Bacteriophages (phages) are a promising anti-infective option for human disease. Major gaps remain in understanding their potential utility.Pseudomonas aeruginosa airway colonization. The single dose of phage consists of a mixture of four anti-pseudomonal phages. Six sentinel participants will be sequentially enrolled with dose escalation of the phage mixture by one log10 beginning with 4 × 107 plaque-forming units in an unblinded stage 1. If no serious adverse events related to the study product are identified, the trial will proceed to a double-blinded stage 2. In stage 2a, 32 participants will be randomly assigned to one of three phage dosages or placebo in a 1:1:1:1 allocation. An interim analysis will be performed to determine the phage dosage with the most favorable safety and microbiological activity profile to inform phage dosing in stage 2b. During stage 2b, up to 32 additional volunteers will be randomized 1:1 to the phage or placebo arm. Primary outcomes include (1) the number of grade 2 or higher treatment-emergent adverse events, (2) change in log10P. aeruginosa total colony counts in sputum, and (3) the probability of a randomly selected subject having a more favorable outcome ranking if assigned to receive phage therapy versus placebo. Exploratory outcomes include (1) sputum and serum phage pharmacokinetics, (2) the impact of phage on lung function, (3) the proportion of P. aeruginosa isolates susceptible to the phage mixture before and after study product administration, and (4) changes in quality of life.This is a randomized, placebo-controlled, double-blind study of a single dose of intravenous phage in approximately 72 clinically stable adult cystic fibrosis volunteers recruited from up to 20 US sites with P. aeruginosa colony counts and provide insights into the safety profile of phage therapy.This trial will investigate the activity of phages in reducing ClinicalTrials.gov NCT05453578. Registered on 12 July 2022. The order of the items has been modified to group similar items  bacterial infections remain an international public health concern . The cysrbations . Becausee needed .Bacteriophages  are viruses that target and kill bacteria . Since tUnique features that make phages attractive for clinical use include their bactericidal activity , 8, highP. aeruginosa in expectorated sputumDescribe the safety of a single dose of intravenous (IV) phage therapy in CF volunteers with P. aeruginosa in expectorated sputumDescribe the microbiological activity of a single dose of IV phage therapy in CF volunteers with P. aeruginosa in expectorated sputumDescribe the benefit-to-risk profile of a single dose of IV phage therapy in CF volunteers with P. aeruginosa in expectorated sputumCharacterize the serum and sputum pharmacokinetic profiles of a single dose of IV phage therapy in CF volunteers with Describe changes in lung function in CF volunteers after the administration of a single dose of IV phage therapyP. aeruginosa isolates from CF volunteersCharacterize phage susceptibility among geographically diverse Describe CF volunteers’ quality of life (QoL) before and after receiving phagesP. aeruginosa in expectorated sputum. The primary study outcomes include the safety and microbiological activity of IV phage therapy. The single dose of IV phage will consist of a mixture of four anti-pseudomonal phages.This is a phase 1b/2, multicenter, randomized, placebo-controlled, double-blind study of a single dose of IV phage in approximately 72 clinically stable adult CF volunteers colonized with 10 starting at 4 × 107 plaque-forming units (PFU). In each dosing arm, two volunteers will be enrolled. Each sentinel subject will receive a single dose of IV phage therapy. If no serious adverse events (SAEs) related to the study product are identified during the 96 h after phage administration in stage 1 volunteers, the study will proceed to stage 2.Stage 1 will involve six volunteers assigned to one of three IV phage dosing arms  at the time of screeningCF diagnosis based on a compatible clinical syndrome confirmed by either abnormal sweat chloride testing or cystic fibrosis transmembrane conductance regulator gene variationsAbility to produce approximately 2 mL of sputum over a 30-min periodP. aeruginosa isolated from a sputum culture, throat culture, or other respiratory specimens in the past 12 monthsP. aeruginosa isolation from a sputum sample at the screening visitConfirmed Capable of providing informed consentCapable and willing to complete all study visits and perform all procedures required by the protocolVolunteers must meet all inclusion criteria to be eligible to enroll in the study:Body weight <30 kgForced expiratory volume in 1 s (FEV1) <20% of predicted at screeningElevated liver function tests obtained at screeningAcute clinical illness requiring a new oral, parenteral, or inhaled antibiotic(s) ≤30 days prior to the baseline visitPregnant, planning to become pregnant during the study period, or breastfeedingActive treatment of any mycobacteria or fungal organisms ≤30 days prior to the baseline visitAnticipated need to change chronic antibiotic regimens during the study periodKnown allergy to any component of the study productAny significant finding that, in the opinion of the investigator, would make it unsafe for the volunteer to participate in the studyEnrollment in a different clinical trial ≤30 days of the baseline visit, or while enrolled in the current clinical trialPrevious enrollment in the current trialVolunteers who meet any of the following exclusion criteria will not be enrolled in the study:Upon identification of a potentially eligible participant, study procedures, risks, and potential benefits will be presented by the local study team during a screening visit. Participants will receive a copy of the study consent and will have the opportunity to ask questions. Before any study procedures are performed, informed consent will be obtained and documented.P. aeruginosa isolates to identify changes that occur after exposure to phages, and the impact of phages on the respiratory microbiome. No human genetic testing  will be performed.The information and specimens collected for this study may be used for future research. The research may include, but is not limited to, investigating the role of serum neutralizing antibodies, whole genome sequencing of P. aeruginosa airway colonization. Phage therapy is not currently approved as an anti-infective for use in humans by the US Food and Drug Administration (FDA). As such, most humans who have received phages as anti-infectives have received them in conjunction with, rather than in place of, antibiotic therapy [This trial will compare the safety and microbiological activity of phages versus placebo in clinically stable CF volunteers with chronic  therapy . This co therapy . An undeP. aeruginosa. These phages are naturally occurring and free of known deleterious genes, including genes essential for lysogeny , antibiotic resistance genes, and toxin genes. Each phage lot has been manufactured by Adaptive Phage Therapeutics (APT) in accordance with current Good Manufacturing Practices. Endotoxin levels in the phage lots are below acceptable limits set by the FDA (5 endotoxin units/kg/h). The phage mixture is administered as a total of 4 × 107 PFU, 4 × 108 PFU, or 4 × 109 PFU, depending on the target dose. The final phage combination to be administered to a trial participant is diluted to the target dose in normal saline. The minimum and maximum phage doses that are being investigated were informed by previous case reports, case series, and clinical trials, which generally used IV doses ranging from approximately 107 PFU to 1010 PFU per dose [The product used in this study is WRAIR-PAM-CF1. WRAIR-PAM-CF1 is a cocktail of four phages in a 1:1:1:1 combination: PaWRA01Phi11, PaWRA01Phi39, PaWRA02Phi83, and PaWRA02Phi87 , all of which are lytic against per dose , 7. All Volunteers may withdraw consent for study participation at any time without penalty. An investigator may also withdraw a volunteer from receiving the study product if it is determined that participation in the study is not in the best interest of the subject. Follow-up safety evaluations will be conducted, if the volunteer agrees.If any of the following events occur, enrollment and dosing for all volunteers will be suspended until the event is assessed by the Data Safety and Monitoring Board (DSMB): (1) any volunteer develops an SAE related to the study product through the last study visit, (2) two or more volunteers in the study experience a grade 3 or higher AE that is related to study product and is of the same type through the last study visit, (3) any volunteer develops anaphylaxis within 24 h after receiving the study product, or (4) any volunteer reports two or more pulmonary exacerbations, from the time of study product administration through day 8. Moreover, an individual infusion will be stopped if a drug-related hypersensitivity reaction is suspected. Study withdrawal could also occur if the study or study site is terminated by the sponsor for any reason.Recruitment of volunteers will occur by site investigators who are clinicians who care for persons with CF. Although there are no prespecified strategies that have been developed to increase adherence, only volunteers willing and able to participate in all planned follow-up visits will be selected for trial inclusion.P. aeruginosa after receipt of the study product, the volunteer will continue in the trial and be included in an intent-to-treat analysis. Receipt of antibiotic therapy with activity against P. aeruginosa will be documented as a concomitant medication and the underlying condition for which the antibiotic was taken will be reported as an AE. Chronic medications, rescue medications, and over-the-counter medications are allowed.Concomitant medications will be reviewed during each trial visit. If a volunteer is prescribed antibiotic therapy with activity against All volunteers will be followed for 30 days. AEs will be assessed and followed from initial recognition of the AE through the end of the 30-day follow-up period. SAEs will be followed through resolution even if the duration of follow-up goes beyond the planned follow-up period.10P. aeruginosa total colony counts in sputum cultures after administration of study product, and (3) desirability of outcome ranking (DOOR) using the greatest reduction by the day 8 visit. More specifically, volunteers will be placed into one of four DOOR categories  the number of grade 2 or higher treatment-emergent AEs through the day 30 visit, (2) change from baseline to day 30 logP. aeruginosa isolates susceptible to the four individual phages and the phage mixture before and after exposure to study product, and (4) changes in QoL of participants based on responses documented in the Cystic Fibrosis Questionnaire-Revised and the Cystic Fibrosis Respiratory Symptom Diary before and after exposure to the study product.Several exploratory endpoints will also be investigated: (1) sputum and serum pharmacokinetics of phage therapy, (2) impact on FEV1 from the administration of study product through day 30, (3) the proportion of The participant timeline is illustrated in Fig. The sample size was calculated to provide desired precision of the estimate of the DOOR probability in order to describe the benefit-to-risk profile of a single dose of IV phage. If the DOOR probability comparing IV phage and placebo is 70%, when the total sample size in each arm is 20 (combining volunteers from stages 2a and 2b), the two-sided normal approximate 95% confidence interval for DOOR probability is calculated at 51% and 89%, respectively, with the lower limit larger than 50%. Superiority will be considered to have been achieved if the 95% confidence interval for the probability does not cross 50%.Based on the interim analysis after stage 2a, the planned sample size for stage 2b will be re-evaluated as to whether it provides the desired precision of estimates of the DOOR probability for a selected phage dose and placebo. The total estimated sample size for the trial is 72 participants. In the intention-to-treat population, it is estimated that there will be up to 25 volunteers in the phage arm  and 25 volunteers in the placebo arm.CF volunteers will be recruited from up to 20 outpatient clinics in the USA. There will be no enrollment from international sites. It is anticipated that approximately three patients will be enrolled per month. The anticipated enrollment period is from October 3, 2022, until January 31, 2024.In stage 2a, volunteers will be randomized to either one of three IV phage doses or placebo with a 1:1:1:1 allocation as per a computer-generated randomization stratified by site, using a permuted block design. In stage 2b, volunteers will be randomly assigned to one of two arms: the selected phage dose  or placebo in a 1:1 allocation. The block size will be concealed until the primary endpoint is analyzed.Volunteers will be randomized using the Advantage eClinical data management system, a centralized, web-based enterprise resource developed and maintained by the Emmes Company. Allocation concealment will be ensured, as the randomization code will not be released until the patient has been recruited into the trial. The codes will be kept confidential and allocation communicated to sites electronically via a separate online enrollment module.The randomization process will be managed via an online enrollment module within the Advantage eClinical data management system.In stage 2, the subject and the investigators will be unaware of treatment group assignments. The three phage doses and placebo will be packaged identically so that treatment blinding is maintained. Specimens provided to the laboratory for analyses will be blinded to participant identification and visit number in addition to treatment assignment. Only the site pharmacist preparing the study product will be unblinded.Randomization code breaks will occur only when knowledge of the actual treatment is essential for further management of the subject. Site investigators are encouraged to discuss with the study team and DMID if they believe that unblinding is necessary. In the case of a medical emergency, if the site investigator believes that unblinding would benefit the medical care of the volunteer, the unblinding process can occur on-site by contacting the unblinded pharmacist.Data will be entered electronically by site study staff into Advantage eClinical. Instructions for use of the system and completion of the electronic case report forms (eCRFs) for each study will be included in the Advantage eClinical User’s Guide and the eCRF Instructions. Quality assurance reports will be generated to ensure study data are clean, accurate, and complete. Quality assurance reports will include, but are not limited to, the following: missing forms, missing and out-of-range values, automated data queries, and targeted manual reviews of study data. The schedule of events is described in Table Volunteers may voluntarily withdraw their consent for study participation at any time without penalty. An investigator may also withdraw a subject from receiving the study product for any reason. Follow-up safety evaluations will be conducted if the subject agrees. Volunteers who withdraw, are withdrawn, or are lost to follow-up after administration of the study product will not be replaced.DMID’s monitoring staff will either conduct site visits or remote source verification to assure appropriate quality and completeness of data. Discrepancies in data entry in eCRFs will trigger data re-entry requirements and/or site retraining for the relevant data fields.Personal health information will be collected and stored securely within the electronic study database maintained by the Emmes Company for a minimum of 2 years after study completion.P. aeruginosa by culture and determination of phage concentrations. Phage susceptibility testing (PST) will be performed on cultured P. aeruginosa strains. More specifically, sputum will be collected before and after study product administration at the baseline visit, as well as all follow-up visits and processed within 48 h of collection. The total CFU/mL of all morphotypes of P. aeruginosa identified in sputum cultures will be evaluated at each visit. As persons with CF are often colonized with more than one morphotype of P. aeruginosa, colony size, color, mucoid phenotype, antimicrobial susceptibility testing results, and other morphological features will be documented for each P. aeruginosa colony type to determine whether isolates might represent the same P. aeruginosa strain over time from the same patient.Processing of serum chemistries, liver function tests, and hematology to monitor for AEs will occur at local laboratories, in accordance with local standard operating procedures. Sputum specimens will be shipped to a central laboratory for quantification of P. aeruginosa quantitative cultures are performed, phage quantitative polymerase chain reaction (qPCR) will be performed to understand the pharmacokinetics of phage in sputum. Phages are expected to amplify upon infecting a susceptible bacterial host (in this case P. aeruginosa); therefore, it is hypothesized that the quantity of phage identified in the sputum should increase in the days after a dose of phage is administered.From the same sputum specimens and after P. aeruginosa in their bloodstream, the identification of phage in serum is expected to be short-lived; serum will be analyzed prior to infusion of the study product, and at 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, and 3.5 h post-administration. Serum PK studies will also be performed at a single timepoint during the day 2 visit .As the study product is being administered IV, serum pharmacokinetics will also be evaluated. As trial participants would not be expected to have P. aeruginosa obtained from sputum specimens will undergo PST using both index P. aeruginosa isolates  and sequential P. aeruginosa isolates identified in sputum samples collected at six timepoints after study product administration, for each subject. PST results will inform the proportion of trial participants with P. aeruginosa isolates susceptible to both individual phages and the phage mixture.Each morphotype of P. aeruginosa isolate “susceptible” to the phages. Replicate testing will occur for any P. aeruginosa isolates with discrepant PST results. Briefly, the plaque assay utilizes a modification of the double agar overlay plaque assay [As no reference standard testing method for assessing bacterial susceptibility to phages exists, PST will occur using two approaches: (1) plaque assay and (2) liquid assay. Susceptibility to both tests will qualify a ue assay . Phages ue assay . The liqAnalyses will be performed on the basis of the intention-to-treat principle. Grade 2 or higher treatment-emergent AEs will be summarized descriptively. The number of events and number and percentage of volunteers with events in each arm will be tabulated and presented by system organ class and likelihood of being related/unrelated. The difference in the proportion of the number and percentage of volunteers with events between phage and placebo arms will be calculated with the corresponding 95% confidence interval.10P. aeruginosa CFU/mL  in quantitative sputum cultures from administration of the study product through day 30 will be summarized descriptively, by treatment arm. The area under the curve (AUC) calculated using the trapezoidal rule will be used to summarize log10P. aeruginosa CFU/mL over time. Differences in mean changes in log10P. aeruginosa CFU/mL and AUC between the phage and placebo arms will be calculated with the corresponding 95% confidence interval.Changes in logThe DOOR will be summarized using the DOOR probability . The DOOThere will be one planned interim analysis of the primary endpoints after volunteers in stage 2a complete their day 30 follow-up visits. The interim analysis will be performed to select the phage dose with the most favorable benefit-to-risk profile compared to placebo for stage 2b. The interim analysis will consist of a quantitative evaluation of potential effect sizes and associated precision using a predicted interval plots approach that will be generated for a range of assumptions . The resP. aeruginosa isolates susceptible to each phage and the phage mixture will be evaluated. The difference between phage and placebo will be calculated, along with a 95% confidence interval.As previously stated, there will be four exploratory outcomes. Phage pharmacokinetics will be analyzed using compartmental population pharmacokinetic models to obtain population mean estimates of clearances and volumes as well as estimates of inter-individual variability. Individual estimates of clearances and volumes will be obtained from post hoc estimates and used to estimate exposure in the central compartment and a peripheral compartment representing the sputum. The change in lung function measured by FEV1 will be summarized descriptively. The proportion of The Cystic Fibrosis Questionnaire-Revised will be administered at the baseline and final visit for stage 2 participants. This questionnaire contains a series of questions regarding several physical health and ability domains. The Cystic Fibrosis Respiratory Symptom Diary will be collected at all visits for stage 2 participants. Change from baseline through day 30 follow-up visit in Cystic Fibrosis Respiratory Symptom Diary score will be presented by treatment group.The primary outcome will be analyzed on the intention-to-treat population. Endpoints may be missing for volunteers who withdraw from the trial. The reasons for withdrawal will be reported and compared by arm. The effect that any missing data might have on results will be assessed via a sensitivity analysis. Baseline characteristics of participants with missing data will be compared to participants without missing data.The datasets analyzed during the current study and statistical code will be available from the corresponding author, upon reasonable request. The full protocol will be available on clinicaltrials.gov.DMID within the NIH serves as the overall study sponsor, responsible for trial conduct and safety oversight. The DMID Clinical Research Operations and Management Support team will conduct site training, monitoring, and close oversight of study visits to assure proper adherence to trial protocol and research standards. Monitoring visits will include periodic review of data submission forms, source data verification, adverse event reporting, and consent documentation.Safety oversight will be conducted by a DSMB that is an independent group with the necessary expertise. The DSMB will monitor subject safety and advise DMID. The DSMB members will be separate and independent of research personnel participating in this study and will not have scientific, financial, or other conflicts of interest related to this trial. The DSMB will review data at prespecified intervals during the trial and will conduct ad hoc reviews, as appropriate when a halting rule is met or for immediate concerns regarding observations during the trial.All AEs will be graded for severity according to the Common Terminology Criteria for Adverse Events Version 5.0 and assessed for the relationship to the study product. The assessment of the AE’s relationship to the study product will be performed by a licensed clinician. An AE is considered an SAE if, in the view of either the site principal investigator or sponsor, it results in any of the following outcomes: death, a life-threatening AE; inpatient hospitalization, a persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions; or a congenital anomaly/birth defect.Monitoring for this study will be overseen by the DMID Clinical Research Operations and Management Support monitoring contractor. The Clinical Research Operations and Management Support team will conduct periodic site visits throughout the trial, including a review of data collection forms, source data verification, adverse event reporting, and consent documentation. The monitors will also count and view the study product to verify the number of phage vials.Protocol modifications will be submitted for review by participating institutional review boards (IRBs) for approval prior to implementation. Should any amendments alter the study conduct for participants, participants will be notified of the changes and will be requested to sign updated consent forms.Study results will be reported in accordance with Consolidated Standards of Reporting Trials guidelines for randomized controlled trials. Results will be submitted to clinicaltrials.gov within 1 year of study completion. The authors plan to submit study results for publication in a peer-reviewed scientific journal and/or present at relevant conferences. No participant-protected health information will be revealed in any publication or presentation.Phage usage in human medicine in the West currently remains experimental. In recent years, phage administration as an adjunct therapy to systemic antibiotics for the treatment of highly resistant and/or recalcitrant infections under compassionate use conditions has significantly expanded . SeveralP. aeruginosa otitis media in 24 patients in the UK [P. aeruginosa strains with susceptibility to at least one phage in a 6-phage mixture. A single topical application of the phage mixture was applied to the affected areas of patients randomized to the treatment arm. The trial was terminated early due to a 30% greater detectable efficacy signal in the treatment arm, without any treatment-related AEs observed.To summarize some notable trials: Wright and colleagues conducted a randomized, controlled trial evaluating the efficacy of phages for the treatment of chronic n the UK . EnrollmEscherichia coli diarrhea in 120 Bangladeshi children [E. coli susceptibilities to phage were not evaluated. Moreover, it was unclear if E. coli was the causative diarrheal pathogen in participating children and protection of oral phage during gastric transit was not provided. No treatment-related AEs were identified.Another trial evaluated the efficacy of oral phage therapy to reduce the severity and duration of children . The triP. aeruginosa using a 12-phage cocktail was investigated in 220 patients in France and Belgium [The treatment of burn-related infections caused by  Belgium . Patient4–105 PFU and the unexpected effectiveness of the mechanical bladder irrigations.Finally, a clinical trial was conducted on males prone to urinary tract infections awaiting transurethral resection of the prostate . One hunAlthough several trials have sought to investigate the efficacy of phage therapy, they have all had notable limitations. Important gaps in knowledge that need further investigation include the efficacy of phage therapy; optimal frequency, dosage, and duration of phage administration; limitations in rapid and accurate laboratory testing platforms—including a reference standard that reliably predicts phage susceptibility; an understanding of the frequency of the emergence of phage resistance; the role of the human immune system in phage efficacy; and the comprehensive safety profile of phage therapy. With continued advancements in medical care, complicated bacterial infections will remain challenging. It is essential to determine if the limitless supply of phages is an important adjunct to traditional anti-infectives. The current trial will provide important insight into the efficacy and safety of phage therapy and provide the foundation for future larger, multi-dose phage studies.The trial enrolled its first subject on October 3, 2022, and recruitment remains active as of the time of printing. It is estimated that recruitment will be completed by January 31, 2024. The most current protocol version number is 20-0001, version 4.0 dated 7 October 2022.


To study the effect of the birthing room design on nulliparous women’s childbirth experience up to 1 year after birth.Although it is known that the birth environment can support or hinder birth processes, the impact of the birthing room design on maternal childbirth experience over time is insufficiently studied.n = 406) to either a regular birthing room (n = 202) or a new birthing room designed with more person-centered considerations (n = 204). Childbirth experiences were measured 2 hr, 3 months, and 12 months after birth by using a Visual Analogue Scale of Overall Childbirth Experience (VAS-OCE), the Fear of Birth Scale (FOBS), and the Childbirth Experience Questionnaire (CEQ2).The Room4Birth randomized controlled trial was conducted at a labor ward in Sweden. Nulliparous women in active stage of spontaneous labor were randomized (p = .002) and 12 months (p = .021) after birth compared to women randomized to a regular room. Women in the new room also scored higher in the total CEQ2 score (p = .039) and within the CEQ2 subdomain own capacity after 3 months (p = .028). The remaining CEQ2 domains and the FOBS scores did not differ between the groups.Women randomized to the new room had a more positive childbirth experience reported on the VAS-OCE 3 months (These findings show that a birthing room offering more possibilities to change features and functions in the room according to personal needs and requirements, positively affects the childbirth experience of nulliparous women 3 and 12 months after they have given birth. The experience of childbirth is of significant meaning for women and their families, both short and long term . In the Contextual and psychosocial elements within the birth environment significantly influence childbirth experiences and physiological birth processes . For insContextual and psychosocial elements within the birth environment significantly influence childbirth experiences and physiological birth processes.Having less clinical-like indoor hospital environments with controllable sensory stimuli, aromas, sounds, lights, comfortable furniture, and views of nature are beneficial for the health of persons admitted to hospitals . For birThere is also a lack of research concerning the effect of the built environment on women’s childbirth experience over time . TherefoWe hypothesized that when the physical design supports physiological birth by offering women enhanced freedom of changing features and functions in the birthing room, the likelihood of a positive childbirth experience will increase.ClinicalTrial.gov (NCT03948815), and ethically approved by the Regional Ethics Board (Dnr:478-18). The study procedures for the RCT followed the CONSORT guidelines and are described in detail in a study protocol  in Sweden . Women wprotocol .Randomization was undertaken between January 2019 and October 2020 at a labor ward located in western Sweden and with an annual birth rate of around 4,000 births . All ran>1 cm). Furthermore, only women who understood either Swedish, English, Arabic, or Somali or had access to an interpreter could be recruited. Women in induced or latent phase of labor or with a planned caesarean birth were excluded.Eligible participants for randomization were women aged 18 or more, classified as Robson 1 ; nullipaParticipant recruitment was carried out by the midwives and assistant nurses at the labor ward. Eligible women were asked to participate after labor ward arrival and confirmed active stage of labor, if both a regular room and the new room were vacant. All women gave signed, written consent to participate, and the time interval from labor ward arrival to randomization was set to be as short as possible to ensure an early allocation to the randomized room. The randomly computer-generated allocation list was managed by an independent agency. Information about the allocated room was printed in sealed, opaque envelopes sequentially placed in a study box at the labor ward. The recruiting care provider was not aware of the randomization sequence, and the independent agency ensured that the allocation list was followed. The women were provided with a four-digit ID code, which was printed on the sealed envelope.Follow-Up 1), while they were still in the birthing room. The questionnaire included a Visual Analogue Scale of Overall Childbirth Experience (VAS-OCE 1-10) and a modified Fear of Birth Scale (FOBS 0-100)   and was lication  but wereFollow-Up 2) and 12 months (Follow-Up 3), all participants were asked to complete another online questionnaire (Supplementary Information 2) sent to their email address with two reminders to nonresponders. These questionnaires included VAS-OCE, FOBS, and the Childbirth Experience Questionnaire version 2 (CEQ2) (After 3 (2 (CEQ2) . A data 2 (CEQ2) , 2004. I2 (CEQ2) .Follow-Up 1, since it was in congruence with the linear scales in the other instruments (CEQ2 and FOBS). Since VAS most often is used to measure pain, we chose to name the variable VAS-OCE to clarify that we measure overall childbirth experience.Assessing overall childbirth experience by using a VAS (continuous data) ranging from 1 to 10, where 10 is most positive, is routinely used in Sweden . In the How do you feel right now about the approaching birth? Answers are rated from calm to worried (0–100) and from no fear to strong fear (0–100), and the scales are summed and averaged to calculate a score. Since participants for this study answered the questionnaire after birth, we modified the FOBS to report the question retrospectively and prospectively: How do you rate worry and fear when you think about your completed labor and birth? and How do you rate worry and fear when you think about giving birth again?. Higher scores in the FOBS represent stronger childbirth fear. As suggested by the creators, a cutoff point of 60 and above was used to define the fear of childbirth  concerning the childbirth experience. For 19 of the CEQ2 items, the response format is a 4-point Likert-type scale ranging from 1 = totally disagree, 2 = mostly disagree, 3 = mostly agree to 4 = totally agree. For the remaining three items , answers are assessed on a linear analogue scale (0–100), which are subsequently transformed to categorical variables: 1 = 0–40, 2 = 41–60, 3 = 61–80, and 4 = 81–100. The items produce mean scores in the four domains as well as a total CEQ2 score. Higher scores in the CEQ2 reflect more positive experiences. The few women who chose to answer the self-reported questionnaire at 2 hr in Arabic (two women in the new room and none in the regular room) or Somali (one woman in the new room and none in the regular room) received the questionnaires at 3 and 12 months in Swedish, since there were no validated translations for FOBS and CEQ2 in these languages.The CEQ2  is a val2 test was used for nonordered categorical variables and Fisher’s exact tests for dichotomous variables. For group comparisons of the continuous variables included in the instruments , Mann Whitney U test was used due to nonnormally distributed data. We also estimated effect sizes r, as proposed by r> .1 is considered a small effect, r> .3 medium effect, and r> .5 large effect (n (%), and for continuous outcome variables as mean (standard deviation), median, quartile 1 and 3, and 95% confidence intervals for mean. Cronbach’s α was used to evaluate the internal consistency reliability of the domains included in the CEQ2 questionnaire.To compare differences between the two randomized groups, χe effect . ResultsA linear mixed effects model was used to describe the group differences in childbirth experience over the three time points. This regression model was chosen since it can provide information about the women’s individual change in childbirth experience over time but is also flexible since it has the capability to handle missing observations in the repeated measures data . The modn = 3) answered Follow-Up 2, but two of them answered Follow-Up 3 in Swedish. Of the total study sample, 397 participants were included in the per protocol population. Among the nine not included according to protocol, eight were recruited in latent phase of labor and one was erroneously allocated to neither the regular room nor the new room. The per protocol population included 396 responders to Follow-Up 1, 285 responders to Follow-Up 2, and 286 responders to Follow-Up 3.The study sample consisted of 406 women, where 204 were randomized to the new room and 202 to the regular room . ResponsThe demographic characteristics of the responders to the follow-up questionnaires did not differ between the randomized groups . Respondr = .18, p = .002) and 12 months  after birth, compared to women in the regular room  between the groups were not statistically significant on either of the follow-up questionnaires (p > .05).The women’s overall childbirth experience on a VAS-OCE 2 hr after birth was similar in both randomized groups, as previously presented . Women ilar room . The difown capacity  and in the total CEQ2 score  3 months after birth  at this time point (p > .05). There were no significant differences in any of the domains or in the total CEQ2 score between the groups 12 months after birth. Cronbach’s α coefficients for each of the four CEQ2 domains indicated internal consistency for the 3-month follow-up (new room vs. regular room); own capacity: α = .81 versus .80, perceived safety: α = .85 versus .86, professional support: α = .75 versus .85, and participation: α = .71 versus .74. Internal consistency was also seen for the 12-month follow-up; own capacity: α = .84 versus .84, perceived safety: α = .88 versus .89, professional support: α = .85 versus .82, and participation: α = .73 versus .85.Women in the new room scored higher within the CEQ2 domain er birth . No signB = −0.36, p = .005) and 12 months  after birth among women in both randomized groups (Supplementary Information 3). Women allocated to the new room scored higher in overall childbirth experience over time than women in the regular room  and these levels of fear remained after 12 months. In contrast, the reported fear of a potential future birth increased in the total cohort after 3 months  and remained after 12 months. This increase in reported fear did not differ between the randomized groups , participation , and for the total CEQ2 score . The change over time did not differ between the randomized groups in the total CEQ2 score or in any of the subdomains , but not for oxytocin augmentation . These adjusted analyses also showed slightly reduced B-coefficients considering the effect of the room on the VAS-OCE among all responders randomized to the new room. The difference regarding time and the interaction between time and the randomized group did not change after the subsequent analyses in any of the outcome variables .The significant effect of the new room in the CEQ2 subdomain own capacity and total CEQ2 score 3 months after birth, which additionally indicates more positive overall childbirth experiences. The statistically significant differences between the groups regarding the CEQ2 variables did not remain in the 12-month follow-up.This randomized trial shows that a hospital birthing room designed with person-centered considerations offering women possibilities to change features and functions in the room has a positive impact on the self-reported childbirth experience 3 and 12 months after birth. Women randomized to the new room had a more positive overall childbirth experience as reported on a VAS-OCE, 3 and 12 months after birth, compared with women randomized to a regular room. Study participants in the new room also had significantly higher scores in the CEQ2 domain This randomized trial shows that a hospital birthing room designed with person-centered considerations offering women possibilities to change features and functions in the room has a positive impact on the self-reported childbirth experience 3 and 12 months after birth.To our knowledge, this is the first quantitative evaluation of the effect of a specially designed hospital birthing room on women’s childbirth experience over several time points during the first year after birth. The study findings emphasize that the physical design of the room is one essential contributing factor for subsequent childbirth satisfaction, which supports our hypothesis. This is also in accordance with previous findings about the experiential value of a conscientiously designed birthing room . An explown capacity after 3 months, which indicates that they experienced a greater sense of self-efficacy and control during birth than women in the regular room. This may have been due to women in the new room being provided with more opportunities to modify physical aspects of the birth environment. Women may experience this as being allowed to prepare the environment for giving birth or, when expressing it from a biological perspective, to prepare the nest for the baby via expression of archaic patterns of behavior. This procedure is essential for birthing women and most certainly, in a subtle way, increases the sense of familiarity with the room, thereby enhancing oxytocin release and the experience of positive emotions . Nevertheless, it is challenging to measure experiences quantitatively, since aspects such as context, social support, interpersonal relationships, and physical and emotional senses need to be taken into consideration . A studyThe place and space for birth is not just created through the physical design but also through those who inhabit and interact within the room . AlthougThe findings of this study show the benefit of acknowledging the physical design of hospital-based birthing rooms. Nulliparous women randomized to a birthing room designed with person-centered considerations aimed at supporting the physiology of birth reported more positive overall childbirth experiences 3 and 12 months after birth than women randomized to a regular birthing room. When designing interventions and environments in hospital settings, it is essential to understand the psychological and existential dimensions of childbirth, since the dynamics and changes in labor and birth experiences influence outcomes. It is also essential to understand how remnants of archaic nest-building behaviors and experiences are still present in birthing women of today. The findings of the study confirm that the physical design matters to women in their assessment of childbirth experience, but we were unable to show the actual effect of the new room on the variables explored. Therefore, additional research is needed that qualitatively explores the impact of the birth environment design on women’s long-term childbirth experiences.From a mental health perspective, it is essential to understand the importance of emotional well-being in the transition to parenthood.A birthing room designed with person-centered considerations, where women and companions can adapt features and functions in the room in accordance with personal needs and requirements, positively impacts women’s birth experiences 3 and 12 months after birth.When striving to optimize care during labor and birth, both the physical and the psychosocial environment need to be improved.To value emotions, birth physiology and person-centered perspectives in labor ward design may be one strategy to help reverse the rising trend of medical interventions in labor that is observed in many hospital settings.Click here for additional data file.Supplemental Material, sj-pdf-1-her-10.1177_19375867221124232 for Impact of Birthing Room Design on Maternal Childbirth Experience: Results From the Room4Birth Randomized Trial by Lisa Goldkuhl, Hanna Gyllensten, Cecily Begley, Christina Nilsson, Helle Wijk, Göran Lindahl, Kerstin Uvnäs-Moberg and Marie Berg in HERD: Health Environments Research & Design Journal


More applications of 2-[18F]-FDG simultaneous PET/MRI in low back pain and other musculoskeletal diseases should be further investigated in the future.Failed back surgery syndrome (FBSS) is a highly prevalent condition in patients after spine surgery. Although magnetic resonance imaging (MRI) is the gold standard for the diagnosis of epidural fibrosis, it is sometimes difficult to determine if epidural fibrosis contributes to radiculopathy. Herein, we share our experience in locating radiculopathy lesions using simultaneous positron emission tomography (PET)/MRI. 2-[ Failed back surgery syndrome (FBSS) is defined as persistent back or leg pain after one or more lumbar disk surgeries . Treatme18F]-FDG PET-MRI scans were arranged and performed on a 3T PET/MRI scanner , which combines 3T MRI with time-of-flight capable silicone photomultiplier-based PET. The result revealed a faint FDG uptake  over the right lateral epidural scar at the level of L5/S1; the scar was located along the course of the right S1 root, which revealed a nodular FDG uptake . Multiple epidural scars were seen over the sides of L4 to S1, bilaterally; however, no abnormal FDG uptake other than at the right S1 root was observed. The tentative diagnosis was entrapment neuropathy secondary to epidural fibrosis  was noted after the operation; it correlated to the L5 or S1 dermatome. MRI revealed extensive scars over the sides of L4 to S1, bilaterally. However, the decision for surgical intervention was difficult to make because the patient only had symptoms on the right side. She underwent right pedicle screw removal and decompression but did not have any improvement after surgery. She was also treated with radiofrequency ablation and nerve block, but the effect was only transient and it lasted less than one day. The lower back pain and right leg claudication worsened after 3 months, with a visual analog scale (VAS) score of 9. The patient visited our outpatient department due to persistent symptoms. 2-[fibrosis . We arrafibrosis . There wFailed back surgery syndrome has become a common problem in recent years, given the increasing rate of spine operations to treat back pain due to the aging population. It was found in a systematic review that the number of patients reporting recurrent back or leg pain after discectomy ranged from 5 to 36% . The posPositron emission tomography is a sensitive tool for identifying hypermetabolic activity in cells, with broad utility in cancer surveys . PET hasIn our case, contrast enhanced MRI was not performed. According to previous study , althougOur case is the first to identify neural inflammation due to extensive epidural fibrosis after laminectomy. Using PET/MRI maps, body glucose metabolism detected using PET to correlate anatomical details provided by MRI can offer a very clear picture of the neural inflammation caused by extensive epidural fibrosis . In our The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Review Board of Tri-Service General Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Y-HT and W-CC reviewed the literature and contributed to manuscript drafting and revision. G-SH analyzed and interpreted the imaging findings. C-TT and Y-CH were responsible for the revision of the manuscript. All authors issued final approval for the version to be submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study describes the design and synthesis of mimetic peptides modelled on the catalytic active site of the fructose-1,6-bisphosphate aldolase (FBPA) enzyme. The synthesized peptides consisting of the turn motifs and catalytic site amino acids of FBPA enzyme were evaluated for catalytic activity in direct asymmetric aldol reactions of ketones and aldehydes. The influence of substrate scope, catalyst loading and solvents including water, on the reaction were also investigated. Nuclear magnetic resonance (NMR) and circular dichroism (CD) were used to determine the secondary structure of the peptides to provide an understanding of the structure–activity relationship. The peptides showed catalytic activity and the aldol products were obtained in low yields (up to 44%), but excellent enantioselectivity (up to 93%) and moderate diastereoselectivity (65 : 35). Novel asymmetric aldol reaction catalysing fructose-1,6-bisphosphate aldolase peptide mimics with secondary structural motifs. Type I aldolases follow an enamine mechanism which uses the epsilon-amino group of a lysine residue in the active site to activate the donor by forming a Schiff base as an intermediate.4,5 Type I aldolase, have inspired many chemists to design short peptides that mimic its natural mechanism for asymmetric catalysis. For many decades enzymes have gained considerable attention as useful catalysts for the aldol reaction, due to their fascinating properties such as their high selectivity and mild reactions conditions. Although enzymes are efficient catalysts, they still suffer from limitations such as narrow substrate tolerance, extremes of pH specificity and incompatibility with organic solvents;5,6 there is therefore a need for the design of catalysts that mimic enzyme activity but have fewer drawbacks.The aldol reaction is regarded as one of the most prominent carbon–carbon bond forming organic reactions.7–9To date, various catalysts for the aldol reactions have been reported, which include enzymes, catalytic antibodies and organocatalysts. In recent years there has been discoveries of efficient selective catalysts for a wide range of liquid- and multi-phase organic transformations.3,7 In particular proline and its derivatives have also received wide application as organocatalysts for the asymmetric aldol reaction with the goal to improve selectivity and catalytic efficiency.10–12 Despite the ability of proline-related compounds to carry out a vast array of asymmetric reactions, it is still limited by poor results with certain substrates and low solubility in most organic solvents.1The majority of these advances are also in sync with attempts made by pharmaceutical and chemical industries, who have been in pursuit of rationalizing the cost of manufacturing and waste disposal in an increasing ecologically aware and economically competitive market. The area of organocatalysis, which has witnessed an immense growth over the years has received a great deal of attention owing to its green chemistry advantages such as its high efficiency, economic feasibility and ecological sustainability and its analogy to eon-perfected enzyme catalysis.4,13–15 The structural and functional diversity of peptides provides multiple sites for modifications that can be easily attained by changing sequences of amino acids residues and acid monomers to produce optimized catalysis.16–18 Peptides are often regarded as simplified enzymes because of their unique structural features similar to that of enzymes, while consisting of only a fraction of the molecular mass. The catalytic function of enzyme is based on their folded states and therefore an understanding of their three dimensional folded structures presents an opportunity to design small molecules to mimic the recognition surface involved in selectivity and efficiency.19,20 Most of the work done on peptides as catalysts has been on simple peptide structures, usually consisting of few residues which are unlikely to adopt to a well-defined structures;21,22 and few studies have been conducted on well-designed peptide catalysts that incorporate secondary structures such as α-helices that may enable enzyme-like asymmetric organocatalytic reactions.On the other hand, the use of peptides as catalysts in organocatalytic reactions has been of interest.15,23 combinatorial and library based methods.24,25However, one major challenge in the development of these peptide catalysts is the incorporation of appropriate amino acids with suitable catalytic properties a specified peptide to mimic natural enzymes. Hence there has been some effort made in the design of peptide catalysts to mimic the fine structures of enzymes using different approaches such as rational design,et al.20,26,27 reported a series of resin supported catalytic peptides which can efficiently catalyse various asymmetric reactions, yielding products with excellent enantiomeric excess and catalytic efficiency. Their successful design incorporated a β-turn and helical motif. The turn structure consists of five terminal residues and controls the enantioselectivity, while hydrophobic helical part enhances the reactions and stabilizes the whole peptide structure under aqueous conditions. Being inspired by this approach, we became interested in applying the similar technique for the mimic of fructose-1,6-bisphosphate aldolase enzyme (EC 4.1.2.13) to afford a peptides catalyst possessing secondary structural units.Akagawa 28 Catalytic reactions performed in aqueous systems are of interest since these systems are associated with enzymatic reactions under physiological conditions.27 Herein we report on the design and synthesis of peptides mimics based on the fructose-1,6-bisphosphate aldolase (FBPA) enzyme for the catalysis of direct asymmetric aldol reaction.The use of water as a ‘solvent’ or co-solvent in organocatalytic reactions is of current interest due to its potential and advantages such as safety, low cost, availability, efficiency, reactivity when compared to common organic solvents.2.2.1Drosophila melanogaster fructose-1,6-bisphosphate aldolase (FBPA) enzyme structure obtained from the UniProt protein database . The alignment of the selected amino acids into different peptide sequences was guided by the interactions between the active site amino acid residues with the substrate fructose-1,6-bisphosphate (FBP)  into the peptide sequences can impose local conformational restrictions and improve selectivity. Studies have also shown that a peptide sequence consisting of both l- and d-amino form ambidextrous helical structures.31The amino acid sequence for the catalytic and turn motifs were adopted from the te (FBP) . In addiWe hypothesize that the peptide sequence (CRYKDGASGKDYRC) with β-turn, and α-helix motifs from the active site of the FBPA enzyme will yield a peptide with a secondary structure that can interact with most organic substrates in an aqueous media to yield aldol products.2.2th and 9th amino acids (AA) were not completely coupled. Studies have shown that the coupling of glycine (a hydrophobic amino acid) into a peptide sequence as the peptide chain grows can be problematic, due to its small structural nature which induces agglomeration.32,33 At the end of the synthesis, the peptides were cleaved from the resin using cocktail mixtures of trifluoroacetic acid (95%), ethanedithiol (2.5%), triisopropylsilane (1.0%) and water (2.5%). The peptides were further purified by preparative RP-HPLC prior to aldol reaction catalysis screening, and were >85% pure as confirmed by LC-MS. The results of the two synthesized peptides are outlined in The peptides were synthesized manually and automatically using the Fmoc solid phase peptide synthesis strategy. Peptides were synthesized on Rink amide resin, and the sequential deprotection of the Fmoc protecting group from the amine to allow for the coupling of the next amino acid was performed until the full peptide was completed. Double coupling was successfully carried out for all the amino acids in the peptide sequence for 45 minutes per coupling. However, it was observed that the 6p-nitrobenzaldehyde  residues into the peptide sequences is also postulated to impose conformational restrictions,31 and to possibly improve the activity of the peptides. Taking into account the solubility of the peptide in 60% water/40% acetonitrile, the effect of water (addition) on the reaction medium and its influence on the catalyst was also investigated.Initially, the catalytic activity of the two selected peptides denoted by TP_Asp and TP_ADLys; having the sequence CYRKDGSAGDKYC, were examined for the aldol reaction of acetone and substituted aldehyde . Both sep-nitrobenzaldehyde was selected in the initial test using 4 mol% of the peptide catalyst. The results revealed poor conversions when reactions were conducted in acetone/water , using both TP_Asp and TP_ADLys peptides. However, good enantioselectivities of up to 70 and 80% were achieved under these conditions.The aldol reaction between acetone and 18,51 of aldol reactions proceeding slowly affording low yields and excellent ee's in acetone. Córdova et al. also reported low yields (25%) with good ee's of up to (65%) for the same reaction conditions.34 When o-nitrobenzaldehyde was used as substrate (entries 2 and 5), there was a notable decrease in enantioselectivity with low yields still being observed. The aldol reactions performed using p-chlorobenzaldehyde (entries 3 and 6) as a donor, demonstrated low reactivity but good ee's of up 85%. In another attempt to improve the reaction conditions while using the same catalysts, acetone/water was replaced with DMSO/H2O (entries 7–12). Unfortunately, only trace amounts of the product were detected by TLC and increasing the reaction time did not improve the yield. Although DMSO is commonly used for studies on aldol reactions involving acyclic ketones,35,36 studies have shown that acyclic ketones may be poor substrates in DMSO.37,38 This indicates inefficient formation of active enamine intermediates by acyclic ketones.7 There was no formation of product in the catalyst free control experiments under the same reaction conditions reported in Similar results are reported in literature,6,28 The experimental results in 39 Another attempt was made with reduced amount of water and a positive outcome resulted from this modification (entries 9 and 10). The beneficial influence of reducing water, allows the dissolution of cyclohexanone consequently creating a monophasic system and a homogenous aldol reaction.39,40 In addition, this also proved that the partition coefficient has an influence in establishing the optimal reaction conditions. Even though this attempt gave considerably better results, the products were obtained in low yields with poor selectivities for both aldehydes. Cheng and Luo did similar work whereby the ketone was used in excess as a co-solvent and they obtained low selectivities. They further revealed that an acid additive is needed to attain good stereoselectivity.41 Furthermore, it was also observed that aromatic aldehydes bearing a halogen groups (entries 6–7 and 11–12) required longer reaction time or more catalyst loading as only trace amounts of the product was observed for both peptides. Similar results were also observed in literature,7,37 where the halogenated aldehydes yielded no product or required longer reaction time. Between the two nitrobenzaldehydes , it can be seen that p-nitrobenzaldehyde was higher than o-nitrobenzaldehyde. This suggests that due to steric hindrance, the nitro-group which is located at the ortho position might be unfavourable during the nucleophilic attack.42 The effect of various organic solvents was examined on peptide catalyzed aldol reaction between p-nitrobenzaldehyde and cyclohexanone and the results are summarized in 3) and acetonitrile (MeCN) were not suitable for the peptide catalyzed aldol reactions , proceeded at a slow rate as only trace amounts of the product were observed after several days (entries 3 and 12). This outcome may suggest longer reaction times are required for the formation of the product. The aforementioned solvents were selected in this study because they are readily available in the lab and they have been reported in literature,3,45 to yield aldol products with high yields and good selectivity. Although high-boiling organic solvents such as DMSO and DMF are commonly used in aldol reactions studies they are often considered problematic for large-scale synthesis due to inconvenient work-up, solvent removal and recovery steps.15 The influence of these solvents on a small-scale reaction synthesis was then investigated. As shown in 46,47 previous studies also demonstrated no reaction or longer reaction time when anhydrous DMSO was applied.37 When the same reaction was performed in hydrous/wet DMSO, good yields and high selectivities (with both yield and ee) were obtained as reported in literature.7,15,34,46 Such findings would imply the mode of action of the peptide in this system is quite different. Moreover, it can be noted that some promising results were observed when DMSO/H2O was used (entries 6 and 15), with the yields of 16 and 15% and it was also intriguing to observe a considerable improvement in the ee's, to 14 and 20% respectively and moderate diastereoselectivities.The scope of the catalytic activity of the peptides was further investigated using cyclohexanone and substituted aromatic aldehydes . Cyclic 48 Certain enzyme catalysts are reported to be less reactive in anhydrous solvents than in water due to constrained conformational stability.44 For this reason, water is essential in binding to the surface of the enzyme, to exhibit both conformational flexibility and enzymatic activity. It is also believed that water affects the hydrogen bonding of the acidic moiety and the backbone of the peptide transition state, which facilitate highly ordered transition states in aqueous conditions.34,44 Therefore, we postulate that the role of water in this reaction medium is of significant importance in influencing the reaction rate, and therefore improving the hydrogen bonding for the stabilization of the transition state and ground state of the substrate.8,49These observations also illustrated that the addition of water to peptide promoted aldol reaction has some effect on influencing both the yields and stereoselectivities. The presence of small amount of water is proposed to influence the hydrogen bonding, which then alters the reaction mechanism and stereochemistry.50,51 Gryko and co-workers also demonstrated that the use of NaCl can accelerate the organocatalysed aldol reaction in water by accelerating the aggregation of the organic material in concentrated hydrophobic pockets.52The reactions were then carried out using higher equivalence of water in DMSO (entries 8 and 17). The reaction still proceeded slowly, causing a decrease in both the reaction rate and yields, as only trace amounts of the product was obtained. Brine is often applied as an alternative to MilliQ water when investigating the aqueous environment for reaction optimizations, and to also explore tolerance of catalyst towards the complex aqueous environment. It is reported that brine influences the reaction to proceed faster than in concentrated organic phase due to salting-out-effect.calculated) which is the pH where the net charge of the peptide is zero found to be 9.6 when determined using a peptide calculator. It is also understood that most enzymatic catalytic reactions are dependent upon the pH of the reaction medium to function efficiently and for obtaining optimal enantioselectivity.4,53 Based on this, the aldol reaction in buffered aqueous media, was performed using 200 mM phosphate buffer at a pH of 8.0.54To examine these hypotheses, the reactions were performed in brine solution in order to further optimize the reactions conditions to improve the yield and the influence of catalyst loading was then investigated and results are represented in 4 A pH of 8 was selected because previous studies have shown that for the best results the pH must be kept above the peptide's pI for better stability. In this study the use of a buffer solutions greater than a pH of 9 is restricted because the aldol reaction may become base-catalysed around this pH. In order to ascertain the influence of the buffer, a reaction with the buffer in the absence of the peptide  of 80% and a diastereomeric ratio of 57 : 43. High stereoselectivity has been previously reported for biocatalysts facilitating asymmetric aldol reactions in buffer solutions (pH = 4.0–8.0).p-nitrobenzaldehyde, as illustrated in 2O) to the combined water layers.Finally, we next explored the reusability of the catalyst for the aldol reaction between cyclohexanone and The catalyst recovery studies were performed for the TP_ADLys peptide selected aldol reactions. The results indicate that the reusability of catalyst was unsatisfactory in DMSO, brine or ketone, as only a trace amount of the product was observed after 72 hours (entries 1–2 and 3–4). This suggests the peptide cannot be efficiently reused as there is loss of catalytic activity. However, when the reaction was performed in a buffered solution, promising results were observed as yield of 13% was obtained (entry 5), and as already mentioned above, the buffer at this pH might have an effect in influencing the favourable structural conformation. Despite a longer reaction time and low yield when compared to the 38% that was obtained from the first use , entry 93.et al.7 and Hernández et al.55 , with nearly equal percentages of random (15.88%) and beta structures (12.09%). A peptide structure is proposed to adopt an extended conformation in water due to extensive solvation.58,59 In addition, studies suggest that helices are preferred when CD experiments are conducted in water, since hydrogen bonding promotes favourable chain–solvent interactions.58 In buffered solution the CD experiment for TP_ADLys shows an absorption minima around 202 and 194 nm  that results in different spectra patterns. Each specific residue possesses a unique pattern connecting an amide to its backbone Cα proton and the remaining sidechain protons. The long spin system residues such as Arg and Lys; which consists of γ and δ protons in their side chains, were unambiguously identified from TOCSY spectra. Moreover, Gly residues, which have only two α-protons whose chemical shift appears around 3.5–4 ppm, were also not difficult to identify. However, it was challenging to identify the residues corresponding to an AMX spin system  based on TOCSY spectra only, since their backbone consist of one α-protons and two β-protons.The spectroscopic analyses were conducted using circular dichroism (CD) and nuclear magnetic resonance (NMR), to acquire information about the secondary structures of the peptide catalysts. The CD experiments were performed both in water and phosphate buffer solution. The obtained results in water for TP_ADLys, depict random coil structures going to a helix conformation . A relatd 194 nm , and a mspectrum . This te61 some residues could not be unambiguously located on the TOCSY. Based on these difficulties, ROESY spectrum was then used for identifying neighbouring amino acids and to complete sequential spin specific assignment. The NOEs of the observed neighbouring amino acid residues,  in the 2D 1H-ROESY spectrum were utilized as an alternative to “sequential walk” technique to complete assignment (1H–13C HSQC (heteronuclear single quantum coherence) spectrum  was also used to assign Cα of the corresponding spin system.Furthermore, due to severe overlaps and some missing cross peaks in the fingerprint region, which is attributed to low intensity resulting from small NH–αCH coupling constant or large line width;signment . All the62,63 Of particular interest, a long-range correlation NOEs between the amide proton of Ala8, Ala8(HN – 8.10 ppm) and Arg13(Hβ – 1.23) was observed for TP_Asp. The presence of a medium range correlation between Ala8(HN – 8.10) and Asp11(Hβ – 1.24) NOE crosspeaks was observed for TP_ADLys These findings suggests that the structures are not linear but adopt a certain conformation. Unfortunately, due difficulties mentioned above, such as severe overlaps and missing cross-peaks, as well as finding virtually identical chemical shifts on the ROESY spectrum, other long/medium correlation could not be resolved.The complete sequential assignment of the amino acids residues is summarized in 4.In conclusion, the aldol addition catalytic peptides were designed and successfully synthesised using the solid phase peptide synthesis strategy. The peptides were tested for asymmetric aldol reaction catalytic activity, and they showed activity towards a selected range of substrates including aliphatic and aromatic ketone/aldehydes. The reaction between acetone and aromatic aldehydes afforded aldol products with low yields but good enantioselectivities (85–93%). Aldol products between aromatic aldehydes with cyclohexanone were obtained with low yields and selectivities. Selected organic solvents were found to provide aldol products with low yields, poor enantioselectivities and moderate diastereoselectivities. The addition of water proved to significantly improve the reaction rate and selectivities, and it was further demonstrated that a buffered water solution achieved aldol products with very high ee and moderate diastereoselectivity. Both the CD and NMR results provided summative confirmation that the peptides are not linear but possesses the evidence of secondary structural elements. The used approach for the design of the peptides might provide further advancement of the next generation of organocatalysts with improved catalytic properties. Further studies involving more detailed mechanistic studies and a broader scope of the catalyst-aqueous media systems are currently under investigation in our laboratory.MM was involved in the conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, writing-review & editing and making available resources needed for the chemistry part of the project. TP acquired and curated all the data, he wrote the original draft, performed the formal analysis, investigation, and methodology in the lab. DB was involved in funding acquisition, supervision, the writing-review and editing and provided the resources needed for the biocatalysis part of the project. WJ was involved in the determination of selectivity using liquid chromatography.There are no conflicts to declare.RA-011-D1RA06616A-s001


Studies assessing the efficacy of therapies for neovascular age-related macular degeneration (nvAMD) have demonstrated that aflibercept may have a longer treatment interval than its less-expensive alternative, bevacizumab. However, whether this benefit justifies the additional cost of aflibercept remains under debate. We have recently reported that a treat-and-extend-pause/monitor approach can be used to successfully wean 31% of patients with nvAMD off anti-VEGF therapy. Here, we examined whether the choice of therapy influences the outcomes of this approach.n = 70) or bevacizumab (n = 52), followed by a treat-and-extend protocol, in which the decision to extend the interval between treatments was based on visual acuity, clinical exam, and the presence or absence of fluid on optical coherence tomography. Eyes that remained stable 12 weeks from their prior treatment were given a 6-week trial of holding further treatment, followed by quarterly monitoring. Treatment was resumed for worsening vision, clinical exam, or optical coherence tomography findings.In this retrospective analysis, 122 eyes of 106 patients with nvAMD underwent 3 consecutive monthly injections with either aflibercept  compared with eyes receiving aflibercept. However, eyes treated with aflibercept were almost 3 times more likely to be weaned off treatment (43% vs. 15%) compared with eyes treated with bevacizumab at the end of 1 year.These observations expose an advantage of aflibercept over bevacizumab and have important clinical implications for the selection of therapy for patients with nvAMD.This work was supported by the National Eye Institute, NIH grants R01EY029750 and R01EY025705, Research to Prevent Blindness, the Alcon Young Investigator Award from the Alcon Research Institute, and the Branna and Irving Sisenwein Professorship in Ophthalmology. Aflibercept (Eylea) and bevacizumab (Avastin) are the two most frequently used therapies for the treatment of neovascular age-related macular degeneration (nvAMD) . Both thGiven the anticipated increase in the number of patients with nvAMD worldwide, the sustainability of indefinite intraocular injections with either therapy is unclear. Moreover, many patients are understandably hesitant to commit to a lifetime of monthly (or bimonthly) clinic visits and intraocular injections. This has motivated some clinicians to reduce the number of treatments and visits. This may help explain why the success of anti-VEGF therapies in real-world clinical practice has been less impressive than that observed in clinical trials . This haOne approach to reduction of the treatment burden is to monitor patients with nvAMD using a fixed interval but to only treat patients as needed or pro re nata (PRN). This approach can reduce the total annual number of injections but would not effect either the frequency of patient visits or the number of imaging studies performed. This reactive approach aims to capture most relapses promptly, while minimizing the number of treatments. An alternative approach to optimize the efficacy of a drug for each patient while minimizing the number of injections is the treat-and-extend (TAE) protocol, in which the response of an individual patient to treatment is used to determine whether the interval between treatments can be extended for that particular patient; this approach provides mandatory dosing but at a personalized schedule. TAE is a proactive approach that assumes that patients manifest a regular pattern of disease activity . TAE, therefore, can reduce annually both the total number of visits as well as the total number of injections. However, the TAE approach may still result in overtreatment of patients during the extension phase, and patients may be treated unnecessarily during the maintenance phase.Both PRN and TAE approaches have been shown to be noninferior compared with monthly or bimonthly treatment with anti-VEGF therapy in multiple randomized multicentered clinical trials –10. We rReviewing patient charts from the clinic of a single vitreoretinal surgeon at a tertiary care center from 2013 to 2020 identified 227 eyes of insured patients with a diagnosis of nvAMD who underwent intravitreal injections with either aflibercept or bevacizumab . One hunP = 0.337) 4–6 weeks following their third treatment (P = 0.021) but a similar mean decline in vision of 5 letters or greater  in patients receiving aflibercept compared with those receiving bevacizumab, respectively . The mean change in CST  and vision  was similar in patients receiving aflibercept compared with patients receiving bevacizumab at the end of month 6 (P = 0.012) by month 6  of fluid, and no hemorrhage was extended by 2 weeks , as has previously been described . Six mon month 6 . There w month 6 .P = 0.001) by the end of year 1 (P = 0.002). Compared with traditional bimonthly treatment with aflibercept , the number of treatments using the TEP/M protocol was reduced by 10% (from 8.0 to 7.2). Compared with traditional monthly treatment with bevacizumab, the number of treatments using the TEP/M protocol was reduced by 33% (from 13.0 to 8.7) at 12 months (P = 0.312) and vision (1 ± 2 ETDRS letters vs. 4 ± 2 ETDRS letters) was similar in patients receiving aflibercept compared with patients receiving bevacizumab at the end of year 1. There was also a similar percentage of patients with a mean improvement or decline in vision of 5 letters or greater (43% vs. 42% and 23% vs. 14%) in patients receiving aflibercept compared with those receiving bevacizumab, respectively  was longer for patients receiving aflibercept than for patients receiving bevacizumab  after 24 months (P = 0.001). The mean change in CST  was similar between patients receiving aflibercept and those receiving bevacizumab at the end of year 2. Compared with traditional bimonthly treatment with aflibercept , the number of treatments using the TEP/M protocol was reduced by 27% (from 14.0 to 10.2) at 24 months (There were 31 eyes (27 patients) from the aflibercept group and 34 eyes (31 patients) from the bevacizumab group that were followed under the TEP/M protocol for at least 2 years (without deviations from the protocol as described for year 1). The mean interval between treatments  remained higher in the patients receiving aflibercept compared with the patients receiving bevacizumab . At their first 6-week monitoring visit, 90% (30 of 33) of eyes of patients receiving aflibercept remained quiescent; the other 3 eyes of 3 patients required maintenance treatment every 12 weeks. Eight of 8 eyes of 8 patients receiving bevacizumab remained quiescent at their 6-week and 18-week monitoring visits  of eyes (30 of 60 patients) receiving aflibercept were quiescent 12 weeks after their prior treatment compared with 15% (8 of 52) of eyes (8 of 46 patients) receiving bevacizumab  by the end of year 1  requiring treatment every 4 weeks compared with eyes of patients receiving aflibercept ; this was not statistically significant . The mean treatment interval at the end of 12 months for the fellow eye was similar to that of the first eye (We next examined whether closer monitoring of the fellow eye while treating the first eye may have led to a bias in the data. We found that 10 of 60 (17%) eyes treated with aflibercept and 6 of 46 (13%) eyes treated with bevacizumab developed choroidal neovascularization (CNV) in their fellow eye over the duration of the study ; this diirst eye . Howeverirst eye ; this waThe majority of eyes of patients included in this study had newly diagnosed (treatment-naive) nvAMD. However, a subset of eyes had reactivated CNV , and at 1, 2, 3, 6, and 12 months after initiating treatment for the presence of fluid. Each SD-OCT was classified as having no fluid, subretinal fluid (SRF), intraretinal fluid (IRF), or SRF and IRF by 2 independent masked graders; disagreements were reconciled by a third grader. Patients were then divided based on if they received aflibercept or bevacizumab and whether they could be successfully weaned from treatment . InteresBetween 2013 and 2016, almost one-third of treated patients with nvAMD received aflibercept and approximately one-half received bevacizumab . While aHere, we compared aflibercept and bevacizumab using a hybrid of the as-needed PRN protocol and the personalized TAE protocol, in which we optimized the efficacy of each drug for a specific patient, while minimizing the number of treatments needed. After a mandatory 3 consecutive monthly treatments, we observed a modest advantage of aflibercept over bevacizumab for the percentage of patients with a 5 or more letter gain in vision. This was consistent with findings in prior studies, demonstrating that aflibercept may be more effective than bevacizumab when used at a similar treatment interval (P = 0.001) by the end of year 1. The advantage of aflibercept over bevacizumab for the prolonged treatment interval was maintained at the end of year 2 . This, in turn, resulted in a decrease in the number of injections by 27% in eyes of patients receiving aflibercept compared with eyes of patients receiving bevacizumab using the TEP/M protocol  at the end of year 2. Collectively, these results demonstrate that TEP/M with aflibercept is particularly effective in reducing the number of treatments and extending the interval between visits for patients with nvAMD.The treatment interval for eyes of patients receiving aflibercept was 44% longer compared with that for eyes receiving bevacizumab  of eyes of patients with nvAMD treated with aflibercept entered a treatment pause compared with one-quarter (27%) of eyes of patients treated with bevacizumab. The potential long-term cost-savings for patients with nvAMD successfully weaned off treatment — even temporarily — raises questions as to the cost-effectiveness of bevacizumab compared with aflibercept and have implications for the current management of these patients. The advantage of aflibercept over bevacizumab in weaning patients off treatment was particularly notable in patients with nvAMD with reactivated CNV (62% vs. 11%). This suggests an additional benefit of aflibercept over bevacizumab in the treatment of patients with reactivated CNV.Of note, it has previously been reported that second eyes fare better than first eyes in patients with nvAMD . HoweverThe observation that the early response to treatment with aflibercept was significantly greater than the response to bevacizumab, and that this correlated with the improved efficacy of aflibercept over bevacizumab at weaning patients off treatment, suggests that there may be an unexpected long-term advantage to starting patients on treatment with aflibercept. If the early treatment phase using the TEP/M approach influences whether the CNV enters quiescence, during which time a patient can safely be followed while their treatment is paused, initiating treatment with bevacizumab may ultimately limit the efficacy of the TEP/M approach to wean patients off therapy. Indeed, after 2 years of treatment, patients treated with bevacizumab were still half as likely to be weaned off treatment compared with patients treated with aflibercept. This raises questions about the long-term impact of requiring patients to initiate treatment with bevacizumab and switch to aflibercept only after patients fail treatment with bevacizumab. Ultimately, the advantages of aflibercept over bevacizumab in terms of its duration of action  and improved efficacy at weaning patients off anti-VEGF therapy needs to be balanced by the substantial cost differences between the 2 drugs. Future studies modeling the cost-effectiveness of each medication will need to account for these factors as well as the theoretical benefits to patients who are spared frequent intraocular injections.We speculate that early treatment strategies that more effectively quench the angiogenic drive in nvAMD eyes may be necessary to achieve CNV quiescence; this emphasizes the importance of early detection and treatment of CNV lesions. We postulate that the advantage to patients with nvAMD who receive aflibercept over bevacizumab in successfully weaning off treatment may be attributed to the increased efficacy of aflibercept in promoting CNV quiescence. It is important to note that, in patients with nvAMD with quiescent CNV, the CNV likely remains present but is inactive. Close monitoring is therefore essential for the detection of CNV reactivation, at which point treatment needs to be resumed. Whether quarterly monitoring by a vision care provider (as was performed in this study) is sufficient for the early detection of CNV reactivation, or additional measures  VEGF-A. While we did not observe a difference in the aqueous levels of VEGF-A following treatment with aflibercept compared with bevacizumab, we cannot state that this reflects the efficacy of the 2 drugs at neutralizing VEGF-A in the retina/choroid. However, in addition to targeting VEGF-A, aflibercept also binds to other members of the VEGF family, including VEGF-B/C/D and placental growth factor, which have collectively been implicated in the promotion of pathological angiogenesis, vascular leakage, neurodegeneration, and inflammation, independent of VEGF . We receThe observation that 43% of eyes of patients with nvAMD treated with aflibercept could achieve a treatment pause within 1 year  further suggests many patients with nvAMD may not require — or benefit from — the anticipated introduction of second-generation, longer-acting anti-VEGF therapies  or alterOne of the major criticisms raised about protocols in which anti-VEGF therapy is paused is that this approach may expose patients with nvAMD to a recurrence of CNV (and vision loss), which could otherwise have been prevented if the patient received continued  treatment. Although we did not observe an increased risk of vision loss for patients in whom treatment was paused compared with the patients who were treated every 8–12 weeks , this waThe importance of including a control arm with patients receiving maintenance therapy in studies examining the potential risks and benefits of holding treatment is emphasized by recent reports that continuing treatment with anti-VEGF therapy after achieving functional and morphological stability does not prevent recurrence of CNV. Two recent studies both concluded that patients continue to have a lifetime risk of recurrence that accumulates with each additional year of treatment, despite ongoing maintenance therapy with anti-VEGF , 37. IndCollectively, these data do not support the hypothesis that maintenance therapy more effectively prevents either recurrent CNV or the vision loss that accompanies recurrence compared with a PRN approach once CNV activity is no longer detectable. This assumption is further weakened by the observation from prospective clinical studies that PRN therapy for nvAMD, which also includes a treatment pause, has been shown to be noninferior to continued treatment , 8. ConvThe limitations of our study include that it is a retrospective cohort study. The study design introduced biases that need to be weighed when assessing outcomes measured. However, a recent review summarizing the results of methodological reviews that compared the outcomes of observational studies (including retrospective cohorts) with randomized trials addressing the same question demonstrated that there is little evidence for significant estimate differences between observational studies and randomized control trials, regardless of the specific observational study design . The autAnother important limitation of this study is that the study arms were not randomized. However, the choice between treatment with bevacizumab and aflibercept was made by the patient, rather than the treating physician . We prevTo our knowledge, there has not been any prior published report — retrospective or prospective — comparing the 2 most frequently used anti-VEGF therapies, aflibercept and bevacizumab, in a head-to-head comparison using a protocol designed to wean patients with nvAMD off treatment. A prospective randomized clinical trial assessing the safety and efficacy of the TEP/M approach compared with TAE with maintenance therapy or monthly/bimonthly treatment will ultimately be needed to assess the risks and benefits of each of these approaches. Studies incorporating newer therapies that target multiple vasoactive mediators  may further improve our understanding of the factors that influence the ability for therapies to promote CNV quiescence. Until then, considerations for current management of patients with nvAMD should consider the potential advantage of more rapid resolution of fluid and, in turn, greater success at weaning patients off therapy using the TEP/M approach with anti-VEGF therapies.Insured patients at a satellite office (primary cohort) or a hospital-based clinic (secondary cohort) of a tertiary care center with a diagnosis of nvAMD were treated with bevacizumab or aflibercept . Please see Please see Please see SD-OCT images were obtained and analyzed from all eligible patients enrolled in the study who underwent the TEP/M approach for at least 12 months. SD-OCT images were graded after initiation of protocol for the presence of fluid by 2 independent and masked graders at the following time points: 0, 1, 2, 3, 6, and 12 months; OCTs performed at the time points closest to 6 and 12 months  were included in this analysis. Each image was classified as having no fluid, SRF, IRF, or both. Differences in image grading between the 2 blinded graders were reconciled, and if an agreement could not be reached, a third investigator cast the tie-breaker vote. The location of fluid for each patient and at each time point was graphically represented in a table with different color cells representing no fluid, SRF, IRF, or both. Patients were grouped based on frequency of treatment at 12 months to compare those who were able to successfully pause treatment with those who were unable to reach treatment pause.Aqueous samples (0.1–0.2 mL) were collected via limbal paracentesis using a 30-gauge needle attached to a tuberculin syringe from consenting patients at the Wilmer Eye Institute immediately after performing intravitreal injection for active CNV. Aqueous samples were immediately processed and stored at –80°C prior to analysis.Human VEGF ELISA kits  were purchased from R&D Systems. Aqueous samples were analyzed for VEGF (10 μL of aqueous diluted 1:10) using ELISAs, which were performed according to the manufacturer’s protocols. All ELISAs were performed in duplicate (VEGF), and quantitation was performed using the standard curve constructed with the standards included in the kit.2 test, with significance set at P < 0.05. Patient best corrected visual acuities were converted from Snellen visual acuity into a logarithm of the minimum angle of resolution (logMAR) score for statistical analysis using the following formula for conversion: logMAR = –1 × log10(snellen fraction)  . This scInstitutional review board approval from the Johns Hopkins University School of Medicine was obtained for all patient samples, including OCT images and aqueous samples, used in this HIPAA-compliant study. Patients provided voluntary, written consent and were not provided a stipend.A Sodhi was the primary contributor to the research design. XC, JCS, TPP, ZY, CG, DM, A Sopeyin, and A Sodhi were responsible for research execution. XC, JCS, TPP, ZY, CG, and DM were responsible for data acquisition. XC, JCS, TPP, CG, SM, and A Sodhi were the primary contributors to data analysis and interpretation. A Sodhi prepared the manuscript, with revisions provided by XC, TPP, and SM. A Sodhi had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis