HESI/FDA workshop on immunomodulators and cancer risk assessment: Building blocks for a weight-of-evidence approach.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

Nonclinical aspects of biopharmaceutical development: discussion of case studies at a PhRMA-FDA workshop.

Robust assessments of the nonclinical safety profile of biopharmaceuticals are best developed on a scientifically justified, case-by-case basis, with consideration of the therapeutic molecule, molecular target, and differences/similarities between nonclinical species and humans (ICH S6). Significant experience has been gained in the 10 years ensuing since publication of the ICH S6 guidance. In a PhRMA-FDA-sponsored workshop, "Nonclinical Aspects of Biopharmaceutical Development," industry and US regulatory representatives engaged in exploration of current scientific and regulatory issues relating to the nonclinical development of biopharmaceuticals in order to share scientific learning and experience and to work towards establishing consistency in application of general principles and approaches. The proceedings and discussions of this workshop confirm general alignment of strategy and tactics in development of biopharmaceuticals with regard to such areas as species selection, selection of high doses in toxicology studies, selection of clinical doses, the conduct of developmental and reproductive toxicity (DART) studies, and assessment of carcinogenic potential. However, several important aspects, including, for example, appropriate use of homologues, nonhuman primates, and/or in vitro models in the assessment of risk for potential developmental and carcinogenic effects, were identified as requiring further scientific exploration and discussion.

Distance stereoacuity in intermittent exotropia.

BACKGROUND: Studies of distance stereoacuity in intermittent exotropia suggest that normal stereoacuity corresponds to good control of the deviation and that reduced or negative stereoacuity signifies poorer control. AIM: : To evaluate distance stereoacuity in intermittent exotropia using the Frisby Davis Distance stereo test (FD2). METHODS: Children with intermittent exotropia where the near angle was less than or equal to distance were eligible for recruitment. Standardised prospective data collection included FD2 distance stereoacuity. This was a longitudinal study in which outcomes are reported for baseline, last follow-up (> or =6 months before any surgery) or preoperative and last postoperative visits for those undergoing surgery. RESULTS: 110 children with intermittent exotropia had FD2 stereoacuity tested at baseline: 70 comprehended the test. Mean (standard deviation (SD)) age was 4.6 (1.7) years (range 2-10 years). 41/70 (59%) showed positive responses: mean (SD) stereoacuity 30 (12) s of arc. The mean follow-up period before any surgery was 13 months (range 6-27 months). At follow-up, mean (SD) stereoacuity was 24 (11) s of arc. Preoperative and postoperative stereoacuity were not significantly different from those not undergoing surgery. CONCLUSION: This study was the first to report distance stereoacuity in intermittent exotropia using the FD2 stereo test: patients with intermittent exotropia can achieve normal levels of distance stereoacuity, but a considerable proportion, despite comprehending, showed a negative response. This suggests that using the FD2, distance stereoacuity in intermittent exotropia is either absent or normal rather than reduced. Possible reasons for this and its implications are discussed.

The Newcastle Control Score: a new method of grading the severity of intermittent distance exotropia.

AIM: To describe the development and application of a novel scoring system for grading the severity of intermittent distance exotropia (IDEX) and its potential application as an intervention criterion for surgical intervention. METHODS: The Newcastle Control Score (NCS) for IDEX was developed by incorporating both subjective (home control) and objective (clinic control) criteria into a scale to grade severity. The score structure described was evaluated for interobserver and test-retest reliability. To determine an optimal score threshold for surgical intervention, 170 cases of IDEX were scored retrospectively. Cure rates for surgical and non-surgical cases were then compared according to preoperative or presenting scores. RESULTS: Interobserver and test-test reliability were good (r = 0.82 and r = 0.89 respectively). Total cure rate with surgery was 54% and without surgery 18% (chi(2) = 23.093, df = 1, p<0.001). Significantly fewer patients with NCS >/=3 achieved cure without surgery than those with NCS 2 (chi(2) = 3.362, df = 1, p<0.047). CONCLUSIONS: The NCS is a reliable method for grading the severity of IDEX and aids decisions regarding intervention. Patients with a score of 3 or more are unlikely to attain a cure without surgery.

Role of metabolism in dimethylnitrosamine-induced immunosuppression: a review.

Dimethylnitrosamine (DMN) has been characterized as a potent hepatotoxin, carcinogen and mutagen. As described below, immunotoxicity should be added to its profile of activity. Although a broad spectrum of immune parameters is affected by DMN, humoral immunity is particularly sensitive. In order for DMN to produce its traditional profile of toxicity it requires metabolic activation to reactive intermediates which alkylate macromolecules. Similarly, DMN also must be metabolized to produce its immunological effects. However, as this review suggests, the metabolism of DMN to an intermediate capable of suppressing the humoral immune response may be qualitatively and/or quantitatively different from that which mediates hepatotoxicity and genotoxicity.

Statistical quantification of 24-hour and monthly variabilities of spontaneous otoacoustic emission frequency in humans.

Previous evidence has suggested a relationship between spontaneous otoacoustic emissions (SOAEs) and established, biological cycles, although detailed statistical quantifications of the suggested relationships do not exist in the literature. In an attempt to statistically quantify the purported circadian and monthly influences on this phenomenon, two experiments were undertaken. The first experiment was conducted over eight weeks, investigating 31 SOAEs recorded from eight women and two men. Time series statistical analysis examined whether daily, weekly, and/or monthly cycles characterized SOAE frequency variability. Results yielded a significant monthly cycle for the majority of SOAEs recorded from the women but for none of the SOAEs recorded from the men. These results suggest the possibility that SOAE frequency fluctuation in women may be entrained to the monthly menstrual cycle. In the second experiment, hourly SOAE frequency stability was examined over a 24-h period to ascertain the nature of the daily frequency variation as precisely as possible. Four SOAEs from two subjects were examined, and time series analysis of these data included (1) modelling the autocorrelation structure of the measurements, (2) resolving each 24-h series of measurements into cyclical components of various periodicities, and (3) testing the statistical significance of given cycles within the spectrum of each series. Findings included a significant 24-h variability of frequency for each SOAE, suggesting the possibility of a circadian influence on frequency fluctuation. Results from the two experiments provide quantitative evidence supporting a hypothetical relationship between SOAEs and established, biological cycles.

Characterization of the effects of direct alkylators on in vitro immune responses.

Although their mechanism of degradation may differ, both the SN1 alkylators, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-nitroso-N-methylurea (MNU), and the SN2 alkylators, dimethyl sulfate (DMS) and methyl methanesulfonate (MMS), spontaneously decompose under aqueous conditions to the methyldiazonium ion or a direct methylating intermediate, respectively. Thus, these agents serve as useful probes to investigate the immunosuppressive potential of the putative primary reactive intermediate of dimethylnitrosamine (DMN) metabolism, the methyldiazonium ion. The effects of these direct alkylating agents on the in vitro immune response were characterized. Direct addition of both the SN1 and SN2 alkylators to naive B6C3F1 murine splenocytes produced a dose-dependent suppression of the in vitro antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes (sRBC), T-independent antigen, dinitrophenyl (DNP)-Ficoll, and the polyclonal activator, lipopolysaccharide (LPS). The T-dependent and T-independent responses proved to be more sensitive than the polyclonal response to the effects of these compounds, except for MNNG in which all 3 antibody responses were equally affected. The suppression of the AFC response for all antigens was unaffected by the addition of 2-ME, and was observed at concentrations below those affecting viability, although at the highest concentrations an effect on viability was often observed. The addition of MNNG to the T-dependent AFC response at any time within the first 96 h produced a marked suppression, while the addition of DMS to cultures was only effective in suppressing the AFC response if added within the first 24 h. MNNG and DMS suppressed the proliferative responses to both B-cell (LPS) and T-cell (Concanavalin A; Con A) mitogens, as well as in the mixed lymphocyte response (MLR). In addition, a positive correlation between immunosuppression and DNA damage, as measured by single-strand breaks, was observed. Although these compounds produced suppression of in vitro immune responses, their profile of activity on immunocompetence and DNA damage was different from that associated with DMN and thus, the direct alkylators may not prove to be useful models to elucidate the mechanism of the DMN-induced immunosuppression.

Production of DNA single-strand breaks in unstimulated splenocytes by dimethylnitrosamine.

In an attempt to elucidate the mechanism whereby primary hepatocytes, but not liver S9 homogenates, generate immunosupprssive metabolites of dimethylnitrosamine (DMN), the production of DNA single-strand breaks (SSB) in unstimulated splenocytes was investigated with alkaline-elution analysis. Both hepatocytes and S9 homogenates induced SSB in cultured splenocytes by DMN - minimum detectable doses with the two metabolic activation systems (MAS) were 1 microM and 5 mM, respectively. DNA elution profiles were linear in splenocytes co-cultured with DMN and hepatocytes and convex in splenocytes incubated with DMN and S9 homogenates. Aminoacetonitrile (AAN; 10 mM), a DMN demethylase inhibitor, reversed SSB in splenocytes when incubated with either MAS. Addition of exogenous calf-thymus DNA to the hepatocyte co-culture medium did not affect the production of SSB. Rocking the hepatocyte-splenocyte cultures changed the elution profile from linear to convex. All of these treatments have been previously shown to block the immunosuppression by DMN in the hepatocyte co-culture system. These results indicate that the immunosuppression by DMN is not related to DNA damage, as measured by the production of SSB, and suggest that the metabolism of DMN to intermediates capable of producing genotoxicity and immunotoxicity may be qualitatively and/or quantitatively different.

Solving audiometric masking dilemmas with an insert masker.

For some bilateral hearing losses, standard audiometric techniques cannot provide sufficient data for correct otologic diagnosis. These hearing losses are termed "masking dilemmas" because sound attenuation across the head is inadequate to prevent unwanted masking of the test cochlea. In most cases, the masking dilemma can be eliminated by presenting masking through an insert phone in the ear canal, rather than through a standard earphone and cushion. Insert earphones are not standard on most audiometers, but the minimal necessary equipment and alterations are inexpensive, readily available, and require little time to implement. Test time is not increased significantly. Some masking dilemmas will not be solved by the insert masker, but, when properly constructed and used, an insert masker will provide an accurate measurement of cochlear reserve not otherwise obtainable for most cases.

Alveolar soft part sarcoma of the head and neck. A disease of children and young adults.

Alveolar soft part sarcoma (ASPS) is a rare malignancy of uncertain histologic origin that predominantly afflicts female children and young adults. Forty-three cases occurring in the head and neck have been identified, to which we add a case. The most frequent sites of occurrence in the head and neck are orbit and tongue. Treatment is universally surgical, with limited roles for adjuvant radiotherapy and chemotherapy. Representative populations of patients with orbital, non-orbital and extremity ASPS are compared statistically. Improved disease-free intervals in orbital ASPS vs non-orbital ASPS and head and neck ASPS vs extremity ASPS are documented. Debate exists regarding the histologic origin of ASPS. The additional case presented does not corroborate recent immunohistochemical studies suggesting myogenic origin. Nevertheless, the clinical pattern of disease offers additional support to the myogenic hypothesis of histologic origin.

Histological reaction to polyimide films in the cochlea.

The biocompatibility of commercially available polyimides was examined in relation to the sensory and neural structures of the cochlea. Thin films of four different polyimides were prepared as substrates for photolithographically produced scala tympani electrode arrays and implanted in 32 cat cochleae. Following at least three months implantation time, the animals were sacrificed and the cochlea were evaluated histologically for toxicity, differences in the severity of ototoxic effects among the four polyimide groups, and relationships among the different kinds of histopathology secondary to implantation. Findings included the following. Inflammatory reaction to the implants, when present, was generally mild and confined to the immediate vicinity of the implant. Additionally, ototoxic effects were essentially the same across the four groups. Finally, a clear interdependence among the different kinds of cochlear damage secondary to implantation was demonstrated.

A mutant of the WEHI-231 B lymphocyte line that is resistant to phorbol esters is still sensitive to antigen receptor-mediated growth inhibition.

In order to gain a better understanding of the pathways linking receptor immunoglobulin (sIg) crosslinking to downstream B lymphocyte responses, mutants were generated that were defective in sIg-associated signaling pathways. The murine B lymphoma WEHI-231 has proven to be a useful model for studies of sIg-mediated signal transduction. Signaling through sIgM using anti-receptor antibodies (anti-mu) leads to growth arrest and apoptosis of this continuously proliferating cell line. Direct activation of protein kinase C (PKC) with phorbol esters also can mediate this response in WEHI-231. This negative growth response is a useful characteristic that can be exploited to generate signaling mutants that are resistant to the growth-inhibiting effects of anti-mu or phorbol ester. Using this approach, we selected a mutant, PR30-3, in which signaling was blocked downstream of the phorbol diester response element, presumably PKC. Although no longer responsive to phorbol ester stimulation, PR30-3 is not defective in PKC expression or function. Western blot analyses of cellular lysates shows the mutant to express the PKC isoforms alpha, beta, and delta, at levels not markedly different from wild-type WEHI-231. PR30-3 expresses active PKC as shown by its ability to phosphorylate a PKC-specific peptide in vitro. PR30-3 and WEHI-231 express equivalent levels of sIgM expression and nearly indistinguishable second messenger responses in the form of increases in [Ca2+]i and inositol phospholipid hydrolysis. Both PR30-3 and WEHI-231 demonstrate rapid induction of tyrosine kinase activation following sIgM signaling, although there is a reproducible difference in the ability to phosphorylate a 40-kDa substrate in PR30-3. Interestingly, tyrosine phosphorylation of this substrate is induced by phorbol ester stimulation in the wild-type but not the mutant PR30-3. We observed that phorbol ester stimulation of PR30-3 induces the expression of the early response gene c-fos, previously shown to be PKC dependent in this cell line. These results indicate that the signaling component(s) defective in PR30 lie downstream of PKC but upstream of the commitment point for growth inhibition and cell death. Finally, although PR30-3 is resistant to the inhibitory effects of phorbol ester, proliferation is nonetheless still inhibited in response to anti-mu stimulation. These results suggest that the growth inhibitory response of WEHI-231 to anti-mu and phorbol ester involves different pathways.

Clarion cochlear implant: phase I investigational results.

Under phase I of an FDA-approved Investigational Device Exemption (IDE), six patients at the University of California, San Francisco (UCSF) have been implanted with the Clarion, a new multichannel, multiprogrammable cochlear implant system. Over a period of 5 years, the Clarion has been collaboratively developed by UCSF, the Research Triangle Institute (RTI) of North Carolina, and the device manufacturer and sponsor, MiniMed Technologies of Sylmar, California. For UCSF, it represents the culmination of 21 years of cochlear implant research. The Clarion incorporates the 16-contact electrode array developed at UCSF. This electrode is designed with a gentle curve and an elastic memory that allow it to hug the modiolar wall of the scala tympani. In addition, the electrode has mechanical features that prevent it from rotating upward and injuring the basilar membrane. One of the innovations necessary to allow broad clinical application of this device has been the development of an electrode insertion tool to assist the surgeon in safe placement. In each of the six UCSF cases, the insertion tool was used and electrode placement was achieved with ease. With the availability of a variety of speech processing strategies, the Clarion has also provided the opportunity for all of the patients tested to date to achieve some degree of open-set, auditory-only speech recognition. Surgical considerations and preliminary audiologic data demonstrate that the design rationale and objectives of the Clarion have been met.

Differential effects of coadministration of aminoacetonitrile on immunosuppression and hepatotoxicity produced by dimethylnitrosamine.

Aminoacetonitrile (AAN) has been reported to be a dimethylnitrosamine (DMN) demethylase inhibitor which prevents the metabolic activation of DMN to a hepatotoxin. The purpose of this investigation was to determine if AAN pretreatment, which ameliorates hepatotoxicity, would also prevent the immunotoxicity associated with DMN exposure. Female B6C3F1 mice were exposed to either 3 or 6 mg/kg of DMN (in saline) or saline i.p. for 7 consecutive days. The animals were also treated (i.p.) twice daily, 1 hr before and 6 hr after DMN exposure, with either, 10, 30 or 100 mg/kg of AAN (in saline) or saline. Mice were sensitized with sheep red blood cells i.v. on day 8. On day 12, body and organ weights were determined, serum chemistry and histopathology were evaluated and day 4 immunoglobulin M antibody response was measured. Hepatotoxicity caused by DMN, as reflected by an increase in body weight attributed to the production of ascites, a 485.7% increase in the serum glutamic pyruvic transaminase levels and histopathology was reversed by doses of AAN as low as 10 mg/kg. Conversely, doses of AAN as high as 100 mg/kg were unable to reverse the suppression of the antibody forming cells response to sheep red blood cells produced by DMN. The results of this investigation indicate that DMN-induced immunosuppression and hepatotoxicity can be separated and that an immunosuppressive metabolite of DMN is produced by an AAN-insensitive pathway.

Endogenous expression of delta on the surface of WEHI-231. Characterization of its expression and signaling properties.

WEHI-231 is a murine B cell lymphoma that has been used extensively as a model for the immature stage B cell and its functional response to Ag receptor cross-linking as a model for immature B cell tolerance. This cell line expresses sIgM, CD5, and FcR gamma, but lacks the B cell-specific isoform of CD45 (B220). This study demonstrates for the first time that WEHI-231, in contrast to classically defined immature B cells, expresses delta on its surface. Analysis of delta on WEHI-231 revealed structural differences with respect to that on BAL-17 or primary splenic B cells. Although the m.w. of delta on the latter two B cell populations was similar, delta on WEHI-231 manifested a marked increase in its apparent m.w. deduced by SDS-PAGE. This difference was found to be due primarily to differential N-linked glycosylation. Signal transduction through the endogenous sIgD on WEHI-231 was investigated. In contrast to cross-linking of sIgM, cross-linking of the endogenous surface IgD on WEHI-231 was unable to generate a negative growth response in these cells. This inability may be due to uncoupling from normal surface Ig signaling pathways. The signaling properties of the endogenous sIgD on WEHI-231 differ from that on primary B cells and other sIgD-expressing cell lines. Whereas sIgD on splenic B lymphocytes or the mature B cell line BAL-17 is coupled to inositol phospholipid hydrolysis and calcium mobilization, cross-linking of sIgD on WEHI-231 failed to elicit these events, although induced changes in tyrosine phosphorylation were observed. Thus, endogenous expression of surface IgD on WEHI-231 is inconsistent with its representing the classically defined immature stage B cell. The structural and signaling differences associated with delta on these cells suggest the potential for developmentally regulated delta function and model for study of sIgD signal transduction.

Neurotrophic factors modulate hair cells and their potassium currents in chick otocyst explants.

Neurotrophins, retinoids and their receptors are present in the sensory epithelia of the inner ear during development. We show that these factors modulate the proliferation of hair cells and their K+-currents when the embryonic day 3 (ED 3) presumptive inner ear (i.e. otocyst) is maintained in organ culture. All trans-retinoic acid (RA) increases hair cell differentiation and enhances the acquisition of outward currents, including a delayed rectifier and a fast activating, transient type, voltage-gated potassium current. In contrast, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) decrease ionic current activity, and the addition of RA with the neurotrophins enhances this inhibitory response in an age-dependent manner. We measured the total number of cells per explant over time to determine precisely when and how these factors inhibit explant growth. We found that high concentrations of BDNF and NT-3 administered together, and low concentrations of both neurotrophins combined and administered with RA suppress otocyst cell numbers after 24 h in vitro. This suppressive response is induced by RA and NT-3, not by RA and BDNF. The suppressive or inhibitory influence of NT-3 and RA is the result of NT-3 binding to the low affinity receptor, p75NTR, not the result of RA increasing mRNA levels for the high affinity receptor, trkC. However, trk may act with p75NTR, as disruption of trk signalling alleviates the inhibitory response induced by NT-3 and RA. Our data suggest that various combinations and/or concentration gradients of these factors can differentially regulate inner ear development and hair cell excitability.

Prevention of immunotoxin-induced immunogenicity by coadministration with CTLA4Ig enhances antitumor efficacy.

Immunotoxins have shown promise as antitumor agents in clinical trials. However, they have not become part of standard cancer therapy because of factors that include their inherent immunogenicity, which limits the duration of therapy. To address this issue, we evaluated in preclinical models the concomitant use of the immunosuppressive agent CTLA4Ig and BR96 sFv-PE40, a single-chain immunotoxin that binds to carcinoma cells expressing Le(y). Cotreatment with CTLA4Ig, an inhibitor of the CD28/CTLA4-CD80/CD86 costimulation pathway, blocked the production of Abs against BR96 sFv-PE40 in immunocompetent rodents and dogs. It also blocked hypersensitivity reactions in rats carrying colon carcinoma allografts during a second course of BR96 sFv-PE40 therapy, and the cotreatment with CTLA4Ig resulted in enhanced antitumor activity. Cotreatment with CTLA4Ig also prevented hypersensitivity reactions induced by repeat dosing of BR96 sFv-PE40 (q3dx5) in dogs. The production of anti-BR96-sFv-PE40 Abs was decreased in CTLA4Ig-cotreated rodents and dogs resulting in increased plasma levels of BR96 sFv-PE40 relative to non-CTLA4Ig-cotreated animals. These data show that cotreatment of immunotoxins with CTLA4Ig, by inhibiting the production of anti-immunotoxin Abs, can extend the duration of BR96 sFv-PE40 therapy to give greater exposure, reduced toxicities, and increased efficacy.

BR96 sFv-PE40 immunotoxin: nonclinical safety assessment.

BR96 sFv-PE40, a recombinant DNA-derived fusion protein composed of the heavy- and light-chain variable region domains of the monoclonal antibody BR96 and the translocation and catalytic domains of Pseudomonas exotoxin A, is being developed for the treatment of solid tumors expressing cell surface Lewis(y)-related antigens. Single- and repeat-dose intravenous toxicity studies in rats and dogs and a comparative ex vivo tissue-binding study with rat, dog, and human tissues were conducted to assess the toxicity of BR96 sFv-PE40 and to estimate a safe starting dose in humans. Additional studies were performed to investigate the prevention of pulmonary vascular-leak syndrome, the dose-limiting toxicity of BR96 sFv-PE40 in rats, and the immunogenicity of BR96 sFv-PE40. In single-dose studies in rats, the vascular leak appeared to be primarily confined to the lungs; however, with a repeat-dose regimen (every other day for 5 doses) other organs including the brain and heart were involved at lethal doses (12-15 mg/m2 cumulative). Single doses of 1.8 mg/m2 and a cumulative 3.8 mg/m2 dose (0.75 mg/m2, every other day for 5 doses) were generally well tolerated in rats. These doses are significantly greater than doses required to cure rodents bearing human tumor xenografts. In dogs, the major target organ following single or repeated doses (every 3 days for 5 doses) was the pancreas. Morphologic changes in the exocrine pancreas ranged from atrophy with single-cell necrosis to diffuse acinar necrosis. After a 1-mo dose-free observation period, no residual pancreatic toxicity was observed in dogs given single doses up to 6.0 mg/m2 or 5 doses of 2.4 mg/m2 (12 mg/m2 cumulative). No significant pancreatic toxicity was observed at doses <0.6 mg/m2 in high Lewis(y)-expressing dogs. Assessment of trypsinlike immunoreactivity was useful in monitoring changes in pancreatic function. The immunogenicity of BR96 sFv-PE40 could be inhibited by combined treatment with an immunosuppressant in dogs, thus maintaining exposure to BR96 sFv-PE40.