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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 12 4044-4048
Copyright © 1997 by The Endocrine Society

Original Studies

A Novel Cyclic Adenosine Monophosphate Analog Induces Hypercalcemia via Production of 1,25-Dihydroxyvitamin D in Patients with Solid Tumors

Mark P. Saunders, Amanda J. Salisbury, Kenneth J. O’Byrne, Louise Long, Ruth M. Whitehouse, Denis C. Talbot, E. Barbara Mawer and Adrian L. Harris

Imperial Cancer Research Fund, Medical Oncology Unit, Churchill Hospital (M.P.S., A.J.S., L.L., K.J.O., D.C.T., R.M.W., A.L.H.), Oxford, OX3 7LJ; and Department of Medicine, Manchester Royal Infirmary (E.B.M.), Manchester, M13 9WL, United Kingdom

Address all correspondence and requests for reprints to: Adrian L. Harris, Imperial Cancer Research Fund, Medical Oncology Unit, University of Oxford, Oxford Radcliffe Hospital, Headington, Oxford, United Kingdom OX3 7LJ.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
The treatment of cancer patients with conventional chemotherapy is sometimes associated with severe systemic toxicity and only a minimal survival benefit. Because of this, new less toxic and more efficacious treatments have been sought. 8-Chloro-cAMP (8-Cl-cAMP) is one of a new generation of anticancer drugs that act at the level of signal transduction. In preclinical models, 8-Cl-cAMP modulates protein kinase A (PKA) leading to growth inhibition and increased differentiation of cancer cells. 8-Cl-cAMP was given to 16 patients with advanced cancer as an infusion via an indwelling subclavian venous catheter. We showed that 8-Cl-cAMP had a parathyroid hormone-like effect leading to increased synthesis of renal 1,25-dihydroxyvitamin D [up to 14 times the baseline value, median 3.6 times; P = 0.00001 (Student’s paired t test)]. This produced the dose-limiting toxicity of reversible hypercalcemia that could not be controlled by the administration of either pamidronate or dexamethasone. The treatment was otherwise well tolerated, and other cAMP-dependent pathways (cortisol and TSH) were not affected, emphasizing the marked differences between organs in their sensitivity to this cAMP analog. Our results have shown that 8-Cl-cAMP is biologically active, and it is feasible that if the hypercalcemia can be controlled, then this drug may have a role as a single agent, or as a short infusion between cycles of chemotherapy.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
BECAUSE of the narrow therapeutic window, cytotoxic chemotherapy is often associated with considerable toxicity. Attempts therefore have been made to develop drugs with novel actions with minimal side effects. 8-chloro-cAMP (8-Cl-cAMP) is one of a new generation of drugs that act at the level of signal transduction to modulate the activity of cAMP-dependent protein kinase (PKA).

PKA is tetrameric having two catalytic and two regulatory subunits. There are two main isoenzymes (PKAI and PKAII), each sharing a common catalytic unit but differing by virtue of distinct regulatory subunits, termed RI and RII. Both regulatory subunits have {alpha} and ß forms, which are present in varying proportions in different tissues. Two molecules of cAMP are able to bind to each regulatory subunit, one at each of two separate regions, termed site 1 and site 2 (1, 2). Enhanced expression of PKAI (or RI-{alpha}) has been associated with cellular proliferation, whereas up-regulation of PKAII (or RII) has been correlated with growth inhibition and cellular differentiation. In vitro studies in tumor cell lines have shown that an increase in the RI/RII ratio can stimulate rapid uncontrolled growth (2, 3). Overexpression of R1-{alpha} has also been shown to be an indicator of a poor prognosis in patients with breast cancer (4, 5). The ratio of RI to RII is therefore an important factor in normal cell growth. By manipulating this ratio, cAMP has been considered as a growth control switch, in that it can stimulate RI leading to cellular proliferation, or RII resulting in morphological changes and cytostasis.

In the past, cAMP analogs lacked selectivity for the regulatory subunits. Recently however, more discriminatory site 1 analogs have become available of which 8-Cl-cAMP is the most potent. By up-regulation of RII and down-regulation of RI, it is able to inhibit the growth of cancer cells and stimulate their differentiation both in vitro and in vivo (1, 2, 6, 7).

A preclinical study in beagle dogs has shown that 8-Cl-cAMP reaches a stable concentration in a few hours and is rapidly excreted by the kidneys. Gastrointestinal and renal toxicity were noted (8). In view of the limited toxicity and encouraging preclinical results, a phase 1 study in humans was undertaken. A parallel phase I study carried out by Tortora et al. (9) showed evidence of hypercalcemia with decreased PTH values in several patients. We confirmed this unexpected finding and also elucidated the cause. We show in this study that the analog had a PTH-like action, causing profound elevation of 1,25-dihydroxyvitamin D leading to hypercalcemia.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
All patients had a Eastern Co-operative Oncology Group (ECOG) performance status of 0–2 (ECOG performance status 2: ambulatory, up and about for more than 50% of the day, capable of self-care, unable to carry out any work activities) and had either failed conventional therapy or had tumors for which there was no standard treatment (i.e. mesothelioma, thymoma). A histological diagnosis of cancer was obtained in every case. A complete history, full examination, chest x-ray, and 12-lead electrocardiograms were obtained before entry into the study. Ethical approval for this study was granted by the Central Oxford Research Committee, and all patients gave informed consent before entry.

Other eligibility criteria included a life expectancy of greater than 3 months, age greater than 16 yr, and no radiotherapy or chemotherapy within 28 days of commencing treatment. All patients were required to have acceptable hematological, renal, and liver function for a phase 1 study.

8-Cl-cAMP was kindly supplied by Dr. K Miki of Fundamental Research Labs. (Tonen Corp., Japan). The dose level 1 was 0.005 mg·kg·h, a dose approximately 50 times lower than the one that caused toxicity in preclinical studies. Escalation to 0.045 mg-kg-h was achieved in three incremental rises. At least two patients received the drug at each dose level (Table 1Go). During the study it became evident that a continuous infusion for 3 weeks/month at the highest dose level was not possible. The schedule was therefore changed to 5 days/week for 3 weeks followed by a 1-week rest. This 4-week period was considered as one course of treatment.


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  		Table 1. 8-Cl-cAMP dose schedule

 
The drug was dissolved in 5% dextrose and infused into the patient via an indwelling subclavian venous catheter, using a 10-ml syringe driver. Each patient remained an inpatient for the first 24 h, after which all treatment was given on an outpatient basis. No dietary restrictions were stipulated throughout this study.

Because it acts at the level of signal transduction, 8-Cl-cAMP may have many modulatory effects on different endocrine and cellular systems. Pharmacodynamic end points were therefore chosen based on the physiological sites in which cAMP is a known mediator. Blood or urine samples for the following assays were collected on days 1, 3, and 5 of each 5-day period of treatment.

Assays

Serum samples were analyzed for calcium, phosphate (normal range: 2.45–4.34 mg/100 ml), albumin, and creatinine (0.77–1.65 mg/100 ml) on a Bayer-Technicon Axon (London, UK). The serum calcium level was corrected to a reference serum albumin of 4 g/100 ml using a correction factor of 0.96 mg/g albumin (10) (normal range: 8.48–10.48 mg/100 ml).

Calcium excretion per liter of glomerular filtrate (CaE). This was measured in the fasting state from a urine sample and venous blood sample and calculated as (11): CaE = (urine calcium/urine creatinine) x serum creatinine (mg/L glomerular filtrate).

Renal threshold phosphate concentration [TmPO4/glomular filtration rate (GFR)]. This is an estimate of renal phosphate reabsorption and is elevated in a hypoparathyroid state. It was measured in a fasting state from a urine sample and a venous blood sample and calculated as: CPO4/CCr = (urine PO4 x plasma Cr)/(urine Cr x plasma PO4).

A nomogram can then be used to derive TmPO4/GFR, using the plasma phosphate concentration and the CPO4/CCr as calculated above (all concentrations should be expressed in consistent units) (12, 13) (normal range: 2.5–4.2 mg/L).

25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D. A 10-ml clotted sample was collected, and the serum stored at -70 C before analysis. Serum vitamin D metabolites were extracted for assay as previously described (14). Briefly, 25OHD was quantified by competitive protein binding assay using normal human serum as the source of vitamin D binding protein at a dilution of 1:20,000 (15). The fractions containing 1,25-(OH)2D2 and 1,25-(OH)2D3 were combined and measured by RIA using monoclonal antibody 5F2 (16). All samples from each patient were measured on the same assay. (normal ranges: 25OHD: 5–25 ng/ml, 1,25(OH)2D: 20–50 pg/ml).

PTH. A 10-ml clotted sample was collected and the serum stored at -70 C before analysis. Serum PTH was measured using the intact 1–84 PTH assay by the magic-lite immunochemiluminometric system (CIBA-Corning diagnostics LTD, Halstead, Essex) (normal range: 0.9–5.4 pmol/L).

PTH-related peptide (PTHrP). A 10-ml sample was collected and the plasma stored at -70 C before analysis. A two-site immunoradiometric assay was performed for human parathyroid-related peptide 1–86 (PTHrP 1–86) in plasma using a mouse monoclonal antibody to PTHrP 1–34 coupled to cellulose particles for immunoextraction of N-terminal immunoreactivity, and a rabbit antiserum to PTHrP 37–67 that is indirectly labeled with 125I-labeled PTHrP 37–67 for quantifying the bound analyate (17) (normal range: <2.6 pmol/L).

Pyridinium cross-links in urine. Pyridinoline cross-links [pyridinoline (PYR) and deoxypyridinoliine (DPYR)] present in the urine in peptidic and free forms were released by acid hydrolysis from peptides separated from other urinary metabolites on fibrous cellulose by partition chromatography before separating by isocratic reverse-phase chromatography on high-performance liquid chromatography and detection of the natural fluorescence of pyridinium cross-links (18) (normal range: PYR, 19–190; DPYR, 3.65–54.80 µg/g creatinine).

Cortisol and TSH. For cortisol estimation, a 10-ml blood sample was collected before and 30 min after iv tetracosactrin (250 µg; Ciba, Sussex, UK), at the start and end of the first course of treatment. The plasma was stored at -70 C before RIA using a diagnostic systems laboratory kit (Metro Biosystems, Oxford, UK) (normal morning range: 90–230 ng/ml with at least a 2x rise after tetracosactrin). TSH levels were measured using the DPC Coat-A-Count TSH IRMA kit (DPC Ltd., Caernasfon, UK) on the pretetracosactrin blood sample (normal range 0.3–5.0 mU/L).

Statistic analyses

The Student’s paired t test was used for all statistical analysis with the exception of the calcium data. This data was slightly skewed, and so a nonparametric test (Wilcoxan’s signed rank test) was used. All results were expressed as mean values ± SEM.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
8-Cl-cAMP was administered to 16 patients with advanced local or metastatic cancer. Patient characteristics are presented in Table 2Go. The biochemistry and hormone results for the patients who received the analog at the highest dose level for 5 days/week are shown in Table 3Go.


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  		Table 2. Patient characteristics

 

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  		Table 3. Effect of treatment for 5 days/week at dose level 4 (0.045 mg·kg·h) on blood and urine biochemistry and hormone levels (day 0, start of cycle; day 20, cessation of treatment ± SEM)

 
The dose-limiting toxicity (DLT) was reversible hypercalcemia. At 0.045 mg·kg·/h for 3 weeks/month, patients became symptomatic from hypercalcemia, requiring drug cessation. Hypercalcemia was also the DLT at a revised level of 0.045 mg•kg•h for 5 days/week for 3 weeks/month (Fig. 1AGo). Urinary calcium excretion became significantly elevated above its baseline value at this dose level (Fig. 1BGo). Serum phosphate levels fell during the infusion, but remained within the normal range throughout treatment.



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  		Figure 1. A, Serum calcium levels over two cycles of treatment with 8-Cl-cAMP; 0.045 mg·kg·h for 5 days/week for 3 weeks followed by 1-week rest. Dotted line shows upper limit of reference range. Solid line indicates 8-Cl-cAMP infusion (mean values from eight patients ± SEM). B, Urinary calcium excretion over two cycles of treatment with 8-Cl-cAMP; 0.045 mg·kg·h for 5 days/week for 3 weeks followed by 1-week rest. Solid line indicates 8-Cl-cAMP infusion (mean values from seven patients ± SEM).

 
Impaired renal function did occur, but the peak serum creatinine remained lower than twice the upper limit of normal, and no permanent renal damage was demonstrated. Cortisol and TSH levels were not significantly altered during the infusion, and no other major side effects were noted. Patient weights remained relatively stable throughout this treatment, unless there was rapid progressive disease.

To investigate the mechanism of hypercalcemia, we measured PTH levels before, during, and after treatment in patients who received 8-Cl-cAMP at dose level 4 (0.045 mg•kg•h). They were found to be suppressed by the 8-Cl-cAMP-induced hypercalcemia (Fig. 2Go), whereas PTHrP levels were undetectable in all patients at the same time points. In a hypoparathyroid state, the renal phosphate reabsorption would be expected to be high (13). It did however, fall as the analog was infused, but returned to its baseline value on discontinuation of the drug (Fig. 2Go). The serum concentration of 25OHD remained unchanged, whereas the physiologically active metabolite, 1,25-(OH)2D, rose markedly during the 8-Cl-cAMP infusion (up to 14 times the baseline value, median 3.6 times; Fig. 3Go).



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  		Figure 2. Effect of 8-Cl-cAMP on PTH (•) and renal phosphate reabsorption (TmPO4/GFR, {circ}) at dose level 4 (0.045 mg·kg·h for 5 days/week for 3 weeks followed by 1-week rest). Solid line indicates 8-Cl-cAMP infusion (mean values from 8 patients ± SEM).

 


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  		Figure 3. Effect of 8-Cl-cAMP on vitamin D metabolites; 25OHD ({circ}) and 1,25-(OH)2D (•) at dose level 4 (0.045 mg·kg·h for 5 days/week for 3 weeks followed by 1-week rest). Solid line indicates 8-Cl-cAMP infusion (mean values from seven patients ± SEM).

 
Urinary pyridinium cross-links were also measured, but they remained within the normal range in all cases, suggesting that bone turnover was not increased. Pamidronate, an osteoclastic inhibitor, was given to three patients both as a treatment for hypercalcemia and also prophylactically (60 mg/week) to prevent its recurrence. It had no significant effect on control of the hypercalcemia (Table 4Go). Three other patients were given dexamethasone 4 mg bid prophylactically to try to limit the calcium rise. Again, this addition did not help reduce the hypercalcemia (Table 4Go).


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  		Table 4. Effect of continuous dexamethasone (4 mg bid) or pulsatile pamidronate (6 pulses of 60 mg) on serum calcium rise between start and end of each week of treatment with 8-Cl-cAMP (0.045 mg·kg·h) (mean calcium concentrations in mg/100 ml ± SEM)

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
This study showed that the DLT of 8-Cl-cAMP was hypercalcemia. To investigate this, we measured the blood and urinary concentrations of various factors that have an influence on calcium homeostasis. One of the mechanisms of action of PTH is by stimulation of renal 1-{alpha}-hydroxylation of 25OHD by a cAMP-dependent kinase mechanism (19, 20). PTH levels were found to fall, yet PTH-like effects were produced. These included a drop in renal phosphate reabsorption and elevated levels of 1,25-(OH)2D leading to hypercalcemia. We postulated that this stimulus was because of the infusion of 8-Cl-cAMP mimicking the action of PTH. 8-Cl-cAMP is probably acting directly on the site 1 binding site of the RII regulatory subunit of PKA in renal tissue, resulting in stimulation of 1-{alpha}-hydroxylase and subsequently elevated levels of 1,25-(OH)2D. This vitamin D metabolite and the profound hypercalcemia would cause suppression of PTH (Fig. 4Go).



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  		Figure 4. Postulated mechanism of 8-Cl-cAMP-induced hypercalcemia.

 
The principal action of 1,25-(OH)2D is on the intestine to promote calcium absorption (21) and also directly on bone to promote normal growth and development. To determine whether increased osteoclastic activity was the cause of the hypercalcemia, we monitored the levels of urinary pyridinium cross-links as the analog was infused. These remained within the normal range in all cases, and the osteoclastic inhibitor pamidronate did not have any beneficial effect.

Hypercalcemia is sometimes a feature of sarcoidosis caused by synthesis of 1,25-(OH)2D3 in granulomatous tissue. When steroids are administered, the hypercalcemia and hypercalciuria both return to normal (22). There is evidence that nonrenal 1-{alpha}-hydroxylation is reduced by steroid treatment, probably because of an antiinflammatory effect on the relevant cells. To test whether the excess 1,25-(OH)2D may have been formed extrarenally, three patients were given dexamethasone. This proved ineffective, suggesting that the elevated levels of 1,25-(OH)2D were likely to be caused by renal 1-{alpha}-hydroxylation.

In summary, 8-Cl-cAMP has a PTH-like effect causing increased synthesis of the active metabolite of vitamin D leading to hypercalcemia. The markedly elevated levels of 1,25-(OH)2D may result in increased net renal calcium absorption, but it is likely to have a much greater effect on the intestine to promote calcium uptake. Because 1,25-(OH)2D itself has been shown to cause tumor cell differentiation (23), increased synthesis of this metabolite by 8-Cl-cAMP may provide further therapeutic advantage in the treatment of cancer if the hypercalcemia can be controlled.

A recent report on active mutations of the PTH-PTHrP receptor in a patient with Jansen-type metaphyseal chondrodysplasia, showed accumulation of cAMP in cells possessing the mutant receptor (24). The authors suggest that this may explain the profound hypercalcemia seen in this rare form of short-limbed dwarfism. 8-Cl-cAMP may therefore cause hypercalcemia directly via exogenous activation of this pathway, as well as indirectly via production of 1,25-(OH)2D.

It was rather surprising that 8-Cl-cAMP was able to regulate one specific cAMP pathway, whereas it did not appear to affect other pathways controlled by cAMP such as cortisol and TSH. This demonstrates that there may be marked tissue specificity in the responsiveness to cAMP analogs.

It is possible that 8-Cl-cAMP could gain a role in the treatment of a range of metabolic disorders that are treated with oral vitamin D metabolites and/or calcium supplementation such as hypoparathyroidism, pseudohypoparathyroidism (type 1), and vitamin D-dependent rickets (type 1). Presently however, 8-Cl-cAMP is only available intravenously, and its effects varies between patients. Unless these can be controlled and there is a distinct advantage over the standard treatment modalities, this analog should not replace vitamin D.

Recently it has been shown that tumor cells with high RI levels are differentially sensitive to topoisomerase II inhibitors (e.g. adriamycin and etoposide) as well as 8-Cl-cAMP (25). Thus roles for this analog may either be as a single agent, or as a short, relatively nontoxic infusion between courses of chemotherapy. It may also be possible to design drugs with different tissue profiles of cAMP modulation to provide specific biochemical end points.


   Acknowledgments
 
The following assays were kindly performed by the following individuals. Pyridinium cross-links, Dr. J. L. Baron, Department of Clinical Pathology, St. Helier Hospital, Carshalton, Surrey, UK; PTHrP, Dr. W. Ratcliffe, Department of Clinical Chemistry, Queen Elizabeth Medical Centre, Birmingham, UK; PTH, Dr. R. John, Department of Medical Biochemistry, University Hospital of Wales, Cardiff, UK; and cortisol and TSH, Dr. M. Dowsett, Academic Biochemistry, Royal Marsden Hospital, London, UK.

Received November 11, 1996.

Revised March 20, 1997.

Revised August 13, 1997.

Accepted August 26, 1997.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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