A prospective study of oral 5-aminolevulinic acid to prevent adverse events in patients with localized prostate cancer undergoing low-dose-rate brachytherapy: Protocol of the AMBER study

Background Radiotherapy is one of the most frequently selected treatment options for patients with prostate cancer. However, adverse effects related to the irradiated surrounding normal organs are significant clinical concerns. Specifically, genitourinary and gastrointestinal toxicities can lead to a dramatically reduced quality of life. The aim of this clinical trial is to determine the efficacy of oral 5-aminolevulinic acid (ALA) phosphate with sodium ferrous citrate (SFC) in patients treated with low-dose-rate brachytherapy (LDR-BT) using an iodine-125 seed source. Methods The AMBER study is a prospective, single-center trial in patients with localized prostate cancer undergoing LDR-BT. Patients who undergo supplementary extra-beam radiotherapy are excluded, whereas those who undergo pre-implantation short-term (4–6 months) androgen deprivation therapy to decrease the prostate volume and/or improve oncological outcomes are included. After the screening and registration, the patients will be instructed to take capsules of ALA-SFC twice a day (200 mg and 229.42 mg per day) for 6 months from the day of seed implantation (prescribed radiation dose of 160 Gy). Patient data will be collected before the implantation; during oral ALA-SFC treatment; and 1, 3, 6, 9, and 12 month(s) after seed implantation. The primary endpoint of this trial is the urinary frequency 3 months after seed implantation. At each visit, the 24-h urinary frequency, total voided volume, and mean voided volume on a frequency volume chart and other patient-reported outcomes are recorded. The data of the trial cases will be compared with those of historical controls, who are consecutive patients undergoing LDR-BT without supplementary extra-beam radiotherapy between January 2016 and January 2019. The number of subjects has been set to be 50 for trial cases and 150 for the historical control cases. Pre- and post-treatment clinicopathologic factors are compared between two groups. Discussion The goal of this trial is to determine the potential benefit of ALA-SFC in patients who undergo LDR-BT. To the best of our knowledge, this is the first study investigating the potential clinical benefit of oral ALA-SFC after radiotherapy. More evidence from a further randomized controlled trial is needed to change the standard of care and lead to better post-radiotherapy management. Trial registration This clinical trial was prospectively registered with the Japan Registry of Clinical Trials on 5 December 2019. The reference number is jRCTs051190077, nara0013 (Certified Review Board of Nara Medical University).


. Introduction
Ra dio ther apy is one of the most fre quently se lected treat ment options for pa tients with prostate can cer (PCa). Ac cord ing to a Japan ese na tion wide prospec tive co hort study (J -POPS), thou sands of pa tients un dergo low -dose -rate brachyther apy (LDR -BT) us ing an io dine -125 (I -125) seed source per year [ 1 ]. Cur rently, brachyther apy is a wellestablished treat ment modal ity for lo cal ized PCa in terms of or gan preser va tion and prefer able long -term on co log i cal out comes [ 2 ]. How ever, ad verse ef fects re lated to the ir ra di ated sur round ing or gans are sig nif i cant clin i cal con cerns in the man age ment af ter im plan tation. Specif i cally, the gas troin testi nal (GI) and gen i touri nary (GU) com pli ca tions can lead to a dra mat i cally re duced qual ity of life (QOL) [ 3 -5 ].
Stud ies over the past sev eral decades have in ves ti gated the pathophys i o log i cal and mol e c u lar mech a nisms un der ly ing ra dio ther apyinduced ef fects on both tu mor tis sue and nor mal tis sue [ 6 , 7 ]. Although sev eral phar ma co log i cal in ter ven tions that po ten tially pre vent ra di a tion -induced dam age in nor mal tis sues have been ex plored in pre clin i cal stud ies, only a cou ple of them have pro gressed to clin i cal use [ 6 ]. Our pre vi ous study was the first to demon strate the dual bene fit of sup ple men tary oral 5 -aminolevulinic acid (ALA) and pelvic radio ther apy in a syn genic PCa model, i.e., MyC -CaP cells in FVB mice [ 7 ]. The "dual ben e fit" in cludes ra diosen si ti za tion of PCa tu mor tissues and ra dio pro tec tion of nor mal pelvic or gans from ra dio ther apy. ALA is dis trib uted ubiq ui tously in mam malian cells and is a pre cur sor of por phyrins and heme pro tein, which play es sen tial roles in aer o bic en ergy me tab o lism and the elec tron trans port sys tem [ 8 ]. Pre clin i cal stud ies have demon strated that ALA can con fer a broad range of cy topro tec tive ef fects against cis platin -induced nephro tox i c ity, rhab domyol y sis -induced acute kid ney in jury, hy poxia -induced car diomy ocyte in jury, and neu ro tox i c ity in pre clin i cal stud ies [ 9 -12 ]. Based on the re sults, an in ter ven tional sin gle -arm clin i cal trial is on go ing to eval uate whether oral ALA phos phate with sodium fer rous cit rate (ALA -SFC) pre vents cis platin -induced re nal in jury in pa tients with un resectable gas tric can cer (trial ID: UMIN000024642). More over, Japanese dou ble -blinded, ran dom ized, placebo -controlled tri als are on go ing to in ves ti gate the clin i cal ben e fit of oral ALA -SFC for pa tients with mi to chon dr ial dis ease (JMA -IIA00358), Alzheimer dis ease (jRCT -s041180135), and autism spec trum dis or der (jRCT s051190017).
This prospec tive, open -label, sin gle -center, sin gle -arm pi lot trial eval u ates the short -term tox i c ity, such as uri nary fre quency, and other types of com pli ca tions of LDR -BT. The in ter ven tion in this trial is oral ad min is tra tion of ALA -SFC, which is widely ac cepted as a health supple ment and food with func tional claims. Based on the re sults obtained in this trial, we will de sign a dou ble -blinded, ran dom ized, placebo -controlled trial to con sol i date the clin i cal value of this in terven tion. To the best of our knowl edge, this is the first study in ves tigat ing the po ten tial clin i cal ben e fit of oral ALA -SFC af ter ra dio ther apy and is ex pected to lead to bet ter post -radiotherapy man age ment.

1 . Inclusion criteria, patient recruitment, and study design
This prospec tive, sin gle -center trial in pa tients with lo cal ized PCa is on go ing at Nara Med ical Uni ver sity (a Japan ese aca d e mic hos pi tal).
The trial de sign and pro to col ad here to the Rec om men da tions for Inter ven tional Tri als (SPIRIT) cri te ria. The com pleted SPIRIT check list can be found in Sup ple men tary data. In ves ti ga tors and pa tients will be aware of the in ter ven tion of ALA -SFC. The flow chart, in clu sion crite ria, pri mary end point, and sec ondary end points are de picted in Fig.  1 . Pa tients who un dergo sup ple men tary ex tra -beam ra dio ther apy (EBRT) are ex cluded, whereas those un der go ing short -term (4 -6 months) neoad ju vant an dro gen de pri va tion ther apy (ADT) to de crease the prostate vol ume and/ or im prove on co log i cal out comes are included. As a gen eral rule of our hos pi tal, the com bi na tion of luteinizing hor mone -releasing hor mone ag o nist/ an tag o nist and oral bi ca lutamide (80 mg/ day) are used for neoad ju vant ADT. The ex clu sion crite ria are listed in Fig. 2 . In formed con sent and writ ten con sent forms of pa tients are manda tory be fore study par tic i pa tion. Af ter the screening and reg is tra tion, the pa tients will be en rolled to a sin gle treat ment group of a fixed dose of oral ALA -SFC (200 mg and 229.42 mg per day). Sub jects will un dergo the seed im plan ta tion un der spinal anesthe sia and be in structed to take cap sules of ALA -SFC twice a day for 6 months from the day of seed im plan ta tion.
The use of α1 -adrenoceptor an tag o nists to ame lio rate post -seed uri nary dis com fort or pre vent uri nary re ten tion is per mit ted. Pro hibited med ica tions and pro ce dures are not set up in this trial. Med ications be fore and af ter seed im plan ta tion should be recorded.

2 . Procedure of LDR -BT
As of No vem ber 30, 2019, we have an LDR -BT treat ment ex per iment of more than 1400 pa tients. The de tailed pro ce dure of the seed im plan ta tion has been pre vi ously de scribed [ 13 , 14 ]. All the pa tients are hos pi tal ized for 3 nights and 4 days. The pre scribed ra di a tion dose is 160 Gy for the monother apy. For pa tients with out any con traindi cations, SpaceOAR™ hy dro gel is in jected be tween the rec tum and prostate to min i mize high -dose ra di a tion to the rec tum and con sequent bowel side ef fects. Ex pe ri enced ra di a tion on col o gists (I. Asakawa and K. Ya maki) cal cu late the post -implant dosi met ric pa rame ters based on a pelvic com puted to mog ra phy scan per formed about 1 month af ter LDR -BT.

3 . Follow -up, data collection, and data protection
Pa tient data will be col lected be fore the im plan ta tion; dur ing oral ALA -SFC treat ment; 1, 3, 6, 9, and 12 months af ter the im plan ta tion; and at the end of the trial ( Fig. 3 ). Af ter the ini ti a tion of this trial, we pro vide a spe cific di ary book let to the par tic i pants, by which they record tak ing the in ter ven tion drug. At each visit, the 24 -h uri nary fre quency, to tal voided vol ume, and mean voided vol ume are recorded on a fre quency vol ume chart (FVC) over 3 days [ 15 ]. The assess ment in cludes pa tient re ported out comes (PROs) such as the SF -8™, Ex panded Prostate Can cer In dex Com pos ite (EPIC), In ter na tional Prostate Symp tom Score (IPSS), over ac tive blad der symp tom score (OABSS), and Sex ual Health In ven tory for Men (SHIM) ques tion naire. The data will be doc u mented in spe cific Case Re port Forms re gard ing GU/ GI, hema to log i cal, and other pos si ble tox i c i ties us ing the Common Tox i c ity Cri te ria for Ad verse Events (CT CAE v 5.0), com plete blood count, serum chem istry, and serum prostate -specific anti gen (PSA) lev els. The in ves ti ga tors will con duct as sess ments for po ten tial new or wors en ing ad verse events as in di cated in the trial sched ule In clu sion cri te ria and end points. Pa tients should ful fill the in di cated in clu sion cri te ria to be el i gi ble for this trial. * he mo globin, ≥9.0 g/ dL; white blood cell count, ≥12,000/ mm 3 ; ab solute neu trophil count, ≥2000 cells/ mm 3 ; platelet count, ≥100,000 cells/ mm 3 ; nor mal kid ney and liver func tions as de ter mined by cre a ti nine, to tal biliru bin, as par tate transam i nase (AST), and ala nine transam i nase (ALT) ≤2 × the up per limit of nor mal for the ref erence lab o ra tory. and more fre quently if clin i cally needed. Bio chem i cal re cur rence is de fined as the Phoenix de f i n i tion of a rise in PSA level by 2 ng/ mL or more above the nadir PSA [ 16 ]. When clin i cal re cur rence is suspected, imag ing ex am i na tions, such as com puted to mog ra phy, magnetic res o nance imag ing, and/ or bone scans, are per formed. Af ter discon tin u a tion or close -out of this trial, pa tients will be fol lowed up every 6 months for 5 years and an nu ally there after. Dis con tin u a tion/ dropout cri te ria are listed in Fig. 4 .
To pro tect ing the pa tient data, unique iden ti fi ca tion codes will be pro vided to all pa tients. All the data will be pro tected in pass wordaccessible files, and only in ves ti ga tors will be able to ac cess the files. All data and doc u ments will be deleted and dis carded 5 years af ter the end of the trial un less the data are un der sec ondary use for another study.

4 . Determining the target sample size
To date, we have been work ing on sev eral clin i cal stud ies re garding on co log i cal out comes, ad verse events, com pli ca tions, and changes in uri nary fre quency and PROs among pa tients un der go ing LDR -BT [ 3 , 4 , 14 , 17 , 18 ]. In this study, the data of the trial cases will be compared with his tor i cal con trol data. Data of pa tients who un der went seed im plan ta tion monother apy be tween Feb ru ary 2016 and Feb ru ary 2019 will be ex tracted for the his tor i cal con trol data. The pri mary  Fig. 3 . In ter ven tion and as sess ment sched ule of the AM BER study ac cord ing to the Rec om men da tions for In ter ven tional Tri als (SPIRIT). Full phys i cal ex am i nations are re quired at screen ing and every visit. Fol low -up vis its and data col lec tion should oc cur ap prox i mately 1, 3, 6, 9, and 12 months from the seed im plan tation. Pa tients will com plete a set of ques tion naires at every visit, and fol low -up in for ma tion may be col lected via med ical charts. In case of in for ma tion be ing unavail able in chart re views, pa tients may be con tacted via tele phone or in per son. Sub ject iden ti fi ca tion cards may be pro vided af ter the in formed con sent process and prior to seed im plan ta tion. The Case Re port Form will in clude in for ma tion re gard ing past his tory, con comi tant med ica tions, and any med ica tions taken af ter the treat ment. Chestabdomenpelvis X -ray will be per formed to ver ify seed mi gra tion. X, manda tory; (X), op tional. * He mo glo bin, hema t ocrit, white blood cell count and frac tions, platelet count; ** as par tate transam i nase (AST), ala nine transam i nase (ALT), g -glutamyl transpep ti dase (g -GTP), to tal biliru bin, al ka line phos phatase (ALP), lac tate de hy dro ge nase (LDH), to tal pro tein, al bu min, serum cre a ti nine, uric acid, to tal cho les terol, low -density lipopro tein (LDL) -cholesterol, high -density lipopro tein (HDL) -cholesterol, triglyc eride, C -reactive pro tein (CRP), cal cium, phos pho rus, potas sium, chlo ride; *** Urine dip stick test (spe cific gravity, pH, pro tein, glu cose, biliru bin, uro bilino gen, ke tone body, oc cult blood) and urine sed i ment test; † 3 days and nights record. Pa tients pick days that will be con ve nient for them to mea sure and record every thing; † † Ac cord ing to the Com mon Tox i c ity Cri te ria for Ad verse Events (CT CAE v 5.0) trans lated to Japan ese; † † † Ques tion naires: SF -8™ and Ex panded Prostate Can cer In dex Com pos ite (EPIC). Ab bre vi a tions: LDR -BT, low -dose -rate brachyther apy; BW, body weight; BT, body tem per a ture; BP, blood pres sure; ECOG -PS, East ern Co op er a tive On col ogy Group -performance sta tus scale; PSA, prostate -specific anti gen; QOL, qual ity of life; IPSS, In ter na tional Prostate Symp tom Score; OABSS, over ac tive blad der symp tom score; SHIM, Sex ual Health In ven tory for Men. end point of this trial is uri nary fre quency 3 months af ter the im planta tion. One of our pre vi ous stud ies showed that a sig nif i cant in crease in uri nary fre quency and sig nif i cant de crease in mean voided vol ume per sisted for 12 months af ter seed im plan ta tion [ 14 ]. The av er age times per day of uri na tion in creased from 9.2 at base line to 12.2 at 3 months af ter seed im plan ta tion be cause of blad der ir ri tabil ity. This ob ser va tion in di cated that the in crease in uri nary fre quency was 3 times per day at 3 months, which was the peak point. To de ter mine the re quired sam ple size of this trial, we ex pect that oral ALA -SFC can sup press the in crease in uri nary fre quency to only 1 time per day and es ti mate the stan dard de vi a tion of uri nary fre quency to be 3.5 times both in trial cases and his tor i cal con trol cases. The ra tio of case numbers is set to 1:3 for trial cases and his tor i cal con trol cases, re spectively. Given the 10% pro por tion of in el i gi ble pa tients, the re quired sam ple size was de ter mined to be at least 37 for trial cases and 109 for his tor i cal con trol cases to pro vide 80% power (β = 0.20) and an α level of 0.05 (two -sided). Based on the es ti ma tion, a to tal of 50 trial cases and 150 his tor i cal con trol cases will be en rolled in this study. The his tor i cal data will be ob tained from con sec u tive pa tients un dergo ing LDR -BT with or with out short -term ADT be tween Jan u ary 2016 and Jan u ary 2019. Pa tients who re ceived pro phy laxis with 50 mg of mirabegron (a β3 -adrenoceptor ag o nist) just af ter seed im plan ta tion for de creas ing un de sired ur gency and uri nary fre quency will be excluded from the his tor i cal con trol cases. Pa tients who re ceived sup plemen tary ex tra -beam ra dio ther apy will be ex cluded from the his tor i cal con trol cases. Pre -and post -treatment clin i co patho logic fac tors are com pared be tween two groups: age, body mass in dex, co mor bidi ties, pre treat ment uri nary fre quency, pre treat ment PSA level, pre treat ment prostate vol ume, biopsy Glea son score, clin i cal tu mor stage, D'Am ico risk strat i fi ca tion, and the post -implant dosi met ric pa ra me ters.

5 . Interim analysis and monitoring
Be cause this is a short -term pi lot study with a 12 -month fol low -up, we will not con duct in terim analy sis for the clin i cal ef fi cacy of oral ALA -SFC. How ever, the safety of ALA -SFC will be in de pen dently evalu ated by the Data and Safety Mon i tor ing Com mit tee at the time as fol lows: i) Critical modification of the study protocol is required ii) Any serious adverse event associated with this agent occurs iii) A critical problem is observed while monitoring iv) The principal investigator needs the judgement of this committee More over, the mon i tor ing com mit tee in de pen dently eval u ates whether the study is im ple mented in com pli ance with the study pro tocol, and the data are ap pro pri ately cor rected ac cord ing to a prearranged mon i tor ing plan at the time when the first pa tients have been en rolled and once per year.

6 . Statistical analysis
De scrip tive sta tis tics will be com puted for all study vari ables. Com par i son of the two groups, the trial cases and his tor i cal con trol cases, will be con ducted us ing the Mann -Whitney U test or Wilcoxon signed rank test for con tin u ous vari ables, and Fish er's ex act test or Tarone test for cat e gor i cal vari ables as ap pro pri ate. Sur vival analy sis will be con ducted us ing the Ka plan -Meier method and log -rank test. P < 0.05 will be con sid ered sta tis ti cally sig nif i cant.

7 . Trial registration
This clin i cal trial was prospec tively reg is tered with the Japan Registry of Clin i cal Tri als on 5 De cem ber 2019. The ref er ence num ber is jRCT s051190077, nara0013 (Cer ti fied Re view Board of Nara Med ical Uni ver sity). The URL of trial reg istry record is found in https:// jrct. niph. go. jp/ en -latest -detail/ jRCTs051190077 .

8 . Dissemination
The re sults will be sub mit ted for a peer -reviewed jour nals for publi ca tion and pre sented at lo cal and in ter na tional sci en tific con ferences. Also, the re sults will be avail able to in ter ested par tic i pants.

. Discussion
It has been more than 15 years since LDR -BT was in tro duced in Japan [ 2 ]. A com pre hen sive re view re ported ex cel lent on co log i cal out comes show ing equiv a lent or su pe rior ef fi cacy to rad i cal prosta te ctomy for low -or in ter me di ate -risk PCa and su pe ri or ity for high -risk PCa based on the ex pe ri ence of more than 52,000 pa tients un der go ing LDR -BT with or with out EBRT [ 2 ]. How ever, there are still sig nif i cant con cerns about side ef fects, com pli ca tions, and con se quently re duced QOL af ter ra dio ther apy. One of our long -term in ter ests is wors en ing lower uri nary tract symp toms. Most uri nary ad verse events are cat ego rized into grade 1 to 2, while higher -severity ad verse events are rare. More than 70% of pa tients ex pe ri ence in creased uri nary frequency (pol lak i uria) dur ing the first 6 months af ter seed im plan ta tion [ 19 ]. To over come this clin i cal is sue, our group and other groups have con ducted ran dom ized con trolled tri als to ex plore med ica tions pre vent ing post -treatment uri nary ad verse events. How ever, the true ben e fits of α1 -adrenoceptor an tag o nists, anti -cholinergic drugs, β3adrenoceptor ag o nists, phy tother a peu tic drugs, and non -steroidal antiinflammatory drugs have not been proven yet [ 14 , 20 -22 ].
We plan to eval u ate whether oral ALA -SFC pre vents GU tox i c i ties af ter seed im plan ta tion. ALA -SFC is widely ac cepted as a health supple ment and food with func tional claims in Japan. This prod uct report edly in hibits el e va tions in blood glu cose level, im proves sleep qual ity, and sup ports mus cle strength and ex er cise ef fi ciency [ 23 , 24 ]. More over, pre clin i cal stud ies have demon strated that ALA con fers cyto pro tec tive ef fects in the blad der, rec tum, kid ney, heart, and neural sys tem from var i ous types of stress [ 7 , 9 -12 ]. An other po ten tial ben efit of adding ALA is an in crease in the ra diosen si ti za tion of ma lig nant cells. Pre vi ous re search has shown that ALA sup ple men ta tion sen sitizes ma lig nant cells in clud ing glioma, melanoma, and colon ade nocar ci noma cells to ra dio ther apy via en hanced gen er a tion of pro to porphyrin IX and re ac tive oxy gen species [ 7 ]. Our pre vi ous pre clin i cal study con firmed that adding in vitro sin gle and in vivo re peated ad minis tra tion of ALA sen si tized PCa cells to ra dio ther apy. Based on this evi dence, this trial in cludes on co log i cal out comes such as bio chem i cal re cur rence -free sur vival as sec ondary end points.
As sess ments based on the CT CAE are the gold stan dard for eval uat ing ad verse events re lated to treat ment. How ever, pre vi ous stud ies have demon strated that agree ment be tween the CT CAE as sess ment and PROs is poor to mod er ate [ 25 ]. Ben nett et al. re ported that the rate of pa tients re port ing se vere neu ropa thy is 30%, which is higher than that iden ti fied by the CT CAE as sess ment (10%) [ 26 ]. This suggests that tox i c i ties are of ten un de tected or un der es ti mated by clin icians. This trial uti lizes FVCs and PROs as well as the CT CAE for tox ic ity as sess ment. Our tar get pri mary end point in this trial is pre ven tion of in creas ing uri nary fre quency af ter seed im plan ta tion. We sup pose that the ben e fit of this in ter ven tion will be more pre cisely and ob jectively eval u ated us ing FVCs.
The goal of this prospec tive, sin gle -center trial is to de ter mine the po ten tial ben e fit of tak ing ALA -FSC in pa tients who un dergo LDR -BT. We an tic i pate fa vor able con trol of GU tox i c i ties and ac cept able tol erance. The po ten tial vast util ity of ALA -SFC is fur ther en cour ag ing because this drug can sen si tize can cer cells to ra dio ther apy. Ul ti mately, the promis ing re sults will pro vide pa tients with sup ple men tary in terven tion and po ten tially chang ing the stan dard of care.

Trial status
The study be gan in De cem ber 2019. Pa tient re cruit ment has not yet been com pleted and the in ter ven tion pro gram is on go ing. A follow -up and data col lec tion will be com pleted in March 2022. The final re sults are ex pected in Sep tem ber 2022.

Ethics approval and consent to participate
This clin i cal trial com plied with the De c la ra tion of Helsinki regard ing in ves ti ga tion in hu mans. Its eth i cal clear ance, pro to col (version 1.0 on No vem ber 11th, 2019), and as so ci ated doc u ments were ap proved by the Cer ti fied Re view Board of Nara Med ical Uni ver sity (in sti tu tion ID: CR B5180011). In formed con sent and writ ten con sent forms of pa tients are manda tory be fore study par tic i pa tion.

Consent for publication
Not re quired

Availability of data and materials
The col lected datasets used dur ing this clin i cal trial are avail able from the cor re spond ing au thor (N. Tanaka) on rea son able re quest.

Authors' contributions
MM is the prin ci pal in ves ti ga tor and con ceived the study. NT, MK, and KF are the ad vi sors of the study and par tic i pated in the de sign and in ter ven tion of the study. IA, KY, SH, YN, SA, KT, and MH are ma jor con trib u tors of the data ac qui si tion. TF and CO per form the histo log i cal ex am i na tion of the prostate. SS and TI will sta tis ti cally an alyze and in ter preted the pa tient data re gard ing the ad verse events and other as so ci ated out comes. MT, HN, and ST are ma jor con trib u tors of the mon i tor ing and data pro tec tion. MM wrote the first draft of the man u script and SS sub stan tively re vised it. All au thors pro vided in put into the study de sign, pro vided in tel lec tual in put to the man u script and ap proved the fi nal ver sion of the man u script.

Funding
The AM BER study is funded by SBI Phar ma ceu ti cals Co., Ltd . The role of the fund ing body is pro duc tion and pro vi sion of the in ter vention drug (ALA -SFC).

Declaration of competing interest
MM, NT, and KF have re ceived a joint re search fund from SBI Phar ma ceu ti cals Co., Ltd, Tokyo, Japan, that pro duces ALA -SFC capsules and par tially sup ported this clin i cal trial.