peptides 28 (2007) 1891–1895
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Intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) in a patient with multiple myeloma and myeloma kidney: A case study Min Li *, Jerome L. Maderdrut, Juan J.L. Lertora 1, Vecihi Batuman Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA
We have recently shown significant renoprotective effects with the administration of
Received 1 February 2007
pituitary adenylate cyclase-activating polypeptide (PACAP) in models of myeloma kidney.
Received in revised form
PACAP markedly inhibited the production of proinflammatory cytokines stimulated by
11 April 2007
immunoglobulin light chains in human renal proximal tubule epithelial cells and in the
Accepted 7 May 2007
kidneys of rats infused with myeloma light chains. PACAP was also shown to suppress the
Published on line 22 May 2007
proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. In this case study, an 81-year-old male patient with active multiple myeloma and
myeloma kidney was infused intravenously with synthetic human PACAP38 at a rate of
4 pmol/kg/min for 120 min. The continuous infusion increased the level of PACAP38 in
blood, with a plateau at about 0.2 nM during the infusion. The level of PACAP in the blood
rapidly declined after the cessation of administration with a half-life of about 5–10 min. The
Mitogen-activated protein kinase
continuous infusion did not significantly alter the basal glucose level, blood gases or blood
pressure. There was a large reduction in free lambda light chains in urine after the start of the
treatment with PACAP. These studies show that PACAP can be safely used in humans and
suggest that it could be used as a novel therapeutic agent for the treatment of multiple myeloma and myeloma kidney. # 2007 Elsevier Inc. All rights reserved.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide that was originally isolated from ovine hypothalami in our laboratories based on its ability to stimulate adenylate cyclase-activity in rat anterior pituitary cell cultures . It exists as two a-amidated peptides with 38 (PACAP38) or 27 (PACAP27) amino acids . PACAP is a member of the secretin/vasoactive intestinal peptide (VIP)/ growth hormone-releasing hormone (GHRH) family, and PACAP27 has 68% sequence identity with VIP. Our previous studies have shown that PACAP is synthesized as a prohormonal precursor and is processed mainly by prohormone
convertase (PC) 1 and PC2 in neuroendocrine cells  and by PC4 in the testes [18–20]. PACAP binds to three distinct, but closely related, G-protein-coupled receptors: the PAC1, VPAC1 and VPAC2 receptors . PACAP and VIP bind to both the VPAC1 and VPAC2 receptors with similar affinities, but PACAP binds to the PAC1 receptor with more than 100-fold higher affinity than VIP. One or more of these three receptors have been identified on almost every known type of tumor cell [28,34]. These receptors belong to a family of glycoprotein receptors that are coupled to multiple signal transduction pathways . PACAP inhibits cell proliferation through a cAMP-dependent pathway, whereas the growth-stimulatory action of PACAP is via a phospholipase C-dependent
* Corresponding author. Tel.: +1 504 988 1361; fax: +1 504 988 1909. E-mail address: [email protected]
(M. Li). 1 Present address: Clinical Pharmacology Program, NIH Clinical Center, Bethesda, MD 20892. 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.05.002
peptides 28 (2007) 1891–1895
mechanism and via a mitogen-activated protein kinase (MAPK) pathway . PACAP functions as a hypophysiotropic factor, neurotransmitter, neuromodulator, neurotrophic factor, and cytokine [1,36]. Recently, PACAP has been shown to have potent immunomodulatory effects on the MAPKmediated immune responses by suppressing or stimulating the production of inflammatory cytokines [4,8]. Multiple myeloma is the sixth most common cancer in the United States. The disease can cause serious medical complications, including bone resorption, anemia and kidney failure. Renal involvement is seen in about half of the patients, and the most common type of kidney impairment is myeloma kidney. The major pathological change is a tubulointerstitial nephropathy that is associated with immunoglobulin light chaincontaining casts in the tubule lumens. This neuropathy is caused by the interaction of overproduced light chains and Tamm-Horsfall proteins [14,22,32]. There is no efficacious therapy for myeloma nephropathy except for the limited use of corticosteroids and dialysis . We have recently found that PACAP prevents the renal tubular injury induced by myeloma light chains both in vitro and in vivo by suppressing the expression of proinflammatory cytokines [2,3,29,32]. Experiments in our laboratories demonstrated that subnanomolar concentrations of PACAP inhibit the production of myeloma light chain-induced proinflammatory cytokines, including tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6), with a considerably greater potency than dexamethasone, and attenuate the resulting kidney cell injury in human renal proximal tubule cell cultures and suppress cytokine production in the kidneys of rats infused with myeloma light chains [2,3]. Our results also indicated that these effects are mediated by inhibition of the phosphorylation of MAPK and the nuclear translocation of nuclear factor kappa B (NF-kB) via both PAC1 and VPAC1 receptors within the kidney epithelial cells. Cytokines have long been known to play a major role in the proliferation of myeloma cells. The binding of myeloma cells to stromal cells in the bone marrow triggers both adhesionand cytokine-mediated myeloma cell proliferation, survival, and drug resistance [12,13,26,27]. Many of these cytokines also contribute to osteolytic bone resorption. The cells involved in bone resorption in multiple myeloma produce large amounts of cytokines that are capable of stimulating myeloma proliferation [30,31,33]. The disease remains incurable despite the use of conventional and high-dose chemotherapies with hematopoietic stem cell transplantation. Therefore, the establishment of new strategies that increase the specificity of the treatment and minimize undesirable toxicity to normal cells is necessary to improve patient outcome. In our most recent studies, we presented preliminary evidence that PACAP inhibits myeloma cell proliferation not only directly but also indirectly by suppressing the production of cytokines from bone marrow stromal cells . Specifically, PACAP suppressed the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. The addition of PACAP suppressed light chain-producing myeloma cellstimulated IL-6 secretion by the bone marrow stromal cells. A specific antagonist for either the human PAC1 receptor or the VPAC receptors attenuated the suppressive effect of PACAP on IL-6 production during the adhesion of human myeloma cells to bone marrow stromal cells. The signaling cascades involved
in the inhibitory effect of PACAP on IL-6-mediated paracrine stimulation of light chain-secreting myeloma cell proliferation were the suppression of p38 MAPK phosphorylation as well as activation of the transcription factor NF-kB . These findings justify evaluating PACAP as an effective and safe renoprotective agent in myeloma kidney, and also as a new antitumor agent for multiple myeloma. Based on these observations, we designed a clinical protocol to study the effects of PACAP38 in a patient with active multiple myeloma and myeloma kidney. We infused synthetic human PACAP38 intravenously at a dose rate of 4 pmol/min/kg in a male patient. This dose was selected because a previous report had shown that the highest tolerable infusion rate of PACAP is 10 pmol/min/kg; higher doses caused intense flushing [6,9]. To determine the circulating level of PACAP during infusion, we also measured plasma PACAP38-like immunoreactivity with an enzymelinked immunosorbent assay (ELISA).
2. Infusion of PACAP38 in a patient with multiple myeloma 2.1.
Synthetic human PACAP38 was purchased from American Peptide Co., Inc. (Sunnyvale, CA, United States). The peptide was dissolved in sterile distilled water and then diluted in 0.9% saline with the addition of 1% human serum albumin and filtered through an 0.2-mm nitrocellulose filter, lyophilized and stored at 20 8C until used. Net peptide content rather than gross weight was used for dose calculations.
The patient was 81-years old at the time of these studies. Earlier he had been diagnosed clinically and by bone marrow aspiration studies to have lambda light-chain multiple myeloma and myeloma kidney. He volunteered for the study. He weighed 58 kg, his body mass index was 21.6, and his blood glucose concentration ranged between 90 and 104 mg/dL. The patient had a past history of pulmonary tuberculosis and his right lung had been treated by collapse in 1954; and he also had hypothyroidism and was on a 75 mcg/day thyroxine replacement regimen. His blood pressure was normal and he was on no other medication. His serum creatinine ranged between 3.5 and 4.0 mg/dL, with an estimated glomerular filtration rate between 14 and 17 cc/min. His 24-h urine creatinine clearance was 17 cc/ min. The patient had no urinary abnormalities, and there was no proteinuria by routine dipstick analysis. However, quantitative immunoelectrophoresis showed 21,240 mg of monoclonal lambda light chains in his urine. A kidney biopsy was not performed. The subject received oral and written information concerning the aims and methods of the study and signed a consent form before the start of the study. The study protocol and the consent form were approved by the Institutional Review Board of the Tulane University Human Research Subject Protection Program after obtaining approval for a Single Patient Investigational New Drug protocol from the United States Food and Drug Administration.
peptides 28 (2007) 1891–1895
Infusion of PACAP38
After an overnight fast, PACAP38 was initially infused intravenously at a dose rate of 4 pmol/kg/min for 60 min and the patient’s response was evaluated. As the patient tolerated this ‘‘test–dose’’, a treatment protocol consisting of infusion of PACAP38 at 4 pmol/min/kg for 120 min, 3 days a week was initiated. The infusions were given at Louisiana State University/Tulane University General Clinical Research Center under direct supervision by a physician and the patient’s vital signs were monitored every 15 min. Samples for determination of routine clinical chemistries, complete blood count (CBC), and PACAP38 were taken before, during and after the infusion of PACAP38. They were immediately centrifuged at 4 8C, and plasma or serum was stored at 70 8C until analyzed. Blood samples for analysis of plasma glucagon and plasma PACAP38 were put into prechilled test tubes containing 0.084 mL of ethylenediaminetetraacetic acid (0.34 mol/L) and aprotinin (250 kallikrein inhibitor units/ mL blood).
Clinical responses and PACAP38 levels
The PACAP38 infusion was well tolerated at all time points, although the patient experienced flushing of the facial skin and a peripheral paleness. The flushing occurred 20–30 min after the initiation of the infusion and was visible or was felt as heating of the face that lasted for 2–3 days after the end of the infusion. The peripheral paleness was mainly observed in the distal parts of the extremities. It occurred immediately at the start of the peptide infusion, but vanished after discontinuation of PACAP administration. Blood pressure was measured every 15 min. Immediately after the start of the peptide infusion, both diastolic and systolic blood pressures dropped by 2–3 mmHg. After approximately 30 min of PACAP38 infusion, the blood pressures had stabilized at approximately 120 mmHg (range: 110–125) systolic, and 50 mmHg (range 47– 52) diastolic. Simultaneously, heart rate ranged between 98 and 112 during the PACAP38 infusion. The patient received a total of 11 infusions over 6 weeks, after a 2-week interruption
due to an intervening viral infection of the upper respiratory tract. During the 5th infusion day, blood samples were drawn for pharmacokinetic measurements. The concentration of PACAP38 in human plasma was determined with a sandwich ELISA. The standard curve for the assay of PACAP38 in plasma was prepared in normal human plasma, which has an undetectable amount of PACAP38. Synthetic human PACAP38 was used as the standard. All measurements were made in quadruplicate. The recovery of PACAP38 added to unknown plasma samples was approximately 95%. The PACAP38 assay prepared in plasma had a detection limit of approximately 25 pg/mL. The ELISA showed no cross-reactivity with any other peptide of the secretin/VIP/ GHRH family. The plasma samples were stored at 70 8C after collection. The plasma level of PACAP38 increased after infusion of the peptide and was stable throughout the infusion. The mean concentration of plasma PACAP38 reached 0.197 nmol/L at 90 min during peptide infusion (Fig. 1(A)), and rapidly declined soon after the cessation of administration (Fig. 1(B)). Plasma PACAP38 remained near or below 0.01 nmol/L at 100 min after the infusion. The half-life in the blood is about 5–10 min. The patient reported an improvement in his overall condition, felt less fatigued and had an improved sense of well-being. The patient continued to receive dexamethasone and zoledronic acid treatment during the same period that he received his PACAP38 infusions. His laboratory parameters including CBC, and blood urea nitrogen (BUN) and creatinine did not change at the end of the infusions. However, there was a marked decrease in his 24-h urinary excretion of lambda light chains after the infusions were completed (Table 1). We are unable to attribute this improvement to the treatment with PACAP because the patient continued to receive dexamethasone during this period. It could be argued that PACAP did not make the patient’s multiple myeloma worse, and it is even conceivable that the marked decline in the urinary excretion was induced by PACAP. However, a multi-patient placebocontrolled clinical trial will be necessary to make any meaningful conclusions. Other members of the secretin/VIP/GHRH family also have similar short half-lives [7,15]. The members of the secretin/
Fig. 1 – PACAP38 in physiological saline was infused intravenously into a male patient with active multiple myeloma and myeloma kidney at a rate, 4 pmol/kg/min for 2 h. The continuous intravenous infusion did not appear to alter plasma glucose level, blood gases or blood pressure and heart rate except for the subject experienced a transient flushing on the face and upper portion of the body, but no unpleasant feeling. The blood level of the peptide reached about 0.2 nM during the infusion (A), and rapidly declined soon after the cessation of administration (B). The half-life in the blood is about 5– 10 min.
peptides 28 (2007) 1891–1895
Table 1 – Lambda light-chain immunoglobulins and total protein in the urine of a patient with multiple myeloma and myeloma kidney Date
Free lambda light chains in urine (mg/day)
12/6/03 1/18/04 3/15/04 10/11/04 1/14/05 3/29/05a 8/1/05b 1/31/06 1/27/07 a b
21,347 17,784 4,968 4,665 4,092 13,200 751 1,013 520
Total protein (mg/day) 21,584 17,952 5112 4873 4312 ND 1068 1331 781
Before PACAP infusion. After PACAP infusion.
VIP/GHRH family are degraded in plasma mainly by aminopeptidases, especially dipeptidyl peptidase IV [7,15,23,37]. It is interesting to observe that flushing with a feeling of ‘‘well being’’ lasted 2–3 days after cessation of the peptide administration. It is possible that the genomic effects of PACAP38 last long after the peptide has disappeared from the circulation. This is important from a practical point in the clinical use of the peptide. Some, albeit not all, of the effects of a single large intraperitoneal dose of PACAP38 on serum cytokine levels in mice have been reported to last at least as long as 12 h . We have found that a single daily intraperitoneal injection of PACAP38 can extend the survival of mice infected orally with ten times the LD50 dose of Salmonella enterica by 5.5 days (unpublished observations). Therefore, it is essential to test whether infrequent short-term intravenous or intraperitoneal administration of the PACAP38 induces a prolonged suppressive effect on cytokine production by the human renal proximal tubule epithelial cells and prevents the renal injury.
This is the first study in a patient with multiple myeloma who has been infused systemically with PACAP. Infusion of PACAP38 elevated its concentration in the blood to about 0.2 nM during the infusion, and the levels rapidly declined soon after the cessation of administration, without obviously altering the physical conditions of the patient. The half-life of PACAP38 in the blood is about 5–10 min. During this shortterm single-patient study, we did not observe an improvement in kidney function or changes in hematological parameters. An apparent reduction in the urinary excretion of light chains could be a coincidence. A long-term multi-patient study will be necessary to determine whether PACAP is a useful therapeutic for multiple myeloma.
Acknowledgements This work was supported in part by grants from the Veterans Administration (Merit Review Award), the Louisiana Cancer Research Consortium, the National Institutes of Health (#2M01RR005096), the Multiple Myeloma Research
Foundation (2005 Fellows Award), and the Kaken American Foundation.
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