Pre-clinical evaluation of a novel class of anti-cancer agents, the Pyrrolo-1, 5-benzoxazepines.

Microtubules are currently ranked one of the most validated targets for chemotherapy; with clinical use of microtubule targeting agents (MTAs) extending beyond half a century. Recent research has focused on the development of novel MTAs to combat drug resistance and drug associated toxicities. Of particular interest are compounds structurally different to those currently used within the clinic. The pyrrolo-1, 5-benzoxazepines (PBOXs) are a structurally distinct novel group of anti-cancer agents, some of which target tubulin. Herein, we review the chemistry, mechanism of action, preclinical development of the PBOXs and comparisons with clinically relevant chemotherapeutics. The PBOXs induce a range of cellular responses including; cell cycle arrest, apoptosis, autophagy, anti-vascular and anti-angiogenic effects. The apoptotic potential of the PBOXs extends across a wide spectrum of cancer-derived cell lines, by targeting tubulin and multiple molecular pathways frequently deregulated in human cancers. Extensive experimental data suggest that combining the PBOXs with established chemotherapeutics or radiation is therapeutically advantageous. Pre-clinical highlights of the PBOXs include; cancer specificity and improved therapeutic efficacy as compared to some current first line therapeutics.

PBOX-15, a novel microtubule targeting agent, induces apoptosis, upregulates death receptors, and potentiates TRAIL-mediated apoptosis in multiple myeloma cells.

BACKGROUND: In recent years, much progress has been made in the treatment of multiple myeloma. However, a major limitation of existing chemotherapeutic drugs is the eventual emergence of resistance; hence, the development of novel agents with new mechanisms of action is pertinent. Here, we describe the activity and mechanism of action of pyrrolo-1,5-benzoxazepine-15 (PBOX-15), a novel microtubule-targeting agent, in multiple myeloma cells. METHODS: The anti-myeloma activity of PBOX-15 was assessed using NCI-H929, KMS11, RPMI8226, and U266 cell lines, and primary myeloma cells. Cell cycle distribution, apoptosis, cytochrome c release, and mitochondrial inner membrane depolarisation were analysed by flow cytometry; gene expression analysis was carried out using TaqMan Low Density Arrays; and expression of caspase-8 and Bcl-2 family of proteins was assessed by western blot analysis. RESULTS: Pyrrolo-1,5-benzoxazepine-15 induced apoptosis in ex vivo myeloma cells and in myeloma cell lines. Death receptor genes were upregulated in both NCI-H929 and U266 cell lines, which displayed the highest and lowest apoptotic responses, respectively, following treatment with PBOX-15. The largest increase was detected for the death receptor 5 (DR5) gene, and cotreatment of both cell lines with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), the DR5 ligand, potentiated the apoptotic response. In NCI-H929 cells, PBOX-15-induced apoptosis was shown to be caspase-8 dependent, with independent activation of extrinsic and intrinsic apoptotic pathways. A caspase-8-dependent decrease in expression of Bim(EL) preceded downregulation of other Bcl-2 proteins (Bid, Bcl-2, Mcl-1) in PBOX-15-treated NCI-H929 cells. CONCLUSION: PBOX-15 induces apoptosis and potentiates TRAIL-induced cell death in multiple myeloma cells. Thus, PBOX-15 represents a promising agent, with a distinct mechanism of action, for the treatment of this malignancy.

Novel pyrrolo-1,5-benzoxazepine compounds display significant activity against resistant chronic myeloid leukaemia cells in vitro, in ex vivo patient samples and in vivo.

BACKGROUND: Imatinib is a direct and potent inhibitor of the constitutively active tyrosine kinase, breakpoint cluster region-Abelson (Bcr-Abl), which is central to the pathogenesis of chronic myeloid leukaemia (CML) patients. As such, imatinib has become the front-line treatment for CML patients. However, the recent emergence of imatinib resistance, commonly associated with point mutations within the kinase domain, has led to the search for alternative drug treatments and combination therapies for CML. METHODS: In this report, we analyse the effects of representative members of the novel pro-apoptotic microtubule depolymerising pyrrolo-1,5-benzoxazepines or PBOX compounds on chemotherapy-refractory CML cells using a series of Bcr-Abl mutant cell lines, clinical ex vivo patient samples and an in vivo mouse model. RESULTS: The PBOX compounds potently reduce cell viability in cells expressing the E225K and H396P mutants as well as the highly resistant T315I mutant. The PBOX compounds also induce apoptosis in primary CML samples including those resistant to imatinib. We also show for the first time, the in vivo efficacy of the pro-apoptotic PBOX compound, PBOX-6, in a CML mouse model of the T315I Bcr-Abl mutant. CONCLUSION: Results from this study highlight the potential of these novel series of PBOX compounds as an effective therapy against CML.

Caspase-activated DNase (CAD)-independent oligonucleosomal DNA fragmentation in chronic myeloid leukaemia cells; a requirement for serine protease and Mn2+-dependent acidic endonuclease activity.

We have previously reported that the pro-apoptotic pyrrolobenzoxazepine, PBOX-6, induces apoptosis in chronic myelogenous leukaemia (CML) cells which is accompanied by oligonucleosomal DNA fragmentation. In this study we show that PBOX-6-induced oligonucleosomal DNA fragmentation occurs in the absence of caspase and CAD activation in CML cells. Dissection of the signalling pathway has revealed that induction of apoptosis requires the upstream activation of a trypsin-like serine protease that promotes the phosphorylation and inactivation of anti-apoptotic Bcl-2. In addition, in this system chymotrypsin-like serine proteases are dispensable for high molecular weight DNA fragmentation, however are required for the activation of a relatively small manganese-dependent acidic endonuclease that is responsible for oligonucleosomal fragmentation of DNA. Furthermore, we demonstrate mitochondrial involvement during PBOX-6-induced apoptosis and suggest the existence of unidentified mitochondrial effectors of apoptosis.

In vitro cytotoxicity of a composite resin and compomer.

AIM: This work was designed to investigate the potential cytotoxicity of two of the newer dental restorative materials. Spectrum composite resin and Dyract AP compomer. METHODOLOGY: Cultured human endothelial cells (ECV-304) were exposed to each of the restorative materials through a 70-microm dentine barrier to simulate the in vivo clinical situation. Cell viability was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay and lactate dehydrogenase release assay. The effects of different extents of light-curing were also examined by microscopic examination of stained human promyelocytic leukemia cells (HL-60). Caspase-3 activation was determined as a measure of apoptotic cell death. RESULTS: Assessment of cellular viability indicated that both materials cause cell death, with Spectrum being the more toxic. The cytotoxicity was considerably increased in the absence of the dentine barrier. Direct exposure to Spectrum for 12 h resulted in the death of 69% of the cells after full light-curing (78% of total death was by apoptosis) and 96% after partial light-curing (73% of total death was by necrosis). Assessment of caspase activation, in the absence of the dentine barrier, showed that longer curing-times resulted in an increase in the proportion of the cells dying through apoptosis, rather than necrosis, for both materials tested. CONCLUSIONS: These results indicate the restorative materials to be potentially toxic, particularly if the degree of light-cure is inadequate.

Pyrrolo-1,5-benzoxazepines: a new class of apoptotic agents.

Some members of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) potently induce apoptosis in a number of human cancerous cell lines including HL-60 cells and the drug-resistant chronic myelogenous leukaemia cell line, K562. The apoptotic induction seems to be independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR), which binds these PBOXs with high affinity, due to a lack of correlation between their affinities for the receptor and their apoptotic potencies and their high apoptotic activity in PBR-deficient cells. PBOX-6, a potent member of the series, induces a transient activation of c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which correlates with induction of apoptosis. Expression of a cytoplasmic inhibitor of the JNK signal transduction pathway, Jip-1, prevents JNK activity and significantly reduces the extent of apoptosis induced by PBOX-6. This demonstrates the requirement for JNK in the cellular response to this apoptotic agent. In addition, PBOX-6 activates caspase-3-like proteases in K562 and HL-60 cells. The caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk), blocks caspase-3-like protease activity in both cell types but only prevents PBOX-6-induced apoptosis in HL-60 cells, suggesting that the requirement for caspase-3-like proteases in the apoptotic pathway is dependent on the cell type.

Flexible estrogen receptor modulators: design, synthesis, and antagonistic effects in human MCF-7 breast cancer cells.

Although many series of estrogen receptor antagonists continue to be produced, the majority are direct structural analogues of existing modulators. To examine the tolerance of the estrogen receptor toward flexible ligands, a series of novel flexible estrogen receptor antagonists were prepared and their antiproliferative effects on human MCF-7 breast tumor cells investigated. Each of these compounds deviated from the traditional triphenylethylene backbone associated with common tamoxifen analogues through the introduction of a flexible methylene (benzylic) spacing group between one of the aryl rings and the ethylene group and through variations in the basic side chain moiety. The compounds prepared, when assayed in conjunction with a tamoxifen standard, demonstrated high potency in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity and high binding affinity. A computational study was undertaken to investigate the compounds' potential interactions with specific residues within the human estrogen receptor alpha ligand-binding domain (ER-LBD), predicting these compounds bind in an antiestrogenic fashion within the ER-LBD and interact with those important residues previously identified in the structures of ER-LBD agonist/antagonist cocrystals. These compounds further illustrate the eclectic nature of the estrogen receptor in terms of ligand flexibility tolerance.

Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor bcr-abl.

Expression of the transforming oncogene bcr-abl in chronic myelogenous leukemia (CML) cells is reported to confer resistance against apoptosis induced by many chemotherapeutic agents such as etoposide, ara-C, and staurosporine. In the present study some members of a series of novel pyrrolo-1,5-benzoxazepines potently induce apoptosis, as shown by cell shrinkage, chromatin condensation, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage, in three CML cell lines, K562, KYO.1, and LAMA 84. Induction of apoptosis by a representative member of this series, PBOX-6, was not accompanied by either the down-regulation of Bcr-Abl or by the attenuation of its protein tyrosine kinase activity up to 24 h after treatment, when approximately 50% of the cells had undergone apoptosis. These results suggest that down-regulation of Bcr-Abl is not part of the upstream apoptotic death program activated by PBOX-6. By characterizing the mechanism in which this novel agent executes apoptosis, this study has revealed that PBOX-6 caused activation of caspase 3-like proteases in only two of the three CML cell lines. In addition, inhibition of caspase 3-like protease activity using the inhibitor z-DEVD-fmk blocked caspase 3-like protease activity but did not prevent the induction of apoptosis, suggesting that caspase 3-like proteases are not essential in the mechanism by which PBOX-6 induces apoptosis in CML cells. In conclusion, this study demonstrates that PBOX-6 can bypass Bcr-Abl-mediated suppression of apoptosis, suggesting an important potential use of these compounds in the treatment of CML.

Oxidative stress induces apoptosis in C6 glioma cells: involvement of mitogen-activated protein kinases and nuclear factor kappa B.

Excessive oxidative stress has been implicated in the induction of cell death in a variety of neurodegenerative diseases. In the present study, hydrogen peroxide (H2O2)-induced cell death in rat C6 glioma cells was used as a model system for studying the molecular events associated with oxidative stress-induced cell death in glial cells. We demonstrate that exposure of C6 glioma cells to H2O2 results in apoptotic cell death in a concentration-dependent manner, and caused activation of a member of the caspase-3-like family of proteases resulting in cleavage of the DNA repair enzyme poly(ADP-ribose)polymerase, PARP. Furthermore, H2O2 induced a transient activation of the transcription factor, nuclear factor kappa B (NF(Kappa)B). Pre-treatment of cells with the antioxidant N-acetylcysteine, (NAC), prevented both the activation of NF(Kappa)B and the induction of apoptosis by H2O2, suggesting a possible role for this transcription factor in oxidant-induced apoptosis in glial cells. Exposure of the cells to H2O2 led to transient activation of both c-Jun N-terminal kinase (JNK) and p38 kinase but has no effect on extracellular regulated kinase (ERK) activity. Inhibition of p38 by SB203580 did not protect the cells against H2O2-induced apoptosis suggesting that activation of p38 is not essential for H2O2-mediated cell death in C6 glioma cells.

Ethyl side-chain modifications in novel flexible antiestrogens--design, synthesis and biological efficacy in assay against the MCF-7 breast tumor cell line.

To examine the efficacy of ethyl side-chain modifications in a family of flexible non-steroidal modulators of the estrogen receptor, a series of novel compounds was prepared and their antiproliferative effects on human MCF-7 breast tumor cells evaluated. These flexible antiestrogens consisted of members wherein the ethyl portion of the parent compound, a flexible analogue of tamoxifen, had been modified so as to introduce halogens or a nitro group, or to extend the side-chain length from ethyl to propyl or butyl. The compounds demonstrated potency at low micromolar concentrations in antiproliferative assays against an MCF-7 human breast cancer cell line with low associated cytotoxicity. Tested compounds exhibited nanomolar binding affinity (Ki) for the estrogen receptor (ER) as determined through displacement of radiolabelled estradiol. Semiempirical calculations predict an inherent lower oxidative potential at the allylic position, similar to that calculated for the established analogue toremifene, indicating a lesser propensity of such compounds towards metabolic oxidative carbocation generation and consequent DNA adduct formation. Computational studies predict these compounds to bind in a typical estrogen antagonist mode within the ER-ligand binding domain (LBD). Ethyl side-chain modification in this compound class is well tolerated within the ER and is not detrimental to compound efficacy, with additional potential anti-carcinogenic properties imbued to the molecule.

Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents.

Some, but not all, of a series of novel pyrrolo-1,5-benzoxazepines (PBOXs) induce apoptosis as shown by cell shrinkage, chromatin condensation, and DNA fragmentation in three human cell lines, HL-60 promyelocytic, Jurkat T lymphoma, and Hut-78 s.c. lymphoma cells. This chemical selectivity, together with the lack of apoptotic activity against rat Leydig cells, argues against a general cell poisoning effect. PBOX-6, a potent member of the series, caused activation of a member of the caspase-3 family of proteases. In addition, the caspase-3-like inhibitor z-DEVD-fmk, but not the caspase-1-like inhibitor z-YVAD-fmk prevented PBOX-6-induced apoptosis, suggesting that caspase 3-like proteases are involved in the mechanism by which PBOX compounds induce apoptosis. The release of cytochrome c into the cytosol in HL-60 cells in response to PBOX-6 suggests that this cellular response may be important in the mechanism by which PBOX-6 induces apoptosis. However, reactive oxygen intermediates do not play a key role in PBOX-6-induced apoptosis because neither the free radical scavenger TEMPO nor the antioxidant N-acetylcysteine had any effect on PBOX-6-induced apoptosis. The apoptotic induction seems independent of the mitochondrial peripheral-type benzodiazepine receptor (PBR) that binds these pyrrolobenzoxazepines with high affinity, due to the lack of correlation between their affinities for the receptor and their apoptotic potencies, their high apoptotic activity in PBR-deficient cells such as Jurkats, and their lack of apoptotic induction in PBR-rich rat Leydig cells. These PBOXs also can overcome nuclear factor-kappaB-mediated resistance to apoptosis. This suggests an important potential use of these compounds in drug-resistant cancers.

Antiproliferative action of pyrrolobenzoxazepine derivatives in cultured cells: absence of correlation with binding to the peripheral-type benzodiazepine binding site.

Three novel peripheral-type benzodiazepine binding site (PBBS) ligands, NF 182, 213 and 262, along with the classically used PBBS ligands, PK 11195 and Ro5-4864, were found to inhibit, at micromolar concentrations and in dose-dependent manner, the proliferation of rat C6 glioma and human 1321N1 astrocytoma, without being cytotoxic. This antiproliferative effect is mediated by arrest in the G1 phase of the cell cycle and does not appear to be mediated by a specific interaction of these ligands with the peripheral-type benzodiazepine binding site.

Peripheral-type benzodiazepine receptors.

1. The pharmacological effects of benzodiazepines are mediated through a class of recognition sites associated with the gamma-aminobutyric acid A receptor. A second class of benzodiazepine binding sites is found in virtually all mammalian peripheral tissues and is therefore called the peripheral type benzodiazepine receptor (PBR). 2. The first section of this review describes the tissue and subcellular distribution of the PBR in mammalian tissues and analyzes its many putative endogenous ligands. 3. The next section deals with the pharmacological, structural and molecular characterization of the PBR that has taken place in the past few years. 4. The final section describes the possible physiological role(s) of the PBR and identifies future work that would help deepen our understanding of the PBR and its function.

Calmidazolium and other imidazole compounds affect steroidogenesis in Y1 cells: lack of involvement of the peripheral-type benzodiazepine receptor.

Calmidazolium potently stimulated steroidogenesis in a mouse adrenocortical Y1 cell line, in a Ca(2+)-independent manner, an effect similar to that reported by Choi and Cooke [1] for rat primary adrenocortical and Leydig cells. Calmidazolium analogues, econazole and miconazole, were shown to inhibit both this calmidazolium-stimulated rate and the endogenous rate of steroidogenesis. In determining the mechanism by which imidazole compounds affect steroidogenesis, they were found not to act directly on the mitochondrial Cyt P-450scc enzyme, making it likely that they act instead on the intramitochondrial transport of cholesterol. Using competition binding studies, calmidazolium, econazole and miconazole were subsequently identified as novel ligands for the peripheral-type benzodiazepine receptor (PBR). Econazole and miconazole were found to inhibit stimulation by PK 11195 (a specific PBR ligand) of steroidogenesis, whereas treatment of the cells with calmidazolium and PK 11195 at the same time resulted in an additive stimulatory effect on steroidogenesis. These results suggest that the effects of these substituted imidazoles on steroidogenesis in Y1 cells is not mediated through their interaction with the PBR.

Synthesis, biological activity, and SARs of pyrrolobenzoxazepine derivatives, a new class of specific "peripheral-type" benzodiazepine receptor ligands.

The "peripheral-type" benzodiazepine receptor (PBR) has been reported to play a role in many biological processes. We have synthesized and tested a novel series of PBR ligands based on a pyrrolobenzoxazepine skeleton, in order to provide new receptor ligands. Several of these new compounds proved to be high affinity and selective ligands for PBR, and benzoxazepines 17f and 17j were found to be the most potent ligands for this receptor to have been identified to date. The SAR and the molecular modeling studies detailed herein delineated a number of structural features required for improving affinity. Some of the ligands were employed as "molecular yardsticks" to probe the spatial dimensions of the lipophilic pockets L1 and L3 in the PBR cleft and to determine the effect of occupation of L1 and L3 with respect to affinity, while other C-7 modified analogues provided information specifically on the hydrogen bonding with a putative receptor site H1. The new pyrrolobenzoxazepines were tested in rat cortex, a tissue expressing high density of mitochondrial PBR, and exhibited IC50 and Ki values in the low nanomolar or subnanomolar range, as measured by the displacement of [3H]PK 11195 binding. A subset of the highest affinity ligands was also found to have high affinities for [3H]PK 11195 and [3H]Ro 5-4864 binding in rat adrenal mitochondria. All the ligands in this subset are stimulators of steroidogenesis having similar potency and extent of stimulation as PK 11195 and Ro 5-4864 of steroidogenesis in the mouse Y-1 adrenocortical cell line.

Two cellular and subcellular locations for the peripheral-type benzodiazepine receptor in rat liver.

Determination of ligand binding properties of the peripheral benzodiazepine receptor (PBBS) in liver, in hepatocytes, and in nonparenchymal cells demonstrated the presence of receptor-specific high-affinity binding in both hepatocyte and nonhepatocyte cells. Density gradient centrifugation showed that the high-affinity receptor in hepatocytes was localised to mitochondria, whereas in nonhepatocytes it was not mitochondrial, but with a possible biliary epithelial cell plasma-membrane location. Both receptors showed the peripheral-type specific high-affinity binding of PK 11195 and Ro5 4864 and could be photolabelled as 18 kDa proteins with [3H]PK 14105. Immunocytochemistry showed the presence of acyl-CoA binding protein, a putative endogenous ligand for the receptor, in both cell locations. Some other properties of the PBBS were investigated in liver. Diphosphatidyl glycerol had a strong inhibitory effect on receptor binding in both liver and adrenal, with Ro5 4864 more sensitive to inhibition than PK 11195. However, whereas soybean lipid and phosphatidyl serine increased the binding of both ligands to adrenal receptor, these lipids had no effect on liver, suggesting that liver PBBS may differ from the well-characterised adrenal PBBS in some of its protein conformation. Modulators of mitochondrial respiration that also influence intermembrane contact site formation were found to elicit no marked stimulatory or inhibitory effects on PBBS ligand binding in liver, a result also found for adrenal mitochondria, suggesting that the extent of contact site formation does not influence ligand binding and that the hepatocyte receptor may not play a role in regulating mitochondrial respiration. These two cellular and subcellular locations of the PBBS in liver and the different effect of phospholipids compared to other peripheral tissues may be important for the role(s) of PBBS in liver and also for the multiple roles ascribed to the receptor and to peripheral-type benzodiazepine ligands.

Hexachlorocyclohexanes inhibit steroidogenesis in Y1 cells. Absence of correlation with binding to the peripheral-type benzodiazepine binding site.

Lindane, the gamma-isomer of hexachlorocyclohexane (HCH), and two other HCH-isomers, alpha and delta-HCH, inhibit steroidogenesis in a Y1 adrenocortical cell line. In determining the mechanism by which HCH isomers inhibit steroidogenesis, they were found not to act directly on the mitochondrial Cyt P-450scc enzyme, making likely that they act, instead, on intramitochondrial transport of cholesterol. gamma-HCH, but not alpha or delta-HCH, is a potent and selective inhibitor of ligand binding to the peripheral-type benzodiazepine binding site (PBBS) which, in turn, is reported to regulate the rate-limiting step in steroidogenesis. Although these results demonstrate that alpha and delta-HCH do not inhibit steroid production through the PBBS, the possibility that the interaction of gamma-HCH with the PBBS is responsible for its inhibitory effect on steroid ogenesis could not be excluded.

PK 11195 reduces the brain availability of lindane in rats and the convulsions induced by this neurotoxic agent.

The effect of pretreatment with PK 11195, a ligand of the 'peripheral-type' benzodiazepine receptor (PBR), on convulsions induced by lindane (gamma-hexachlorocyclohexane, gamma-HCH) in rats was examined, to determine whether the mechanism of this convulsant activity may be mediated through the PBR. PK 11195 elicited a protective effect against the convulsant activity of orally administered lindane. It reduced the frequency of animals exhibiting convulsions and delayed the time to onset of these seizures. The concentration of lindane in the brain was found to be significantly lower in PK 11195 pretreated rats and a high correlation between blood and brain lindane concentrations was obtained. When similar experiments were repeated with alpha-HCH, a non-convulsant isomer of HCH, brain and blood concentrations were again found to be significantly reduced in PK 11195 pretreated animals. We conclude that the 'anticonvulsant' action of PK 11195 was not due to an interaction of PK 11195 and lindane on common CNS target sites, but by an action of PK 11195 on the gastrointestinal tract of the animal, delaying the absorption of lindane into the bloodstream.

The effects of the peripheral-type benzodiazepine acceptor ligands, Ro 5-4864 and PK 11195, on mitochondrial respiration.

The role of the peripheral-type benzodiazepine acceptor is unclear. It has been suggested that the acceptor ligands, Ro 5-4864 and PK 11195, stimulate mitochondrial respiration by binding to the peripheral-type benzodiazepine acceptor. We determined the effect of the benzodiazepine Ro 5-4864 and of the isoquinoline carboxamide PK 11195 on the respiration rates of liver, kidney and adrenal mitochondria during coupled, uncoupled and phosphorylating respiration. These ligands inhibited uncoupled and phosphorylating respiration, but only at concentrations substantially greater than their KD values for binding to the acceptor. There was a slight stimulation of coupled respiration by these ligands at concentrations similar to their KD values for the acceptor, but this stimulation was markedly greater at higher concentrations. These results suggest that the ligands Ro 5-4864 and PK 11195 affect respiration in a non-specific way, independently of binding to the acceptor. There was no correlation between the effect of these ligands on respiration and the density of the acceptor in mitochondria from liver, kidney and adrenals. We suggest that the earlier reported alteration of respiration by these ligands was due to non-specific effects and was not mediated by the peripheral-type benzodiazepine acceptor.