Intrahepatic Reinfusion of CD133+ Stem Cells in Cirrhotic Patients
Intrahepatic Reinfusion of CD133+ Stem Cells in Cirrhotic Patients
OBJECTIVE(S): Primary:

 To assess the safety of the intrahepatic reinfusion of increasing numbers of autologous
 highly purified CD133+ stem cells (SCs) to patients with end-stage liver disease. Safety will
 be evaluated as the incidence of adverse event (graded according to WHO) and clinically
 significant abnormal laboratory value following reinfusion of SCs.

 Secondary:

 To assess the feasibility of the immunomagnetic selection of autologous CD133+ cells
 collected with leukapheresis from the peripheral blood (PB) of patients with end-stage liver
 disease, previously mobilized with G-CSF. To assess the effects of the intrahepatic
 reinfusion of highly purified CD133+ cells on residual hepatic function of the patients.

 STUDY DESIGN:

 Twelve patients will be enrolled. At first, G-CSF at 7.5µg/Kg/b.i.d. will be administered
 subcutaneously (sc) from day 1 until the completion of peripheral blood stem cells (PBSC)
 collection. Harvest of bone marrow (BM)-derived PBSC will begin on day + 4 only if the
 concentration of CD133+ cells is > 8/uL and will be continued until the collection of the
 target cell dose: 0.5 x 106 CD133+ cells/Kg for the first 2 cohorts of patients; 1 x 106
 CD133+ cells/Kg for cohort 3 and 2 x 106 CD133+ cells/Kg for cohort 4 (see below for
 definitions). PB mononuclear cells obtained from mobilized standard-volume leukapheresis will
 be incubated with Macs colloidal superparamagnetic CD133 microbeads and CliniMacs device will
 be used for the positive selection of CD133+ cells under good manufacturing practice (GMP)
 conditions. Cryopreservation and storage in liquid nitrogen will be performed according to
 standard procedures. At least 4 weeks after SC mobilization and collection, up to 40 mL of
 single cell suspension of highly purified autologous CD133+ cells, obtained after rapid
 thawing, will be infused through the hepatic artery by transfemoral or transbrachial
 arteriography. Infusion time will be lower than 15ml/min to avoid thrombi formation. The
 entire procedure will be performed under anesthesiological control. According to modified
 Fibonacci's increment rule, highly purified G-CSF-mobilized CD133+ cells will be administered
 to patients starting from 5x104/Kg patient's body weight and increased every 3 patients. The
 maximum infused cell dose will be 1x106/kg. G-CSF at 5µg/Kg/day will be administered sc for 3
 days after the reinfusion of SCs (day 0 to day +2).
TITLE: Phase I study of intrahepatic reinfusion of highly purified CD133+ stem cells in
 patients with end-stage liver disease.

 PRINCIPAL INVESTIGATOR: Roberto M. Lemoli, MD CO-INVESTIGATORS: Pietro Andreone, MD,
 Francesca Bonifazi, MD; Lucia Catani, BS; Antonia D'Errico, MD; Michelangelo Fiorentino, MD;
 Valeria Giudice, MD; Annagiulia Gramenzi, MD; Elisabetta Loggi, BS; Stefania Lorenzini, MD;
 Francesco Losinno, MD; Maria Rosa Motta, BS; Simonetta Rizzi, BS; Cristina Rossi, MD.

 STUDY CENTER: Azienda Ospedaliero-Universitaria di Bologna, Policlinico S.Orsola-Malpighi,
 Bologna - Italia, Unità Operativa di Ematologia in collaboration with:

 Unità Operativa di Semeiotica Medica Unità Operativa di Anatomia e Istologia Patologica Unità
 Operativa di Immunoematologia e Trasfusionale Unità Operativa di Radiologia

 CLINICAL PHASE: Phase I

 OBJECTIVE(S): Primary:

 To assess the safety of the intrahepatic reinfusion of increasing numbers of autologous
 highly purified CD133+ stem cells (SCs) to patients with end-stage liver disease. CD133+ SCs
 will be previously collected and cryopreserved after mobilization with granulocyte-colony
 stimulating factor (G-CSF) and leukapheresis. Safety will be evaluated as the incidence of
 adverse event (graded according to WHO) and clinically significant abnormal laboratory value
 following reinfusion of SCs.

 Secondary:

 To assess the feasibility of the immunomagnetic selection of autologous CD133+ cells
 collected with leukapheresis from the peripheral blood (PB) of patients with end-stage liver
 disease, previously mobilized with G-CSF.

 To assess the effects of the intrahepatic reinfusion of highly purified CD133+ cells on
 residual hepatic function of the patients. In particular, the following values will be
 monitored: synthesis of proteins (albumin), synthesis of factors of the coagulation
 (prothrombin time, INR, fibrinogen, antithrombin III, thromboplastin time), bilirubinemia,
 proliferative activity, biosynthetic capacity (cholesterol, pseudocholinesterase).

 To assess the effects of the treatment on the production of selected cytokines during the
 administration of G-CSF and after the intrahepatic reinfusion of highly purified CD133+ SCs
 to patients with end-stage liver disease.

 STUDY DESIGN:

 Twelve patients will be enrolled within 18-24 months in the dose-finding phase. The study has
 been designed as follows: at first, G-CSF (Lenograstim, Aventis Pharma or Filgrastim, Amgen)
 at 7.5µg/Kg/b.i.d. will be administered subcutaneously (sc) from day 1 until the completion
 of peripheral blood stem cells (PBSC) collection. Harvest of bone marrow (BM)-derived PBSC
 will begin on day + 4 only if the concentration of CD133+ cells is > 8/uL and will be
 continued until the collection of the target cell dose: 0.5 x 106 CD133+ cells/Kg for the
 first 2 cohorts of patients; 1 x 106 CD133+ cells/Kg for cohort 3 and 2 x 106 CD133+ cells/Kg
 for cohort 4 (see below for definitions). PB mononuclear cells obtained from mobilized
 standard-volume leukapheresis will be incubated with Macs colloidal superparamagnetic CD133
 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and CliniMacs device will be used
 for the positive selection of CD133+ cells under good manufacturing practice (GMP) conditions
 (see attachments). Cryopreservation and storage in liquid nitrogen will be performed
 according to standard procedures (see attachments).

 At least 4 weeks after SC mobilization and collection, up to 40 mL of single cell suspension
 of highly purified autologous CD133+ cells, obtained after rapid thawing, will be infused
 through the hepatic artery by transfemoral or transbrachial arteriography. Infusion time will
 be lower than 15ml/min to avoid thrombi formation. The entire procedure will be performed
 under anesthesiological control. The intrahepatic route of administration is deemed necessary
 to avoid the potential massive spleen trapping of CD133+ cells after conventional intravenous
 reinfusion in cirrhotic patients. Within 24 hours from the procedure, a doppler
 ultrasonography of the celiac trunk will be performed to exclude thrombotic events of the
 mesenteric district.

 According to modified Fibonacci's increment rule, highly purified G-CSF-mobilized CD133+
 cells will be administered to patients starting from 5x104/Kg patient's body weight and
 increased every 3 patients. The maximum infused cell dose will be 1x106/kg (see below).
 Lenograstim or Filgrastim at 5µg/Kg/day will be administered sc for 3 days after the
 reinfusion of SCs (day 0 to day +2) for their expansion and to induce a selective
 proliferative advantage of reinfused cells in vivo.

 NUMBER OF PATIENTS AND TIME FRAME: Twelve patients will be enrolled within 18-24 months in
 the dose-finding phase.

 DIAGNOSIS AND INCLUSION CRITERIA: Signed informed consent. Age >18. Karnofsky score > 70% or
 WHO <1. Adequate renal (serum creatinine < 2 mg/dl) and pulmonary (Sat O2 > 96%) function.
 Diagnosis of advanced, end-stage liver disease defined by a Mayo Model for End Stage Liver
 Disease (MELD) score between 17 and 25.

 Patients with the following liver disease etiologies will be enrolled in the study: chronic
 viral hepatitis B, chronic viral hepatitis C, chronic viral hepatitis D, alcoholic cirrhosis,
 primary sclerosing cholangitis, primary biliary cirrhosis, Wilson's disease, genetic
 haemochromatosis or iron over-load cirrhosis, cirrhosis due to non alcoholic
 steato-hepatitis. Patients with alcoholic liver cirrhosis should have withdrawn alcohol
 active consumption to be enrolled in the study.

 Eligibility for orthotopic liver transplantation (OLT) is not a contraindication to enter in
 the clinical study. Patients in waiting list for OLT transplantation will not be withdrawn
 and will be transplanted as soon as a suitable donor will become available. Their MELD score
 will remain that recorded prior SCs infusion in case of improvement following the
 experimental procedure.

 The presence of cirrhosis related symptoms, like ascites, peripheral edema, recurrent
 gastrointestinal tract bleeding, recurrent encephalopathy do not represent major
 contraindications to be enrolled into the study. The patients may be considered eligible when
 clinically stable.

 EXCLUSION CRITERIA: HIV positivity. Pregnant or nursing females. Current uncontrolled
 infection. Intercurrent organ damage. Diagnosis of hepatocarcinoma. Complete portal
 thrombosis. Severe impairment of coagulative function (PT< 30%, INR>2.5, Platelets <
 40x109/L) that would contraindicate artheriography. Grade IV splenomegaly.

 Budd-Chiari Syndrome with sovrahepatic vein thrombosis and cirrhosis of unknown origin will
 not be included in the study.
Inclusion Criteria:

 -  Signed informed consent

 -  Age >18 years

 -  Karnofsky score >70% or WHO <1

 -  Adequate renal (serum creatinine < 2 mg/dl) and pulmonary (Sat O2 >96%) function

 -  Diagnosis of advanced, end-stage liver disease defined by a Mayo Model for End Stage
 Liver Disease (MELD) score between 17 and 25. Patients with the following liver
 disease etiologies will be enrolled in the study: chronic viral hepatitis B, chronic
 viral hepatitis C, chronic viral hepatitis D, alcoholic cirrhosis (without alcohol
 active consumption), primary sclerosing cholangitis, primary biliary cirrhosis,
 Wilson's disease, genetic hemochromatosis or iron over-load cirrhosis, cirrhosis due
 to non alcoholic steato-hepatitis.

 Eligibility for orthotopic liver transplantation (OLT) is not a contraindication to enter
 in the clinical study. Patients in waiting list for OLT transplantation will not be
 withdrawn and will be transplanted as soon as a suitable donor will become available.Their
 MELD score will remain that recorded prior SCs infusion in case of improvement following
 the experimental procedure.

 The presence of cirrhosis related symptoms, like ascites, peripheral edema, recurrent
 gastrointestinal tract bleeding, recurrent encephalopathy do not represent major
 contraindications to be enrolled into the study.

 The patients may be considered eligible when clinically stable.

 Exclusion Criteria:

 -  HIV positivity

 -  Pregnant or nursing females

 -  Current uncontrolled infection

 -  Intercurrent organ damage

 -  Diagnosis of hepatocarcinoma

 -  Complete portal thrombosis

 -  Severe impairment of coagulative function (PT< 30%, INR>2.5, Platelets < 40x109/L)
 that would contraindicate arteriography

 -  Grade IV splenomegaly

 -  Budd-Chiari Syndrome with sovrahepatic vein thrombosis and cirrhosis of unknown origin
 will not be included in the study