence the elucidation of the immunogenicity of biologics is required for drug approval by health regulatory authorities worldwide. Published ADA 'incidence' rates can vary greatly between same-class products and different patient populations. Such differences are due to disparate bioanalytical methods and interpretation approaches, as well as a plethora of product-specific and patient-specific factors that are not fully understood. Therefore, the incidence of ADA and their association with clinical consequences cannot be generalized across products. In this context, the intent of this review article is to discuss the complex nature of ADA and key nuances of the methodologies used for immunogenicity assessments, and to dispel some fallacies and myths.

Raised haematocrit concentration and the risk of death and vascular complications after major surgery.

BACKGROUND: Preoperative anaemia is associated with adverse postoperative outcomes. Data on raised preoperative haematocrit concentration are limited. This study aimed to evaluate the effect of raised haematocrit on 30-day postoperative mortality and vascular events in patients undergoing major surgery. METHODS: This was a cohort study using the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database. Thirty-day mortality and vascular events, demographics and perioperative risk factors were obtained for adults undergoing major surgery. The adjusted effect of raised (over 0·50) compared with normal (0·41-0·50, American Medical Association reference range) preoperative haematocrit concentration on postoperative outcomes was assessed. Separate sex-specific analyses were also conducted, using haematocrit concentration thresholds commonly used in the diagnosis and management of apparent or absolute erythrocytosis. RESULTS: Some 3961 (2·0 per cent) of 197 469 patients had a raised haematocrit concentration before surgery. After adjustment, the 30-day postoperative mortality rate was higher in patients with raised haematocrit than in those without (odds ratio (OR) 2·23, 95 per cent confidence interval 1·77 to 2·80). Thirty-day rates of deep vein thrombosis (OR 1·95, 1·44 to 2·64) and pulmonary embolism (OR 1·79, 1·17 to 2·73), but not myocardial infarction or stroke, were also higher in patients with a raised haematocrit concentration. The effect on mortality was noted beyond the haematocrit thresholds of 0·48 in women and 0·52 in men; the effect estimates were considerably higher for values exceeding 0·54. Values between 0·41 and 0·45 were not associated with increased mortality risk. Similar observations were noted for venous thrombosis, although with apparent sex differences. CONCLUSION: A raised haematocrit concentration was associated with an increased risk of 30-day mortality and venous thrombosis following major surgery.

A randomized single-blind trial of standard diet versus fiber-free diet with polyethylene glycol electrolyte solution for colonoscopy preparation.

BACKGROUND AND STUDY AIMS: Colonoscopy preparation usually involves the intake of large volumes of polyethylene glycol electrolyte solution (PEG-ES) in combination with a clear-liquid diet (CLD). Liberalization of the diet might enhance the tolerance to PEG-ES without compromising the quality of the preparation. The primary aims of this study were to evaluate the efficacy and tolerability of PEG-ES given with a CLD compared with a fiber-free diet (FFD) for colonoscopy preparation. The incidence of adverse events among patients in the two diet groups was also assessed as a secondary outcome. METHODS: This was a single-center randomized, prospective, single-blind study. A total of 200 patients undergoing colonoscopy were randomized to either CLD or FFD in addition to PEG-ES. RESULTS: Patients in the FFD group were able to drink more PEG-ES (mean +/- SD, 3.9 +/- 0.3 L) compared with those in the CLD group (3.3 +/- 0.7 L) ( P < 0.01). The quality of the preparation was significantly better in the FFD group, with more patients having satisfactory preparations than those in the CLD group (81.4 % vs. 52.0 %; P < 0.001). Tolerance to the preparation was higher in the FFD group compared with the CLD group, with significantly more patients adhering to the FFD regimen ( P < 0.001). There were more adverse events experienced in the CLD group, with odds ratios of 1.9 for nausea (95 % confidence interval [CI] 1.0 - 3.6), 3.8 for vomiting (95 % CI 1.3 - 11.3), and 3.0 for headache (95 % CI 1.5 - 5.9). CONCLUSION: FFD given with PEG-ES on the day before colonoscopy is a more effective regimen than the standard CLD regimen, and is better tolerated by patients.

Effects of angiotensin II blockade on inflammation-induced alterations of pharmacokinetics and pharmacodynamics of calcium channel blockers.

BACKGROUND AND PURPOSE: Inflammation elevates plasma verapamil concentrations but diminishes pharmacological response. Angiotensin II is a pro-inflammatory mediator. We examined the effect of angiotensin II receptor blockade on the pharmacokinetics and pharmacodynamics of verapamil, as well as the binding properties and amounts of its target protein in calcium channels, in a rat model of inflammation. EXPERIMENTAL APPROACH: We used 4 groups of male Sprague-Dawley rats (220-280 g): inflamed-placebo, inflamed-treated, control-placebo and control-treated. Inflammation as pre-adjuvant arthritis was induced by injecting Mycobacterium butyricum on day 0. From day 6 to 12, 30 mg kg(-1) oral valsartan or placebo was administered twice daily. On day 12, a single oral dose of 25 mg kg(-1) verapamil was administered and prolongation of the PR interval measured and plasma samples collected for verapamil and nor-verapamil analysis. The amounts of the target protein Ca(v)1.2 subunit of L-type calcium channels in heart was measured by Western blotting and ligand binding with (3)H-nitrendipine. KEY RESULTS: Inflammation reduced effects of verapamil, although plasma drug concentrations were increased. This was associated with a reduction in ligand binding capacity and amount of the calcium channel target protein in heart extracts. Valsartan significantly reversed the down-regulating effect of inflammation on verapamil's effects on the PR interval, and the lower level of protein binding and the decreased target protein. CONCLUSIONS AND IMPLICATIONS: Reduced responses to calcium channel blockers in inflammatory conditions appeared to be due to a reduced amount of target protein that was reversed by the angiotensin II antagonist, valsartan.

Rapidly dissolving formulations for quick absorption during pain episodes: ibuprofen.

OBJECTIVE: Pain or its associated trauma impairs oral absorption of drugs due, perhaps, to a diminished disintegration and dissolution rate secondary to suppression of the vagal nervous system. We first examined whether ibuprofen absorption is impaired on suppressing vagus nerve activity in a rat model. Secondly, we examined if ibuprofen absorption in rats during vagal suppression and in humans experiencing dental pain is improved by enhancing disintegration and dissolution rate of the administered formulation. METHODS: Vagal suppression was achieved in rats by administering 20 mg/kg propantheline i.p. 2 h and 1 h before gastric gavage of 20 mg/kg ibuprofen as small crushed pieces of a regular release and a fast-dissolving ibuprofen caplets. In humans, after surgical removal of wisdom teeth and emergence of pain, 2 A 200 mg ibuprofen as regular released (n = 14) and fast-dissolving (n = 12) caplets were administered. Serial blood sample were collected for bioavailability studies. RESULTS: Vagal suppression resulted in significantly decreased absorption rate of ibuprofen enantiomers following administration of the regular release but not after fast-dissolving formulation. Human dental pain was associated with significantly slower absorption of ibuprofen enantiomers from the regular released as compared with the fast-dissolving caplets. Within the first hour post-dose the area under the plasma drug concentration-time curve was raised to 2.7-fold (p < 0.05) after the fast-dissolving as compared with the regular release formulations. There was a 1-h difference in the peak concentration time (tmax) between the two caplets. CONCLUSION: Impaired drug absorption appears to be due to slow disintegration and dissolution encountered during pain episodes.

A wandering spleen presenting as a pelvic mass: case report and review of the literature.

Wandering spleens are rare clinical entities found more commonly in females. We report a young female patient found to harbour a pelvic spleen. The literature regarding this rare ectopia is reviewed. The wandering spleen should be considered in the differential diagnosis of pelvic masses.

Trauma epidemiology and outcome in a developing country: perspectives from a university teaching hospital in Beirut.

This study aims at evaluating trauma care at the American University of Beirut Medical Centre (AUB-MC) and comparing it to the norms established by the Major Trauma Outcome Study (MTOS). From January 2001 until January 2003, data necessary to calculate probability of survival using the Trauma Injury Severity Score methodology were collected. M, W, Z, Ws and Zs statistics were calculated to compare outcome at AUB-MC to the MTOS dataset. A total of 873 patients were included in the study. W statistics was calculated at 0.35 with Z score for the overall sample of 0.081 indicating that there was no statistically significant difference in survival between this group and the MTOS group. In a developing country a hospital achieves trauma outcomes similar to the MTOS dataset. Further studies looking at trauma care in Lebanon as a whole are needed.

Solid pseudopapillary neoplasm of the pancreas in a 12-year-old female: case report and review of the literature.

Solid pseudopapillary neoplasms of the pancreas (SPNP) are rare pancreatic tumors that occur predominantly in young women, with very few cases reported in men. While the origin of the tumor may be unclear, it is characterized by a distinct histological appearance and a clinical course highlighting its low malignant potential. SPNP have an excellent prognosis and are potentially curable provided they are managed appropriately by complete surgical resection. In the rare instances where metastatic disease is encountered, surgical debulking has been shown to prolong survival. The role of chemotherapy and radiation therapy in the management of SPNP is still controversial. We report here on an unusual occurrence of SPNP in the area of the head of the pancreas in a 12-year-old female treated by pancreatico-duodenectomy, together with a review of the literature.

Low-dose methotrexate kinetics in arthritis.

Twelve patients with either rheumatoid or psoriatic arthritis were injected with a 10-mg bolus dose of methotrexate (MTX) either intramuscularly (n = 6) or intravenously (n = 6) and the MTX concentration in their sera was determined by radioimmunoassay. MTX concentration--time data fitted triexponential equations. Doses injected intramuscularly were rapidly and completely absorbed. There were no significant intergroup differences in drug mean t 1/2, volume of distribution, and total body clearance. In nine patients serum MTX concentrations remained above the suggested critical level of 0.01 mumol throughout the 24-hr study irrespective of the route of administration, but MTX did not cumulate in the serum. We conclude that the behavior of low doses of MTX in patients with arthritis closely resembles the pattern in patients receiving intermediate and high doses for the treatment of neoplasia.

Lack of pharmacokinetic interaction between cimetidine and pirenzepine.

The oral administration of pirenzepine (PRZ), an antimuscarinic agent, has a variable effect on gastric acid secretion in patients with duodenal ulcer, and it seems to potentiate cimetidine-induced inhibition of secretion. The possibility of a pharmacokinetic interaction between these drugs was examined in eight patients who received cimetidine and PRZ alone and in combination in a crossover fashion. Cimetidine, 600 mg b.i.d., PRZ, 50 mg b.i.d., or combination therapy were each given for 1 week before the study. Serum samples were serially drawn during each dosing interval for determination of cimetidine and PRZ concentrations by HPLC and RIA, respectively. Cimetidine given alone or with PRZ exhibited diurnal variation, as the peak serum concentration was lower after the nighttime dose than after the morning dose. PRZ showed intersubject variation. However, each drug failed to alter the pharmacokinetic indices of the other. The times to attain the peak serum concentration were not significantly different for cimetidine alone or with PRZ, arguing against an effect of PRZ on gastric motility in the doses we used. The greater and prolonged acid inhibition with the combination of cimetidine and PRZ, therefore, may stem from a synergistic action of both drugs on receptor sites on gastric parietal cells.

Dose-response relationship of doxapram in the therapy for refractory idiopathic apnea of prematurity.

Eighteen infants with idiopathic apnea of prematurity refractory to therapeutic levels of aminophylline were treated with incremental doses of doxapram beginning at 0.5 mg/kg/h. Continuous recording of heart rate, thoracic impedance, and transcutaneous PO2 demonstrated that 47% of the infants satisfied objective response criteria at the lowest dose, 53% responded at 1.0 mg/kg/h, 65% at 1.5 mg/kg/h, 82% at 2.0 mg/kg/h, and 89% at the highest allowed dose of 2.5 mg/kg/h. The mean serum doxapram concentration at the response dose was 2.9 +/- 1.3 micrograms/mL, and all infants who responded had levels greater than 1.5 micrograms/mL. BP was significantly elevated at doses higher than 1.5 mg/kg/h (P less than .05). Minute ventilation significantly increased and PCO2 significantly decreased as the doxapram dosage was increased (P = .02). Terminal elimination half-life was 9.9 +/- 2.9 hours. When doxapram is used for treatment of refractory neonatal apnea the starting dosage should be no more than 0.5 mg/kg/h.

Bioequivalence of chiral drugs. Stereospecific versus non-stereospecific methods.

Guidelines for bioequivalence of non-racemic pharmaceuticals are abundant in the literature. However, few guidelines exist for the bioequivalence of racemic drugs which consist of 2 or more stereoisomers. The aim of this article is to address the question of whether the bioequivalence of racemic drugs should be based on the measurement of the individual enantiomers or that of the total drug. Several pharmacokinetic-pharmacodynamic cases are examined to test the validity of extrapolating the bioequivalence of racemic drugs to that of their individual enantiomers after administration of the racemate; simulation and experimental data are presented to support these cases. It is shown that for drugs which exhibit non-linear pharmacokinetics, the results of bioequivalence studies based on the total drug may differ from those based on the individual enantiomers. Similar discrepancies can be shown for a racemic drug with linear pharmacokinetics whose enantiomers substantially differ from each other in their pharmacokinetic parameters. Therefore, it is suggested that stereospecific assays be used for these drugs. Additionally, it is recommended that for racemic drugs which undergo chiral inversion, and for most products with modified release characteristics, the bioequivalence be assessed using stereospecific assays. Conversely, for racemic drugs with linear pharmacokinetics and minimal to modest stereoselectivity in their kinetic parameters, and for those with non-stereoselective pharmacodynamics, the use of stereospecific analytical methods are not warranted. Finally, the limited, controversial literature in favour of or against the use of stereospecific assays in bioequivalence of chiral drugs are reviewed and a preliminary guideline is proposed.

Clinical pharmacokinetics of ketoprofen and its enantiomers.

Ketoprofen, a potent nonsteroidal anti-inflammatory drug (NSAID) of the 2-arylpropionic acid class, has been used clinically for over 15 years in Europe, and has recently been introduced in the United States. Although it possesses a chiral centre, with only the S-enantiomer possessing beneficial pharmacological activity, all ketoprofen preparations to date are marketed as the racemate. Ketoprofen exhibits little stereoselectivity in its pharmacokinetics. The enantiomers have similar plasma time-courses and do not seem to interact with one another. Hence, the data generated using nonstereospecific assays may be used to explain the pharmacokinetics of individual enantiomers. The absorption of ketoprofen is rapid and almost complete when given orally. Sustained release dosage forms are available, which may be beneficial due to the short terminal phase half-life of ketoprofen (1 to 3h). They may also decrease local gastrointestinal side effects. Although with these preparations the peak plasma drug concentration is reduced and time to peak is prolonged, the bioavailability is the same as that with regular release counterparts. Ketoprofen binds extensively to plasma albumin, apparently in a stereoselective manner. Substantial concentrations of the drug are attained in synovial fluid, the proposed site of action of NSAIDs. It is eliminated following extensive biotransformation to inactive glucuroconjugated metabolite. There is about 10% R to S inversion upon oral administration. Conjugates are excreted in urine, and virtually no drug is eliminated unchanged. The excretion of conjugates is closely tied to renal function; accumulation of conjugates occurs in the elderly, but not in young subjects or patients. Significant drug interactions have been demonstrated for probenecid, aspirin and methotrexate. There appears to be circadian variation, particularly in the absorption of ketoprofen. The relationship between concentration and anti-inflammatory effect has yet to be elucidated for this drug.

Clinical pharmacokinetics of drugs used in juvenile arthritis.

Juvenile arthritis is defined as the occurrence of objective evidence of arthritis for a minimum of 6 weeks, in a child 16 years of age or younger. With a reported incidence of 9 to 19.6 per 100,000 children, juvenile arthritis is considered to be a rare disease. There is no known cure; however, up to 75% of patients will undergo remission by late adolescence. Drugs used in the treatment of juvenile arthritis are divided into 2 major classes: (a) the nonsteroidal anti-inflammatory drugs (NSAIDs) including salicylates, naproxen, ibuprofen, fenoprofen, ketoprofen, flurbiprofen, indomethacin, sulindac, tolmetin and diclofenac, and (b) disease modifying agents which encompass drugs such as antimalarial agents, gold, methotrexate, penicillamine and sulfasalazine. In almost all the reports dealing with the pharmacokinetics of NSAIDs, the level of disease activity has not been noted. The level of activity is important since, during a flare, the plasma albumin may fall to the point that it causes a substantial and clinically significant increase in the unbound serum concentration of highly bound drugs. The relationship between the concentration of these drugs in the systemic circulation and their efficacy is not clear. However, for many of them, therapeutic drug monitoring is recommended as a means of reducing the possibility of toxic reactions. Further pharmacokinetic and -dynamic evaluations are needed for many of these drugs in juvenile arthritis.

Clinical pharmacokinetics of ketorolac tromethamine.

Ketorolac is a new chiral nonsteroidal anti-inflammatory drug (NSAID) which is marketed for analgesia as the racemate. The drug is administered as the water soluble tromethamine salt and is available in tablets or as an intramuscular injection. The absorption of ketorolac is rapid, Cmax being attained between 20 to 60 min. Its oral bioavailability is estimated to range from 80 to 100%. The drug is extensively bound (> 99%) to plasma proteins and has a volume of distribution (0.1 to 0.3 L/kg) comparable with those of other NSAIDs. Only small concentrations of ketorolac are detectable in umbilical vein blood after administration to women in labour. The elimination half-life is between 4 and 6h and is moderate in comparison with other NSAIDs. The area under the plasma concentration-time curve of ketorolac is proportional to the dose after intramuscular administration of therapeutic doses to young healthy volunteers. Ketorolac is extensively metabolised through glucuronidation and oxidation; little if any drug is eliminated unchanged. Most of the dose of ketorolac is recovered in the urine as conjugated drug. Although ketorolac is excreted into the breast milk, the amount of drug transferred comprises only a small fraction of the maternal exposure. Little stereoselectivity was present in the pharmacokinetics of ketorolac in a healthy volunteer receiving single intravenous or oral doses. The elderly exhibit reduced clearance of the drug. Renal insufficiency appears to cause an accumulation of ketorolac in plasma, although hepatic disease may not affect the pharmacokinetics.

Etodolac clinical pharmacokinetics.

Etodolac is a chiral nonsteroidal anti-inflammatory drug (NSAID) that is marked as the racemate. Currently, the drug is available in several countries for the treatment of arthritis and the alleviation of pain. Etodolac possesses several unique disposition features mainly due to its stereoselective pharmacokinetics. In plasma, the concentrations of the 'inactive' R-enantiomer are about 10-fold higher than those of the active S-enantiomer, an observation that is novel among the chiral NSAIDs. In common with other NSAIDs, the drug is highly plasma protein bound, and undergoes virtually complete biotransformation to oxidised metabolites and acyl-glucuronides. Etodolac is well absorbed, with maximal plasma concentrations attained within 1 to 2 hours in healthy volunteers. The area under the plasma concentration-time curve of racemic etodolac increases linearly with doses used clinically. The elimination half-life of etodolac is between 6 and 8 hours in plasma, and is similar for both enantiomers. The volume of distribution (Vd) of racemic etodolac is higher than that of most other NSAIDs mainly because of the extensive distribution of the S-enantiomer. The very large Vd of the S-enantiomer, compared with its antipode is, at least in part, due to its less extensive plasma protein binding. In addition to the unchanged drug, substantial concentrations of the acyl-glucuronides of etodolac are found in both plasma and the synovial fluid of patients with arthritis. A limited amount of conjugated etodolac is found in the bile of patients following cholecystectomy. Hepatic cirrhosis has no effect on the pharmacokinetics of racemic etodolac, although the effect of hepatic dysfunction on the pharmacokinetics of the individual enantiomers has yet to be determined. In elderly non-arthritic individuals with excellent kidney function, aging does not affect the pharmacokinetics of etodolac. The pharmacokinetics of the drug in patients with renal failure have not been published, and may be important because the acyl-glucuronides are renally cleared.

Drug-disease interactions: reduced beta-adrenergic and potassium channel antagonist activities of sotalol in the presence of acute and chronic inflammatory conditions in the rat.

Inflammation may influence response to pharmacotherapy. We investigated the effect of inflammation on response to sotalol, a beta-adrenergic receptor and potassium channel antagonist. Racemic sotalol (40 mg kg(-1)) was administered to healthy, acutely (interferonalpha 2a-induced) and chronically (Mycobacterium butyricum-induced adjuvant arthritis) inflamed male Sprague-Dawley rats (n=4 - 6/group). Another group of interferon-treated rats received 3 mg kg(-1) of anti-TNF antibody infliximab. Electrocardiogram (ECG) recorded and plasma sotalol concentration monitored for 6 h. The study was repeated in acutely inflamed rats following administration of stereochemically pure individual sotalol enantiomers [40 mg kg(-1) S (potassium channel blocker) or 20 mg kg(-1) R (beta-adrenergic/potassium channel blocker)]. Chronic arthritis was readily evident. Acute arthritis was associated with elevated segmented neutrophils and increased plasma nitrite and tumour necrosis factor (TNF) concentrations. Sotalol affected ECG in all rats. In both inflamed groups, however, response to sotalol in prolongation of QT interval (potassium channel sensitivity) was reduced. The effect of PR interval (beta-adrenergic activity) was also reduced following administration of the racemate and R-enantiomer. No significant differences in pharmacokinetics were observed between control and inflamed rats. Infliximab reduced nitrite and TNF concentrations and reversed the effect of acute inflammation on both PR and QT intervals. The reduced electrocardiographic responses to sotalol is likely due to the influence of inflammation on the action of the drug on both beta-adrenergic and potassium channel receptors secondary to over-expression of pro-inflammatory cytokines and/or nitric oxide. Our observation may have therapeutic consequences in all conditions where inflammatory mediators are increased.