Pharmacology Journal

Troglitazone, 4-thiazolidinedione, is an insulin action enhancing agent under clinical development for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). Administration with, or up to 30 min after, food significantly improves absorption of troglitazone due probably to enhanced bile solubilisation and dissolution time.

Cholestyramine is an anion exchange resin with lipid-lowering properties. Taken orally, it is not absorbed from the gastrointestinal tract but is excreted unchanged in the faeces. Cholestyramine lowers LDL-cholesterol by binding to bile acids and preventing their enterohepatic circulation.

Therefore, because of the physicochemical properties of troglitazone, investigations were undertaken to determine the influence of concomitant administration of cholestyramine on the pharmacokinetics of troglitazone.

Preclinical studies

An in vitro study using [¹⁴C]-troglitazone, in conditions mimicking the small intestine in vivo, was conducted to determine the adsorption of troglitazone by cholestyramine. [¹⁴C]-troglitazone was dissolved in ethanol to give a solution used to spike 3 and 500  μg  ml⁻¹ incubates of solutions of human serum albumin (HSA) prepared in Sorensen’s buffer pH  6. HSA was used to increase the aqueous solubility of the drug and to mimic food proteins in the gut. The percentage loss (P) of soluble radiolabel from each test incubate compared with the appropriate control was used as a measure of absorption. This was determined from the following equation: where Rc=mean radioactivity concentration in the control incubate supernatant (d  min⁻¹/200  μl) and Rt=mean radioactivity concentration in the corresponding test incubate supernatant (d  min⁻¹/200  μl).

Cholestyramine (4×1  g) was administered once, 1  h prior to receiving a single oral tablet of troglitazone 200  mg, and on three occasions 1, 2 and 4  h post-dose, as an aqueous suspension (1  g in 10  ml water) by gavage to 11 Beagle dogs. Blood sampling was carried out from the cephalic vein at 0.5, 1, 2, 3, 4, 6, 8, 10 and 24  h following administration. Plasma samples were assayed by reversed phase h.p.l.c. with u.v. detection as previously described. A paired t-test was used to compare previously obtained (control) AUC (area under the plasma concentration time curve), C_max (maximum observed plasma concentration) and t_max (time at which C_max was reached) values for each dog with corresponding values obtained from this study.

Clinical study

Study methodology has been described elsewhere. Twelve healthy subjects each received a single oral dose of troglitazone 400  mg alone and with cholestyramine 12  g (3×4  g sachets) in random order as part of an open, two-way crossover study. Troglitazone was taken 30  min after a standardized breakfast and cholestyramine was given as an aqueous solution 1  h after ingestion of troglitazone. Blood samples were taken pre-dose and at 15, 30, 45, 60, and 90  min and 2, 3, 4, 6, 8, 12, 24, and 48  h post-dose.

Pharmacokinetic parameters, C_max, t_max, t_1/2 (elimination half-life) and AUC were calculated for troglitazone and its main metabolites as described previously.

The normal parametric method was not used in these analyses because one subject was reported with no absorption of troglitazone and therefore, a zero value for AUC; thus log transformation was not possible. The pharmacokinetic parameter values for AUC, C_max, and t_max for troglitazone, sulphate and quinone metabolites were analysed using Koch’s non-parametric method based on the Wilcoxon Rank Sum test. An estimate of the median difference between troglitazone with cholestyramine and troglitazone alone, together with a 95% confidence interval was calculated. Subjects for whom it was not possible to calculate t_max due to poor absorption were excluded from the analysis of t_max. The analysis of t_1/2 for troglitazone and sulphate metabolite was performed following log transformation using analysis of variance allowing for subjects, periods and treatments. Subjects for whom it was not possible to calculate t_1/2 due to a poorly defined terminal elimination phase were excluded from the analysis of t_1/2.

Troglitazone, 4-thiazolidinedione, an insulin action enhancing agent, is one of a new class of agents, the thiazolidinediones, currently in clinical development for the treatment of non insulin dependent diabetes (NIDDM). Animal models of NIDDM provide evidence that troglitazone suppresses hepatic gluconeogenesis and improves insulin sensitivity and responsiveness by potentiating insulin-induced peripheral glucose uptake. In vitro studies have shown that troglitazone acts directly on muscle and liver cells to increase glucose utilisation and reduce glucose production. Furthermore, troglitazone may have beneficial effects on abnormal lipid levels such as triglycerides, high density lipoprotein (HDL) cholesterol and non-esterified fatty acids which are implicated as risk factors for cardiovascular disease in NIDDM. In clinical studies, troglitazone is well tolerated and achieves good glycaemic control in NIDDM patients, accompanied by favourable changes in abnormal lipid profiles.

Troglitazone is a white to pale yellow crystalline powder (pK_a 6.1, 12.0) which is practically insoluble in water (solubility approximately 0.02  mg  ml⁻¹ ). It has an oil/water partition coefficient of 2.4 at pH  7 and is absorbed largely via the small intestine. Following oral administration, troglitazone is primarily metabolised to a sulphate conjugate and an oxidative quinone metabolite. A glucuronide conjugate is also formed. Troglitazone has a half-life of approximately 10–15  h and that of its major metabolite is approximately 20  h [unpublished data]. A high degree of variability in area under the plasma concentration-time curve (AUC) occurs (approximate CV of 30%), [unpublished data].

It is important therefore to assess the effect of food on the gastrointestinal absorption of troglitazone. This study was designed to investigate the effect of concomitant food intake and food intake 30  min prior to dosing on the pharmacokinetic profile of troglitazone.

hirteen healthy males (mean age 31 years, range 20–41 years) were enrolled in this open, randomized, three-way, cross-over study. They weighed between 64.4  kg and 95.2  kg (mean 79.1  kg), and ranged in height from 1.70–1.86  m (mean 1.76  m). Each subject received one tablet of troglitazone (400  mg) following an overnight fast 1) alone, 2) concomitantly at the start of a standardized diabetic breakfast and 3) 30  min after a standardized diabetic breakfast. All treatments were given with water (200  ml) and were separated by a minimum of 7 days. Apart from the standardized breakfast (approximately 64  g carbohydrate, 25  g fat, 20  g protein and 600 calories) of cornflakes (35  g), skimmed milk (150  ml), a slice of wholemeal toast (25  g) and butter (25  g) and an apple, food was not allowed until 4  h after dosing; a light lunch was then provided.

All subjects were free from any significant medical condition, received no regular medication for 4 weeks prior to the study and no drug treatment for 48  h prior to the study. Subjects were not allowed to consume food or drink (apart from water) from 22.00  h on the night prior to the study. Water was permitted up to midnight prior to dosing. Smoking, alcohol and strenuous exercise were not permitted from the evening prior to drug administration to after the last blood sample on each occasion. Subjects returned between 7 and 10 days following the end of the study for laboratory safety tests.

The study protocol was reviewed and approved by the Ethics Review Committee of Glaxo Wellcome Research and Development Ltd. All subjects gave written consent to participate and the study was conducted at the department of Clinical Pharmacology, Glaxo Wellcome Research and Development Ltd, Ware, Hertfordshire, UK in accordance with the provisions of the revised Declaration of Helsinki (1964).

Assessments

Blood samples (5  ml) were taken pre-dose and at the following nominal times post-dose: 15, 30, 45, 60 and 90  min and 2, 3, 4, 8, 12, 24 and 48  h. Blood samples were centrifuged at 1500  g for 10  min and the resultant plasma frozen at −20°  C until required for assay. Plasma samples (300  μl) were assayed by h.p.l.c. with ultraviolet detection (230nm) following liquid/liquid extraction with ethyl acetate:hexane (3ml, 90:10 v/v). The organic phase was dried down under nitrogen and the samples were re-constituted in ethanol (100  μl) prior to injection of an aliquot (30  μl) onto the h.p.l.c. column (YMC-Pack ODS A-314G, 300×6  mm) which was maintained at 35°  C and eluted at a flow rate of 1.2ml  min⁻¹ with a mobile phase consisting of acetonitrile/water/phosphoric acid (60:40:0.08 v/v). Chromatographic peak height ratios of each analyte relative to the internal standard (9-acetylanthracene) were quantified by reference to linear calibration lines prepared freshly on each occasion of assay. A calibration range of 0.1 to 6.4  μg  ml⁻¹ and a limit of quantification of 0.1  μg  ml⁻¹ for both troglitazone and the sulphate metabolite were used. Assay performance was monitored by quantifying three quality control samples in duplicate which were required to be within 15% of their nominal concentrations.

The assay was validated and the intra assay precision ranged from 1.7 to 28% for troglitazone and from 1.1 to 13% for the sulphate with a bias of −12 to +3% for troglitazone and +0.8 to +22% for the sulphate. The inter assay variation was determined for three analyte concentrations on seven occasions and precision varied from 13 to 14.8% for troglitazone (bias +0.4 to +7.8%) and from 4.2 to 16.2% for the sulphate (bias −0.4 to +9.5%). Troglitazone and the sulphate metabolite were stable in plasma for up to 12 months and three freeze-thaw cycles had little effect on their concentrations.

Data analysis

The following parameters were derived for each subject from the plasma troglitazone and sulphate metabolite concentration data. The maximum observed plasma concentration (C_max ) and the time at which C_max was reached (t_max ) were noted directly. For troglitazone the AUC to the last measurable time point (AUC_last ) was calculated by the log-linear trapezoidal rule and the lag-time before appreciable absorption of troglitazone occurred (t_lag ) was recorded as the last timepoint with a value below the limit of quantification prior to absorption. For the sulphate metabolite, the terminal elimination rate constant (λ_z ) was calculated by linear least-square regression using logarithmically transformed points in the terminal phase. The terminal phase plasma half-life (t_1/2 ) was calculated by ln(2)/λ_z. AUC was extrapolated to infinity (AUC_∞ ) by adding the ratio of the last measurable concentration divided by the elimination rate constant. The plasma concentrations of troglitazone were not adequately defined in the terminal phase and therefore λ_z and the corresponding t_1/2 and AUC parameters could not be measured.

The pharmacokinetic parameter values, C_max and AUC for troglitazone and the sulphate metabolite were analysed using analysis of variance allowing for subjects, periods and treatments. Separate tests for treatment by period interaction and for carry-over effects were performed for each of the variables analysed parametrically. Where there was evidence of a carry-over effect, this was included in the statistical model. The pharmacokinetic parameter t_1/2 for the sulphate metabolite was therefore analysed using analysis of variance allowing for effects due to subject, period, carryover and treatment. A log transformation was performed for each parameter in order to satisfy the constant variance assumption for the analysis of variance. Geometric mean values for each of the treatments were evaluated together with ranges. Estimates of pairwise treatment differences were calculated together with 95% confidence intervals.

Values of t_max and t_lag were summarized by the treatment medians and compared between pairs of treatments using the Wilcoxon signed rank test, with corresponding 95% confidence intervals for the estimates of treatment difference derived.

Safety

Adverse events occurring during the study and laboratory parameters for clinical chemistry, haematology and urinalysis were recorded at screening, pre-dose, 48  h post-dose and between 7 and 10 days following the end of the study.

The enzyme (CYP2C19) that 4′-hydroxylates S- mephenytoin (S-MP) is a genetically polymorphic cytochrome P450. Based on the activity of CYP2C19, individuals are phenotyped as extensive metabolizers (EM) and poor metabolizers (PM). S-MP is extensively metabolized in EMs; this results in a lower S/R ratio measured in EMs than that in PMs. The PM phenotype is inherited as a Mendelian autosomal recessive trait involving two alleles at a single gene locus. Recently, two genetic mutations (m₁ and  m₂ ) have been found to result in PM of S-MP. The m₁ mutation, caused by a single base pair (G→A) mutation in exon 5 corresponding to base pair 681 of the cDNA, creates an aberrant splice site. This defect accounts for 75–85% of Caucasian and Japanese PMs. The  m₂ mutation consisting of a G→A transition at base 636 has been found only in Oriental populations. It is well known that some of the enzymes in P4502C subfamily (including CYP2C19) can be induced in both animals and humans; also, chronic use of mephenytoin causes autoinduction. Treating EMs and PMs of S-MP with rifampicin, using mephenytoin as a probe, we previously reported that CYP2C19 activity was inducible in EMs but not in PMs. Recently we found that some of the PMs of S-MP whose genotypes were defined as m₁/m₁ or m₁/m₂ excreted as much as 8% of the dose as 4′-OH-MP. In addition, the S/R mephenytoin ratio of those m₁ mutation heterozygous was lower than that of  m₂ mutation heterozygou. These data suggest that the activity of 4′-hydroxylase of those heterozygous with m₁ mutation is different from that of heterozygous with  m₂ mutation. Moreover, the mephenytoin S/R ratio of the heterozygous (wt/m₁ and wt/m₂ ) is slightly higher than that of homozygous (wt/wt), indicating that genetics affects the 4′-hydroxylation of S-MP. Therefore, we hypothesize that the activity of 4′-hydroxylase in PMs with m₁ mutation may not be lost completely and consequently its activity in these PMs should be inducible and the induction may be related to gene dose. The study was designed to test this hypothesis.

Seven male Chinese EMs of S-MP and five PMs with m₁ mutation whose genotype was defined in previous studies, aged 19 to 22 (21±1 years, mean±s.d.), weighing 54 to 75  kg (58.9±6.2  kg) were enrolled in the study. No subject had had recent illness, and none had taken drugs for at least 2 weeks prior to or during the study. No subject had any abnormalities on physical examination or any biochemical evidence of renal or hepatic disorders. Four of the EMs were genotyped as homozygous (wt/wt), three as heterozygous (two as wt/m₁ and one as wt/m₂ ); four of the PMs were genotyped as m₁/m₁, one as m₁/m₂. The study protocol was approved by the Ethics Committee of Hunan Medical University and written consent was obtained from the subjects.

After an overnight fast, all the subjects took 100  mg racemic mephenytoin (Mesantoin^®, Sandoz Pharmaceuticals, Inc.). 0–8  h and 8–24  h urine were collected and the volume was measured. Urine samples were stored at −15°  C until assayed. From the second day on, every subject received 300  mg rifampicin (Rifampicin Capsule, 150  mg/capsule, pitch number: 950106, Xinyang Pharmaceuticals of Henan Province, PRC) daily for 22 days and racemic mephenytoin was again administered along with the final dose of rifampicin, urine samples were collected and stored in the same way as mentioned above.

The 0–8  h urinary mephenytoin S/R ratio was measured by gas chromatography. The amount of 4′-OH-MP excreted in the 0–8  h and 8–24  h urine was determined by high performance liquid chromatography.

The data were analyzed by paired t-test and rank sum test with P<0.05 as the minimal level of statistical significance.

Marimastat (BB-2516) is an inhibitor of the family of enzymes known as matrix metalloproteinases (MMPs). These enzymes are considered to be primarily responsible for the degradation of extracellular matrix proteins in processes of tissue formation and remodelling. Under normal conditions the activity of MMPs is controlled at several levels, including their secretion as latent proenzymes and inhibition by endogenous tissue inhibitor of metalloproteinases (TIMPs). However, excessive MMP activity is now thought to play an important role in the pathogenesis of several diseases including cancer, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, neurodegenerative diseases [7], and cerebral haemorrhage. It is thought that MMP inhibitors may have utility in the treatment of some of these diseases.

The first two MMP inhibitors to be tested in patients were ilomastat (GM6001) and batimastat (BB-94). Neither compound showed good oral bioavailability and indications were sought that allowed alternative routes of administration. Ilomastat was administered as a topical agent in patients with corneal ulceration while batimastat was given as an intraperitoneal or intrapleural suspension in patients with malignant effusions. More recently, marimastat has been identified as one of the first MMP inhibitors to show good absorption following oral administration to animals. It is a broad spectrum reversible inhibitor of MMPs exhibiting IC₅₀s of 5 nm, 6 nm, 3 nm, 16 nm, 230 nm and 5 nm against interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), matrilysin (MMP-7), stromelysin-1 (MMP-3) and metalloelastase (MMP-12), respectively.

Safety, pharmacology and toxicology studies suggest that marimastat has low oral and intravenous toxicity in animals. The only chronic target organ toxicity elicited has been inflammation of the tendons and joint ligaments of marmosets. On the basis of these preclinical data, it was thought that marimastat was a suitable compound to introduce into man with a view to further investigation of its role as an anti-cancer agent. The objectives of these first two studies were to assess the tolerability and pharmacokinetic profiles of single and continuous dosing of oral marimastat in healthy male volunteers.

Follicle stimulating hormone (FSH) is one of the key hormones regulating reproductive function in both genders. FSH, a heterodimeric glycoprotein, is produced in the anterior pituitary gland and then secreted into the general circulation. In the female it stimulates growth and maturation of ovarian follicles; in males it promotes spermatogenesis. One indication for human FSH is the stimulation of multiple follicular development in ovulatory patients undertaking in vitro fertilisation and embryo transfer (IVF-ET). Currently available human FSH preparations are extracted from the urine of postmenopausal women and have low specific activity due to the presence of non-specific co-purified urinary proteins. Biotechnology has made possible production of a high specific activity recombinant human FSH preparation through an in vitro process independent of urine collection (recombinant-human FSH).

The primary objective of the present analysis was to characterize the population pharmacokinetics of intramuscular (i.m.) urinary-human FSH (u-hFSH) and subcutaneous (s.c.) recombinant-human FSH (r-hFSH) in a large group of patients undergoing IVF-ET, and to assess which covariates, if any, influence the variability of FSH pharmacokinetics. The study was a multicentre, randomized, open, parallel group study to compare the efficacy and safety of r-hFSH and u-hFSH. The study was designed with the intention of employing a population approach to the data analysis and thus only sparse numbers of blood samples were taken from each patient during the course of the study.

Administration of an endogenous substance in a pharmacokinetic study gives rise to certain problems that are not normally present with xenobiotic drugs. The resulting plasma concentrations are the result of both endogenous and exogenous parts and these must be distinguished before the pharmacokinetics of the exogenously administered substance can be correctly characterized. The information about many of the model parameters is sparse and therefore the population approach has an advantage over traditional pharmacokinetic methods since it pools the available information across many subjects.

Preliminary analysis of the sparse data indicated that absorption was the rate limiting step for the pharmacokinetics of both u-hFSH and r-hFSH. Since the sampling design in the study did not allow determination of the elimination half-life and volume/bioavailability (V /F ), previously collected experimental data of u-hFSH and r-hFSH were made available. Additional information about the absorption and disposition of u-hFSH and r-hFSH was gained by using deconvolution techniques in independent analyses of the intensively sampled data sets. This information was then utilised in the population analysis.

The notion that the prevention of disease is better than having to cure it is probably as old as the concept of restoration of human health by medical intervention. Many human cancers are preventable because their causes have been identified in the human environment. The finding that regular consumption of certain constituents of fruits and vegetables might protect us from this deadly disease, which was first proposed by Wattenberg, has aroused much interest among the medical establishment and the general public. Chemoprevention or chemoprotection can be defined as the use of specific diets, or natural or synthetic chemicals, to reverse, suppress, or prevent carcinogenic progression to invasive cancer. Minimisation of exposure towards carcinogens in the environment (‘primary prevention’) is undoubtedly an effective strategy in cancer prevention. However, most environmental factors which initiate cancer remain to be identified and, once identified, the avoidance of such factors necessitates life-style changes, which may be difficult to implement. Epidemiological data suggesting that cancer is preventable by intervention with chemicals are based on time trends in cancer incidence and mortality, geographic variations and effect of migration, identificaton of specific causative factors and lack of simple patterns of genetic inheritance for the majority of human cancers. In order to understand how chemopreventive agents exert their activity, one has to recall that epithelial carcinogenesis proceeds via multiple discernible steps of molecular and cellular alterations. Invasive cancer is the ultimate product of this sequence of critical events, many of which can theoretically be prevented. These events can be separated into three distinct phases: initiation, promotion and progression. Initiation is rapid, it involves direct carcinogen binding and damage to DNA, and the resulting mutation is irreversible. Promotion follows initiation and involves clonal expansion of initiated cells induced by agents acting as mitogens for the initiated cell. This stage is generally reversible. The progression stage of carcinogenesis is an extension of promotion and results from it in the sense that cell proliferation caused by promotors allows the cellular damage inflicted by initiation to be further propagated. During tumour progression genotypically and phenotypically altered cells gradually emerge. Both promotion and progression phases are prolonged. Depending on which phase of carcinogenesis chemopreventive agents affect, they can be divided into tumour ‘blocking’ agents, which counteract cancer by interfering with initiation, and tumour ‘suppressors’, which intercept promotion or progression. Blocking agents probably play a significant role in reducing the accumulation of initiating mutations, but the fact that initiation can occur very early in life confounds clinical chemoprevention strategies based on anti-initiation only. Hence suppression of the development of the initiated cell to a full-blown tumour is undoubtedly the strategy of choice in human cancer chemoprevention, and tumour-suppressing agents are the focus of this review. Its aim is to outline mechanisms by which such substances suppress tumours and to highlight the importance of the understanding of these mechanisms for the discovery and clinical development of novel and safe chemopreventive substances.

Nonsteroidal anti-inflammatory drugs (NSAIDS) are widely used at a low dosage as an analgesic treatment. They are very efficient analgesic and anti-inflammatory drugs but may cause gastrointestinal damage by two independent mechanisms: a systemic effect (mediated by cyclo-oxygenase 2 inhibition) and a local effect that is pH and pKa related.

Low dose ibuprofen is the reference treatment in analgesic indications, thanks to its analgesic effect without anti-inflammatory activity and the lowest gastrotoxicity.

Diclofenac-K (Cataflam^®) is a new pharmaceutical formulation of diclofenac that is not enteric-coated, unlike Voltarene^® (diclofenac-Na). Cataflam^® looks very attractive in analgesic indications because of its bioequivalence in terms of AUC to diclofenac-Na and a more rapid onset of the analgesic action related to a shorter t max (0.7 ± 0.5 h vs. 2.3 ± 0.9 h). Before generalising its use however, gastric tolerance must be evaluated. Upper fibroscopy is the validated method for visualising and scoring the gastric lesions.

The aim of the present study is to compare short-term gastro-duodenal tolerance of diclofenac-K 12.5 mg (simple analgesic dose) with acetylsalicylic acid 500 mg and ibuprofen 200 mg; all three at the maximal dose recommended in pain indications. Read more…

Budesonide is a synthetic glucocorticosteroid used in the topical treatment of diseases such as rhinitis, asthma and psoriasis , which has also been introduced for the treatment of inflammatory bowel disease. This drug is characterized by a high local potency at the site of application with relatively low systemic activity after absorption. This high therapeutic ratio is probably explained by a combination of high affinity to the glucocorticoid receptor and rapid clearance from the systemic circulation, due to rapid biotransformation in the liver.

Budesonide contains an asymmetric 16α,17α-acetal group, resulting in an equal mixture of two epimers with 22R and 22S configuration. The 22R epimer has two to three times the anti-inflammatory potency of the 22S epimer. The oxidative metabolism of budesonide has been widely studied. The main metabolites formed from budesonide in human liver microsomes by isoenzymes of the cytochrome P450 3 A (CYP3A) subfamily have been identified as 6β-hydroxybudesonide and 16α-hydroxyprednisolone. The 6β-hydroxylation is a common metabolic pathway for glucocorticoids that proceeds equally with both epimers of budesonide; by contrast, the unusual ‘acetal splitting’ pathway is stereoselective for the 22R epimer. The anti-inflammatory potency of these two metabolites of budesonide is approximately two orders of magnitude lower than that of the parent drug. Although the oxidative metabolism constitutes the quantitatively more important elimination route of synthetic corticosteroids, the contribution of conjugation with sulphate may be also significant; hence, it has been shown that budesonide is metabolized by human liver sulphotransferase yet is not glucoronated. Read more…