How should adverse events be reported in US clinical trials?: ethical considerations.

Research with human subjects, including clinical trials, forms the cornerstone of the development of knowledge on the safety and efficacy of potential drugs and devices. During the conduct of clinical trials, human subjects may experience adverse events ranging from trivial side effects such as mild discomfort to severe complications, including death. Three of the regulatory criteria set forth in the 45 Code of Federal Regulations 46.111 for approval of research(1) focus on risk assessment and minimization of risks. The accurate and full reporting of these adverse events and the assessment of the risk attributable to participation in research are therefore crucial components in the ethical conduct of research.

Adverse events reporting--the tip of an iceberg.

NIH data indicate that annually seven million human subjects are enrolled in research sponsored by NIH alone. In addition, there are sixteen federal agencies and numerous departments outside NIH conducting experiments with human subjects. Moreover, the pharmaceutical industry spends $26 billion on research (compared to $16 billion for NIH), thus, the total number of human subjects enrolled in research for both the public and private sectors can be estimated as high as nineteen million. I present data on the potential magnitude of adverse events in the United States among human subjects enrolled in research that appear to be unreported and unaccounted for. We obtained data from the Office for Human Research Protections (OHRP) through the Freedom of Information Act for the years 1990 to August 2000 regarding all Institutional Incident Reports (IRPTs) and a list of Compliance Oversight Branch Investigations (COBIs) involving Multiple Project Assurances (MPAs). In the ten years of reporting for nearly seventy million human subjects, there were only 878 IRPTs and 41 investigations. From the incident reports to OHRP, 44% involved adverse events. Those projects investigated for Multiple Project Assurances violations (41 such investigations) showed that 51% were suspended or terminated. The number of deaths reported to OHRP in ten years for the seventy million human subjects is merely eight. The anticipated number of deaths among the general population in seventy million (assuming subject's duration in trials is one month) is 51,000. The number of suicides and attempted suicides alone among the seventy million expected research subjects can be anticipated to be about 5,000. Therefore, the number of expected deaths should have been between 5,000 and 51,000. These numbers and percentages represent minimal numbers since they are not a result of random audits or investigations, but a result of self-reporting or an exogenous complaint. Despite the fact that these are conservative estimates, they represent a significant problem. The purpose of this paper is to present the potential boundaries and magnitude of the problem in the current use of human subjects in research. This is a call for responsible institutions to undertake a thorough evaluation of the problem in order to obtain accurate information. For the immediate future, strong actions need to be taken to increase the protection of human subjects enrolled in research. This precautionary policy is prudent in light of recent revelations and data from this investigation.

Identification of the major transport pathway for the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium.

1-Methyl-4-phenylpyridinium is a potent parkinsonism-inducing neurotoxin which has become a valuable tool for the examination of the mechanisms and therapeutic treatment strategies for Parkinson's syndrome. Recently, it has been found that physiological levels of extracellular ATP (0.1-1 mM) stimulate dopamine uptake into both rat and bovine brain synaptosomes and rat pheochromocytoma cells in a dose-dependent manner. In this study we report that physiological levels of extracellular ATP (0.1-2 mM) stimulate the transport of 1-methyl-4-phenylpyridinium into the pheochromocytoma cell line by 270% over basal levels. Kinetically, the presence of ATP increases both the K(m) and Vmax of 1-methyl-4-phenylpyridinium transport. In addition, 1-methyl-4-phenylpyridinium is far more effective at inhibiting ATP-stimulated dopamine transport (IC50 = 11 microM) than basal dopamine transport (IC50 100 microM) into pheochromocytoma cells. These data show that the ATP-regulated 1-methyl-4-phenylpyridinium transport pathway is the major component (approximately 95%) of total 1-methyl-4-phenylpyridinium transport, and provide the first evidence for the involvement of extracellular ATP in the bulk transport of 1-methyl-4-phenylpyridinium.

Cation requirements of basal and ATP-regulated dopamine transport in rat pheochromocytoma cells.

The transport of dopamine into presynaptic nerve terminals is the primary mechanism for the termination of dopaminergic neurotransmission. This transport process has recently been found to be composed of two components, a basal dopamine transport pathway which exists in the absence of extracellular ATP and an ATP-regulated moiety which comprises approximately 66% of the total transport system [Cao C. J. et al. (1990) Biochem. Pharmac. 39, R9-R14; Cao C. J. et al. (1989) Biochemistry 8, 207-220; Dunigan C. D. and Shamoo A. E. (1995) Neuroscience 65, 1-4; Eshleman A. et al. (1995) Life Sci. 56, 1613-1621]. Using a rat pheochromocytoma cell line and a Krebs bicarbonate buffering system, the present study examined the effect of several cations on both basal and ATP-regulated dopamine transport. In the absence of extracellular ATP, dopamine transport had an absolute dependence on the presence of Na+, but exhibited no requirement for Mg2+. Kinetically, the addition of 120 mM NaCl increased the Vmax of basal dopamine transport by approximately 150%. In contrast, the ATP-regulated dopamine transport pathway displayed a different sensitivity to Na+ and was completely dependent upon the presence of Mg2+. The addition of 1.2 mM MgSO4 increased the Vmax of transport in the presence of 0.7 mM extracellular ATP by 222%. Both basal and ATP-regulated transport were unaffected by the removal of either Ca2+ or K+ from the assay buffer. When the effects of ouabain, a potent inhibitor of Na+, K(+)-ATPase, were tested in the rat pheochromocytoma cell model, it was found that concentrations of ouabain as high as 1 mM were ineffective at inhibiting either the basal or ATP-regulated dopamine transport components. These results imply that the Na+ gradient supplied by Na+, K(+)-ATPase is not the sole provider of energy needed to drive either transport process. The ionic requirements of the basal and ATP-regulated dopamine transport pathways demonstrate the distinction between the two transport processes. In addition, the ionic dependency profile of the ATP-regulated moiety has provided some mechanistic insights into ATP-regulated catecholamine uptake, as the absolute Mg2+ requirement and the ineffectiveness of Ca2+ argues against the involvement of either purinergic receptors or a Ca(2+)-dependent, Mg(2+)-independent ectokinase in the ATP-regulated transport system.

Li+ stimulates ATP-regulated dopamine uptake in PC12 cells.

Increased levels of dopamine have been associated with schizophrenia and mania; conversely, decreased levels of dopamine are associated with depression. Since the main mechanism for the termination of dopamine's pharmacological action is by re-uptake into the presynaptic cell, the speed of dopamine transport dramatically influences the concentration of dopamine present in the synaptic cleft, which in turn could determine brain disorders. Our preliminary studies have found that ATP can stimulate dopamine transport in rat synaptosomal preparation. We have also observed this ATP-regulated moiety of the dopamine uptake system when tested in PC12 cells. The large magnitude of ATP stimulation suggests that this dopamine uptake pathway may be important in the etiology and treatment of brain disorders. In order to test the relevance of ATP-stimulated dopamine uptake, we tested the effect of lithium salts on this system. Lithium chloride, one of the first drugs used in the treatment of mania, has become one of the most important agents utilized for the treatment of manic-depression and many schizoeffective disorders. Unfortunately, despite all efforts that have been made to explain lithium's mode of action, a clear cut biochemical mechanism has not been defined. We have found that lithium chloride, at therapeutic levels, is able to stimulate the ATP-regulated component of the dopamine uptake system by 49%. The further enhancement of dopamine re-uptake by lithium ions is consistent with its therapeutic effect. It is suggested that any substance that facilitates dopamine re-uptake could be of great importance in defining a useful treatment for mania and schizophrenia, as well as depression.

Uncoupling of occlusion from ATP hydrolysis activity in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.

Ca2+ permeability of rat parotid gland basolateral plasma membrane vesicles is modulated by membrane potential and extravesicular [Ca2+].

This study examines the Ca2+ permeability of basolateral plasma membrane vesicles (BLMVs) isolated from the rat parotid gland by monitoring the rate of 45Ca2+ efflux from actively-loaded (via the Ca(2+)-ATPase) inside-out BLMVs. Ca2+ efflux from BLMVs into a K(+)-gluconate medium which hyperpolarizes the cytoplasmic side (i.e. outside) of the inside-out BLMVs resulted in a faster rate of Ca2+ efflux compared with a control medium containing N-methyl-D-glucamine (NMDG)-gluconate. Conversely, Ca2+ efflux into a medium which depolarizes the cytoplasmic side of the BLMVs (NMDG-chloride) resulted in slower rates of efflux compared with those observed with the control medium. This increased rate of 45Ca2+ efflux from the hyperpolarized BLMV was inhibited by 1 mM Ni2+, yielding a rate of efflux similar to the rate observed in depolarized BLMVs. The rate of Ca2+ efflux from BLMVs was affected by [Ca2+]o ([Ca2+] on the extravesicular, cytoplasmic side of the vesicle). When [Ca2+]o was kept > 200 nM during efflux, the rate of Ca2+ efflux from both hyper- and depolarized BLMVs was slow and relatively unresponsive to changes in [Ca2+]o, despite sizeable changes in the Ca2+ gradient across the BLMV. However, when [Ca2+]o was lowered < 200 nM, there was an abrupt increase in the rate of Ca2+ efflux from both hyper- and depolarized BLMVs. Additionally, when [Ca2+] was < 200 nM, the rate of Ca2+ efflux appeared to be more sensitive to driving force changes. These data suggest that Ca2+ permeability across the rat parotid gland basolateral plasma membrane is modulated by membrane potential and [Ca2+] on the cytoplasmic side.

Quality assurance.

The use of the terms "quality," "quality control," and "quality assurance" has been thrust recently upon us in descriptions of products and services. There is a great deal of confusion and inappropriate use of these terms. A historical search of the use of these terms in the past five decades indicated that quality is a subjective term that connotes excellence and reliability of performance. Quality control is concerned with ensuring compliance with prior agreed upon specifications. Quality assurance is concerned with reviewing the performance of products and services after the use of products and services. In this paper, I propose a process by which quality assurance can be achieved. Also, I list a few values for compliance with quality assurance programs.

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

In reconstituted rabbit skeletal muscle (Ca2+ + Mg2+)-ATPase proteoliposomes, Ca(2+)-uptake is decreased by more than 90% with T2 cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T3 cleavage, which represents the cleavage of A1 fragment to A1a + A1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca2+ efflux between control SR and tryptically digested SR in the absence of Mg2+ + ruthenium red or in the presence of ATP. However, the passive Ca2+ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg2+ + ruthenium red. These results show that the Ca2+ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca2+ channel inhibitors, Mg2+ and ruthenium red. In the reconstituted proteoliposomes, the Ca2+ efflux rates are the same regardless of digestion (T2); also, efflux is not affected by the presence or absence of Mg2+ + ruthenium red. These results indicate that T2 cleavage causes 'uncoupling' of the 'Ca(2+)-pump' from ATP hydrolytic activity. A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca2+ + Mg2+)-ATPase polypeptide with the loss of Ca(2+)-transport. Fits of the theoretical equations to the data are consistent with that Ca(2+)-transport system appears to require a dimer of the polypeptide (Ca2+ + Mg2+)-ATPase.

Tertiary structure and energy coupling in Ca2(+)-pump system.

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8-9 A and the average distance from the Ca2+ sites to CrATP is about 18 A. Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2(+)-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.

Identification of a spectroscopic marker for the Ca2(+)-binding site of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum in the occluded state.

The 7F0----5D0 excitation spectrum of Eu3+ bound to the high-affinity calcium sites of SR (Ca2+ + Mg2+)-ATPase diminishes upon occlusion of the Eu3+ into the interior of the enzyme. This "quenching" was found to be caused by the enzyme itself because trypsin digestion could relieve it. The level of digestion needed to relieve the quenching is beyond the level needed to eliminate occlusion; thus, the two processes are not related. Ca2+ is required during digestion to preserve the quenching, indicating close proximity between the Ca2+ site(s) and the quenching segment. Synthetic peptides were found that could mimic the native enzyme's ability to quench the Eu3+ fluorescence, although no native sequence has yet been identified that could emulate the enzyme.