Moderate Colitis Not Requiring Intravenous Steroids Is Associated with Improved Survival in Stage IV Melanoma after Anti-CTLA4 Monotherapy,But Not Combination Therapy

BackgroundFor patients with melanoma, gastrointestinal immune-related adverse events are common after receipt of anti-CTLA4 therapy. These present difficult decision points regarding whether to discontinue therapy. Detailing the situations in which colitis might predict for improved survival and how this is affected by discontinuation or resumption of therapy can help guide clinical decision-making.Materials and MethodsPatients with stage IV melanoma receiving anti-CTLA4 therapy from 2008 to 2019 were analyzed. Immune-related colitis treated with ≥50 mg prednisone or equivalent daily or secondary immunosuppression was included. Moderate colitis was defined as receipt of oral glucocorticoids only; severe colitis was defined as requiring intravenous glucocorticoids or secondary immunosuppression. The primary outcome was overall survival (OS).ResultsIn total, 171 patients received monotherapy, and 91 received dual checkpoint therapy. In the monotherapy group, 25 patients developed colitis and a nonsignificant trend toward improved OS was observed in this group. Notably, when colitis was categorized as none, moderate or severe, OS was significantly improved for moderate colitis only. This survival difference was not present after dual checkpoint therapy. There were no differences in known prognostic variables between groups, and on multivariable analysis neither completion of all ipilimumab cycles nor resumption of immunotherapy correlated with OS, while the development of moderate colitis did significantly affect OS.ConclusionThis single-institution retrospective series suggests moderate colitis correlates with improved OS for patients with stage IV melanoma treated with single-agent anti-CTLA4, but not dual agent, and that this is true regardless of whether the immune-checkpoint blockade is permanently discontinued.     

De novo mapping of α-helix recognition sites on protein surfaces using unbiased libraries

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered “undruggable”, such as protein–protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein–protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Recent advances in identifying human disease targets have not been matched by advances in the ability to drug these targets. This actionability gap is largely due to the fact that neither of the two main classes of approved therapeutics – biologics and small molecules – can simultaneously address target accessibility and selective target engagement. Biologics, despite an impressive ability to engage diverse target proteins, are largely restricted to an extracellular operating theater, as their size and polarity render them unable to cross biological membranes. Small molecules, in contrast, can access the intracellular space, but cannot bind with high affinity and specificity to the vast majority of proteins that are found there (1).This disconnect between the ability to identify disease targets and the ability to drug them with high strength and specificity has created an impetus to develop new classes of drugs – ones that can engage intracellular proteins that lack the deep hydrophobic pocket ordinarily required for small-molecule binding. In nature, such “undruggable” proteins are often targeted with macrocyclic molecules, frequently peptidic in structure, whose large size compared with small molecules enables them to bind with high affinity and specificity to protein surfaces.Significant efforts have been made to elucidate the mechanisms of cell entry for these natural products, which possess molecular weights of 700 to 1,200 Da or higher, well beyond the typical range for cell penetration in small-molecule drug discovery (2). While the mechanisms of cell entry are complex and vary from molecule to molecule, a substantial body of research on peptidic macrocycles has highlighted the importance of desolvating amide protons and reducing their exposure to the membrane interior as a key driver in passive, thermal diffusion across the lipid bilayer (2, 3) – a phenomenon we refer to as amide-proton cloaking. The amide proton, present between every residue in a polypeptide chain, is highly electropositive and forms a strong hydrogen-bonding interaction with water. This poses a substantial hurdle for membrane permeability, since tightly bound solvent water molecules must be shed prior to entering the lipid bilayer. Exposed amide groups incur a further energetic penalty upon membrane entry due to unfavorable electrostatic interactions with the low-dielectric environment of the membrane interior. Consequently, most peptides and proteins are unable to cross membranes.For peptide macrocycles that are able to permeate the membrane, these problematic amide protons are typically removed either by replacing the amide with an ester, replacing it with a methyl group, or cloaking it from solvent water through the formation of intramolecular hydrogen bonds between the amide proton groups and a hydrogen bond-accepting group elsewhere in the molecule, often a carbonyl. Indeed, the paradigmatic example of a natural peptide macrocycle that exhibits robust cytosolic exposure, cyclosporine A (CsA), employs both N-methylation and cloaking through transannular hydrogen bonding (4). Extensive work by several research groups has shown that these strategies can be applied as design principles to endow artificial macrocycles with the ability to cross membranes (5–7).In the context of folded proteins, nature has offered an alternative structural solution to the problem of amide proton cloaking: the α-helix, a protein secondary structure that is defined by repeating intramolecular hydrogen bonds between the amide proton group of one residue and the carbonyl of the amino acid located four residues N terminal to it. The intrinsic ability of α-helices to cloak their own amide protons explains their widespread prevalence in natural transmembrane proteins (8). Nuclear-encoded transmembrane proteins in eukaryotes are almost exclusively α-helical, and the only alternative transmembrane fold found in nature is the bacterially derived β-barrel, a helical structure that also cloaks amide protons via an intramolecular hydrogen bonding network, albeit in a significantly larger structure than single α-helices that is impractical for the development of synthetic drugs.Just as CsA has served as the inspiration for the design of mimetic head-to-tail cyclized peptide ligands, so have proteinaceous α-helices inspired efforts to recapitulate nature’s design features in small, synthetic, α-helically constrained peptides (Helicons) that are hyperstabilized through the incorporation of a structural brace, also known as a “staple” (9–12). One of these, the all-hydrocarbon staple formed by ring-closing metathesis, has been extensively studied and is the basis for a drug candidate that targets the challenging proteins MDM2 and MDMX, currently undergoing Phase II clinical trials (13, 14).Rational design of Helicons is difficult given the inability to systematically define the α-helix binding sites on a protein’s surface, and to identify Helicons that bind to those sites. This limitation has restricted research on Helicons to only those protein targets for which naturally occurring or previously characterized α-helical binders were known, with the Helicons generated from fragments of the known binders (3). Here, we report a rapid, high-throughput screening platform utilizing phage display that enables an unbiased mapping of the α-helical interactome of a given protein without any prior knowledge of its structure or known binding partners. We show that this platform is capable of identifying α-helix binding sites on the surfaces of a range of protein folds, including many for which no α-helical binders are known to exist. Helicons that bind these sites are able to impact diverse protein functions, including inhibiting protein–protein interactions, inhibiting enzymatic activity, inducing dimerization, and inducing conformational changes. Analysis of 14 high-resolution crystal structures of Helicon–protein complexes across six different protein domains reveals a range of binding modes, all of which are “side-on”, i.e., mediated exclusively by Helicon side-chains rather than involving main chain amide interactions. This screening platform significantly expands the universe of proteins that can be bound by Helicons, and furthers the pursuit of targeting undruggable proteins.     

J USTIFICATION OF P ULSE O XIMETER C OSTS FOR P ARAMEDIC P REHOSPITAL P ROVIDERS

Objective. To assess the potential cost savings of decreasing prehospital oxygen utilization by using pulse oximetry to identify those patients who do not require supplemental oxygen. Methods. A prospective, controlled trial was performed comparing rates of oxygen utilization by paramedics with and without access to pulse oximetry. Consecutive patient encounters over a ten-week period were randomized by day of presentation. Pulse oximeters were made available on alternate days. On those days, patients whose oxygen saturations were less than 95% were treated with supplemental oxygen. Results. The use of pulse oximeters incurred a saving of 0.14 “D”-size oxygen cylinders per call. For the authors' service, this translates to a potential saving of $2,324 (C)/vehicle/year. Conclusion. For regions with patient demographics similar to the authors', the initial cost of providing paramedics with pulse oximeters may be offset by savings in oxygen consumption. A formula is provided to allow individual ambulance services to calculate the potential savings for their service.     

Evaluation of platelet-rich plasma in combination with anorganic bovine bone in the rabbit cranium: a pilot study

PURPOSE: Platelet-rich plasma (PRP) is potentially useful as an adjunct to allograft and xenograft materials in oral and maxillofacial bone and implant reconstructive surgery. This study compares bone healing and formation in 4 cranial defects in rabbits grafted with autogenous bone, xenograft, and xenograft with PRP (with a no-graft group as a control). MATERIALS AND METHODS: Fifteen New Zealand white rabbits were included in this randomized, blind, prospective pilot study. Four identical 8-mm-diameter defects were created in each rabbit cranium and immediately grafted with the above materials. Five rabbits were evaluated at 1 month, 5 at 2 months, and 5 at 4 months. Radiographs were used to evaluate bone density. RESULTS: Radiographically, sites at which Bio-Oss, autogenous bone, and Bio-Oss + PRP were grafted showed a significant increase in bone density at 1 month (P = .05 for Bio-Oss, P = .02 for autogenous bone, P = .008 for Bio-Oss + PRP) and at 4 months (P = .02 for Bio-Oss, P = .04 for autogenous bone, P = .05 for Bio-Oss + PRP). Autogenous bone sites (P < .001) and Bio-Oss + PRP sites (P < .001) also showed significant increases at 2 months. Histomorphometrically, autogenous bone sites showed a significantly greater increase than control sites (P = .08 at 1 month, P = .03 at 2 months, P = .01 at 4 months), Bio-Oss sites (P < .001 at all 3 evaluation points), or Bio-Oss + PRP sites (P = .009 at 1 month, P = .02 at 2 months, P = .01 at 4 months). Furthermore, Bio-Oss + PRP sites showed a significantly greater increase in bone area at 1, 2, and 4 months than Bio-Oss alone (P = .003 at 1 month, P = .02 at 2 months, P = .006 at 4 months). DISCUSSION: Radiographs showed significantly greater bone density at the Bio-Oss, autogenous bone, and Bio-Oss + PRP sites than at control sites at nearly every point in time evaluated; however, clinical significance is difficult to determine, since all materials appeared dense on the radiograph. Histomorphometry showed that the increase in bone area at autogenous sites was significantly greater than that seen with other grafting materials or at the control sites. CONCLUSION: This study showed a histomorphometric increase in bone formation with the addition of PRP to Bio-Oss in non-critical-sized defects in the rabbit cranium.     

Cardiomyopathies and Genetic Testing in Heart Failure: Role in Defining Phenotype-Targeted Approaches and Management

Cardiomyopathies represent an important cause of heart failure, often affecting young individuals, and have important implications for relatives. Genetic testing for cardiomyopathies is an established care pathway in contemporary cardiology practice. The primary cardiomyopathies where genetic testing is indicated are hypertrophic, dilated, arrhythmogenic, and restrictive cardiomyopathies, with left ventricular noncompaction as a variant phenotype. Early identification and initiation of therapies in patients with inherited cardiomyopathies allow for targeting asymptomatic and presymptomatic patients in stages A and B of the American College of Cardiology/American Heart Association classification of heart failure. The current approach for genetic testing uses gene panel–based testing with the ability to extend to whole-exome and whole-genome sequencing in rare instances. The central components of genetic testing include defining the genetic basis of the diagnosis, providing prognostic information, and the ability to screen and risk-stratify relatives. Genetic testing for cardiomyopathies should be coordinated by a multidisciplinary team including adult and pediatric cardiologists, genetic counsellors, and geneticists, with access to expertise in cardiac imaging and electrophysiology. A pragmatic approach for addressing genetic variants of uncertain significance is important. In this review, we highlight the indications for genetic testing in the various cardiomyopathies, the value of early diagnosis and treatment, family screening, and the care process involved in genetic counselling and testing.     

The efficacy and toxicity of immune checkpoint inhibitors in a real-world older patient population

ImportanceImmunotherapy has emerged as an effective treatment option for the management of advanced cancers. The effects of these immune checkpoint inhibitors in the older patient population has not been adequately assessed.ObjectiveTo understand the impact of aging on CTLA-4 and PDL-1 inhibitors efficacy and immune-related adverse events (irAE) in the context of real-world management of advanced solid cancers.Design, Setting, and ParticipantsThis retrospective study involved all non-study patients with histologically-confirmed metastatic or inoperable solid cancers receiving immunotherapy at Kingston Health Sciences Centre. We defined ‘older patient’ as age ≥ 75. All statistical analyses were conducted under SPSS IBM for Windows version 24.0.Main Outcomes and MeasuresStudy outcomes included immunotherapy treatment response, survival, as well as number, type, and severity of irAEs.ResultsOur study (N = 78) had 29 (37%) patients age <65, 26 (33%) patients age 65–74, and 23 (30%) patients age ≥75. Melanoma, non-small cell lung cancer, and renal cell carcinoma accounted for 70%, 22%, and 8% of the study population, respectively. Distributions of ipilimumab (32%), nivolumab (33%), and pembrolizumab (35%) were similar in the study. The response rates were 28%, 27%, and 39% in the age <65, age 64–74, age ≥75 groups, respectively (P = 0.585). Kaplan-Meier curve showed a median survival of 28 months (12.28–43.9, 95% CI) and 17 months (0–36.9, 95% CI) in the age <65 and age 64–74 groups, respectively; the estimated survival probability did not reach 50% in the age ≥75 group (P = 0.319). There were no statistically significant differences found in terms of irAEs, multiple irAEs, severity of grade 3 or higher, types of irAEs, and irAEs resolution status when comparing between different age groups.Conclusion and RelevanceOur results suggest that patients age ≥75 are able to gain as much benefit from immunotherapy as younger patients, without excess toxicity. Our findings suggest that single agent immunotherapy is generally well-tolerated across different age groups with no significant difference in the type, frequency or severity of irAEs. Future studies evaluating aging and combination immunotherapy are warranted.