The BALB/c Mouse Model for the Evaluation of Therapies to Treat Infections with Aerosolized Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of the disease melioidosis, has been isolated from the environment in 45 countries. The treatment of melioidosis is complex, requiring lengthy antibiotic regimens, which can result in the relapse of the disease following treatment cessation. It is important that novel therapies to treat infections with B. pseudomallei be assessed in appropriate animal models, and discussions regarding the different protocols used between laboratories are critical. A 'deep dive' was held in October 2020 focusing on the use of the BALB/c mouse model and the inhalational route of infection to evaluate new antibiotic therapies.

Efficacy of finafloxacin in a murine model of inhalational glanders.

Burkholderia mallei, the causative agent of glanders, is principally a disease of equines, although it can also infect humans and is categorized by the U.S. Centers for Disease Control and Prevention as a category B biological agent. Human cases of glanders are rare and thus there is limited information on treatment. It is therefore recommended that cases are treated with the same therapies as used for melioidosis, which for prophylaxis, is co-trimoxazole (trimethoprim/sulfamethoxazole) or co-amoxiclav (amoxicillin/clavulanic acid). In this study, the fluoroquinolone finafloxacin was compared to co-trimoxazole as a post-exposure prophylactic in a murine model of inhalational glanders. BALB/c mice were exposed to an aerosol of B. mallei followed by treatment with co-trimoxazole or finafloxacin initiated at 24 h post-challenge and continued for 14 days. Survival at the end of the study was 55% or 70% for mice treated with finafloxacin or co-trimoxazole, respectively, however, this difference was not significant. However, finafloxacin was more effective than co-trimoxazole in controlling bacterial load within tissues and demonstrating clearance in the liver, lung and spleen following 14 days of therapy. In summary, finafloxacin should be considered as a promising alternative treatment following exposure to B. mallei.

Histopathological and Immunological Findings in the Common Marmoset Following Exposure to Aerosolized SARS-CoV-2.

There is an enduring requirement to develop animal models of COVID-19 to assess the efficacy of vaccines and therapeutics that can be used to treat the disease in humans. In this study, six marmosets were exposed to a small particle aerosol (1-3 µm) of SARS-CoV-2 VIC01 that delivered the virus directly to the lower respiratory tract. Following the challenge, marmosets did not develop clinical signs, although a disruption to the normal diurnal temperature rhythm was observed in three out of six animals. Early weight loss and changes to respiratory pattern and activity were also observed, yet there was limited evidence of viral replication or lung pathology associated with infection. There was a robust innate immunological response to infection, which included an early increase in circulating neutrophils and monocytes and a reduction in the proportion of circulating T-cells. Expression of the ACE2 receptor in respiratory tissues was almost absent, but there was ubiquitous expression of TMPRSS2. The results of this study indicate that exposure of marmosets to high concentrations of aerosolised SARS-CoV-2 did not result in the development of clear, reproducible signs of COVID-19.

Comparison of Experimental Middle East Respiratory Syndrome Coronavirus Infection Acquired by Three Individual Routes of Infection in the Common Marmoset.

Two strains of Middle East respiratory syndrome coronavirus (MERS-CoV), England 1 and Erasmus Medical Centre/2012 (EMC/2012), were used to challenge common marmosets (Callithrix jacchus) by three routes of infection: aerosol, oral, and intranasal. Animals challenged by the intranasal and aerosol routes presented with mild, transient disease, while those challenged by the oral route presented with a subclinical immunological response. Animals challenged with MERS-CoV strain EMC/2012 by the aerosol route responded with primary and/or secondary pyrexia. Marmosets had minimal to mild multifocal interstitial pneumonia, with the greatest relative severity being observed in animals challenged by the aerosol route. Viable virus was isolated from the host in throat swabs and lung tissue. The transient disease described is consistent with a successful host response and was characterized by the upregulation of macrophage and neutrophil function observed in all animals at the time of euthanasia. IMPORTANCE Middle East respiratory syndrome is caused by a human coronavirus, MERS-CoV, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Humans typically exhibit fever, cough, shortness of breath, gastrointestinal issues, and breathing difficulties, which can lead to pneumonia and/or renal complications. This emerging disease resulted in the first human lethal cases in 2012 and has a case fatality rate of approximately 36%. Consequently, there is a need for medical countermeasures and appropriate animal models for their assessment. This work has demonstrated the requirement for higher concentrations of virus to cause overt disease. Challenge by the aerosol, intranasal, and oral routes resulted in no or mild disease, but all animals had an immunological response. This shows that an appropriate early immunological response is able to control the disease.

Comparison of the Pharmacokinetics of RIPK1 Inhibitor GSK2982772 in Healthy Western and Japanese Subjects.

BACKGROUND AND OBJECTIVES: GSK2982772 is an oral small-molecule RIPK1 inhibitor with potential therapeutic efficacy in immune-mediated inflammatory diseases (IMIDs). An inter-ethnic comparison of GSK2982772 pharmacokinetics was conducted based on data from Western (Study 1) and Japanese subjects (Study 2). METHODS: Both studies were single-centre, randomised, double-blind, placebo-controlled studies with objectives to assess the safety and characterise the pharmacokinetics of GSK2982772. Western subjects in Study 1 (NCT03305419), Part A (N = 15), were randomly assigned to receive 120 mg three times daily (TID), 240 mg TID, or 360 mg twice daily (BID) doses of GSK2982772, or placebo (TID or BID) for 1 day. Part B subjects (N = 47) received GSK2982772 120 mg TID, 240 mg TID, or placebo TID for 14 days. Japanese subjects in Study 2 (N = 13) (NCT03590613) were randomly assigned to receive TID doses of GSK2982772 60, 120, 240 mg TID or placebo TID for 1 day. RESULTS: GSK2982772 was well tolerated and adverse events were generally mild. Maximum observed plasma drug concentration (Cmax), time to reach Cmax (Tmax), area under the plasma drug concentration versus time curve after the first GSK2982772 dose (AUC(0-7)) of 120 and 240 mg, and (AUC(0-24)) values for the 120 and 240 mg TID doses over a single day were similar in Japanese and Western subjects. CONCLUSIONS: The pharmacokinetics and tolerability of GSK2982772 were similar between Western and Japanese subjects, justifying inclusion of Japanese subjects in future global clinical studies to assess the therapeutic potential of RIPK1 inhibition for the treatment of IMIDs. Clinical Trials: NCT03305419 and NCT03590613 available from http://www.clinicaltrials.gov .

Aerosol infection of Balb/c mice with eastern equine encephalitis virus; susceptibility and lethality.

BACKGROUND: Eastern equine encephalitis virus is an alphavirus that naturally cycles between mosquitoes and birds or rodents in Eastern States of the US. Equine infection occurs by being bitten by cross-feeding mosquitoes, with a case fatality rate of up to 75% in humans during epizootic outbreaks. There are no licensed medical countermeasures, and with an anticipated increase in mortality when exposed by the aerosol route based on anecdotal human data and experimental animal data, it is important to understand the pathogenesis of this disease in pursuit of treatment options. This report details the clinical and pathological findings of mice infected with EEEV by the aerosol route, and use as a model for EEEV infection in humans. METHODS: Mice were exposed by the aerosol route to a dose range of EEEV to establish the median lethal dose. A pathogenesis study followed whereby mice were exposed to a defined dose of virus and sacrificed at time-points thereafter for histopathological analysis and virology. RESULTS: Clinical signs of disease appeared within 2 days post challenge, culminating in severe clinical signs within 24 h, neuro-invasion and dose dependent lethality. EEEV was first detected in the lung 1 day post challenge, and by day 3 peak viral titres were observed in the brain, spleen and blood, corresponding with severe meningoencephalitis, indicative of encephalitic disease. Lethality follows severe neurological signs, and may be linked to a threshold level of virus replication in the brain. Effective medical countermeasures for EEEV may necessitate early inoculation to inhibit infection of the brain in zoonotic incidents, and be able to traverse the blood-brain barrier to sufficiently interrupt replication in the brain in cases of aerosol infection. CONCLUSIONS: There is little human data on the hazard posed by aerosol infection with encephalitic alphaviruses, and use of EEEV as a bioweapon may be by the aerosol route. A well characterized model of aerosol exposure that recapitulates some of the most severe human clinical features is necessary to evaluate the efficacy of putative medical countermeasures, and to increase our understanding about how this route of infection induces such rapid neuro-invasion and resulting disease.

An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology Is Protective in an Inhalational Rat Model of Tularemia.

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.

Susceptibility and Lethality of Western Equine Encephalitis Virus in Balb/c Mice When Infected by the Aerosol Route.

Western equine encephalitis virus (WEEV) naturally cycles between mosquitos and birds or rodents, with a case fatality rate of up to 15% in humans during epizootic outbreaks. There are no medical countermeasures to treat WEEV infection, and accidental aerosol exposure increases the case fatality rate up to 40%. Understanding the pathogenesis of infection is required to develop and assess medical countermeasures. This study describes the clinical and pathological findings of mice infected with WEEV by the aerosol route, and use as a model for WEEV infection in humans. Balb/c mice were infected by the aerosol route with a dose range of high-virulence WEEV strain Fleming to establish the median lethal dose (MLD). The disease course was acute, culminating in severe clinical signs, neuroinvasion, and dose-dependent mortality. Further groups of mice were exposed by the aerosol route, periodically sacrificed, and tissues excised for histopathological examination and virology. Viral titres peaked four days post-challenge in the brain and lungs, corresponding with severe bilateral lesions in rostroventral regions of the encephalon, especially in the olfactory bulb and piriform cortex. Recapitulation of the most serious clinical presentations of human WEEV disease in mice may prove a useful tool in the evaluation of medical countermeasures.

Inhaled Liposomal Ciprofloxacin Protects against a Lethal Infection in a Murine Model of Pneumonic Plague.

Inhalation of Yersinia pestis can lead to pneumonic plague, which without treatment is inevitably fatal. Two novel formulations of liposome-encapsulated ciprofloxacin, 'ciprofloxacin for inhalation' (CFI, Lipoquin®) and 'dual release ciprofloxacin for inhalation' (DRCFI, Pulmaquin®) containing CFI and ciprofloxacin solution, are in development. These were evaluated as potential therapies for infection with Y. pestis. In a murine model of pneumonic plague, human-like doses of aerosolized CFI, aerosolized DRCFI or intraperitoneal (i.p.) ciprofloxacin were administered at 24 h (representing prophylaxis) or 42 h (representing treatment) post-challenge. All three therapies provided a high level of protection when administered 24 h post-challenge. A single dose of CFI, but not DRCFI, significantly improved survival compared to a single dose of ciprofloxacin. Furthermore, single doses of CFI and DRCFI reduced bacterial burden in lungs and spleens to below the detectable limit at 60 h post-challenge. When therapy was delayed until 42 h post-challenge, a single dose of CFI or DRCFI offered minimal protection. However, single doses of CFI or DRCFI were able to significantly reduce the bacterial burden in the spleen compared to empty liposomes. A three-day treatment regimen of ciprofloxacin, CFI, or DRCFI resulted in high levels of protection (90-100% survival). This study suggests that CFI and DRCFI may be useful therapies for Y. pestis infection, both as prophylaxis and for the treatment of plague.

p21(WAF1) modulates drug-induced apoptosis and cell cycle arrest in B-cell precursor acute lymphoblastic leukemia.

p21(WAF1) is a well-characterized mediator of cell cycle arrest and may also modulate chemotherapy-induced cell death. The role of p21(WAF1) in drug-induced cell cycle arrest and apoptosis of acute lymphoblastic leukemia (ALL) cells was investigated using p53-functional patient-derived xenografts (PDXs), in which p21(WAF1) was epigenetically silenced in T-cell ALL (T-ALL), but not in B-cell precursor (BCP)-ALL PDXs. Upon exposure to diverse cytotoxic drugs, T-ALL PDX cells exhibited markedly increased caspase-3/7 activity and phosphatidylserine (PS) externalization on the plasma membrane compared with BCP-ALL cells. Despite dramatic differences in apoptotic characteristics between T-ALL and BCP-ALL PDXs, both ALL subtypes exhibited similar cell death kinetics and were equally sensitive to p53-inducing drugs in vitro, although T-ALL PDXs were significantly more sensitive to the histone deacetylase inhibitor vorinostat. Transient siRNA suppression of p21(WAF1) in the BCP-ALL 697 cell line resulted in a moderate depletion of the cell fraction in G1 phase and marked increase in PS externalization following exposure to etoposide. Furthermore, stable lentiviral p21(WAF1) silencing in the BCP-ALL Nalm-6 cell line accelerated PS externalization and cell death following exposure to etoposide and vorinostat, supporting previous findings. Finally, the Sp1 inhibitor, terameprocol, inhibited p21(WAF1) expression in Nalm-6 cells exposed to vorinostat and also partially augmented vorinostat-induced cell death. Taken together, these findings demonstrate that p21(WAF1) regulates the early stages of drug-induced apoptosis in ALL cells and significantly modulates their sensitivity to vorinostat.

Liposome encapsulation of ciprofloxacin improves protection against highly virulent Francisella tularensis strain Schu S4.

Liposome-encapsulated ciprofloxacin for inhalation (CFI) was investigated as a putative postexposure therapeutic for two strains of Francisella tularensis. The efficacies of oral ciprofloxacin and intranasally instilled CFI could not be distinguished in a mouse model of infection with the F. tularensis live vaccine strain (LVS), where a single dose of either formulation offered full protection against a lethal challenge. However, mouse studies with the more virulent Schu S4 strain of F. tularensis demonstrated that a higher level of protection against a lethal aerosol infection is provided by CFI than by oral ciprofloxacin. In addition, using this infection model, it was possible to discriminate the efficacy of intranasally instilled CFI from that of aerosolized CFI, with aerosolized CFI providing full protection after just a single dose. The improved efficacy of CFI compared to oral ciprofloxacin is likely due to the high sustained concentrations of ciprofloxacin in the lung. In summary, CFI may be a promising therapy, perhaps enabling the prophylactic regimen to be shortened, for use in the event of a deliberate release of F. tularensis. The prophylactic efficacy of CFI against other biological warfare (BW) threat agents also warrants investigation.

Trimethoprim/sulfamethoxazole (co-trimoxazole) prophylaxis is effective against acute murine inhalational melioidosis and glanders.

Burkholderia pseudomallei is the causative agent of the disease melioidosis, which is prevalent in tropical countries and is intractable to a number of antibiotics. In this study, the antibiotic co-trimoxazole (trimethoprim/sulfamethoxazole) was assessed for the post-exposure prophylaxis of experimental infection in mice with B. pseudomallei and its close phylogenetic relative Burkholderia mallei, the causative agent of glanders. Co-trimoxazole was effective against an inhalational infection with B. pseudomallei or B. mallei. However, oral co-trimoxazole delivered twice daily did not eradicate infection when administered from 6h post exposure for 14 days or 21 days, since infected and antibiotic-treated mice succumbed to infection following relapse or immunosuppression. These data highlight the utility of co-trimoxazole for prophylaxis both of B. pseudomallei and B. mallei and the need for new approaches for the treatment of persistent bacterial infection.

Silencing of ASXL1 impairs the granulomonocytic lineage potential of human CD34⁺ progenitor cells.

The ASXL1 gene encodes a chromatin-binding protein involved in epigenetic regulation in haematopoietic cells. Loss-of-function ASXL1 mutations occur in patients with a range of myeloid malignancies and are associated with adverse outcome. We have used lentiviral-based shRNA technology to investigate the effects of ASXL1 silencing on cell proliferation, apoptosis, myeloid differentiation and global gene expression in human CD34(+) cells differentiated along the myeloid lineage in vitro. ASXL1-deficient cells showed a significant decrease in the generation of CD11b(+) and CD15(+) cells, implicating impaired granulomonocytic differentiation. Furthermore, colony-forming assays showed a significant increase in the number of multipotent mixed lineage colony-forming unit (CFU-GEMM) colonies and a significant decrease in the numbers of granulocyte-macrophage CFU (CFU-GM) and granulocyte CFU (CFU-G) colonies in ASXL1-deficient cells. Our data suggests that ASXL1 knockdown perturbs human granulomonocytic differentiation. Gene expression profiling identified many deregulated genes in the ASXL1-deficient cells differentiated along the granulomonocytic lineage, and pathway analysis showed that the most significantly deregulated pathway was the LXR/RXR activation pathway. ASXL1 may play a key role in recruiting the polycomb repressor complex 2 (PRC2) to specific loci, and we found over-representation of PRC2 targets among the deregulated genes in ASXL1-deficient cells. These findings shed light on the functional role of ASXL1 in human myeloid differentiation.

Mutation patterns of 16 genes in primary and secondary acute myeloid leukemia (AML) with normal cytogenetics.

Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.

p53-independent epigenetic repression of the p21(WAF1) gene in T-cell acute lymphoblastic leukemia.

The p53 protein is a primary mediator of cellular apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has shown that the majority of childhood acute lymphoblastic leukemia (ALL) cases express a wild type p53 gene, although the functionality of the p53 pathway has rarely been validated. In the present study, the integrity of the p53 pathway was investigated in a panel of ALL cell lines and xenografts established from direct patient explants in immune-deficient mice. A focused real-time quantitative reverse transcription PCR array of known p53-regulated genes identified p21(WAF1) (CDKN1A) as the highest ranked gene to be differentially expressed between B-cell precursor (BCP)-ALL and T-ALL xenografts following exposure to the DNA-damaging drug etoposide. Lack of p21(WAF1) induction was observed in six of seven T-ALL xenograft lines, as well as primary T-ALL cells following irradiation exposure, despite an otherwise functional p53 response. Repression of p21(WAF1) in T-ALL cells was associated with decreased acetylated H3K9 localized at its promoter compared with BCP-ALL cells, together with increased CpG methylation within the first exon and intron. Although the histone deacetylase inhibitor vorinostat failed to induce p21(WAF1) in T-ALL samples, the combination of vorinostat and the demethylating agent decitabine reactivated expression of the silenced p21(WAF1) gene in the Molt-4 T-ALL cell line. Considering the known anti-apoptotic function of p21(WAF1), our findings have significant implications for the responses of T- versus BCP-ALL cells to chemotherapeutic drugs that induce p21(WAF1).

Influence of particle size on the pathology and efficacy of vaccination in a murine model of inhalational anthrax.

Deposition of Bacillus anthracis endospores within either the lungs or nasal passages of A/J mice after aerosol exposure was influenced by different particle sized aerosols and resulted in different infection kinetics. The infection resulting from the inhalation of endospores within a 12 µm particle aerosol was prolonged compared to that from a 1 µm particle aerosol with a mean time-to-death of 161 ± 16.1 h and 101.6 ± 10.4 h, respectively. Inhalation of endospores within 1 µm or 12 µm particle aerosols resulted in a median lethal dose of 2432 and 7656 c.f.u., respectively. Initial involvement of the upper respiratory tract lymph nodes was observed in 75-83% of mice exposed to either the 1 µm or 12 µm particle inhalational infections. Lung deposition was significantly greater after inhalation of the 1 µm particle aerosol with pronounced involvement of the mediastinal lymph node. Gastrointestinal involvement was observed only in mice exposed to 12 µm particle aerosols where bacteriological and histopathological analysis indicated primary gastritis (17%), activation of the Peyer's patches (72%) and colonization and necrosis of the mesenteric lymph nodes (67%). Terminal disease was characterized by bacteraemia in both inhalational infections with preferential dissemination to spleen, liver, kidneys and thymus. Immunization with 1 µg recombinant protective antigen vaccine was equally efficacious against B. anthracis infections arising from the inhalation of 1 and 12 µm particle aerosols, providing 73-80% survival under a suboptimum immunization schedule.

Epigenetic silencing of BIM in glucocorticoid poor-responsive pediatric acute lymphoblastic leukemia, and its reversal by histone deacetylase inhibition.

Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor. Although a comprehensive assessment of BIM CpG island methylation revealed no consistent changes, glucocorticoid resistance in xenografts and patient biopsies significantly correlated with decreased histone H3 acetylation. Moreover, the histone deacetylase inhibitor vorinostat relieved BIM repression and exerted synergistic antileukemic efficacy with dexamethasone in vitro and in vivo. These findings provide a novel therapeutic strategy to reverse glucocorticoid resistance and improve outcome for high-risk pediatric ALL.

Development and characterization of mouse models of infection with aerosolized Brucella melitensis and Brucella suis.

There is a need to identify vaccines that can protect against Brucella, a potential bioterrorism agent. We have developed mouse models of infection with aerosolized Brucella melitensis and Brucella suis and demonstrated their utility for the evaluation of vaccines using the model live B. melitensis vaccine strain Rev.1.

Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9).

CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wild-type and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5.