Search

The Nanotechnology Risk Assessment Working Group in the Center for Drug Evaluation and Research (CDER) within the United States Food and Drug Administration was established to assess the possible impact of nanotechnology on drug products. The group is in the process of performing risk assessment and management exercises. The task of the working group is to identify areas where CDER may need to optimize its review practices and to develop standards to ensure review consistency for drug applications that may involve the application of nanotechnology. The working group already performed risk management exercises evaluating the potential risks from administering nanomaterial active pharmaceutical ingredients (API) or nanomaterial excipients by various routes of administration. This publication outlines the risk assessment and management process used by the working group, using nanomaterial API by the oral route of administration as an example.

Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE); however, a correlation between blood ammonia levels and HE severity remains controversial. It is believed oxidative stress plays a role in modulating the effects of hyperammonemia. This study aimed to determine the relationship between chronic hyperammonemia, oxidative stress, and brain edema (BE) in two rat models of HE: portacaval anastomosis (PCA) and bile-duct ligation (BDL). Ammonia and reactive oxygen species (ROS) levels, BE, oxidant and antioxidant enzyme activities, as well as lipid peroxidation were assessed both systemically and centrally in these two different animal models. Then, the effects of allopurinol (xanthine oxidase inhibitor, 100 mg/kg for 10 days) on ROS and BE and the temporal resolution of ammonia, ROS, and BE were evaluated only in BDL rats. Similar arterial and cerebrospinal fluid ammonia levels were found in PCA and BDL rats, both significantly higher compared to their respective sham-operated controls (p < 0.05). BE was detected in BDL rats (p < 0.05) but not in PCA rats. Evidence of oxidative stress was found systemically but not centrally in BDL rats: increased levels of ROS, increased activity of xanthine oxidase (oxidant enzyme), enhanced oxidative modifications on lipids, as well as decreased antioxidant defense. In PCA rats, a preserved oxidant/antioxidant balance was demonstrated. Treatment with allopurinol in BDL rats attenuated both ROS and BE, suggesting systemic oxidative stress is implicated in the pathogenesis of BE. Analysis of ROS and ammonia temporal resolution in the plasma of BDL rats suggests systemic oxidative stress might be an important “first hit”, which, followed by increases in ammonia, leads to BE in chronic liver failure. In conclusion, chronic hyperammonemia and oxidative stress in combination lead to the onset of BE in rats with chronic liver failure.

Acute liver failure (ALF) is characterized neuropathologically by cytotoxic brain edema and biochemically by increased brain ammonia and its detoxification product, glutamine. The osmotic actions of increased glutamine synthesis in astrocytes are considered to be causally related to brain edema and its complications (intracranial hypertension, brain herniation) in ALF. However studies using multinuclear (1)H- and (13)C-NMR spectroscopy demonstrate that neither brain glutamine concentrations per se nor brain glutamine synthesis rates correlate with encephalopathy grade or the presence of brain edema in ALF. An alternative mechanism is now proposed whereby the newly synthesized glutamine is trapped within the astrocyte as a consequence of down-regulation of its high affinity glutamine transporter SNAT5 in ALF. Restricted transfer out of the cell rather than increased synthesis within the cell could potentially explain the cell swelling/brain edema in ALF. Moreover, the restricted transfer of glutamine from the astrocyte to the adjacent glutamatergic nerve terminal (where glutamine serves as immediate precursor for the releasable/transmitter pool of glutamate) could result in decreased excitatory transmission and excessive neuroinhibition that is characteristic of encephalopathy in ALF. Paradoxically, in spite of renewed interest in arterial ammonia as a predictor of raised intracranial pressure and brain herniation in ALF, ammonia-lowering agents aimed at reduction of ammonia production in the gut have so far been shown to be of limited value in the prevention of these cerebral consequences. Mild hypothermia, shown to prevent brain edema and intracranial hypertension in both experimental and human ALF, does so independent of effects on brain glutamine synthesis; whether or not hypothermia restores expression levels of SNAT5 in ALF awaits further studies. While inhibitors of brain glutamine synthesis such as methionine sulfoximine, have been proposed for the prevention of brain edema in ALF, potential adverse effects have so far limited their applicability.

Advances in predicting in vivo performance of drug products has the potential to change how drug products are developed and reviewed. Modeling and simulation methods are now more commonly used in drug product development and regulatory drug review. These applications include, but are not limited to: the development of biorelevant specifications, the determination of bioequivalence metrics for modified release products with rapid therapeutic onset, the design of in vitro–in vivo correlations in a mechanistic framework, and prediction of food effect. As new regulatory concepts such as quality by design require better application of biopharmaceutical modeling in drug product development, regulatory challenges in bioequivalence demonstration of complex drug products also present exciting opportunities for creative modeling and simulation approaches. A collaborative effort among academia, government and industry in modeling and simulation will result in improved safe and effective new/generic drugs to the American public.

Ammonia is neurotoxic and believed to play a major role in the pathogenesis of hepatic encephalopathy (HE). It has been demonstrated, in vitro and in vivo, that acute and high ammonia treatment induces oxidative stress. Reactive oxygen species (ROS) are highly reactive and can lead to oxidization of proteins resulting in protein damage. The present study was aimed to assess oxidative status of proteins in plasma and brain (frontal cortex) of rats with 4-week portacaval anastomosis (PCA). Markers of oxidative stress, 4-hydroxy-2-nonenal (HNE) and carbonylation were evaluated by immunoblotting in plasma and frontal cortex. Western blot analysis did not demonstrate a significant difference in either HNE-linked or carbonyl derivatives on proteins between PCA and sham-operated control rats in both plasma and frontal cortex. The present study suggests PCA-induced hyperammonemia does not lead to systemic or central oxidative stress.

Encephalopathy and brain edema are serious complications of acute liver failure (ALF). The precise pathophysiologic mechanisms responsible have not been fully elucidated but it has been recently proposed that microglia-derived proinflammatory cytokines are involved. In the present study we evaluated the role of microglial activation and the protective effect of the anti-inflammatory drug minocycline in the pathogenesis of hepatic encephalopathy and brain edema in rats with ALF resulting from hepatic devascularisation. ALF rats were killed 6 h after hepatic artery ligation before the onset of neurological symptoms and at coma stages of encephalopathy along with their appropriate sham-operated controls and in parallel with minocycline-treated ALF rats. Increased OX-42 and OX-6 immunoreactivities confirming microglial activation were accompanied by increased expression of interleukins (IL-1beta, IL-6) and tumor necrosis factor-alpha (TNF-alpha) in the frontal cortex at coma stage of encephalopathy in ALF rats compared with sham-operated controls. Minocycline treatment prevented both microglial activation as well as the up-regulation of IL-1beta, IotaL-6 and TNF-alpha mRNA and protein expression with a concomitant attenuation of the progression of encephalopathy and brain edema. These results offer the first direct evidence for central proinflammatory mechanisms in the pathogenesis of brain edema and its complications in ALF and suggest that anti-inflammatory agents may be beneficial in these patients.

Publisher Summary This chapter discusses challenges and issues with quality by testing (QbT) and the old pharmaceutical assessment system, and also introduces the concepts and understanding of quality by design (QbD) for generic drugs. In the QbT approach, the Food and Drug Administration (FDA) ensures quality via a fixed manufacturing process, tight specifications, and testing results on raw materials, in-process materials, and finished products. All pharmaceutical products in regulatory evaluation are weighted equally, without regard to the risk to the consumer, for example, chemistry, manufacturing, and controls (CMC) review assessments of complex dosage forms, as well as narrow therapeutic index (NTI) drugs, differ only marginally from those of simple dosage forms. QbD is a systematic approach to development that begins with predefined objectives, and emphasizes product and process understanding, and process control, based on sound science and quality risk management. QbD requires an understanding of how formulation and process variables influence product quality, for example, under QbD, the specifications for raw materials, in-process controls, and finished products are established based on mechanistic understanding of how formulation and process factors impact product, and process performance, while ones under QbT are derived empirically from limited batch data. The specifications under QbD are solely used for the confirmation of product quality, not for assurance of manufacturing consistency and process control.

Encephalopathy and brain edema are serious complications of acute liver failure (ALF). The precise pathophysiologic mechanisms responsible have not been fully elucidated but it has been suggested that oxidative/nitrosative stress is involved. In the present study we evaluated the role of oxidative/nitrosative stress in the pathogenesis of hepatic encephalopathy and brain edema in rats with ALF resulting from hepatic devascularization. We also studied the effect of hypothermia, a treatment previously shown to delay the progression of encephalopathy and the onset of brain edema, on ALF-induced oxidative stress. ALF rats were sacrificed at precoma and coma stages of encephalopathy along with their appropriate sham-operated controls. Hypothermic ALF rats were sacrificed in parallel with normothermic comatose ALF rats. Nitric oxide production in plasma and brain was assessed indirectly by measuring the level of its stable end products, nitrite/nitrate (NOx), using the Griess reagent. Expression of nitric oxide synthase (NOS) isoforms and heme oxygenase-1 (HO-1) were measured using real-time quantitative PCR, Western blot analysis and immunohistochemistry. Increased nitrite/nitrate levels were observed in the plasma and frontal cortex in ALF rats at coma stage of encephalopathy compared to sham-operated controls. Increased expression of HO-1 protein and mRNA was observed in the frontal cortex of ALF rats at both precoma and coma stages of encephalopathy. Significant increases in expression of endothelial and inducible NOS mRNA isoforms also occurred at precoma and coma stages of encephalopathy. Expression of the neuronal nitric oxide synthase isoform (nNOS) was not altered by ALF. Hypothermia normalized nitrite/nitrate levels in brain and significantly attenuated HO-1, eNOS and iNOS expression. These results suggest that, oxidative/nitrosative stress participates in the pathogenesis of brain edema and its complications in ALF and that the beneficial effect of hypothermia depends in part on its ability to inhibit oxidative/nitrosative stress-related mechanisms.

Delivery of vaccine antigens from controlled-release poly(lactic/glycolic acid) (PLGA) microspheres is a novel approach to reduce the number of antigen doses required for protection against infection. A major impediment to developing single-shot vaccines is encapsulated antigen instability during months of exposure to physiological conditions. For example, efforts to control neonatal tetanus in developing countries with a single-dose TT vaccine based on PLGA microspheres have been plagued by poor stability of the 150 kDa formaldehyde-detoxified protein antigen, tetanus toxoid (TT), in the polymer. We examined the denatured states of PLGA-encapsulated TT, revealing two primary TT instability mechanisms: (1) protein aggregation mediated by formaldehyde and (2) acid-induced protein unfolding and epitope damage. Further, we systematically identified excipients, which can efficiently inhibit TT aggregation and retain TT antigenicity under simulated deleterious conditions, i.e., elevated temperature and humidity. By employing these novel additives in the PLGA system, we report the slow and continuous release of high doses of TT for one month with retained antigen stability during bioerosion of PLGA.

Hepcidin is a negative regulator of iron absorption produced mainly by the liver in response to changes in iron stores and inflammation, and its levels have been shown to regulate the intestinal basolateral iron transporter ferroportin1 ( Fp1). Hereditary hemochromatosis patients and Hfe-deficient mice show inappropriate expression of hepcidin but, in apparent contradiction, still retain the ability to regulate iron absorption in response to alterations of iron metabolism. To further understand the molecular relationships among Hfe, hepcidin, and Fp1, we investigated hepcidin and Fp1 regulation in Hfe-deficient mice ( Hfe−/−and β2m−/−) in response to iron deprivation, iron loading, and acute inflammation. We found that whereas basal hepcidin levels were manifestly dependent on the presence of Hfe and on the mouse background, all Hfe-deficient mice were still able to regulate hepcidin in situations of altered iron homeostasis. In the liver, Fp1 was modulated in opposite directions by iron and LPS, and its regulation in Hfe-deficient mice was similar to that observed in wild-type mice. In addition, we found that iron-deprived mice were able to mount a robust response after LPS challenge and that Toll-like receptor 4 (TLR-4)-deficient mice fail to regulate hepcidin expression in response to LPS. In conclusion, these results suggest that although Hfe is necessary for the establishment of hepcidin basal levels, it is dispensable for hepcidin regulation through both the iron-sensing and inflammatory pathways, and hepatic Fp1 regulation is largely independent of hepcidin and Hfe. The inflammatory pathway overrides the iron-sensing pathway and is TLR-4 dependent.

The effects of tacrolimus (FK506) and malononitrilamides (MNA) (FK778 and FK779) monotherapy and combination therapy were examined in prevention of acute heart and kidney allograft rejection and reversal of ongoing acute heart allograft rejection in the rat.Brown Norway (RT1n)-to-Lewis (RT11) and ACI (RT1a)-to-Lewis (RT11) combinations were used, respectively, for heart and kidney transplantation models. Immunosuppressants were administered orally from day 1 to day 14 for preventing acute rejection and from day 4 to day 34 after transplantation for the reversal of ongoing acute rejection.In the prevention of acute heart rejection model, recipient rats treated with monotherapy of tacrolimus or MNA (FK778, FK779) showed a dose-related prolongation of mean survival time (MST) compared with naive control rats (P0.01). The mean survival time in combination therapy of tacrolimus (FK506) and FK778 indicated that an additive to synergistic interaction was produced when compared with the respective monotherapies (combination index [CI]=0.631-1.022). These results were reproducible with tacrolimus and FK779 combination therapy (CI=0.572-0.846). Furthermore, similar results were also found in the prevention of acute kidney allograft rejection in the rat (CI=0.137-0.516). In the reversal of ongoing acute heart allograft rejection, combination therapy of tacrolimus and FK778 demonstrated a strong synergistic interaction (CI=0.166-0.970) compared with monotherapy of tacrolimus or FK778.Combination therapy of tacrolimus and MNA (FK778, FK779) produces synergistic effects in prevention of acute heart and kidney rejection and reversal of ongoing heart allograft rejection in the rat.

Malononitrilamide FK778, an analogue of leflunomide's active metabolite, is a promising novel small molecule with immunosuppressive and immunomodulatory properties. In this study, we evaluated the ability of combination therapy of FK778 with tacrolimus to inhibit lymphocyte proliferation and to prevent acute allograft rejection.Proliferation assay was used to evaluate the effect of FK778 plus tacrolimus on murine splenocytes, monkey lymphocytes, and human peripheral blood mononuclear cells, after activation with T or B cell-specific mitogens. A rat kidney transplantation model was used to evaluate the ability of FK778 combined with tacrolimus to prolong allograft survival. Median-effect principle and combination index (CI) were used to determine synergism, summation, or antagonism.A total of 58 combinations of FK778 plus tacrolimus were evaluated. Of the combinations tested, 82.8% (24/29) produced additive to synergistic effects in B cells, whereas 79.3% (23/29) produced moderate antagonistic effects in T cells. A concomitant 14-day therapy of FK778 (10 mg/kg/day) and tacrolimus (1 mg/kg/day) synergistically prolonged renal allograft survival to 25.5+/-5.9 days (CI=0.458). However, when addition of FK778 to tacrolimus therapy was delayed to day 7 after transplantation, a strong synergism was obtained (mean survival time=74.9+/-14.8 days, CI0.001).This study demonstrates that the combination of FK778 with tacrolimus in vitro produces synergistic inhibition on B-cell proliferation but not on T cell proliferation in mice, nonhuman primates, and humans. When the addition of FK778 treatment was delayed to day 7 after transplantation, a strong synergism was produced in prolongation of renal allograft survival in the rat.

Malononitrilamide 715 (FK778) is a new class of low-molecular-weight immunosuppressant that is a derivative of the active metabolite of leflunomide, A77 1726. In this study, the authors evaluated the combined effect of FK778 with tacrolimus in prevention of renal allograft rejection in Vervet monkeys.Male Vervet monkeys were obtained from Caribbean Primates Ltd. Donor and recipient monkeys were from different breeding colonies. Eleven groups (nor=4 per group) were involved in this study. FK778 and tacrolimus were administered orally for 60 days according to protocol.Naive controls rejected renal grafts, with a median survival time (MST) of 8.0 days in group 1. When recipient monkeys were treated with tacrolimus 1.0 mg/kg/day in group 2 or FK778 2.5 mg/kg/day in group 3, the MST was 16.0 days (P=0.001) and 11.0 days (P=0.266), respectively. Combination therapy of these two agents at the same doses immediately after transplantation resulted in an MST of 25.0 days (P=0.016) in group 4. When tacrolimus was initiated immediately after transplantation and FK778 treatment was delayed until day 7 after surgery in group 5, recipient survivals were significantly prolonged to 38.0 days (P=0.02). These results were repeatable when FK778 5.0 mg/kg/day (9.0 days, P=0.544 in group 6) was combined with tacrolimus 1.0 mg/kg/day immediately after transplantation (8.0 days, P=0.339) in group 7, or when FK778 was delayed 7 days (60.0 days, P=0.002) in group 8. Furthermore, it was also repeatable when FK778 10 mg/kg/day was combined with tacrolimus 1.0 mg/kg/day with a 7-day delay.A significant prolongation of renal allograft survival was produced when FK778 administration was delayed by 7 days combined with tacrolimus in Vervet monkeys.

Improved microsurgical techniques for en bloc vascularized adnexal isograft in the rat is described. The graft of the right ovary together with its fallopian tube and upper third of uterus was transplanted orthotopically with end-to-side anastomoses between the donor aortic segment and recipient aorta and between the donor vena cava segment and recipient inferior vena cava, with end-to-end anastomosis of the donor and recipient uterus in a syngeneic, bilaterally oophorectomized rat. All transplantations were successful in terms of immediate vascular patency rate (10/10, 100%). Evidence of resumed ovarian function was obtained in 9 out of 10 rats (9/10, 90.0%) by histological demonstration of the vaginal smear, in which pregnancies were achieved in six rats (6/10, 60.0%) and six litters of healthy offspring were delivered 9 weeks later after transplantation. These results suggest that microsurgical ovarian transplantation provide a new and potential experimental model for the study of fertility restoration in humans. © 2002 Wiley-Liss, Inc. MICROSURGERY 22:30–33 2002 DOI 10.1002/micr.22006

A formaldehyde-mediated aggregation pathway (FMAP) has been shown to be primarily responsible for the solid-state aggregation of lyophilized formalinized protein antigens [e.g., tetanus toxoid (TT) and formalinized bovine serum albumin (f-BSA)] in the presence of moisture and physiological temperature. Coincorporation of the formaldehyde-interacting amino acid, histidine, strongly inhibits the FMAP. The purpose of this study was to test whether previous solid-state data are applicable toward the stabilization of formalinized antigens encapsulated in poly(lactide-co-glycolide) (PLGA)-based microspheres. Formaldehyde-treated bovine serum albumin (f-BSA) and BSA were selected as a model formalinized protein antigen and a nonformalinized control, respectively. As in the solid state, we found that the FMAP was dominant in the aggregation of f-BSA encapsulated in PLGA 50/50 microspheres, whereas the aggregation mechanism of encapsulated BSA was mostly converted from thiol-disulfide interchange to an acid-catalyzed noncovalent pathway. The lack of noncovalent aggregation in encapsulated f-BSA could be explained by its higher thermodynamic stability after formalinization, which inhibits protein unfolding. Targeting the FMAP, coencapsulation of histidine and trehalose successfully inhibited the aggregation of f-BSA in microspheres. By combining the use of an optimized oil-in-oil (o/o) encapsulation method, coencapsulation of histidine and trehalose, and use of low-acid-content poly(D,L-lactide) (PLA) and poly(ethylene glycol) (PEG) blends, a 2-month continuous release of f-BSA was achieved with the absence of aggregation.