Derivatives of a benzoquinone acyl hydrazone with activity against Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite with global incidence. The acute infection, toxoplasmosis, is treatable but current regimens have poor host tolerance and no cure has been found for latent infections. This work builds upon a previous high throughput screen which identified benzoquinone acyl hydrazone (KG8) as the most promising compound; KG8 displayed potent in vitro activity against T. gondii but only marginal in vivo efficacy in a T. gondii animal model. To define the potential of this new lead compound, we now describe a baseline structure-activity relationship for this chemotype. Several derivatives displayed IC50's comparable to that of the control treatment pyrimethamine with little to no cytotoxicity. The best of these, KGW44 and KGW59, had higher metabolic stability than KG8. In an in vivo T. gondii murine model, KGW59 significantly increased survivorship. This work provides new insights for optimization of this novel chemotype.

Novel Toxoplasma gondii inhibitor chemotypes.

We profiled three novel T. gondii inhibitors identified from an antimalarial phenotypic high throughput screen (HTS) campaign: styryl 4-oxo-1,3-benzoxazin-4-one KG3, tetrahydrobenzo[b]pyran KG7, and benzoquinone hydrazone KG8. These compounds inhibit T. gondii in vitro with IC50 values ranging from 0.3 to 2µM, comparable to that of 0.25 to 1.5µM for the control drug pyrimethamine. KG3 had no measurable cytotoxicity against five mammalian cell lines, whereas KG7 and KG8 inhibited the growth of 2 of 5 cell lines with KG8 being the least selective for T. gondii. None of the compounds were mutagenic in an Ames assay. Experimental gLogD7.4 and calculated PSA values for the three compounds were well within the ranges predicted to be favorable for good ADME, even though each compound had relatively low aqueous solubility. All three compounds were metabolically unstable, especially KG3 and KG7. Multiple IP doses of 5mg/kg KG7 and KG8 increased survival in a T. gondii mouse model. Despite their liabilities, we suggest that these compounds are useful starting points for chemical prospecting, scaffold-hopping, and optimization.

2,5-Dimethyl-2,4-hexadiene induced photodechlorination of 9,10-dichloroanthracene.

Photochemical formation of 9-chloroanthracene (MCA) from 9,10-dichloroanthracene (DCA) is observed in the presence of 2,5-dimethyl-2,4-hexadiene (DMH) in acetonitrile (AN). The mechanism of the reaction was investigated using kinetics, deuterium labeling, and quenching techniques. Contrary to conclusions in a recent publication, our work supports the salient features of the mechanism we had proposed earlier. DCA is photostable in degassed AN in the absence of DMH. When DMH is added, irradiation of DCA at 365 or 404 nm converts it quantitatively to MCA. The photoreaction is strongly inhibited when low concentrations of molecular oxygen or 1,2,4,5-tetracyanobenzene are also present. Results from fluorescence quenching studies along with kinetics parameters from the dependence of DCA loss and MCA formation quantum yields on [DMH] implicate participation of the DCA/DMH singlet exciplex, the DCA/(DMH)(2) triplex and the DCA radical anion (DCA*-) as intermediates in the photodechlorination. Results from experiments using deuterated DMH, deuterated AN, and AN containing D(2)O or H(2)O show that the 10-H of MCA is introduced by protonation of DCA*-. Contrary to a recent report, there is no radical pathway to MCA via dissociation of DCA*- to chloride and MCA radical. Changes in the absorption spectrum of DCA in AN with increasing [DMH] suggest that the static quenching of DCA fluorescence at high [DMH] is due primarily to nearest neighbour quenching instead of DCA/DMH ground state complex formation.

Effect of dietary fatty acids on the intestinal permeability of marker drug compounds in excised rat jejunum.

The aim of this study was to explore the effects of diets containing saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and omega-3 and omega-6 polyunsaturated fatty acids (omega-3 and omega-6 PUFA, respectively) on the passive and active transport properties of rat jejunum using marker compounds. Rats were fed diets supplemented with 18.4% (w/w) lipid (4 groups) or standard rat chow (1 group) for a period of 30 days. At the end of the dietary period, mucosal scrapings were taken for the determination of membrane phospholipids, and the apparent jejunal permeability of radiolabelled marker compounds was determined using modified Ussing chambers. Changes in the phospholipid content of the brush border membrane reflected the different lipid content of the diets. The passive paracellular permeability of mannitol was not significantly affected by the fatty acid composition of the diet, although there was a trend toward decreased mannitol permeability in the rats fed both the omega-3 and omega-6 PUFA diets. In comparison, the transcellular diffusion of diazepam was reduced by 20% (P < 0.05) in rats fed diets supplemented with omega-3 and omega-6 PUFA. In the lipid-fed rats, the serosal to mucosal flux of digoxin, an intestinal P-glycoprotein substrate, was reduced by 20% (P < 0.05) relative to the chow-fed group, however there were no significant differences between the different lipid groups. The active absorption of D-glucose via the Na+-dependent transport pathway was highest in the SFA, MUFA and PUFA omega-3 dietary groups, intermediate in the low-fat chow group and lowest in the PUFA omega-6 group, and was positively correlated with short-circuit current. These studies indicate that dietary fatty acid changes can result in moderate changes to the active and passive transport properties of excised rat jejunum.

Lymphatic transport of proteins after s.c. injection: implications of animal model selection.

Subcutaneous (s.c.) administration continues to be the main route for the delivery of protein drugs due to their poor bioavailability by most non-parenteral routes. While small drug molecules are rapidly and extensively absorbed after s.c. injection, the systemic bioavailability of protein drugs is often incomplete and variable. Given the widespread use of the s.c. route for protein drugs, surprisingly little is known about the factors that govern the rate and extent of protein absorption from the interstitial space and the role of the lymphatic system in the transport of these molecules to the systemic circulation. The few studies that have directly addressed the role of lymphatic transport in protein bioavailability are complicated by the use of methods and models that vary widely. In this review we will evaluate the available literature describing the lymphatic transport of proteins after s.c. injection and more specifically, address the impact of experimental variation (e.g. site of cannulation, animal model, anesthesia) on the interpretation of the data obtained. We will also describe in some detail the sheep model currently in use in our laboratory, which allows both estimation of the extent of uptake of protein drugs into the lymphatics draining the injection site, and quantification of the contribution of lymphatic transport to the absolute bioavailability.

Lymphatic absorption is a significant contributor to the subcutaneous bioavailability of insulin in a sheep model.

PURPOSE: This study was conducted to explore the role of the peripheral lymphatics in insulin absorption following subcutaneous (SC) administration using a sheep model that allows continuous collection of peripheral lymph and simultaneous assessment of systemic bioavailability. METHODS: In a parallel group design, soluble human insulin (0.5 IU/kg) was administered by bolus SC injection into the interdigital space of the hind leg of non-cannulated control sheep, and sheep in which the efferent popliteal lymph duct was cannulated. A separate group received a bolus IV injection (0.15 IU/kg). Blood was sampled from all animals, and lymph was collected continuously over 12 h postdosing. Samples were assayed for insulin by ELISA. RESULTS: The SC bioavailability of insulin in control sheep was 31.5+/-3.2%, which was significantly higher than when the peripheral lymph was continuously collected (18.4+/-1.7%). In the lymph-cannulated animals, 17.3+/-1.0% of the dose was collected in peripheral lymph. CONCLUSIONS: Based on the direct measurement of insulin in regional lymph and on the decrease in the systemic bioavailability when regional lymph was continuously collected, the results demonstrate that lymphatic absorption contributed significantly to the overall insulin bioavailability following SC administration to sheep.

Lymphatic transport of proteins after subcutaneous administration.

This mini-review summarizes the relevant literature regarding the lymphatic transport of proteins after subcutaneous administration. A review of the physiology of the lymphatics and inherent anatomical differences between blood and lymph capillaries is presented followed by a brief overview of the general characteristics of protein absorption and bioavailability following S.C. injection. A description of factors known to directly affect the lymphatic uptake of macromolecules follows and is supported by representative data from this laboratory. A brief perspective on the importance of lymphatic uptake and transport in understanding the biopharmaceutical properties of protein drugs and potentially targeting the lymphatics is presented.

Systemic availability and lymphatic transport of human growth hormone administered by subcutaneous injection.

Degradation of human growth hormone (hGH) at the injection site has previously been implicated as the basis for its reduced systemic availability following subcutaneous (SC) administration. The goal of these studies was to develop an animal model which would allow mass balance calculations to (i) quantify the loss at the injection site and (ii) determine the role of the lymphatics in the transport of subcutaneously-administered hGH. The animal model utilized a sheep and enabled simultaneous sampling of blood and collection of either peripheral lymph (via the efferent duct of the popliteal lymph node draining the injection site) or central lymph (via the thoracic lymph duct). In non-lymph cannulated sheep, the systemic availability of hGH following SC dosing was 58.4 +/- 9.1% (mean +/- SEM) relative to an intravenous (IV) control. The availability of hGH decreased to approximately 30-40% when either peripheral or central lymph was collected indicating that a proportion of the dose was transported via the lymph. The fraction of the administered dose collected in peripheral lymph was 61.7 +/- 8.5% (mean +/- SEM), whereas only 8.6 +/- 1.3% was collected in central lymph. These results suggested that loss of hGH within the lymphatics contributed significantly to its reduced systemic availability following SC administration. The total recovery (sum of the systemic availability and the cumulative amount recovered in lymph) of hGH was approximately 93% of the dose in the peripherally-cannulated group indicating that loss at the injection site was minimal.

Variation in the diffusion and release of ribonuclease through and from esterified hyaluronic acid membranes: effect of changes in matrix characteristics.

50 h) were detected for the transport and release of a model protein (ribonuclease A) compared with that for the translucent region which showed no lag time. The results highlight the importance of carefully controlling matrix formation to ensure reproducible transport and release characteristics from polymer matrices.

A novel prodrug approach for tertiary amines. 3. In vivo evaluation of two N-phosphonooxymethyl prodrugs in rats and dogs.

N-phosphonooxymethyl derivatives of tertiary amine containing drugs have been identified as a novel prodrug approach for improving aqueous solubility. The in vivo reversion of two prodrugs to the corresponding parent compounds following iv and im administration to rats and dogs was investigated. Equimolar doses of parent drugs (loxapine or cinnarizine) and the corresponding prodrugs were each administered via a rapid iv infusion to rats and dogs. Equimolar doses of loxapine and its prodrug were each administered im to rats only. Blood samples were collected over 12 h, and plasma was assayed for both parent drug and intact prodrug by HPLC. Comparison of the plasma AUC for the parent drugs following administration of the parent drugs and prodrugs allowed estimation of the apparent bioavailability of parent drug from prodrug dosing. Plasma levels of the prodrugs fell below the limit of detection 5 min after iv infusion with an approximate half-life of 1 min. The mean AUCs following iv and im dosing of parent drugs were not statistically different from the parent drug AUCs obtained after prodrug dosing. The results are consistent with rapid and quantitative prodrug to parent drug reversion following administration of the phosphonooxymethyl prodrugs to the rats and dogs. This information, together with previous studies on the synthesis and physicochemical evaluation of the prodrugs, suggests that this novel prodrug strategy is a very promising approach for overcoming solubility limitations seen with many tertiary amine containing drugs at physiological pH values.

Mechanisms controlling diffusion and release of model proteins through and from partially esterified hyaluronic acid membranes.

The effects of polymer percent esterification and protein molecular weight on the diffusion of two model proteins, deoxyribonuclease (DNase) and ribonuclease A (RNase A), through and from partially esterified hyaluronic acid membranes were compared. The permeability of the polymer membranes was inversely related to the degree of polymer esterification and the molecular weight of the protein. Transport rates of proteins through the membranes decreased dramatically over narrow ranges of polymer esterification. As expected, the apparent diffusivity of the larger protein in the polymer matrix was more sensitive to changes in membrane hydration than that of the smaller protein. These observations demonstrated the dependence of the mobility of large molecular weight proteins on polymer hydration and chain relaxation. The relationship between protein diffusion through and release from the modified hyaluronate matrices was also investigated using RNase A as a model. The release profiles from fully esterified membranes showed lag behavior and varied with protein load and hyaluronate hydrolysis rates, while release from less esterified membranes was rapid and independent of polymer esterification or hydrolysis. Potential applications of modified hyaluronate matrices in the controlled delivery of proteins are discussed.

The application of capillary electrophoresis for monitoring effects of excipients on protein conformation.

Studies were conducted to assess the utility of free solution capillary electrophoresis (CE) for monitoring the effects of selected excipients on the thermal denaturation of a model protein (Ribonuclease A, RNase A) at low pH. Thermal denaturation/unfolding experiments were conducted via temperature-controlled CE using a run buffer of 20 mM citric acid in the pH range of 2.3-3.1, with a marker peptide incorporated to correct for temperature-induced changes in endoosmotic flow. The effects of selected excipients on the thermal unfolding of RNase A were then evaluated by adding either sorbitol, sucrose, polyethylene glycol 400 (PEG 400) or 2-methyl-2,4-pentanediol (MPD) to the electrophoretic run buffer (pH 2.3). Confirmatory denaturation experiments were conducted under the same solution conditions using circular dichroism (CD) spectropolarimetry. Using temperature-controlled CE, an increase in solution pH from 2.3 to 2.7 and 3.1 resulted in an increase in transition temperatures of RNase A by approximately 8 and 13 degrees C, respectively. Similar shifts in transition temperatures were observed when thermal denaturation transitions were monitored by far-UV CD. Sorbitol (0.55-1.1 M) and sucrose (0.55 M) each shifted the denaturation transition temperatures of RNase A to higher values, whereas PEG 400 and MPD had minimal effect on the unfolding transition midpoint at the concentrations evaluated (0.55 M for each). The observed changes in the transition temperatures for RNase A as a function of pH and selected excipients were similar when measured by either CE or far-UV CD. These results support the utility of CE for monitoring the effects of neutral excipients on the thermal denaturation of a model protein under selected conditions. The widespread utility of the technique may be limited by the narrow temperature range of most commercial CE instruments and the need to use extreme pH conditions to monitor the complete denaturation transition.

Analytical methods and stability assessment of liquid yeast derived sucrase.

Two independent analytical methods for determining the activity and stability profile of liquid yeast derived sucrase (YS) were established and validated in order to conduct preliminary stability studies as a function of temperature. The methods included a hexokinase-based (HK) enzymatic assay for determining the formation of glucose upon hydrolysis of sucrose by YS, and a direct polarimetric procedure to quantitate YS hydrolysis of sucrose. Both assays were validated with respect to YS dilution, incubation time, sucrose or glucose concentration, linearity of response and within- and between-day variability. A preliminary stability study was conducted over a 24 week period with liquid YS samples stored at -20, 4, 30, 40 and 50 degrees C. Enzymatic activity was monitored as a function of time using both the HK and polarimetric assays. Liquid YS samples stored at -20, 4 and 30 degrees C retained 100% activity after 24 weeks storage, while the samples stored at 40 degrees C lost approximately 70% activity over the same storage period and samples stored at 50 degrees C lost approximately 95% activity after 12 weeks storage. The two methods of analysis gave consistent results over the course of the study.

Biphasic drug absorption from the epidural space of the dog may limit the utility of a slow release medium molecular weight hyaluronic acid-lidocaine ionic complex formulation.

Previous epidural studies conducted in rabbits have described a viscous lidocaine-hyaluronate formulation (L-HA) that prolonged the duration of sensory blockade twofold and decreased the rate of drug absorption fourfold relative to a solution formulation. As further evaluation of the L-HA formulation required studies in a larger animal that more closely reflected the characteristic absorption kinetics observed in humans, a conscious dog model was used to functionally and kinetically evaluate the viscous formulation relative to lidocaine solution. In terms of the measured pharmacodynamic end point (loss of weight-bearing ability in hind legs), epidural administration of the L-HA formulation did not prolong the duration of action relative to lidocaine solution in spite of a markedly altered pharmacokinetic profile. For example, administration of L-HA reduced the mean plasma lidocaine Cmax value approximately 50% and increased the Tmax value approximately fivefold relative to lidocaine solution. However, the viscous L-HA formulation did cause a significant prolongation in the latency of onset (P < 0.001) relative to lidocaine solution. The dog exhibited "flip-flop" pharmacokinetics and absorption was biphasic after epidural administration of lidocaine solution (apparent t1/2 of the fast and slow absorption phases were 4 min and 131 min, respectively). The L-HA formulation markedly altered the absorption kinetics such that a single, slow absorption phase was evident (apparent t1/2 of 56 min), although this rate was more rapid than the slow phase observed after lidocaine solution. It is possible that the inability of the hyaluronate-based formulation to further reduce the magnitude of the slow absorption phase resulted in the failure to prolong the duration of action. These data highlight the need to carefully consider the absorption kinetics and pharmacokinetic characteristics of the animal models chosen to evaluate new formulation of epidurally administered local anesthetics.

Lymphatic transport of halofantrine in the triple-cannulated anesthetized rat model: effect of lipid vehicle dispersion.

Halofantrine hydrochloride is an important, highly lipophilic anti-malarial agent. A triple-cannulated anesthetized rat model was used to investigate the potential lymphatic transport of halofantrine (Hf). The effect of formulating Hf in vehicles representative of different physical (digestion) states of triglyceride lipid was also evaluated. The lipid vehicles were either a lipid solution, emulsion, or micellar system comprised of 50 microL of a 2:1 molar ratio of oleic acid:glycerol monooleate containing 2 mg of Hf free base. Lymph was collected from the mesenteric lymph duct, and blood was sampled from the jugular vein following intraduodenal infusion of the different formulations. Lymphatic transport was a major contributor to bioavailability as demonstrated by the recovery of up to approximately 20% of the administered dose in the intestinal lymph. The rank order effect of the vehicles for the promotion of lymphatic transport was micellar > emulsion > lipid solution. Lymphatic drug transport was predominantly associated with chylomicron-based transport. The extent of Hf absorption via the portal blood, estimated from the systemic plasma profiles in the lymph-cannulated rats, was largely independent of the administered formulations. These data indicate that lymphatic transport of the free base of Hf is a major contributor to oral bioavailability when formulated in appropriate lipid vehicles. The data suggest that formulation as increasingly disperse systems facilitates transport in this animal model.

Lymphatic transport of halofantrine in the conscious rat when administered as either the free base or the hydrochloride salt: effect of lipid class and lipid vehicle dispersion.

The intestinal lymphatic transport of halofantrine, an important, highly lipophilic antimalarial drug, has been studied in a conscious rat model after oral administration. In these studies, the lymphatic transport of Hf free base when coadministered with lipid was approximately 20% of the administered dose compared with 5% transport after administration of the HCl salt with or without lipid. These differences in transport can be attributed to the increased lipophilicity of the free base (relative to the HCl salt) thereby facilitating greater association of Hf base with the products of luminal lipid digestion and the subsequent interaction with the intestinally derived chylomicrons responsible for lymphatic drug transport. In contrast to previous results in an anesthetized rat model where lymphatic transport was dependent on the characteristics of the intraduodenally administered lipid formulations, the lymphatic transport of Hf base in the conscious rat was independent of both the class of administered lipid (triglyceride or fatty acid) and the extent of formulation dispersion (micellar lipid or lipid solution). Considering the different lymphatic transport profiles of Hf base in the anesthetized and conscious rat models, it is proposed that the lipid vehicle effects observed in the intraduodenally dosed anesthetized model most likely reflects the lack of gastric processing by preduodenal lipase and the shear action of the stomach otherwise present in the conscious rat model.

Analysis of bovine immunoglobulin G by capillary gel electrophoresis.

A method for the analysis of bovine immunoglobulin G (IgG) using sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE) has been described. Under the electrophoretic conditions employed, monomeric and dimeric IgG were readily resolved, as were light chain and heavy chain subunits, and heavy chain dimers in reduced samples. Molecular weights determined by SDS-CGE compared favourably with those measured by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and published values. Reproducibility of protein quantitation was achieved resulting in a relative standard deviation of approximately 13% and calibration was linear in the range of 0.2-3.5 mg ml-1 protein under the conditions used.

Biopharmaceutical characterisation of a low-dose (75 mg) controlled-release aspirin formulation.

The release of aspirin from a 75 mg controlled-release formulation, designed to inhibit maximally thromboxane A2 production while sparing stimulated prostacyclin biosynthesis, was characterised in healthy subjects. The calculated in vivo release rate of aspirin matched the design goal of approximately 10 mg h(-1). The C(max) of aspirin associated with the controlled-release formulation was lowered 15-fold relative to a solution formulation of the same dose. The bioavailability of aspirin (based on salicylate concentrations) from the controlled-release formulation was approximately 90% relative to the solution, and drug release was not affected by co-administration of a standard breakfast.

Validation of a peptide map for recombinant porcine growth hormone and application to stability assessment.

A reverse-phase HPLC method for the analysis of tryptic digests of recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4 degrees C for a 20-hr period with TPCK-treated trypsin at a 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, was incorporated for separation of the digestion products and peak identification was carried out by mass spectrometry (MS). The digestion procedure and subsequent chromatography were linear in the initial concentration range of 4.55-45.46 microM (100 to 1000 micrograms/mL) pGH. The variability in the fragment retention times was low and the normalized peak area variability was less than 5% for all but three of the fragments. The utility of the trypsin digestion and chromatography procedures has been demonstrated by assessing chemical changes in pGH induced by incubation at elevated pH. Upon incubation of pGH in 0.2 M Tris buffer at pH 9 (ionic strength adjusted to 0.5 with NaCl) and 37 degrees C over a period of 400 hr, significant degradation in the regions corresponding to the digestion fragments T23-T25 (residues 181-182 linked by a disulfide bond to residues 184-191), T9 (residues 96-108), and T5-T18 (residues 43-64 linked by a disulfide bond to residues 158-166) was observed. The disappearance of the peaks corresponding to fragments T23-T25 and T9 both displayed apparent first-order degradation kinetics over the time period investigated with half-lives of 131 and 154 hr, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Techniques for assessing the effects of pharmaceutical excipients on the aggregation of porcine growth hormone.

Three denaturing techniques have been evaluated for their ability to induce irreversible aggregation and precipitation of recombinant porcine growth hormone (pGH). The denaturing stimuli included thermal denaturation, interfacial denaturation through the introduction of a high air/water interface by vortex agitation, and a guanidine (Gdn) HCl technique which involved rapid dilution of a partially unfolded state of pGH to nondenaturing conditions. Soluble and insoluble pGH fractions were evaluated for the presence of covalently modified species and soluble aggregates by size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF). In each of the three denaturation methods, precipitation was found to be irreversible, as the precipitated pellet could not be solubilized upon resuspending in buffer. The soluble pGH fractions consisted of only monomeric material and the insoluble protein pellet could be completely solubilized with Gdn HCl or SDS. There was no evidence of detectable covalent modifications in the precipitated protein pellet following any of the three denaturation techniques. Three excipients, Tween 20, hydroxypropyl-beta-cyclodextrin (HPCD), and sorbitol were evaluated for their stabilizing ability using each of the three denaturation methods and the degree of stabilization was found to be dependent upon the denaturing stimulus incorporated. Tween 20 was found to be highly effective in preventing pGH precipitation using the interfacial and Gdn techniques and was moderately effective using the thermal denaturation method. Inclusion of HPCD in the sample buffer significantly reduced precipitation using the thermal and interfacial methods but was ineffective in the Gdn technique.(ABSTRACT TRUNCATED AT 250 WORDS)