Formulation, stability and thermal analysis of lyophilised wound healing wafers containing an insoluble MMP-3 inhibitor and a non-ionic surfactant.

Lyophilised wafers are being developed as topical drug delivery systems for the treatment of chronic wounds. This study describes the formulation of xanthan wafers containing a selective, insoluble MMP-3 inhibitor (UK-370,106) and a non-ionic surfactant, designed to release accurate doses of UK-370,106 directly to a suppurating wound bed. Stability of UK-370,106 in the wafer compared to a non-lyophilised gel suspension was investigated using a combination of light scattering, thermal and microscopic techniques. Particle size distributions in UK-370,106-loaded wafers were constant throughout an accelerated stability study (12 weeks, 40 degrees C) while the mean particle size in a non-lyophilised suspension increased by 15 microm in the same period. Thermal analysis of UK-370,106-loaded wafers highlighted an unexpected interaction between the drug and the surfactant that was further investigated using simple mixtures of each component. It was concluded that an in situ solvate of UK-370,106 and the non-ionic surfactant can form and that this may have implications towards the stability of UK-370,106 during the formulation process. Further concerns regarding high water contents (14%) in the wafer and its effect on product stability were unfounded and it was concluded that these novel delivery systems provided a viable alternative to gel suspensions.

Gamma-irradiation of lyophilised wound healing wafers.

Lyophilised wafers are being developed as drug delivery systems that can be applied directly to the surface of suppurating wounds. They are produced by the freeze-drying of polymer solutions and gels. This study investigates the possibility of sterilising these glassy, solid dosage forms with gamma-irradiation and determining the rheological properties of rehydrated wafers post-irradiation. One series of wafers was formulated using sodium alginate (SA) modified with increasing amounts of methylcellulose (MC), the other being composed of xanthan gum (XG) and MC. Batches were divided into three lots, two of which were exposed to 25 and 40 kGrays (kGy) of Cobalt-60 gamma-irradiation, respectively, the third being retained as a non-irradiated control. Apparent viscosities of solutions/gels resulting from the volumetric addition of distilled water to individual wafers were determined using continuous shear, flow-rheometry. Flow behaviour on proprietary suppurating surfaces was also determined. Large reductions in viscosity were apparent for irradiated SA samples while those of XG appeared to be largely unaffected. In addition, an increase in the yield stress of xanthan formulations was observed. Xanthan wafers appeared to withstand large doses of irradiation with no detrimental effect on the rheology of reconstituted gels. This offers the possibility of manufacturing sterilisable delivery systems for wounds.

Lyophilised wafers as a drug delivery system for wound healing containing methylcellulose as a viscosity modifier.

Lyophilised wafers have potential as drug delivery systems for suppurating wounds. A dual series of wafers made from low molecular weight sodium alginate (SA) and xanthan gum (XG) respectively, modified with high molecular weight methylcellulose (MC) were produced. The swelling and flow properties of these wafers on model suppurating surfaces were both qualitatively and quantitatively investigated. The wafers instantaneously adhered to the surfaces, absorbing water and transforming from glassy, porous solids to highly viscous gels. The rate at which this occurred varied for the series studied with clear distinctions between the behaviour of SA and XG systems. For SA wafers there was a distinct relationship between the flow-rate and MC content. Increased amounts of MC decreased the rate at which the SA wafers flowed across a model gelatine surface. Flow rheometry was used to quantify the effect of increased MC content on both series of wafers and for the SA series, highlighted a substantial increase in apparent viscosity as a function of incremental increases in MC content. These results reflected those from the gelatine model. Observations of the reluctance of a swollen, unmodified XG wafer to flow compared with the relative ease of unmodified, low molecular weight SA was attributed to the yield stress characteristic of xanthan gels. XG is known to exhibit complex, loosely bound network structures in solution via the association of helical backbone structures. The inclusion of sodium fluorescein as a visible model for a soluble drug highlighted the potential of lyophilised wafers as useful drug delivery systems for suppurating wounds.

Comparison of two azole antifungal drugs, ketoconazole, and fluconazole, as modifiers of rat hepatic monooxygenase activity.

The mechanism of action of azole antifungal agents is believed to involve inhibition of fungal cytochrome P-450, and, therefore, an investigation of the interaction of these drugs with mammalian cytochrome P-450 systems should provide some indication of their selectivity as antifungal agents. The ability of ketoconazole and fluconazole, the latter representing a new generation of triazole antifungal agents, to modify rat mixed function oxidase activity has been investigated in vitro with hepatic microsomes and in vivo using a N-methyl-[14C] antipyrine breath test. As a measure of selectivity the results have been compared with antifungal potency. Ketoconazole is more potent than fluconazole by an order of magnitude in inhibiting metabolism by O-dealkylation of ethoxycoumarin, methoxycoumarin and ethoxyresorufin (IC50 values of 6, 5 and 130 microM for ketoconazole respectively). The effects on the regio- and stereospecific hydroxylation of [14C] testosterone were also measured; the IC50 values for inhibition of total testosterone metabolism were 0.1 mM and greater than 3 mM for ketoconazole and fluconazole respectively. Marked selectivity differences were observed for the two drugs as indicated by ketoconazole being a potent inhibitor of 7 alpha-hydroxylation of testosterone (IC50 20 microM) while fluconazole did not inhibit this activity at 3 mM. In vivo investigations using a range of doses confirmed their ranking for inhibitory potency; the ED50 values for maximum demethylation rate were 17 mumol/kg and greater than 60 mumol/kg for ketoconazole and fluconazole respectively. Thus fluconazole has a lower propensity to interact with rat hepatic cytochrome P-450 and can be considered a more selective antifungal agent as its in vivo antifungal potency is an order of magnitude greater than ketoconazole.

Inhibition of thyroxine binding to serum proteins by fenclofenac and related compounds.

The anti-inflammatory drug fenclofenac lowers the serum concentrations of thyroxine (T4) and triidothyronine (T3) in subjects taking a regular dose of 600 mg twice daily. In eight male volunteers the concentration of T4 in serum was reduced to almost a third of the pre-treatment value while the concentration of T3 fell by about a half. The interaction of fenclofenac, and related compounds, while the thyroid hormone binding proteins of serum was studied in vitro by equilibrium dialysis with measurement of the concentrations of the free T4 and T3 in the dialysate by immunoassay. Fenclofenac, at a concentration of 100 mg/l (of the order achieved therapeutically), increased the concentrations of free T4 and by 131% and free T3 by 62%. The related compounds which increased the free hormone concentrations (although at higher concentrations than achieved during therapy) include diclofenac, monohydroxyfenclofenac and dihydroxyfenclofenac. A second in vitro method was used to study the interaction of these drugs with isolated thyroxine-binding globulin (TBG). An antiserum to TBG, coupled to Sephadex particles, was used to isolate TGB from other serum proteins. The effect of the different drugs on the binding of T4 by the immobilised TBG was then measured. These compounds competitively inhibit the binding of T4 by TBG, and for each an inhibitor constant (Ki) was determined.

The systemic bioavailability of buprenorphine by various routes of administration.

The systemic bioavailability of buprenorphine has been studied in female rats following single doses (200 microgram kg-1) administered by one of six different routes. Relative to the 100% bioavailability from the intraarterial route the mean bioavailabilities were intravenous, 98%; intrarectal, 54%; intrahepatoportal, 49%; sublingual, 13%; and intraduodenal, 9.7%. Area under the curve analysis of buprenorphine concentrations in blood showed the relative fractions of drug extracted (first pass) by gut, liver and lung to be 0.80, 0.50 and 0.02 respectively, In situ absorption studies showed that the poor availability of intraduodenally administered buprenorphine is not due to slow or incomplete absorption.

Gastrointestinal absorption of carbenoxolone in the rat determined in vitro and in situ: deviations from the pH-partition hypothesis.

The absorption of [14C] carbenoxolone from everted rat ileum in vitro and from rat stomach and ileum in situ has been examined. The rate of its mucosal to serosal transfer in vitro increases as pH increases from 5 to 8 whereas the amount bound to ileum tissue decreases with increased pH; absorption closely parallels the drug's solubility. The uptake of carbenoxolone in situ is bi-exponential and the rate constants for the two processes, have been calculated. Absorption in situ, and biliary excretion, of the drug increases with increasing pH from 5.0 to 7.4. Tissue binding to the ileum in situ is not dependent on pH except below pH 5.0 when extensive tissue accumulation of carbenoxolone occurs because of its low solubility. Tissue binding to the stomach increases markedly with decrease of pH from 7.4 to 6.5 and at pH 6.5 is 80 times greater than binding to the intestine. The rate of absorption from the stomach, at pH 6.5-7.4, was much less than that from the intestine in situ. When allowance is made for the binding of carbenoxolone to the stomach, contrary to the pH-partition hypothesis, correlation is apparent between its absorption and the amount present in the ionized form.

Role of metabolism and pharmacokinetic studies in the discovery of new drugs--present and future perspectives.

1. The impact which pharmacokinetics and drug metabolism studies have made to drug discovery programmes is reviewed with examples from the anti-infective, cardiovascular, anti-inflammatory and CNS therapeutic areas. 2. Contributions that advances in analytical technology have made to the early application of pharmacokinetics and drug metabolism are discussed. 3. Some future perspectives are given on the advances being made in basic science and technology and how this will provide the basis for further growth in the contribution to the drug discovery process.

Biliary excretion, metabolism and enterohepatic circulation of buprenorphine.

1. After intramuscular administration of [3H]buprenorphine to rats, dogs, rhesus monkeys and one human volunteer, most of the dosed radioactivity was excreted in the faeces, indicating biliary excretion and a possible enterohepatic circulation of the drug in these species. 2. After intravenous administration of [3H]buprenorphine (100 microgram/kg) to bile-duct cannulated rats, over 90% of the administered radioactivity was excreted in bile within 48 h after dosing. 3. The major drug-related component in rat bile was buprenorphine glucuronide. N-Dealkylated buprenorphine (again conjugated) was also present and a sex difference in the extent of N-dealkylation was apparent. 4. Intraduodenal infusion of rats with bile from rats dosed with [3H]buprenorphine produced a slow, although extensive, excretion of drug-related material in the bile of the recipient animals.

Design of toxicokinetic studies.

1. Toxicokinetics is defined as pharmacokinetic studies in animals during actual toxicity studies or under conditions mimicking them (species, duration, dose level, etc.). 2. Toxicology studies require toxicokinetics to check whether systemic exposure reflects administered dose. In particular, it is important to know whether the absence of toxicity at a given dose is due to the innocuousness of the compound or to its poor bioavailability. 3. Pivotal toxicology studies may require different toxicokinetic support than that of early studies, as more is learned of the compound and its metabolites. Considerations need to be placed on such factors as the choice of biological matrix for drug assay, the relevance of metabolites, and which dose levels require the most pharmacokinetic investigation.

Pharmacokinetic evaluation of UK-49,858, a metabolically stable triazole antifungal drug, in animals and humans.

The pharmacokinetic profile of UK-49,858 (fluconazole), a novel triazole antifungal agent which is being developed for oral and intravenous use, was determined in mice, rats, dogs, and humans. Comparative data following oral and intravenous administration showed that bioavailability was essentially complete in all four species. Peak concentrations in plasma of drug normalized to a 1-mg/kg dose level following oral administration, were relatively high: 0.7, 0.6, 1.1, and 1.4 micrograms/ml in mice, rats, dogs, and humans, respectively. The volumes of distribution ranged between 1.1 liter/kg in mice and 0.7 liter/kg in humans, which are approximate to the values for total body water. Whole body autoradiography studies in mice following intravenous administration of [14C]UK-49,858 demonstrated that the drug was evenly distributed throughout the tissues, including the central nervous system and the gastrointestinal tract. Plasma protein binding was low (11 to 12%) in all species. Marked species differences were observed in elimination half-lives, with mean values of 4.8, 4.0, 14, and 22 h in mice, rats, dogs, and humans, respectively. The major route of elimination of the drug was renal clearance, with about 70% of the dose being excreted unchanged in the urine in each species. Studies with [14C]UK-49,858 on metabolism and excretion (intravenous and oral) in mice and dogs showed that about 90% of the dose was recovered as unchanged drug in urine and feces, confirming the metabolic stability of the drug. This pharmacokinetic profile is markedly different from that of imidazole antifungal drugs and undoubtedly contributes to the excellent efficacy of UK-49,858 in vivo.

Importance of metabolic stability and hepatic distribution to the pharmacokinetic profile of amlodipine.

1. In an isolated perfused rat liver (IPRL) model, the extensive hepatic uptake and subsequent slow redistribution of amlodipine into the perfusate have been demonstrated. The apparent liver volume for amlodipine was 920 ml compared with 38ml for nitrendipine. 2. Metabolism is the major clearance mechanism of amlodipine and nitrendipine in animals and man. In the IPRL, the intrinsic (metabolic) clearance and first-pass extraction values for amlodipine are similar to those of nitrendipine. This is in contrast with in vitro metabolic stability data in rat liver microsomes which indicate about 40-fold greater metabolic stability for amlodipine. 3. The discrepancy between relative clearance rates for the two preparations may be explained by consideration of the hepatic volume of the two compounds, with the higher liver volume of amlodipine amplifying the whole organ clearance.

Metabolism and kinetics of amlodipine in man.

1. The disposition of amlodipine, R,S,2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl- 5- methoxycarbonyl-6-methyl-1,4-dihydropyridine has been studied in two human volunteers using single oral and intravenous doses of 14C-amlodipine. The drug was well absorbed by the oral route while the mean oral bioavailability for unchanged drug was 62.5%. 2. Renal elimination was the major route of excretion with about 60% of the dosed radioactivity recovered in urine. Mean total recovered radioactivity in urine and faeces amounted to 84% for both the oral and intravenous routes. 3. Apart from a small amount of unchanged amlodipine (10% of urine 14C), only pyridine metabolites of amlodipine were excreted in urine. The majority (greater than 95%) of the metabolites excreted in the 0-72 h post-dose period were identified; the major metabolite was 2-([4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl- 2-pyridyl]methoxy) acetic acid and this represented 33% of urinary radioactivity. The data indicate that oxidation of amlodipine to its pyridine analogue is the principal route of metabolism with subsequent metabolism by oxidative deamination, de-esterification and aliphatic hydroxylation. 4. For the two volunteers, amlodipine concentrations in plasma declined with a mean half-life of 33 h, while slower elimination of total drug-related material from plasma was observed, consistent with prolonged excretion (up to 12 days) of metabolites in urine and faeces. Only amlodipine and pyridine metabolites were found in the circulation. As these pyridine derivatives have minimal calcium antagonist activity the efficacy of amlodipine in man can most probably be attributed to the parent drug.

The metabolism and pharmacokinetics of amlodipine in humans and animals.

The disposition of amlodipine, a new calcium-channel blocker with a slow onset and long duration of action, has been investigated in humans and in the animal species used in the evaluation of drug efficacy and safety. Pharmacokinetic studies were conducted with nonlabeled drug using specific high-pressure liquid chromatography or gas chromatographic procedures. The metabolic fate of the drug was investigated in mice, rats, dogs, and humans using [4-14C]-amlodipine. After intravenous administration, the percentages of the dosed radioactivity recovered in urine were 62% in humans, 45% in dogs, 38% in rats, and 25% in mice. The remainder of the doses were recovered in the feces. A similar pattern of excretion was observed after oral dosing indicating complete absorption of the 14C drug. Absorbed drug is extensively metabolized because only approximately 5% of the dose was excreted unchanged in human urine. Metabolism in humans primarily involves oxidation to the pyridine derivative with subsequent oxidative deamination of the 2-aminoethyoxymethyl side chain or deesterification at the 5-methoxycarbonyl group. These metabolites were common to either the rat or dog, although some dihydropyridine derivatives were observed as metabolites in these two species. None of the metabolites identified and then synthesized was found to have any significant calcium antagonist activity relative to amlodipine. Bioavailability of unchanged drug after oral administration was high with values of 63, 88, 100, and 100% in humans, dogs, mice, and rats, respectively. Mean plasma half-life values from single-dose studies were 35 h in humans (cf nifedipine, approximately 2 h), 30 h in dogs, 3 h in rats, and 11 h in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Ketoconazole and fluconazole inhibition of the metabolism of cyclosporin A by human liver in vitro.

The effects of the important antifungal agents, ketoconazole (Ket) and fluconazole (Flu), on the microsomal metabolism of cyclosporin A (CsA) by seven human livers was measured in vitro. A total of eight CsA metabolites were identified by high-performance liquid chromatography, with metabolites AM9 and AM1 predominating. Ket was a stronger inhibitor than Flu for the formation of each of the 8 metabolites; the mean IC50 for the inhibition of total CsA metabolism was 0.26 +/- 0.08 microM and 85.7 +/- 23.9 microM for Ket and Flu, respectively. Inhibition by Ket and Flu was noncompetitive, with Ki = 0.13 microM and 25.1 microM, respectively. There was considerable interindividual variation in the sensitivity of CsA metabolism to inhibition by Ket or Flu and the degree of inhibition was not uniform across the range of individual CsA metabolites. In six of the seven livers tested, Ket and Flu inhibited the aggregate formation of secondary metabolites (AM19, AM49, AM4N9, and AM1c) more than the aggregate formation of primary metabolites (AM9, AM1, and AM4N) and inhibited the formation of AM9 more than AM1. Although the degree of inhibition of total CsA metabolism by Flu correlated directly with the control (uninhibited) rate of total CsA metabolism (r = 0.95), no similar correlation for inhibition by Ket was noted, nor was the magnitude of inhibition by Ket and Flu related. The results are discussed in relation to the inhibition of CsA metabolism by Ket and Flu in patients in vivo and to the possibility of changes in the efficacy and toxicity of CsA as a result of alterations in its metabolite profile.

Species differences in the metabolism and excretion of fenclofenac.

1. The excretion and metabolism of radiolabelled fenclofenac (2-(2,4-dichlorophenoxy)phenylacetic acid, Flenac) has been studied in five species. 2. In the rat, absorption of oral doses of fenclofenac was virtually complete and elimination occurred mainly by the bile and faeces. The guinea-pig excreted equal amounts of radioactivity in urine and faeces, while in rabbit, baboon and man renal excretion was the more important route. 3. In all species the majority of excreted radioactivity was present as fenclofenac ester glucuronide. Amino acid conjunction with fenclofenac was minimal in all species studied. 4. Mono- and di-hydroxylated metabolites have been detected in urine from guinea-pig, baboon and man. The major hydroxylated metabolite in baboon urine has been identified as 2-(2,4-dichlorophenoxy)-5'-hydroxyphenylacetic acid.

The effect of fenclofenac on thyroid function.

The non-steroidal anti-inflammatory agent fenclofenac competitively inhibits the binding of thyroxine(T4) and triiodothyronine (T3) by thyroxine-binding globulin(TBG). Eight male volunteers completed a 4-week study during which they took fenclofenac 600 mg twice daily. The concentration of fenclofenac in serum reached a plateau after 1 week of therapy after which the mean concentration(+/SEM) of the drug in serum was 78.6 o.2 mg/1. During the steady state period on treatment there were reductions of the mean serum concentrations of total T4 to 35% (P less than 0.001), total T3 to 55% (P less than 0.001), free T4 to 69% (P less than 0.001) and free T3 to 90% (NS) of the respective pretreatment values. There were also significant changes in the concentrations of thyrotropin and reverse T3 in serum. After starting treatment with fenclofenac serum concentrations of thyrotrophin fell to a nadir after 2-4 days at which time the mean concentration was 34% (P less than 0.01) of the pretreatment value, whilst reverse T3 values increased to a maximum of 136% (P less than 0.001) of the pretreatment values over 1-2 days. There was subsequently an increase of the thyrotrophin and a reduction in reverse T3 concentrations to normal by 2 weeks of pretreatment. Transient pituitary suppression was also suggested by the response to to thryotrophin-releasing hormone (TRH): 7 days after starting fenclofenac the mean thyrotrophin response was 62% (P less than 9.001) of the pretreatment value. After 4 weeks of fenclofenac the response of TRH had returned to normal. After discontinuing fenclofenac there was a transient increase in the mean concentration of thyrotrophin in serum, to 129% of the pretreatment value (P less than 0.001), with a subsequent return to normal. Four weeks after discontinuing fenclofenac the serum concentrations of thyroid hormones and thyrotrophin were normal.

Disposition of azole antifungal agents. II. Hepatic binding and clearance of dichlorophenyl-bis-triazolylpropanol (DTP) in the rat.

DTP (dichlorophenyl-bis-triazolylpropanol) was evaluated as a probe of drug-cytochromes P450 interactions in vitro and in vivo. Studies with rat liver microsomes demonstrate that DTP shows similar P450 binding affinity to its analog, ketoconazole, as determined by P450 difference spectra and inhibition of the metabolism of methoxycoumarin. As a more polar azole, DTP shows less affinity for rat plasma albumin (fraction unbound 0.56) than ketoconazole (fraction unbound 0.037). DTP metabolism is simpler than that of ketoconazole, with only one pathway, N-dealkylation which removes a triazole ring to yield DTP glycol. This primary metabolite is further metabolised to a carboxylic acid, a glycol glucuronide and a third unknown secondary metabolite (probably an acid glucuronide). Over a dose range of 0.1-24mg/kg there is complete mass balance recovery in urine via the five metabolites and unchanged drug. However DTP metabolism is dose dependent and while the affinity of DTP for the cytochromes P450 carrying out the initial dealkylation is high (1.5 microM based on unbound blood concentration), the capacity of the reaction is low (1 nmole/min). Under linear conditions, metabolic clearance is low (19ml/h), but ten-fold higher than renal clearance. The liver is the major distribution site for both DTP and ketoconazole. At low DTP concentrations, a specific high affinity process dominates the hepatic binding of DTP resulting in a liver:blood partition coefficient of approximately 30. Hepatic binding is concentration dependent and the progressive decrease in partition coefficient observed as the dose of DTP is escalated is coincident with a decrease in volume of distribution. The two saturable processes involved in the disposition of DTP result in an unusual concentration dependency in the blood concentration-time profile of this azole. Following administration of a high dose (10mg/kg) of DTP the log concentration-time profile is sigmoidal. At high concentrations (above 1mg/L) both the N-dealkylation and the hepatic binding of DTP are saturated, but as concentrations fall to approximately 0.05mg/L the former process becomes linear and the time profile is convex over this concentration range. At later times as DTP concentrations decline further, the tissue binding also reaches the linear region and the time profile becomes concave. Only at low concentrations (below 0.05mg/L) do both processes become first order and the true half life is evident.

Formation and pharmacokinetics of the active drug candoxatrilat in mouse, rat, rabbit, dog and man following administration of the prodrug candoxatril.

1. Candoxatrilat, an active neutral endopeptidase inhibitor, was released rapidly from the inactive prodrug candoxatril in vivo in mouse, rat, rabbit, dog and man. 2. Oral doses of [14C]-candoxatril were cleared rapidly, mostly by ester hydrolysis to candoxatrilat, in mouse, dog and man. A complementary intravenous study in man with [14C]-candoxatrilat showed that the active drug was virtually completely renally cleared. Neither candoxatril nor candoxatrilat underwent chiral inversion in man. 3. Systemic availability of candoxatrilat from the oral prodrug was estimated to be 88, 53, 42, 17 and 32% in mouse, rat, rabbit, dog and man respectively. Plasma clearance of candoxatril was too rapid to enable pharmacokinetic parameter calculation in mouse and rabbit; for man, the apparent oral clearance was 57.9 ml/min/kg and the elimination half-life was 0.46 h. 4. For intravenous candoxatrilat, total plasma clearance values were 32, 15, 5.5, 5.8 and 1.9 ml/min/kg for mouse, rat, rabbit, dog and man respectively. Renal clearance values were 8.7, 7.2, 2.9 and 1.7 ml/min/kg for mouse, rat, dog and man and these approximate to the respective glomerular filtration rates. Allometric scaling with respect to bodyweight across the species allowed reasonable prediction of the above two clearance parameters in man.