Toxicology Studies for Inhaled and Nasal Delivery.

This review examines issues related to the toxicological testing of pharmaceuticals delivered by the inhalation or nasal route. The purpose of the toxicology studies is to conduct studies in animals that will aid the assessment of the safety of these agents delivered to patients. Inhalation toxicology studies present some unique issues because the dosing method differs from more standard administration methods such as oral or injection administration. Also, dose determination issues are more complex, particularly for inhalation administration since it is often difficult to determine the amount of material delivered to the lung both for patients and in animal toxicology studies.

Folic acid pathway single nucleotide polymorphisms associated with methotrexate significant adverse events in United States veterans with rheumatoid arthritis.

OBJECTIVES: Methotrexate (MTX) is the cornerstone medication in the treatment of rheumatoid arthritis (RA). We examined whether single nucleotide polymorphisms (SNPs) in enzymes of the folic acid pathway (folylpoly-gamma-glutamate synthetase [FPGS], gamma-glutamyl hydrolase [GGH], and methylenetetrahydrofolate reductase [MTHFR]) associate with significant adverse events (SigAE). METHODS: Patients (n=319) enrolled in the Veterans Affairs RA (VARA) registry taking MTX were genotyped for HLA-DRB1-SE and the following SNPs: FPGS (rs7033913, rs10760503, rs10106), GGH (12548933, rs7010484, rs4617146, rs719235, rs11988534), MTHFR (rs1801131, rs1801133). AE were abstracted from the medical record using a structured instrument. SigAE were defined as an AE leading to MTX discontinuation. Covariates included: age, gender, race, RA antibody status, tobacco, RA disease duration between diagnosis and MTX course, Charlson-Deyo comorbidity index, glucocorticoids, use of prior RA medications, and mean 4-variable disease activity score. Cox regression was performed to determine factors associated with time-to-SigAE. A p-value ≤ 0.005 established significance in the final model. RESULTS: The presence of ≥ 1 copy of the minor allele in MTHFR rs1801131 was associated with an increased hazard ratio (HR) of SigAE (HR 3.05, 95% CI 1.48-6.29, p-value 0.003 and HR 3.88, 95% CI 1.62-9.28, p-value 0.002 for heterozygotes and homozygotes for the minor allele, respectively). An interaction term, between FPGS rs7033913 heterozygotes and GGH rs11988534 homozygotes for the minor allele, had a p-value <0.0001. CONCLUSIONS: RA subjects taking MTX may have decreased time-to-SigAE with ≥ 1 copy of the minor allele in MTHFR rs1801131. Further investigation is warranted, as these SNPs may indicate susceptibility to MTX toxicity.

Genome-wide detection of allelic imbalance using human SNPs and high-density DNA arrays.

Most human cancers are characterized by genomic instability, the accumulation of multiple genetic alterations and allelic imbalance throughout the genome. Loss of heterozygosity (LOH) is a common form of allelic imbalance and the detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes and to characterize tumor stages and progression. Here we describe the use of high-density oligonucleotide arrays for genome-wide scans for LOH and allelic imbalance in human tumors. The arrays contain redundant sets of probes for 600 genetic loci that are distributed across all human chromosomes. The arrays were used to detect allelic imbalance in two types of human tumors, and a subset of the results was confirmed using conventional gel-based methods. We also tested the ability to study heterogeneous cell populations and found that allelic imbalance can be detected in the presence of a substantial background of normal cells. The detection of LOH and other chromosomal changes using large numbers of single nucleotide polymorphism (SNP) markers should enable identification of patterns of allelic imbalance with potential prognostic and diagnostic utility.

Effects of inhalation exposures to an M1-receptor agonist on ventilation in rhesus monkeys.

Information was needed on effects of possible occupational inhalation exposure to an M1-receptor agonist (xanomeline) such as might occur during the manufacturing process. Both acute and repeated inhalation exposures to xanomeline were carried out in six male rhesus monkeys using a head-dome exposure system. Exposure concentrations ranged from 0.3 to 10 mg/m3. The exposure durations were up to 2 weeks. Decreases in tidal volume and increases in respiratory frequency were both time and concentration related during acute exposures. These effects were blocked with atropine pre-treatment. Correlation with pulmonary resistance measurements in two monkeys suggested that these were bronchoconstrictive changes that increased with severity with time at a given concentration and with concentration when measured after a constant exposure time. The dose-response was relatively steep with 10 mg/m3 becoming intolerable to the monkeys after approximately 15 minutes, but no measurable effects were observed at 0.3 mg/m3 after up to 4 hours of exposure. To investigate the effects of repeated exposures, monkeys were exposed for 4 hr/day, 5 days/wk for 2 weeks to 0.0 (air only), 0.3, and 1.2 mg xanomeline/m3 of air. When compared to the air-only exposure, 0.3 mg/m3 caused no significant changes in tidal volume. In contrast, 1.2 mg/m3 caused a rapid and significant decrease in tidal volume that was sustained throughout the 4-hr exposure. A slower rise in breathing frequency also occurred. Repeated exposures did not alter the effects seen after a single exposure. It is concluded that xanomeline, a M1-receptor agonist, can acutely alter normal ventilation in non-human primates at airborne concentrations > or = 0.6 mg/m3 and should be carefully controlled in a manufacturing environment. The no-observed-effect concentration was 0.3 mg/m3.

HFE genotype in patients with hemochromatosis and other liver diseases.

BACKGROUND: Hereditary hemochromatosis is a common inherited disorder of iron metabolism. The gene HFE, which contains two missense mutations (C282Y and H63D), was recently identified. OBJECTIVE: To determine how HFE genotyping for the C282Y and H63D mutations contributes to the diagnosis of hemochromatosis and to determine the prevalence of HFE mutations in a group of patients with liver disease. DESIGN: Cross-sectional study. SETTING: Academic medical center. PATIENTS: 66 patients with hereditary hemochromatosis and 132 referred patients with other liver diseases. MEASUREMENTS: At initial diagnosis, fasting transferrin saturation, ferritin level, routine chemistry panel, and complete blood count were determined. Percutaneous liver biopsy was done on all patients for histologic analysis and measurement of hepatic iron concentration and hepatic iron index. HFE genotyping for the C282Y and H63D mutations was done on all patients by using genomic DNA samples. RESULTS: Of the 66 patients with hemochromatosis diagnosed on the basis of serum iron studies and liver biopsy findings, 60 (91%) were C282Y homozygotes, 2 (3%) were compound heterozygotes, 1 (1.5%) was a C282Y heterozygote, 2 (3%) were H63D heterozygotes, and 1 (1.5%) was negative for both mutations. Of the 132 patients with liver disease, 6 (5%) were C282Y homozygotes, 8 (6%) were compound heterozygotes, 6 (5%) were C282Y heterozygotes, 5 (4%) were H63D homozygotes, 20 (15%) were H63D heterozygotes, and 87 (66%) were negative for both mutations. All 66 C282Y homozygotes had an elevated hepatic iron concentration, and 65 of the 66 patients (98%) had a transferrin saturation of at least 45%. Ten of the 66 patients (15% [95% CI, 7.5% to 26%]) had a hepatic iron index less than 1.9 mmol/kg per year; hemochromatosis was not suspected in 6 of the 10 patients before genotyping. Cirrhosis or substantial hepatic fibrosis was not seen in any (0% [CI, 0% to 18%]) of the 19 patients younger than 40 years of age who were homozygous for the C282Y mutation. CONCLUSIONS: All 66 patients homozygous for the C282Y mutation of HFE had an elevated hepatic iron concentration, but approximately 15% of these patients did not meet a previous diagnostic criterion for hemochromatosis (hepatic iron index > 1.9 mmol/kg per year). Determination of HFE genotype is clinically useful in patients with liver disease and suspected iron overload and may lead to identification of otherwise unsuspected C282Y homozygotes.

Hereditary hemochromatosis in liver transplantation.

A candidate gene, HFE, was recently described in patients with hereditary hemochromatosis (HH) and found to contain a missense mutation leading to a cysteine to tyrosine substitution (C282Y). A second mutation, H63D, was also found in the gene. This study was undertaken to determine the HFE genotype in liver transplant recipients clinically diagnosed with HH and those incidentally found to have increased iron deposition in their explanted livers and to evaluate whether biochemical or histological hepatic iron indices (HIIs) correlated with homozygosity for the C282Y mutation. We identified 15 patients clinically diagnosed with various liver disorders other than HH who had increased liver iron deposits among 918 adult patients who underwent liver transplantation from 1988 to 1995. Four additional patients were clinically diagnosed as having HH. Archival explant liver tissue was evaluated for the histological HII according to the method of Deugnier et al, in which an index greater than 0.15 suggests homozygosity for HH. The HII was computed according to established methods, with a value greater than 1.9 suggesting homozygosity for HH. A portion of liver tissue was subjected to DNA genotyping using polymerase chain reaction-amplified products. Two of 4 patients with clinically suspected HH were homozygous for C282Y, and 2 patients had neither mutation. One of the 15 patients not suspected to have HH was a C282Y homozygote, 1 was a C282Y heterozygote, 6 were H63D heterozygotes, and 7 had neither mutation. The histological HII was consistent with HH in 13 patients, whereas the HII was consistent with HH in 6 patients. Thus, in patients with end-stage liver disease, despite fulfilling the established clinical criteria for HH using biochemical and histological parameters, only a minority of patients were homozygous for the C282Y mutation. Hepatic iron overload may result from other causes, and in end-stage liver disease, an elevated HII may not accurately predict HH. Other factors that either control or lead to iron absorption may explain iron overload in these patients.

Transferrin receptor mutation analysis in hereditary hemochromatosis patients.

The Cys282-->Tyr mutation in the HFE gene is carried by the majority of hereditary hemochromatosis patient chromosomes, yet some patients do not seem to harbor any mutation in this gene. This suggests a possibility that these patients may have a mutation in other genes in the same pathway as HFE. We analyzed the cDNA sequences of transferrin receptor (TFR), which was recently shown to interact with HFE, in twenty-one hereditary hemochromatosis patients including sixteen individuals who did not carry a Cys282-->Tyr mutation. A nucleotide substitution (424A-->G), which resulted in the Ser142-->Gly amino acid substitution, was the only amino acid polymorphism detected in the open reading frame of the TFR gene in these patients. This amino acid substitution was a rather common polymorphism in the general population (49%) and its frequency did not significantly differ in the hereditary hemochromatosis (HH) patients regardless of the HFE genotype. Thus, amino acid changes in the TFR gene do not appear to play a role in HH even when the patients do not have a HFE mutation. However, this study does not rule out the possibility of the involvement of mutations in non-coding regions.

A haplotype and linkage disequilibrium analysis of the hereditary hemochromatosis gene region.

Hereditary hemochromatosis is a recessive disease of iron metabolism widely distributed among people of European descent. Most patients have inherited the causative mutation from a single ancestor. In the course of cloning the hemochromatosis gene, genotypes were generated for these samples at 43 microsatellite repeat markers that span the 6.5-Mb hemochromatosis gene region. The data used to reconstruct the ancestral haplotype across the hemochromatosis gene region are presented in this paper. Portions of the ancestral haplotype were present on 85% of patient chromosomes in this sample and ranged in size from approximately 500 kb to greater than 6.5 Mb. Only one marker, D6S2239, was identical by descent on all of the patient chromosomes containing the ancestral mutation. In contrast, only 3 of the 128 control chromosomes, or 2.3%, carried the ancestral mutation and the surrounding ancestral haplotype. To test new methods for gene finding using linkage disequilibrium we analyzed the genotypic data with a multilocus maximum likelihood method (DISMULT) and a single point method (DISLAMB), both written to analyze data generated from multi-allelic markers. The maximum value from DISLAMB analysis occurred at marker D6S2239, which is less than 20 kb from the hemochromatosis gene HFE, consistent with the haplotype analysis. The peak of the multi-point analysis was 700 kb from HFE, possibly due to the nonuniform recombination rates within this large region. The recombination rate appears to be lower than expected centromeric of the HFE gene.

Safety of inhaled proteins for therapeutic use.

The use of the inhalation route for delivery of inhaled proteins has received increasing attention recently. The purpose of this article is to review the available information related to the safety aspects of inhaled proteins. The review focuses primarily on possible toxicity to the respiratory tract, because usually one is either considering an agent to treat the lung or an agent for which the systemic toxicity has been investigated following subcutaneous (s.c.) administration in its clinical use as a therapeutic agent. Some background is provided on mechanisms of absorption and reasons why inhalation delivery is considered for many proteins. Available data are summarized from clinical trials of proteins and protein-like biomolecules, generally showing minimal, if any, adverse respiratory effects. The results of the animal toxicology studies that have been published are presented. In general, the observed lung toxicity has been relatively low, and it has been difficult to interpret in cases where the animal protein differs considerably from the human protein. Discussion is presented on the possibility of adverse immune reactions, suggesting that this is not likely to be any greater issue than it is for subcutaneously injected materials. Although the safety information is relatively sparse at present, the available data suggest that the inhalation route can be an attractive route to consider for many therapeutic proteins.

The hemochromatosis founder mutation in HLA-H disrupts beta2-microglobulin interaction and cell surface expression.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 --> tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 --> aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds beta2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.

A 1.1-Mb transcript map of the hereditary hemochromatosis locus.

In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A. A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments. As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes. Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein. Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2. The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC. The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC. Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA.

Clone-contig and STS maps of the hereditary hemochromatosis region on human chromosome 6p21.3-p22.

YAC-based and bacterial-clone based STS-content maps were constructed that served as the framework physical maps for the positional cloning of a candidate gene for hereditary hemochromatosis. The YAC-based map comprises 43 YACs and 86 STS and spans approximately 8 Mb of DNA between the class I region of the major histocompatibility complex on human chromosome 6p21.3 and D6S276 in 6p22. Comparison with published maps revealed a hole in the MIT/Whitehead and CEPH YAC maps that includes the immediate region around the hemochromatosis gene itself. Approximately 3 Mb of DNA was covered by a bacterial clone contig that consists of 38 BACs, 45 PACs, 26 PI clones and one lambda phage. The bacterial clone-based STS map comprises 153 STSs. A contiguous block of 8 STSs could be amplified from both human chromosome 6 and 5. Further characterization of selected STSs and bacterial clones by radiation hybrid mapping and fluorescence in situ hybridization, respectively, revealed the presence of a multicopy DNA segment, more than one bacterial clone length in size, which is duplicated near the chromosome-6 centromere and part of which is present in multiple copies on chromosome 5. Possible implications of the incomplete public YAC-contig map and of the multicopy segment for physical mapping and linkage disequilibrium studies of the hemochromatosis candidate region are discussed.

A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

The effects of LY293111Na, a leukotriene B4 receptor antagonist, on the pulmonary neutrophilia and CD11b expression caused by inhalation of a leukotriene B4 aerosol in rhesus monkeys.

The dose-response relationship between aerosolized leukotriene B4 (LTB4 and pulmonary neutrohilia was examined in a group of five rhesus monkeys. The effects of an oral dose of LY293111Na on LTB4-aerosol-induced pulmonary neutrophilia were also examined. Ex vivo expression of CD11b receptors on polymorphonuclear leukocytes from bronchoalveolar lavage fluid and peripheral whole blood were also assessed. Up-regulation of CD11b adhesion receptors by LTB4 was assessed ex vivo on the peripheral whole blood. Pulmonary neutrophilia was linearly associated with dose of inhaled LTB4. LY293111Na, at 10 mg/kg, significantly blocked the profound bronchoalveolar lavage neutrophilia produced by LTB4 aerosol inhalation. A large (48%), but not statistically significant, reduction was seen for CD11b expression on bronchoalveolar lavage polymorphonuclear leukocytes after pretreatment with LY293111Na. LY293111Na did not significantly change the number of white blood cells in peripheral blood. LY293111Na did significantly attenuate the LTB4-induced up-regulation of CD11b receptors on peripheral blood neutrophils. We conclude that LY293111Na may be an effective oral treatment for diseases that involve neutrophilic inflammation.

Use of a head dome system to compare i.v. methacholine-induced bronchoconstriction in conscious vs anesthetized rhesus monkeys.

The use of a newly developed head dome system has allowed measurement of pulmonary function in conscious monkeys. Such information is often desired, so that pharmacological or toxicological effects of administered compounds can be measured in the absence of effects from anesthetic agents. The current study was conducted to gain experience with this method and to allow the determination of the effects of sodium pentobarbital anesthesia (30 mg kg-1 i.v.) on the bronchoconstriction seen during i.v. infusion of methacholine in rhesus monkeys. Bronchoconstriction was measured as changes in respiratory resistance using a Buxco LS20 pulmonary mechanics computer. Four male rhesus monkeys (4.2-5.1 kg) were used. For the anesthetized exposures, the animals were intubated with a 4.0-mm cuffed endotracheal tube attached to a size 'O' Fleisch pneumotachograph. For the conscious exposures, the animals sat in restraining chairs with a custom-built head dome attached to the same pneumotachograph. In both cases, transthoracic pressure was monitored with an intrapleural catheter. Each monkey was infused with methacholine in stepwise doses, while anesthetized and conscious, until a 75% increase in respiratory resistance was seen. The ED50 values of 0.134 and 0.180 mg ml-1 methacholine were not significantly different in anesthetized vs conscious monkeys, respectively.

Generation of aerosolized drugs.

The expanding use of inhalation therapy has placed demands on current aerosol generation systems that are difficult to meet with current inhalers. The desire to deliver novel drug entities such as proteins and peptides, as well as complex formulations including liposomes and microspheres, requires delivery systems of improved efficiency that will target the lung in a reproducible manner. These efforts have also been spurred by the phase out of chlorofluorocarbons (CFCs) and this has included a directed search for alternative propellants. Consequently, a variety of new aerosol devices and methods of generating aerosols are being studied. This includes the use of freon replacement propellants, dry powder generation systems, aqueous unit spray systems and microprocessor controlled technologies. Each approach has advantages and disadvantages depending upon each principle of action and set of design variables. In addition, specific drugs may be better suited for one type of inhaler device vs. another. The extent to which aerosol generation systems achieve their goals is discussed together with a summary of selected papers presented at the recent International Congress of Aerosols in Medicine.

Effects of high exposure concentrations of inhaled low-toxicity dust on pulmonary function in guinea pigs.

The acute pulmonary function response to graded levels of a low toxicity dust was studied in guinea pigs. Four groups of five male guinea pigs each were exposed to mean concentrations of 0, 0.25, 1.01 and 5.39 mg Foundry Hill Clay l-1 air with mass median aerodynamic diameters of 2.6, 4.6 and 6.7 microns, respectively. There was a 15-min pre-exposure period to clean air, a 1-h exposure to the test atmosphere and then a 1-h recovery period with exposure to clean air. Concentration-related changes, compared to the pre-exposure period, occurred with a rapid onset in a number of parameters. Generally, the severity of observed effects increased with exposure time and, therefore, with inhaled dose. Statistically significant changes (P < 0.05) were observed in tidal volume, dynamic compliance, dynamic resistance, flow, pressure and minute volume during the last 15 min of exposure. The observed changes were consistent with acute bronchoconstriction. These effects reversed rapidly and there were no significant changes 1 h post-exposure. These results suggest that adverse physiological responses of short duration can occur when animals are exposed via inhalation to low-toxicity materials in the concentration range 0.25-5 mg l-1.

Toxicologic testing of inhaled pharmaceutical aerosols.

This paper reviews technical issues related to the toxicologic testing of inhaled pharmaceuticals. Although there are commonalities between approaches to general and inhalation toxicity testing, there also are specific challenges in the toxicity testing of inhaled pharmaceuticals. A major issue is that of dose; inhaled dose is more difficult to determine than intravenous or oral doses. Also, it is harder to relate dose in laboratory animals to that in man for inhalation exposure than for other routes of administration. Additionally, in the case of inhaled pharmaceuticals, people generally inhale through the mouth, whereas most laboratory animals inhale primarily through the nose. This presents significant challenges in exposure methodology and technology that often need innovative approaches involving alteration to particle size of the agent or dosing procedure. Because the respiratory tract is the site of deposition, local respiratory toxicity and possible damage to lung cells need to be assessed. Systemic toxicity also needs to be evaluated and may be an issue in some cases. Special studies on pulmonary function, mucociliary clearance, or immune response may be needed, depending on the nature of the inhaled pharmaceutical. This review explores the main issues involved in toxicity testing of inhaled pharmaceuticals, the approaches that have been used, and the current and future challenges.

A radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 locus.

We describe a high-resolution radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 (NF2) gene. Eighty-five hamster-human somatic cell hybrids generated by X-irradiation and cell fusion were used to generate the radiation hybrid map. The presence or absence of 18 human chromosome 22-specific markers was determined in each hybrid by using Southern blot hybridization. Sixteen of the 18 markers were distinguishable by X-ray breakage in the radiation hybrids. Analysis of these data using two different mathematical models and two different statistical methods resulted in a single framework map consisting of 8 markers ordered with odds greater than 1000:1. The remaining nonframework markers were all localized to regions consisting of two adjoining intervals on the framework map with odds greater than 1000:1. Based on the RH map, the NF2 region of chromosome 22, defined by the flanking markers D22S1 and D22S28, is estimated to span a physical distance of approximately 6 Mb and is the most likely location for 9 of the 18 markers studied: D22S33, D22S41, D22S42, D22S46, D22S56, LIF, D22S37, D22S44, and D22S15.