Establishment of Pertussis toxin BRP batch 2 for CHO clustering assay.

Recently, the Chinese hamster ovary (CHO) cell-based clustering assay replaced the in vivo Histamine Sensitisation Test (HIST) in mice in European Pharmacopoeia (Ph. Eur.) general chapter 2.6.33. 'Residual pertussis toxin' as the recommended method to test for residual pertussis toxin in acellular pertussis vaccine intermediates. To support the standardised CHO clustering assay, availability of a reference standard is critical. Ph. Eur. pertussis toxin Biological Reference Preparation (BRP) batch 1 was first calibrated in International Units in 2008 for the HIST and subsequently also calibrated for the CHO clustering assay in 2017. However, its stocks were dwindling and needed to be replaced. In an effort to maintain adequate supply, a project (BSP141) was initiated by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to establish a second pertussis toxin BRP (BRP2). Candidate material was manufactured ad hoc by an acellular pertussis vaccine manufacturer and an optimal formulation for long-term stability was defined. Exhaustive in-process and post-production controls demonstrated that the material was fit for its intended purpose and therefore a collaborative study for calibration and stability assessment of the candidate material was organised, which included 10 laboratories worldwide. As a result of the study, the candidate material was established as Ph. Eur. Pertussis toxin BRP batch 2 with a potency of 130 IU/vial for the CHO clustering assay. Unopened vials must be stored at −20°C. The BRP may be used for up to two weeks after reconstitution if appropriately handled and stored at 2­8°C.

Alternatives to HIST for acellular pertussis vaccines: progress and challenges in replacement.

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST' was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials.

Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines.

Current regulations for acellular pertussis (aP) vaccines require that they are tested for the presence of residual or reversion-derived pertussis toxin (PTx) activity using the mouse histamine sensitisation test (HIST). Although a CHO cell clustering assay can be used by manufacturers to verify if sufficient inactivation of the substance has occurred in-process, this assay cannot be used at present for the final product due to the presence of aluminium adjuvants which interfere with mammalian cell cultures. Recently, 2 modified CHO cell clustering assays which accommodate for the adjuvant effects have been proposed as alternatives to the HIST. These modified assays eliminate the adjuvant-induced cytotoxicity either through dilution of the vaccine (called the Direct Method) or by introducing a porous barrier between the adjuvant and the cells (the Indirect Method). Transferability and suitability of these methods for testing of products present on the European market were investigated during a collaborative study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). Thirteen laboratories participated in this study which included 4 aP-containing vaccines spiked by addition of PTx. This study also assessed the transferability of a standardised CHO cell clustering assay protocol for use with non-adjuvanted PTx preparations. Results showed that the majority of laboratories were able to detect the PTx spike in all 4 vaccines at concentrations of 4 IU/mL or lower using the Indirect Method. This sensitivity is in the range of the theoretical sensitivity of the HIST. The Direct Method however did not show the expected results and would need additional development work.

In search of acceptable alternatives to the murine histamine sensitisation test (HIST): what is possible and practical?

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.

Risk and safety assessment on the consumption of Licorice root (Glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin.

Licorice (or 'liquorice') is a plant of ancient origin and steeped in history. Licorice extracts and its principle component, glycyrrhizin, have extensive use in foods, tobacco and in both traditional and herbal medicine. As a result, there is a high level of use of licorice and glycyrrhizin in the US with an estimated consumption of 0.027-3.6 mg glycyrrhizin/kg/day. Both products have been approved for use in foods by most national and supranational regulatory agencies. Biochemical studies indicate that glycyrrhizinates inhibit 11beta-hydroxysteroid dehydrogenase, the enzyme responsible for inactivating cortisol. As a result, the continuous, high level exposure to glycyrrhizin compounds can produce hypermineralocorticoid-like effects in both animals and humans. These effects are reversible upon withdrawal of licorice or glycyrrhizin. Other in vivo and clinical studies have reported beneficial effects of both licorice and glycyrrhizin consumption including anti-ulcer, anti-viral, and hepatoprotective responses. Various genotoxic studies have indicated that glycyrrhizin is neither teratogenic nor mutagenic, and may possess anti-genotoxic properties under certain conditions. The pharmacokinetics of glycyrrhizin have been described and show that its bioavailability is reduced when consumed as licorice; this has hampered attempts to establish clear dose-effect levels in animals and humans. Based on the in vivo and clinical evidence, we propose an acceptable daily intake of 0.015-0.229 mg glycyrrhizin/kg body weight/day.

Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies.

Green tea extract and its principal active ingredient, epigallocatechin gallate (EGCG), are gaining attention and increased usage due to their healthful properties. Despite the increasing demand for these products, few studies have examined their safety. The toxicity of purified green tea extracts containing high concentrations of EGCG have been evaluated in a series of studies in order to define the safety of Teavigo, a high-concentration EGCG extract produced by the same novel method. Topical EGCG preparations caused minor dermal irritation in rats and guinea pigs, but not rabbits, and was a moderate dermal sensitizing agent in the guinea pig maximization test. A rabbit eye irritation test produced a strong enough response to not warrant any further testing in this assay. An oral dose delivering 2000 mg EGCG preparation/kg was lethal to rats; whereas, a dose of 200 mg EGCG/kg induced no toxicity. The dietary administration of EGCG preparation to rats for 13 weeks was not toxic at doses up to 500 mg/kg/day. Similarly, no adverse effects were noted when 500 mg EGCG preparation/kg/day was administered to pre-fed dogs in divided doses. This dose caused morbidity when administered to fasted dogs as a single bolus dose, although this model was considered an unrealistic comparison to the human condition. From these studies a no-observed adverse effect level of 500 mg EGCG preparation/kg/day was established.

Safety studies on epigallocatechin gallate (EGCG) preparations. Part 3: teratogenicity and reproductive toxicity studies in rats.

Green tea and its principal active ingredient, epigallocatechin gallate (EGCG), have been demonstrated to have anticancer properties through interactions with multiple biochemical processes. Since these processes are often crucial in normal fetal development it is important to evaluate the potential effects of EGCG on the fetus. EGCG preparations of >91% purity were administered to pregnant rats during organogenesis and development in order to define the safety of Teavigo, a high-concentration EGCG extract produced by the same novel method. In an initial preliminary study using subcutaneous and gavage routes, there was no evidence of any direct embryo-fetal toxicity, although some maternal toxicity was seen. In the main teratogenicity study, feeding pregnant rats diets supplemented at 1400, 4200 or 14,000 ppm during organogenesis was non-toxic to dams or fetuses. A two-generation study in rats fed 1200, 3600 or 12,000 ppm EGCG preparation showed no adverse effects on reproduction or fertility. The highest dose reduced the growth rate of offspring, and there was a slight increase in pup loss. A growth effect among pups was also seen at 3600 ppm, but in the second generation only. The lowest dose was considered the overall no-observed adverse effect level (NOAEL). As dams consumed twice the amount of feed during the crucial lactation period, the NOAEL was equivalent to 200 mg/kg/day EGCG preparation.

Safety studies on epigallocatechin gallate (EGCG) preparations. Part 1: genotoxicity.

Public interest in green tea has grown recently due to the potential health benefits from its consumption. Epigallocatechin gallate (EGCG), a principal polyphenolic component of green tea, is considered key to these healthful qualities. Although numerous studies have evaluated the anti-cancer effects of green tea and EGCG, few have examined the safety of EGCG consumption. The genotoxic potential of a concentrated EGCG preparation was tested in Salmonella and L5178Y tk+/- mouse lymphoma cell assays to further define the safety of Teavigo, a high-concentration EGCG extract of Camellia sinensis leaves produced by the same novel method. No mutagenic activity was detected in the bacterial system; however, a clastogenic 'trend' from the formation of hydrogen peroxide was noted in the murine cells. The oral administration of 500, 1000, or 2000 mg EGCG/kg to mice did not induce micronuclei formation in bone marrow cells. Similarly, administering 400, 800, or 1200 mg EGCG/kg/day in their diet for 10 days did not induce bone marrow cell micronuclei and produced plasma EGCG concentrations comparable to those reported in human studies. The intravenous injection of 10, 25 and 50 mg EGCG/kg/day to rats resulted in much higher plasma concentrations and demonstrated an absence of genotoxic effects. From these studies, it is concluded that Teavigo (EGCG) is not genotoxic.

Structure-activity relationship studies of discodermolide and its semisynthetic acetylated analogs on microtubule function and cytotoxicity.

PURPOSE: Discodermolide, a natural product from the marine sponge Discodermia dissoluta, has been previously described as an antimitotic agent with microtubule hyperstabilizing properties similar to those of paclitaxel (Taxol). The clinical success of paclitaxel has led to a growing interest in novel antimitotic compounds and the elucidation of their structure-activity characteristics. Analogs of discodermolide were prepared by acetylation of the hydroxyl groups at carbons 3, 7, 11 and/or 17 and tested for biological activity in human tumor cells to determine the structural requirements for tubulin interaction and cytotoxic effects. METHODS: A549 human lung adenocarcinoma cells were incubated with discodermolide, or its acetylated analogs, and examined for their effects on microtubule architecture, cytotoxicity. and perturbations of the cell cycle. To confirm their direct interaction with tubulin. analogs were assayed for their ability to induce the polymerization of purified bovine brain tubulin. RESULTS: Acetylation of discodermolide at the C-7 hydroxyl group potentiated the cytotoxicity of the molecule to A549 cells, whereas acetylation at the C-3 hydroxyl group had little effect on the cytotoxicity of the parent or C-7-acetylated compounds. The acetylation of the hydroxyl groups at the C-11 and C-17 positions severely abrogated the cytotoxicity of the molecule. Cell cycle analysis by flow cytometry revealed that the more cytotoxic analogs caused the accumulation of cells in the G2/M phase, a mechanism previously reported for discodermolide. All discodermolide analogs with IC50 values below 1000 nM exhibited microtubule effects to varying degrees in cultured A549 cells, yet only the most cytotoxic promoted the polymerization of purified tubulin. CONCLUSIONS: Although the parent compound was more effective at polymerizing purified tubulin, acetylation of the C-3 or C-3 and C-7 hydroxyl groups improved its cytotoxicity in whole cells suggesting that acetylation either enhances accumulation of the molecules within cells or imparts a secondary cytotoxic quality not present in the discodermolide molecule. The study reported here is the first to provide information on the structure-activity relationships of discodermolide using human tumor cells and analogs produced by semisynthetic modification of natural discodermolide.

Acetylated analogues of the microtubule-stabilizing agent discodermolide: preparation and biological activity.

A series of eight discodermolide acetates have been prepared using natural (+)-discodermolide and evaluated for in vitro cytotoxicity against the cultured murine P-388 leukemia cells. The acetylated analogues showed a significant variation of cytotoxicity and suggested the importance of C-11 and C-17 hydroxyl groups for potency. The preparation and structure elucidation of the new analogues are described.

The inhibitory effect of ursodeoxycholic acid and pentoxifylline on platelet derived growth factor-stimulated proliferation is distinct from an effect by cyclic AMP.

This study assessed the ability of ursodeoxycholic acid (UDCA) and one of its metabolites, tauroursodeoxycholic acid (TUDCA), to inhibit platelet derived growth factor (PDGF) stimulated fibroproliferation and compared these results to the effect of pentoxifylline and its metabolite-1 [1-(5-hydroxyhexyl)-3,7-dimethylxanthine] and assessed the potential role of cyclic AMP in this process. Fibroproliferative activity was measured by the tritiated thymidine uptake assay in human fibroblast cultures. All four compounds: pentoxifylline, metabolite-1, UDCA and TUDCA inhibited the fibroproliferative activity stimulated by PDGF (8 ng/ml). Incubation of fibroblasts with dibutyryl cyclic AMP reduced proliferation stimulated by PDGF suggesting that the PDGF stimulated proliferation was sensitive to inhibition by a membrane permeable analogue of cyclic AMP. Incubation of myofibroblasts with dibutyryl cyclic AMP significantly inhibited PDGF stimulated proliferation suggesting that cyclic AMP can regulate PDGF stimulated proliferation in the myofibroblast. To determine if the effect of pentoxifylline on fibroproliferation was mediated by cyclic AMP, we used dideoxyadenosine, a potent inhibitor of adenylyl cyclase. The effect of pentoxifylline on fibroproliferation was not prevented by dideoxyadenosine, which inhibits formation of cyclic AMP, thus suggesting that the inhibitory effect of pentoxifylline on PDGF-stimulated proliferation of fibroblasts was not mediated by cyclic AMP, arguing against a role for cyclic AMP in this process. Combinations of UDCA (250 microM) plus pentoxifylline (120 microM) or UDCA (250 microM) plus TUDCA (250 microM) inhibited fibroproliferative activity stimulated by PDGF to a greater extent than either drug alone. As UDCA has been reported to decrease cyclic AMP these results argue against a role for cyclic AMP in this process. Finally the results suggest that UDCA may inhibit PDGF-stimulated proliferation via an inhibition of C-kinase.

Platelet-derived growth factor and pentoxifylline modulation of collagen synthesis in myofibroblasts.

Fibroblast proliferation and extracellular matrix accumulation are two major events occurring in fibrosis. Hepatic stellate cells are the major collagen-producing cells of the liver and are transformed into proliferative myofibroblasts following activation. Whether proliferation and extracellular matrix production are regulated by the same cytokines is not known. Monocyte-conditioned medium obtained from pigs with yellow phosphorus-induced hepatic fibrosis increased the collagen production by cultured procine myofibroblasts. Liver biopsies from these same fibrotic animals had increased levels of collagen alpha 1(I) and alpha 1(III) mRNA compared to control animals. Preincubation with platelet-derived growth factor (PDGF) B/B antibody significantly reduced the collagen-stimulating ability of the monocyte-conditioned medium. Recombinant PDGF stimulated proliferation in nonconfluent myofibroblasts and stimulated collagen production in confluent cultures of myofibroblasts without increasing cell number, suggesting that these events can occur independent of each other. Pentoxifylline and one of its active metabolites (metabolite-1) inhibited PDGF-stimulated collagen production in cultured porcine myofibroblasts. These results demonstrate the importance of PDGF in the pathogenesis of liver fibrosis and provide evidence that pentoxifylline interferes with PDGF-mediated events during experimental liver fibrosis.

Assessment of arylamine N-acetyltransferase (NAT1) activity in mononuclear leukocytes of cystic fibrosis patients.

The clearance of sulphamethoxazole (SMX), a compound metabolised primarily by the N-acetyltransferase NAT1, is increased in cystic fibrosis (CF) patients. We assessed the activity and kinetic properties of NAT1 in lysates of peripheral blood mononuclear leukocytes (MNL) from CF (n = 17) and control (n = 22) subjects using SMX and p-aminobenzoic acid (PABA) as test substrates. The Km and Vmax values of both substrates in MNL from CF patients and control subjects were not significantly different. The acetylation of PABA (100 microM) by intact MNL from CF patients (n = 4) was not different from the observed in intact MNL from controls (n = 9) (25 +/- 3 pmol h-1 per 10(6) MNL vs 27 +/- 4 pmol h-1 per 10(6) MNL). These results suggest that there are not systemic changes in this enzyme in CF. The increased metabolic clearance of SMX may therefore be related to factors other than alterations in the level of activity of the N-acetyltransferase NAT1.

In vitro effect of platelet-derived growth factor on fibroproliferation and effect of cytokine antagonists.

Platelet-derived growth factor (PDGF) stimulates fibroblast proliferation and increases collagen synthesis. Fibroproliferation, as assessed by tritiated thymidine uptake, was significantly stimulated by platelet-derived growth factor (8 ng/ml). Several drugs including dexamethasone (1 nM-20 microM), cysteamine (1.3-65 mM), N-acetylcysteine (0.6-15 mM), glutathione (1 microM-0.1 mM), glutathione peroxidase (0.1-1 Unit/ml), pentoxifylline (60 microM-36 mM), colchicine (0.025-250 nM) and aurothioglucose (1.15-23 microM) were assessed in the fibroproliferation assay for their ability to block the fibroproliferative effect of platelet-derived growth factor. Dexamethasone and aurothioglucose did not affect PDGF-stimulated fibroproliferation, while pentoxifylline, colchicine, cysteamine and N-acetylcysteine effectively reduced fibroproliferation stimulated by PDGF. The effect of pentoxifylline on PDGF stimulated fibroproliferation was compared to trapidil, theophylline and adenosine to assess mechanism of action. All four methylxanthines effectively inhibited PDGF stimulated fibroproliferation. Pentoxifylline was as effective as trapidil (IC50 = 129 microM and 141 microM, respectively), but pentoxifylline was more potent than theophylline (IC50 = 688 microM) and pentoxifylline was not as potent as adenosine (IC50 = 19 microM) in reducing PDGF-stimulated fibroproliferation.

Acetylator phenotyping: the urinary caffeine metabolite ratio in slow acetylators correlates with a marker of systemic NAT1 activity.

Eighteen healthy Caucasians were evaluated for the systemic acetylation of a caffeine metabolite using the urinary caffeine metabolite ratio 5-acetylamino-6-formylamino-3-methyluracil (AFMU) to 1-methylaxanthine (1X) and for N-acetyltransferase activity in peripheral blood mononuclear leukocytes (MNL) using p-aminobenzoic acid (PABA). These are markers for systemic NAT2 and NAT1 N-acetyltransferase activities, respectively. Fourteen slow acetylators and four fast acetylators (the NAT2 polymorphism) were identified by the caffeine metabolite ratio. In slow acetylators who have decreased levels of hepatic NAT2, the AFMU/1X ratio was significantly correlated with PABA acetylation in MNL (r = 0.8; p = 0.0002). These results suggest that significant variation in the acetylation of arylamine substrates susceptible to the classical acetylator polymorphism is attributable to variation in NAT1 activity in the slow acetylator phenotype.

Fibroproliferation in liver disease: role of monocyte factors.

Fibroproliferation was measured as the uptake of [3H]thymidine into fibroblasts. Human fibroblasts were incubated with 200 microliters monocyte-conditioned medium, the 0.22 microns filtrate from cultured monocytes, in Dulbecco's modified Eagle medium supplemented with controlled process serum replacement 2, a fetal calf serum substitute with low mitogenic activity. Increasing the numbers of fibroblasts resulted in a parallel increase in thymidine uptake to a maximal level. Fibroblasts (2 x 10(3] were plated into microwell plates and incubated with monocyte-conditioned medium for 72 hr. At 16 hr before harvest, 1 muCi [3H]thymidine was added. Cells were harvested with phosphate-buffered saline and washed, and the filters were counted. Fibroblasts incubated with Dulbecco's modified Eagle medium and controlled process serum replacement 2 showed minimal thymidine uptake. Fibroblasts incubated with Dulbecco's modified Eagle medium plus monocyte-conditioned medium from monocytes stimulated with 10 micrograms/ml lipopolysaccharides showed a sixfold increase in thymidine uptake over fibroblasts in Dulbecco's modified Eagle medium and controlled process serum replacement 2 alone. Fibroblasts incubated with Dulbecco's modified Eagle medium plus monocyte-conditioned medium from monocytes of patients with liver disease (n = 20) showed a 10-fold elevation in thymidine uptake compared with Dulbecco's modified Eagle medium and controlled process serum replacement 2. Results indicated that preincubation of monocyte-conditioned medium with either anti-interleukin-1 beta (12.5 half-maximal units, 4 degrees C, 16 hr) or catalase (1,870 IU, 25 degrees C, 1 hr) did not alter the fibroproliferative activity of the monocyte-conditioned medium, suggesting that neither interleukin-1 beta nor activated oxygen intermediates were involved in fibroproliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine mediates calcium-induced antinociception and potentiation of noradrenergic antinociception in the spinal cord.

Intrathecal (i.t.) coadministration of calcium (Ca2+), 50 micrograms potentiates the spinal antinociceptive action of morphine and noradrenaline (NA) but not cyclohexyladenosine, 5'-N-ethylcarboxamido adenosine or 5-hydroxytryptamine in the rat tail flick test. This dose of Ca2+ has no intrinsic effect in this test. Higher doses of Ca2+ (200-400 micrograms) produce antinociception in the tail flick and hot plate tests, which is completely blocked by pretreatment with the adenosine receptor antagonists theophylline, 50 micrograms and 8-phenyltheophylline, 3 micrograms. 8-Phenyltheophylline also eliminates potentiation of the antinociceptive action of NA by 50 micrograms Ca2+. The intrinsic antinociceptive effect of Ca2+ is blocked by i.t. pretreatment with the neurotoxins capsaicin, 50 micrograms and 6-hydroxydopamine, 50 micrograms but not 5,7-dihydroxytryptamine, 50 micrograms. Antinociception also is blocked by pretreatment with phentolamine but not by methysergide. These results suggest that the antinociceptive action of high doses of Ca2+ is due to release of adenosine (or a nucleotide which is metabolized to adenosine) from the spinal cord. At lower doses, the release of adenosine is insufficient to cause antinociception, but potentiates the action of NA. Adenosine appears to originate from capsaicin-sensitive small diameter primary afferent nerve terminals. A subsequent interaction of adenosine with spinal adrenergic receptors contributes to antinociception.